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The role of the inositol phosphatase, SHIP, in the innate immune response to Salmonella Typhimurium Bishop, Jennifer L. 2008

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THE ROLE OF THE INOSITOL PHOSPHATASE, SHIP, IN THE INNATE IMMUNE RESPONSE TO SALMONELLA TYPHIMURIUM  by JENNIFER L. BISHOP B.A. Colby College, 2002  THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August, 2008 ©Jennifer L. Bishop, 2008  ABSTRACT The SH2 domain-containing inositol 5’-phosphatase, SHIP, negatively regulates hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remained unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The focus of this work was to determine the role of SHIP in a murine model of systemic Salmonellosis. Susceptibility of ship+/+and ship-/- mice to S. enterica serovar Typhimurium infection was compared. ship-/- mice displayed an increased susceptibility to both oral and intraperitoneal S. Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship+/+mice, indicating a role for SHIP in the gut and systemic pathogenesis of S. Typhimurium in vivo. Blood cytokine levels showed that infected ship-/- mice produce lower levels of Th1 polarizing cytokines compared to ship+/+ animals, and analysis of supernatants taken from M2 bone marrow derived macrophages correlated with this data. M2 macrophages were the predominant population in vivo during both oral and intraperitoneal infections. Because M2 macrophages are poor defenders against bacterial infection, these data suggest that M2 macrophage skewing in ship-/- mice contributes to ineffective clearance of Salmonella. The role of SHIP in the gut during enteric infections was also explored. ship-/- mice were not susceptible to Citrobacter rodentium infection, yet  ii  developed severe inflammation of the ileum upon infection with this bacterium, with Salmonella, or when challenged orally with LPS. Increased collagen deposition was also observed at early time points post-infection, suggesting that ship-/- mice may be used to study the development of inflammatory bowel diseases characterized by fibrosis, such as Crohn's. Because SHIP is such a critical negative regulator in both innate and adaptive immune cells, it has the potential to significantly alter the outcome of infections. This work highlights the fact that SHIP is important in vivo during Salmonellosis and opens new avenues to explore targeting SHIP in therapies for both systemic infections as well as inflammatory bowel diseases.  iii  TABLE OF CONTENTS Abstract............................................................................................................... ii Table of Contents .............................................................................................. iv List of Tables .................................................................................................... vii List of Figures.................................................................................................. viii List of Abbreviations......................................................................................... ix Acknowledgements........................................................................................... xi Dedication ........................................................................................................ xiv Chapter 1: INTRODUCTION ............................................................................... 1 1.1 HISTORY AND EPIDEMIOLOGY OF SALMONELLOSES.................. 2 1.2 TYPHOID INFECTIONS AND TREATMENT OPTIONS...................... 5 1.3 ANIMAL MODELS OF SALMONELLOSES ......................................... 7 1.4 SALMONELLA PATHOGENESIS IN MURINE TYPHOID ................... 8 1.5 IMMUNITY AGAINST SALMONELLA IN MURINE TYPHOID ........... 12 1.6 NEGATIVE REGULATION OF IMMUNE RESPONSES.................... 14 1.7 REGULATION OF PI3K BY SHIP...................................................... 16 1.8 SHIP BIOCHEMISTRY AND INTERACTORS ................................... 18 1.9 THE SHIP KNOCKOUT MOUSE....................................................... 19 1.10 THE FUNCTION OF SHIP IN INNATE IMMUNE CELLS ................ 20 1.11 THE POTENTIAL ROLE OF SHIP IN IMMUNE RESPONSES TO SALMONELLA................................................................................... 22 1.12 RATIONALE .................................................................................... 23 CHAPTER 2: MATERIALS AND METHODS .................................................... 25 2.1 BACTERIAL CULTURE AND PREPARATION.................................. 26 2.1.1 Growth conditions ................................................................ 26 2.1.2 Opsonization ........................................................................ 26 2.1.3 Bacterial killing ..................................................................... 26 2.1.4 Preparation for infection ....................................................... 26 2.2 IN VIVO INFECTIONS....................................................................... 27 2.2.1 Mice ..................................................................................... 27 2.2.2 Salmonella Typhimurium infections...................................... 27 2.2.3 Citrobacter rodentium infections........................................... 27 2.3 ENUMERATION OF BACTERIAL LOAD FROM INFECTED MICE... 27 2.4 IN VIVO CYTOKINE ANALYSES ...................................................... 28 2.5 IMMUNOHISTOCHEMISTRY............................................................ 29 2.5.1 Preparation of tissues .......................................................... 29 2.5.2. Immunohistochemistry for M2 macrophages ...................... 30 2.5.3 Pathology scoring................................................................. 30 2.6 WESTERN ANALYSIS ...................................................................... 30 2.6.1 Ex-vivo cell preparation........................................................ 30 2.6.2 SDS-PAGE and Western blotting......................................... 30 2.7 FLOW CYTOMETRY......................................................................... 31 2.7.1 Lymphocyte isolation............................................................ 31 2.7.2 Lymphocyte staining for flow cytometry ............................... 31  iv  2.8 MACROPHAGE TISSUE CULTURE ................................................. 32 2.8.1 BMDMs ................................................................................ 32 2.8.2 Raw 264.7 macrophages ..................................................... 32 2.9 IN VITRO S. TYPHIMURIUM INFECTIONS ...................................... 33 2.9.1 Preparation of BMDMs......................................................... 33 2.9.2 Preparation of Raw 264.7 macrophages.............................. 33 2.9.3 Salmonella Typhimurium infections...................................... 33 2.10 GENTAMICIN PROTECTION ASSAY............................................. 33 2.11 CELL DEATH ASSAYS ................................................................... 34 2.12 CYTOKINE ANALYSES ............................................................................. 34 2.13 COLLAGEN ASSAYS................................................................................. 34 2.14 STATISTICAL ANALYSES ......................................................................... 34 CHAPTER 3: THE ROLE OF SHIP IN S. TYPHIMURIUM INFECTIONS IN VIVO .............................................................................................................. 36 3.1 INTRODUCTION ............................................................................... 37 3.2 RESULTS .......................................................................................... 38 3.2.1 SHIP controls susceptibility to Salmonella infection in vivo.. 38 3.2.2 Increased susceptibility is associated with high bacteria load in ship-/- mice................................................................................. 40 3.2.3 ship-/- mice produce levels of inflammatory cytokines typical of M2 macrophages during Salmonella infection .............................. 43 3.2.4 M2 macrophage skewing is associated with increased susceptibility to Salmonella in ship-/- mice..................................... 47 3.2.5 Leukocyte distribution in ship-/- mice .................................... 52 3.3 DISCUSSION .................................................................................... 55 CHAPTER 4: THE ROLE OF SHIP IN S. TYPHIMURIUM INFECTION OF MACROPHAGES IN VITRO .............................................................................. 59 4.1 INTRODUCTION ............................................................................... 60 4.2 RESULTS .......................................................................................... 61 4.2.1 Intracellular Salmonella replication in macrophages is independent of SHIP expression or macrophage phenotype........ 61 4.2.2 M2 skewing of BMDMs provides a model for macrophage function in oral Salmonella infection in ship-/- mice ........................ 64 4.2.3 SHIP deficiency does not influence cell death in vitro during Salmonella infection ...................................................................... 67 4.3. DISCUSSION ................................................................................... 68 CHAPTER 5: THE ROLE OF SHIP IN THE INTESTINAL INFLAMMATORY RESPONSE TO ENTERIC INFECTION............................................................. 70 5.1 INTRODUCTION ............................................................................... 71 5.2 RESULTS .......................................................................................... 72 5.2.1 Salmonella infected ship-/- mice have highly inflamed small intestines....................................................................................... 72  v  5.2.2 Fibrosis is a consequence of inflammation induced during Salmonella infection in the small intestine of ship-/- mice............... 76 5.2.3 Development of inflammation and fibrosis in the illeum of ship/mice is not specific to Salmonella infection................................. 79 5.3 DISCUSSION .................................................................................... 90 CHAPTER 6: DISCUSSION............................................................................... 94 6.1 REGULATION OF ANTI-SALMONELLA IMMUNITY BY SHIP .................... 95 6.2 THE ROLE OF SHIP IN SYSTEMIC SALMONELLOSIS9 ............................. 6 6.3 THE ROLE OF SHIP IN ENDOTOXIN TOLERANCE DURING SYSTEMIC SALMONELLOSIS ............................................................................................. 98 6.4 THE ROLE OF SHIP IN GUT IMMUNOLOGY ............................................. 99 6.5 FUTURE DIRECTIONS.............................................................................. 101 6.6 SIGNIFICANCE .......................................................................................... 103 6.7 CONCLUDING REMARKS......................................................................... 104 REFERENCES ................................................................................................. 105 Appendix 1 Thesis material included in publications....................................... 120 Appendix 2 Contribution of others................................................................... 121 Appendix 3 Publications arising from graduate work....................................... 122 Appendix 4 Animal ethical approvals .............................................................. 124 Appendix 5 Pathology scoring worksheet ....................................................... 125  vi  LIST OF TABLES Table 1 Salmonella secreted effectors and their function in host cells ............... 10 Table 2 Cytokines required for controlling Salmonella infection in mice............. 14 Table 3 Regulation of immune cell functions by SHIP ........................................ 22 Table 4 Antibodies and dyes used in studies ..................................................... 29 Table 5 Statistical Significance of CFU values in ship-/- vs. ship+/+ organs after IP Salmonella infection ........................................................................................... 40 Table 6 Statistical changes of lymphocyte distribution in the spleens of ship-/- vs. ship+/+ mice......................................................................................................... 53 Table 7 Statistical changes of lymphocyte distribution in the MLN of ship-/- vs. ship+/+ mice......................................................................................................... 53 Table 8 Similarities and differences between intestinal inflammation seen in the ship-/- mouse compared to IBD ........................................................................... 93  vii  LIST OF FIGURES Figure 1 History and treatment of salmonellosis................................................... 3 Figure 2 Immune responses to S. Typhimurium in vivo...................................... 11 Figure 3 PI3K signaling and regulation by SHIP................................................. 16 Figure 4 SHIP family proteins............................................................................. 18 Figure 5 SHIP controls susceptibility to Salmonella in vivo ................................ 39 Figure 6 Increased susceptibility to Salmonella is associated with higher bacterial loads in organs of orally infected mice................................................................ 41 Figure 7 Increased susceptibility to Salmonella is associated with higher bacterial loads in organs of IP infected mice ........................................... 42 Figure 8 ship+/+ and ship-/- mice are equally susceptible to colonization by Citrobacter rodentium .................................................................................... 43 Figure 9 SHIP deficiency leads to altered levels of inflammatory cytokine production after oral Salmonella infection in vivo ................................. 45 Figure 10 SHIP deficiency leads to altered levels of inflammatory cytokine production after IP Salmonella infection in vivo .................................................. 46 Figure 11 Cytokine production in uninfected ship-/- and ship+/+ mice................... 47 Figure 12 M2 macrophage markers are found in the gut and peritoneal cavity of ship-/- mice ............................................................................................ 49 Figure 13 Leukoocyte distribution in Salmonella infected ship+/+ and ship-/mice.................................................................................................................... 54 Figure 14 Fold changes in number of S. Typhimurium in M1 vs. M2 macrophages...................................................................................................... 63 Figure 15Salmonella infected BMDMs from ship-/- mice derived under M2 inducing conditions show decreased levels of inflammatory cytokines compared to ship+/+ cells ..................................................................................................... 65 Figure 16 Salmonella infected BMDMs from ship-/- mice derived under M1 inducing conditions show increased levels of inflammatory cytokines compared to ship+/+ cells ......................................................................................................... 66 Figure 17 ship-/- BMDMs are not more susceptible to death in vitro upon infection with Salmonella .................................................................................................. 67 Figure 18 Salmonella infection in ship-/- mice leads to inflammation of the ileum................................................................................................................... 74 Figure 19 TNFα and MCP-1 are increased in ship-/- small intestines during Salmonella infection ........................................................................................... 76 Figure 20 Fibrosis is a consequence of small intestinal inflammation in ship-/- ilea during Salmonella infection ................................................................ 78 Figure 21 Helicobacter infection is not a prerequisite of Salmonella induced intestinal inflammation .......................................................................... 81 Figure 22 Citrobacter rodentium induces intestinal inflammation in ship-/-mice ........................................................................................................... 84 Figure 23 Heat-killed bacteria and LPS can induce intestinal inflammation in ship-/- mice .......................................................................................................... 86  viii  LIST OF ABBREVIATIONS 7AAD apc  APC BPI BMDM CAMP CBA CD CTGF DAPI DC DMEM ECL EDTA ELISA FACS FBS FITC GMCSF HBSS HEPES HRP IBD IF IFN IHC IL IP LB LPS Mφ MCP MCSF MLN MOSF NGS NK NO NTS OMP PAMP PBS PE PFA  7-amino-actinomycin D  allophycocyanin (flow cytometery) antigen presenting cell bacterial permeability inducing protein  bone marrow-derived macrophage cationic antimicrobial peptide cyotmetric bead array Crohn's disease collagen transforming growth factor 4', 6-diamidino-2-phenylindole dendritic cell Dulbecco's Modified Eagle Medium enhanced chemiluminescence ethylenediamine tetraacetic acid enzyme-linked immunoabsorbant assay fluorescence activated cell sorting fetal bovine serum fluorescein isothiocyanate granulocyte colony stimulating factor Hank's balanced salt solution 4-(2-hydroxyethyl1)-1-piperazineethanesulfonic acid horseradish peroxidase inflammatory bowel disease immunofluorescence interferon immunohistochemistry interleukin intraperitoneal Luria-Bertani lipopolysaccharide macrophage monocyte chemotactic protein monoctye colony stimulating factor mesenteric lymph nodes multiple system organ failure normal goat serum natural killer nitric oxide non-typhoidal Salmonella outer membrane protein pathogen-associated molecular pattern phosphate buffered saline  phycoerythrin paraformaldehyde ix  PI3K PIP PMN PRR PTEN RADIL ROS RNS SA SCV SDS SDS-PAGE SH2 SHIP SI SPI TLR TGF TNF TTSS UC WB  phosphytidylinositol 3' kinase phosphytidylinositol polymorphonuclear cell pattern recognition receptor phosphatase and tensin homologue deleted chromosome 10 Research Animal Diagnostic Laboratory reactive oxygen species reactive nitrogen species streptavidin Salmonella-containing vacuole sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis src homology 2 src homology 2 domain-containing 5'inositol phosphatase small intestine Salmonella pathogenicity island toll-like receptor transforming growth factor tumor necrosis factor type three secretion system ulcerative colitis Western blot  x  ACKNOWLEDGEMENTS First and foremost, I'd like to thank my supervisor, B. Brett Finlay for giving me the opportunity to be a part of such a fantastic laboratory and the scientific independence that has made my Ph.D. experience so fulfilling. In addition, I would not have had an ounce of success in this project without the constant guidance, patience and experience of my collaborator in Gerald Krystal's laboratory, Laura Sly, who has acted as my second supervisor and mentor for the last few years. My committee members, Yossef Av-Gay, Rachel Fernandez and Ken Harder have also been an integral part of my training and for all their constructive criticism and support I am grateful. Members of the Finlay lab have been my family for the last five years and no words can express the amount of gratitude and love I have for everyone that has worked at these many benches since I started in 2003. I would especially like to thank Marilyn Robertson and Theresa Charlton for keeping my head out of the clouds and organized, and all the members of Salmonella group that I have relied so heavily upon for scientific guidance for every aspect of my project. In addition, I have special words of thanks for people that have been my best friends and mentors for over the years: Erin Boyle- Since my first day in the lab I've looked up to you as an example to be followed. Every day we shared a bay, I admired you more as a friend and a scientist, I'd be lost in this big lab without you and I definitely wouldn't have laughed so much! Here's another quote for you… "More than you came for, at the bay!" Guntram Grassl- The eternal optimist in all aspects of life; your positive attitude and scientific insight are infectious and permeate everything you do. You have the friendliest, kindest heart around and even found it in you to love our scary little dog and take her hiking. I can't thank you enough! Mark Wickham and Nat Brown- My Aussie friends. You both welcomed me into the lab and took me under your wing when I was an insecure student and always had the time and patience to teach me science, wax on about the world and my worries and most importantly, have a beer (or two). I miss you terribly and wish you could be here for after the defense, but your spirit is everywhere! Olivia Champion- Having you join the lab was the most refreshing thing that's happened to us since I started. You were the best storyteller, a great friend, funny as hell and at the same time a wonderwoman of a mom and scientist. I admire you in so many ways and miss you every day.  xi  Amit Bhavsar- Conscientious, meticulous and hilarious, you are a genuine, and at times, shockingly honest (or just plain shocking) friend. What else can I say? More post-doc than a girl could ask for. Shelley Small: Without you, I wouldn't be at UBC. You helped me navigate the wonders of the international student application process, supported us every step of the way to finding our little girl, Ella, cared for her like she was your own and even helped our little family through an appendectomy! You are a fierce friend and I'm so thankful to have you in my life. And of course, my deepest thanks go to my family. I love you all so, so much. To my Parents: you both instilled in me the self-confidence I needed to push myself towards my goals and fulfill my dreams. Your history together inspired me to go abroad and live in this new city that has been the foundation for my career, marriage and the real start of my adult life. As individuals, you have passed down to me your best qualities that have helped me succeed in this endeavor. Dad, you are the source of my desire to learn and to think deeply and critically about the world we live in. You have taught me to articulate my ideas and my emotions while remembering to never take myself too seriously. Mom, you have instilled in me a devotion to family and faith that has carried me through the hardest times. Your renewed sense of self and accomplishment in your career has paved the way for me to never compromise between my goals and my family and your poise has prepared me for all of those hours of interviews and talks and I've tried my best to emulate it whenever I could. To my siblings, thank you for understanding that I had to go so far to do so much. Being removed from home I know that I've left some burdens for you to carry that have been heavier in my absence. But my time away has helped me realize what amazing individuals each of you are becoming and what qualities you each have that I admire and wish I had. Elyse, you have been there for me every step of the way and for your efforts to keep me close, I can't thank you enough. You've been involved in every aspect of my life here-from our first homesick summer in Vancouver, to our wedding, to my recent interviews at the end of my degree-and it's made every moment so much more special. Hillary, you have kept me on my toes and helped me to remember what it feels like to be in those exciting stages of deciding on your career. Your exuberance and the passion you attack every personal or academic obstacle you face is amazing and I'm channeling that energy now to propel me to the end. Brendan, the transformation I've witnessed in you from afar since being here is tremendous. You've changed from a timid child to an independent young guy who is traveling the world on ski vacations and planning his college career! You've also tried so hard to keep in contact and have kept me grounded to the childhood that I cherished so much. I can't wait to see where the road leads for you!  xii  To Les, Carol, Marcus and Nick: It takes a huge act of selflessness to see your child or brother leave home and travel so far to help their partner pursue their dreams. You have always supported Travis and I and our relationship unconditionally and that is something very few people in the world have experienced in their in-laws. For that, I am forever grateful. You are the most funloving people I know and I am honored that you have welcomed me into your family with such open hearts. I hope that my endeavors have made you proud of me, and even more of Travis, who is the foundation for everything we have built here.  xiii  DEDICATION You may not ever read past this page (actually I'm not sure that you'd want to!), but Travis, there was no question as to who this monster of a document would be dedicated to. Giving you a page in this work may not seem so substantial, but besides our love and life together, I've never wanted to succeed more at anything than this degree. And without you, not one single experiment could have been done, not one single word written. I have no idea how people without such an amazing spouse find the strength to work on their dissertations every day, sifting through piles of paperwork, lab notes scribbled on paper towels and experiments gone wrong. It was knowing that in a few hours I'd be with you, surrounded by your laughter and love that made me come back here each day. That and the promise I've made to you in my heart of accomplishing something that will make our future together that much brighter. And even though LandSea or Cypress seems a far cry from slogging through mouse guts, hearing and learning about your profession has taught me so much that underscored everything other people have tried to teach me in the lab. Most importantly, I've tried to learn the value of being meticulous, thorough and unyielding about the quality of my work from you. The diligence you meet every obstacle with at work or at home is something I will always admire and strive to find in myself. Every step along the way you have been unconditionally supportive of my hopes and dreams-from the first mention of moving out of New England for graduate school to helping me work my head around the many options for jobs to come in the future. That support has extended far beyond work however- in our personal life that we've made here in Vancouver you've been nothing short of a superman when it comes to fulfilling my heart's greatest desires. From building a hearth and home in our little hobbit hole, to the most romantic engagement and marriage a woman could ask for and then extending your love for me to our little Ella, you have succeeded beyond any measure imaginable. So I guess I don't really know what else to say except that somehow, someday, I hope that I can be a wife worthy of the husband you have been to me throughout the years I've worked on my Ph.D. You are the most selfless, loving and funniest man in my life and always will be. I, like this thesis, am dedicated to you. EY, Jenna  xiv  CHAPTER 1: INTRODUCTION  1  1.1 History and epidemiology of salmonelloses Infectious diseases have played a central role in the development of human civilizations since ancient times. Infamous epidemics of plague, smallpox, cholera and influenza have tipped the balance of economies, militaries, politics and populations at the most critical points in world history. In an era of medical miracles and scientific innovation, it is easily forgotten what a profound effect infectious diseases have in the global community. While AIDS often takes center stage as the world's most serious health crisis, other communicable diseases, like enteric fevers and food-borne illness, cause severe morbidity and mortality in millions of people on a daily basis. Enteric fevers are caused by the human-specific pathogen Salmonella enterica serovars Typhi and Paratyphi. Exposure via contact with contaminated water and food leads to systemic dissemination of bacteria and resulting disease characterized by high fever, general malaise and flu-like symptoms. During the 19 and 20th centuries, typhoid fever was a common household illness found in every corner of the globe and was often indistinguishable from other sicknesses with similar symptoms, such as malaria, dengue fever and typhus (Mastroeni and Maskell, 2006). However, with the advancement of microbiological techniques in the late 1800's, contagions and their sources were identified and detailed studies of Salmonella bacteriology and treatment options were explored (Fig. 1).  2  Figure 1. History and treatment of salmonellosis  US Army Typhoid Commission ID’s fecaloral transmission  WHO begins whole cell vaccine efficacy testing  Eberth discovers typhoid bacillus  Alternative vaccine development and trials  Floroquinolone use against MDR strains  Mary Mallon spreads typhoid in NYC  1873 1880  1896 1898  1948 1906  1972 1960 1980’s-90’s  2008 2000  Introduction of chloramphenicol as treatment William Budd postulates contagious nature of typhoid  MDR strains emerge  First inactive whole cell vaccines in use  Chloramphenicol resistance emerges  Single dose oral vaccine field trials underway  3  Today, enteric fevers are most common in the developing world; typhoid is endemic in Asia and Africa, and is a frequent cause of disease in the Middle East, South America and even parts of Southern and Eastern Europe (Mastroeni and Maskell, 2006). Globally, there are an estimated 22 million cases of typhoid fever each year and roughly 10% of these cases are fatal (Crump et al., 2004). High as these numbers are, they do not include the billions of individuals affected by non-typhoidal salmonellosis (NTS), a self-limiting gastroenteritis typically associated with food poisoning. Like typhoid fever, NTS is spread via the fecaloral route but is caused by infection with various S. enterica serovars. These include Typhimurium, Enteriditis, Newport and Heidelberg, all of which have reservoirs in animal populations used for agriculture (Hohmann, 2001; Rabsch et al., 2001). As such, these pathogens are among the most important linked to food-related deaths (Kennedy et al., 2004). Estimates for numbers of NTS cases worldwide vastly exceed those for enteric fevers; there are 1.3 billion cases annually, 300,000 of which are fatal (Pang et al., 1995). While a significant amount of cases are still seen in developed countries like the US, Africa bears the brunt of this burden. For example, NTS is the most common cause of bacteremia in children in Africa, and NTS infection rates can reach up to 60% of HIV/AIDS patients there (Gordon MA, 2004; Graham, 2002). Both typhoid fever and NTS are contracted by ingestion of S. enterica serovars present in contaminated food or water sources. While NTS associated serovars are typically introduced to meat and dairy products from their hosts during processing, S.Typhi is shed in feces or urine from people suffering from typhoid fever, or chronic carriers that show no symptoms of disease. Most S. enterica are quite robust in the environment, being able to survive even in ice, dust or raw sewage for prolonged periods of time (Mastroeni and Maskell, 2006). As such, risk factors for contracting both enteric fevers and NTS are much the same as those identified in the late 20th century-poor hygiene and limited access to sanitized food and water are the main prerequisites for infection. More recent studies have shown that other socio-economic and environmental factors, such  4  as poor housing, inadequate food, and rainfall amounts correlate with disease (Gasem et al., 2001; Luby et al., 1998). 1.2 Typhoid infections and treatment options Infectious doses of S. enterica leading to NTS or typhoid fever have a wide range; ingestion of anywhere from 10-100 bacteria has been associated with food poisoning outbreaks and typically 103-109 S. Typhi are sufficient to cause fever (Mastroeni and Maskell, 2006). Bacteria are swallowed and many resist the harsh environment of the stomach to adhere to, and invade, the epithelium of the small intestine. At this point, the systemic nature of typhoid becomes apparent and distinguished from most NTS infections, as bacteria disseminate to lymphoid follicles, mesenteric lymph nodes (MLN) and liver within phagocytes of the immune system such as macrophages and dendritic cells (DC). After an incubation period of 7-14 days, secondary bacteremia develops and infection spreads to other organs such as the bone marrow and gall bladder (House et al., 2001). During this time, infected patients will develop the common symptoms associated with febrile diseases, such as high fever, chills, malaise and cough. However, if left undiagnosed or untreated, typhoid can progress and serious complications can result, such as gastrointestinal bleeding or perforation, shock, coma, meningitis and encephalitis (Mastroeni and Maskell, 2006). Even without complications, recovery from untreated typhoid can take up to four months and relapses are not uncommon. It has been estimated that up to 10% of patients suffer relapsed infections and between 1-4% of typhoid sufferers will become chronic carriers of the bacteria for up to one year (Caygill et al., 1994; Marmion et al., 1953). Carriage is a serious complication of typhoid infection not only because of the increased risk for transmission, but also because it predisposes patients to bowel, gall bladder and pancreatic cancers (Caygill et al., 1994). Diagnosis and treatment of typhoid, especially in endemic areas, are difficult for a number of reasons. Firstly, typhoid is not easily distinguished from other diseases common in endemic areas, such as dengue, malaria or  5  leishmania, except via blood culture of S. Typhi. This requires that patients have access to timely diagnostic laboratory tests, which may not be readily available (Butler et al., 1978). In addition, serological tests may not prove that a patient is currently infected with S. Typhi; the presence of anti-S. Typhi antibodies from prior infections, vaccinations or cross-reactive epitopes can easily skew the tests towards a false-positive result (Parry et al., 1999). If properly diagnosed, treatment with classic antibiotic regimens is becoming increasingly difficult since resistance to many of the drugs once used to combat typhoid, such as choloramphenicol, amoxicillin and ampiciliin, is widespread (Fig. 1). However, with careful assessment of resistance on a regional level, classic antibiotics and fluoroquinolones can still be effective against susceptible bacteria. In fact, fluoroquinolone treatment can cure up to 96% of typhoid cases and reduce carriage to lower than 2% (Gulig et al., 1993; Parry, 2004). With increasing resistance to fluoroquinolones and limited access to clean water, as well as sanitary agriculture and animal husbandry practices  in  developing nations, it is essential that more efforts go towards prevention rather than treatment of Salmonelloses. Vaccinology therefore, has been a prime target for controlling the spread of Salmonella in both humans and livestock. Whole cell inactivated vaccines against typhoid have been used in humans since the late 1800's (Fig. 1) but their deleterious side-effects prevented widespread use (Engels et al., 1998). Since then, development of more efficacious methods has been an ongoing struggle because i) we have only limited knowledge of immunity and memory to systemic typhoid, ii) many vaccine candidate mutants are either under or over-attenuated, iii) there is little cross-protection for one vaccine against other serovars and strains causing enteric fevers and NTS, and iv) economic pressures often prevent the implementation of vaccine programs (Boyle et al., 2007). Despite these obstacles, two typhoid vaccines have been licensed, the Ty21A live-attenuated vaccine and the Vi antigen subunit vaccine. Because efficacy of these vaccines ranges from 42-92% and they work on a multiple dosing regimen, projects to develop better vaccines are ongoing (Mastroeni and Maskell, 2006). Most promising is a live attenuated ΔaroC/ssaV  6  oral vaccine that can be administered in a single dose. This vaccine has been trialed successfully in the west and the positive results are being verified in both adults and children living in typhoid endemic areas (Boyle et al., 2007; Stratford, 2005). 1.3 Animal models of salmonelloses Because S. Typhi is host-restricted to humans, an animal model system to study typhoid fever must utilize other S. enterica species that have a broader host range yet cause similar symptoms of disease. Why host restriction occurs is a matter of debate, but factors such as host environment as well as genetics of the host and infecting strain most likely play key roles (Mastroeni and Maskell, 2006). Conveniently, S. Typhimurium, one of the major strains associated with NTS, can cause systemic disease in mice that mimics human typhoid. Depending on the expression of the Slc11a1 (Nramp1) gene, mice are either genetically resistant or susceptible to systemic S. Typhimurium infection, allowing researchers to study both acute and persistent forms of the disease (Blackwell et al., 2001). This animal model has been an invaluable tool for the study of Salmonella pathogenesis and the immune responses required to clear infection. The classic murine model of S. Typhimurium infection mimics the systemic disease seen in human typhoid fever and is not a model for the gastroenteritis associated with food poisoning or NTS infections. Bacterial dissemination does not induce enterocolitis or diarrhea in mice, but the animals exhibit classic inflammation at infection foci throughout the body (Santos et al., 2001). To study intestinal disease in relation to S. Typhimurium, two models are commonly used. The first is the more classic model where calves infected with S. Typhimurium develop enterocolitis that closely resembles human disease (Santos et al., 2001). More recently however, a new S. Typhimurium murine model has been developed that also mimics Salmonella induced human enterocolitis. It has been shown that mice treated with the antibiotic streptomycin prior to infection with S. Typhimurium develop severe intestinal pathology, characterized by edema and infiltration of inflammatory cells. While classical diarrhea is still not induced in  7  streptomycin-treated mice, and inflammation is limited to the caecum and colon compared to the small intestinal inflammation seen in humans, it is a large step forward in the search for more relevant animal models used to study Salmonella pathogenesis (Barthel et al., 2003). 1.4 Salmonella pathogenesis in murine typhoid Using the mouse model of systemic typhoid, we have been able to discern specific events key to the pathogenesis of Salmonella and the immune response to infection. As in humans, ingestion of bacteria allows for interaction with intestinal epithelium. Phagocytic M cells located in the Peyer's patches of the small intestine are seen as the canonical method of entry by which Salmonella penetrates the gut to gain entry to systemic sites (Jepson and Clark, 2001). As well, Salmonella can invade enterocytes and be sampled from the lumen by DCs (Rescigno et al., 2001). Systemic spread occurs as S. Typhimurium survives within macrophages (Salcedo, 2001), DCs (Richter-Dahlfors et al., 1997), polymorphonuclear cells (PMNs) (Sheppard et al., 2003), and B Cells (Yrlid et al., 2001) in various organs such as the MLN, spleen and liver. Contradictory to many observations of S. Typhimurium replication in cell culture systems, Salmonella does not replicate to high numbers per cell in vivo and infection foci grow in number rather than in sheer size (Vazquez-Torres et al., 1999). Furthermore it is clear that at late time points in infection Salmonella can exist in the blood in CD18+ cells (Richter-Dahlfors et al., 1997; Sheppard et al., 2003) or extracellularly. Depending on the dose used in the infection and the susceptibility of the mouse strain, immune responses will dictate whether systemic bacteria are cleared or replicate to high enough numbers to cause bacteremia and death (Fig. 2). During the invasion of the gut and spread to systemic sites, Salmonella uses a variety of virulence factors for pathogenesis. While virulence genes can be found anywhere in the genome, in Salmonella they are mostly clustered on Pathogenicity Islands (SPI). Salmonella has 10 SPIs, the first two of which are most fully characterized in the typhoid infection model. Encoded on SPI-1 and 2  8  are type-three secretion systems (TTSS) that are essential for Salmonella pathogenesis. These protein complexes act as "molecular syringes" that inject bacterial encoded proteins, termed effectors, directly into host cells. These effectors modulate cell signaling that promotes either bacterial invasion of host cells (SPI-1 effectors) or intracellular survival and systemic spread of bacteria (SPI-2 effectors) (Gal-Mor and Finlay, 2006; Hensel, 2004). For example, SPI-1 encoded effectors are necessary to induce cytoskeletal rearrangements required for entry into non-phagocytic cells (Schlumberger and Hardt, 2006) and are only required for colonization of the intestinal tract, but not systemic spread, in the mouse typhoid model (Galan and Curtiss, 1989). In contrast, SPI-2 effectors are required for the maintenance of, and replication within, the Salmonella Containing Vacuole (SCV), the specialized compartment Salmonella form when phagocytosed that prevents degradation by the normal endocytic pathway (Galan, 2001) . In addition, effectors of both SPI-1 and SPI-2 encoded TTSS are essential for modulating host immune responses against Salmonella (Table 1).  9  Table 1. Salmonella secreted effectors and their function in host cells Location SPI-1  Effector Target Cell SipA Epithelial  SPI-1  SipB  Epithelial PMN MΦ  Chrom.  SopA  Epithelial PMN  SPI-5  SopB (SigD)  Epithelial PMN MΦ  Chrom. Phage  SopD/E/ E2  Epithelial PMN  SPI-1  SptP  Epithelial  Gifsy-3 Prophage Chrom. SPI-2 SPI-2 Chrom.  Ssph1  Epithelial  SifA SseF SseG PipB2  MΦ Epithelial  SPI-2  SpiC  Phagocytes  Function Invasion Inflammation Invasion Recruitment Apoptosis, increased IL-1β Invasion Recruitment  Reference (Zhang et al., 2003) (Monack et al., 2000)  (Raffatellu et al., 2005; Wood et al., 2000; Zhang et al., 2006) Invasion (Dukes et al., Recruitment 2006; Prevents lysosomal fusion Raffatellu et al., 2005; Zhang et al., 2002; Zhou, 2001) Invasion (Bakshi et al., Recruitment 2000; Raffatellu et al., 2005; Zhang et al., 2002) Decreased IL-8 (Haraga and Miller, 2003) Decreased NF-κβ (Haraga and Miller, 2003) signaling Sif formation (Beuzon et al., SCV integrity 2000; Knodler Intracellular survival and SteeleMortimer, 2005; Kuhle and Hensel, 2002; Stein et al., 1996) Vesicular trafficking (Uchiya et al., Intracellular survival 1999)  10  Figure 2. Immune responses to S. Typhimurium in vivo. Modified from Salmonella infections : clinical, immunological and molecular aspects. Mastroeni and Maskell, 2006. Solid lines represent systemic CFU trends in Salmonella resistant mouse strains. From 0-5 days innate responses are responsible for initial decreases in bacterial load and controlling bacterial replication. Adaptive responses have a bacteriostatic effect between days 5-20. During relapses or chronic infections (dashed lines), antigen specific responses are required to reduce bacterial load. If any immune response is impaired, bacteria replicate to high nubers (dashed lines), leading to death (days 1-10 post-infection) or relapsed and chronic infections.  109  C F U  Uncontrolled Replication Death  Relapse  Innate Responses •Complement •ROS •CAMP  Chronic Infection Adaptive Innate Responses •Th1 Cytokines •RNS •Granuloma formation  104-6  1  5  10  15  Antigen Specific Responses •CD4+ T Cells •B Cell antibody production and APC function  20  25+  Time (days)  11  1.5 Immunity against Salmonella in murine typhoid Immunity against S. Typhimurium during murine typhoid infection can be separated into three distinct types of immune responses, innate, adaptive/innate and antigen-specific (Fig. 2). Innate immunity to Salmonella is required for initial bactericidal actions against colonizing and invading bacteria and is most important in the first days of infection. It begins immediately in the digestive tract with the production of non-specific humoral defenses such as cationic antimicrobial peptides (CAMPs) present in the saliva and gut, as well as opsonins of the complement cascade (Warren et al., 2002; Wehkamp et al., 2007). Once bacteria invade past the epithelium, phagocytes, especially macrophages and DCs, recognize Salmonella via interactions between their surface pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Importantly for Salmonella recognition is the interaction between CD14/TLR4 complexes on the host cell surface and bacterial lipopolysaccharide (LPS). This induces bacterial phagocytosis into the cell, as well as the production of various microbicides, such as acids, enzymes, reactive oxygen and nitrogen species (ROS/RNS), that degrade bacteria within the endocytic pathway (Rosenberger and Finlay, 2002; Vazquez-Torres et al., 2000a). While Salmonella have SPI-2 encoded mechanisms to defend against ROS as well as phago-lysosome fusion (Table 1), these molecules are essential for the rapid killing of bacteria by the cell and protection from infection in vivo (Mastroeni, 2000; Vazquez-Torres et al., 2000b). Central to the ability of phagocytes to initiate these innate responses against Salmonella in mice is the expression of the Slc11a1 (previously known as Nramp1) gene that codes for a divalent cation transporter that localizes to the membrane of phagosomes within macrophages, DCs and PMNs during Salmonella infections (Bellamy, 1999). While it is known that susceptibility of mice to typhoid infection is dependent upon the expression of the dominant Slc11a1 resistant allele, why this occurs is a matter of debate. This is because it is unknown whether the function of Slc11a1 is to change the availability of nutrients, such as iron and manganese, within the phagosome by moving ions in,  12  out, or in either direction (Forbes and Gros, 2003; Goswami et al., 2001; Kuhn et al., 1999). The expression of this transporter is essential for preventing the replication of Salmonella in vitro as well as in PMNs of the liver and spleen in the mouse model (Forbes and Gros, 2001; Richter-Dahlfors et al., 1997). In humans Slc11a1 expression does not seem to control susceptibility to typhoid infections (Dunstan et al., 2001), even though it is required to combat other intracellular pathogens such as Mycobacterium tuberculosis and Leishmania (Blackwell et al., 2001). Recognition of Salmonella by macrophages and other professional phagocytes is essential for the induction of the adaptive/innate immune response, which is responsible for bacteriostasis during murine typhoid infections (Fig. 2). The production of several cytokines by macrophages, DCs, PMNs and natural killer (NK) cells is the hallmark of this stage of immunity; these are required for controlling systemic bacterial replication as well as initiating later antigen-specific responses (Table 2). For example, while the classic proinflammatory mediator TNFα is required to restrict bacterial growth to infection foci within organs (Everest, 1998; Mastroeni, 1995) interleukins (IL) 12, 18 and 15 are essential for the production of IFNγ and the subsequent activation of macrophages to produce ROS and RNS (Kagaya et al., 1989). Elimination of any of these cytokines from the mouse during typhoid infection greatly increases susceptibility to Salmonella (Stoycheva and Murdjeva, 2004). Importantly, the cytokine milieu during Salmonella infection skews CD4+ T helper cells to a Th1 response and IL-2 production by these cells is critical to prevent disease (al-Ramadi et al., 2001; Mittrucker et al., 2002; Srinivasan et al., 2004). In addition, Th1 cells, along with CD8+ cytotoxic lymphocytes, modulate a variety of antigen-specific immune events central to the clearance of Salmonella from systemic sites, including B cell antibody production and isotype profile. B cells can make antibodies against various Salmonella antigens, including LPS, the capsular Vi polysaccharide, outer membrane proteins (OMPs), flagellin and heat shock proteins (Mastroeni, 2002). While B cells are classically known to play an important role in the memory response to secondary Salmonella  13  infections, they also act during initial infections as antigen presenting cells (APC), which is required for the development of Th1 responses (Mastroeni et al., 2000). Table 2. Cytokines required for controlling Salmonella infection in mice. Cytokine TNFα  Producing Cell MΦ  Function Inflammation Lesion formation Activation ROS, RNS production Lysosomal fusion  IFNγ  MΦ, NK, Neutrophils, T Cells  IL-12  MΦ, DC  IFNγ production RNS production  IL-18  MΦ  IL-15  DC  IFNγ production RNS production IFNγ production  IL-4  NK1.1 Regulatory T Cell  IL-10  MΦ  IL-6  MΦ, DC  IL-2  T Cells  Decrease MΦ bactericidal and cytokine responses Decrease MΦ function Enhances PMN killing of bacteria Final clearance from organs  Reference (Everest, 1998; Mastroeni, 1995) (Billiau, 1996; Foster et al., 2003; Jouanguy et al., 1999; Kagaya et al., 1989) (Koch F, 1996; Mastroeni et al., 1996; Mastroeni et al., 1998) (Mastroeni et al., 1999) (Eckmann and Kagnoff, 2001; Ferlazzo et al., 2004; Lucas et al., 2007) (Denich et al., 1993; Enomoto et al., 1997) (Arai, 1995) (Nadeau et al., 2002) (al-Ramadi et al., 2001)  1.6 Negative regulation of immune responses While the production of potent inflammatory mediators, such as TNFα, IFNγ and ROS, are essential in clearing Salmonella infections in humans and mice, severe effects occur if their production is not tightly regulated. Almost 50% of deaths seen in intensive care units in North America are the result of sepsis, which occurs when the intense production of pro-inflammatory products by  14  macrophages and other cell types in response to bacterial LPS leads to multiple system organ failure (MSOF) and death (West, 2002). Thus, it is clear why all cell types of the immune response have multiple regulatory mechanisms to restrict the duration and intensity of their signals during infections. These mechanisms are complex and involve interactions between immunoreceptors, kinases, phosphatases, ubiquitin ligases, adapter proteins and transcriptional regulators, all of which work together in concert to maintain immune homeostasis (Veillette et al., 2002). Besides protecting the host from shock, negative regulation of immune responses has also been shown to be pivotal in determining the outcome of pathogenesis for various infectious diseases. For example, the protein tyrosine phosphatase, SHP-1, is a pluripotent regulator of macrophage function that inhibits signaling from cytokine receptors, chemokine receptors, integrins and immunoreceptors, and it is a negative regulator of phagocytosis for both Neisseria gonorrhoeae and Streptococcus suis (Hauck, 1999; Segura, 2004). In addition, SHP-1 deficiency increases mouse resistance to Leishmania infection by increasing macrophage production of NO and inducing macrophage hypersensitivity to IFN-γ (Forget, 2001). In contrast however, it has been found that a lack of SHP-1 is associated with increased susceptibility in mice to viral infections of the central nervous system (Massa, 2002). In addition, loss of TLR regulators, like IRAK-M or A20, led to severe inflammatory disorders (Kobayashi et al., 2002; Lee et al., 2000). As such, it is clear that depending on the pathogen and immune response required to clear infections, negative regulators can have both positive and negative influences on the outcome of pathogenesis.  15  Figure 3. PI3K signaling and regulation by SHIP  PI3K PI-4,5-P2  PIP3  SHIP  PI-3,4-P2  PTEN  Survival and Proliferation Cytokine gene activation  Cytoskeletal rearrangement  1.7 Regulation of PI3K by SHIP The phosphoinositide 3-kinase (PI3K) is a key master regulator of signaling in innate immune cells. In response to various stimuli during infections, such as TLR or cytokine receptor ligation, this lipid kinase is immediately targeted to the cell membrane and transfers gamma-phosphate from ATP to phosphoinositides  at  the  3'  position,  creating  phosphatidylinositol-3,4,5  trisphosphate (PIP3, Fig. 3). The formation of PIP3 targets multiple signaling complexes that initiate cell activation. This activated state is characterized by cytoskeletal rearrangements, cytokine production and proliferation (Fig. 3), all of  16  which are essential in the control of Salmonella infection (Hazeki et al., 2007; Krystal, 2000). Because PIP3 is such a potent second messenger, there are two distinct mechanisms present in the cell to break the molecule down and thus negatively regulate its signaling capacity. One is the phosphatase and tensin homologue (PTEN), a 53 KDa lipid phosphatase that opposes the function of PI3K and breaks PIP3 down into PI-4,5-P2. In contrast, the SHIP family of src-homology 2 domain-containing (SH2) inositol phosphatases hydrolyze PIP3 into PI-3,4-P2 (Fig. 3) and also can break down inositol-1,3,4,5 tetrakisphosphate (IP4) (Krystal, 2000). PTEN is a classic tumor suppressor gene; up to 50% of human cancers, including prostate, breast and lymphomas, are associated with mutations in PTEN, and mice heterozygous for PTEN are prone to tumors. PTEN is thought to prevent malignancies by suppressing the PIP3-mediated activation of the antiapoptotic AKT pathway (Cantley and Neel, 1999; Suzuki et al., 1998). The SHIPs however, are less associated with cancers and have been implicated in controlling a wide array of functions in both innate and adaptive immune cells, which will be discussed in further detail below. The redundancy in function between the SHIPs and PTEN underscores the importance of regulating PIP3 signaling throughout the immune system.  17  Figure 4. SHIP family proteins. Modified from "The Role of SHIP in macrophages". Sly et al., 2007. A graphic representation of SHIP molecules and domain interactors. Amino terminal src-homology 2 domain (SH2) in SHIP and SHIP2 mediate interactions between these molecules and immuno-tyrosine activating and inhibitory motifs (ITAMs/ITIMs) as well as adapter proteins. A 5' phosphatase (5'Ptase) domain in the center of all SHIPs mediates its inositol phosphatase activity, while proline rich carboxy termini mediate interactions with src-homology 3 (SH3) domain containing adapter proteins.  ITIMs SHP-2 ITAMs Doks Shc Gabs C-Cbl Cas SHIP 145 KDa  SH2  Shc, Dok1,2 Grb2, Src 5’PTase  Proline rich Grb2,Src  SHIP 2 150 KDa  SH2  sSHIP 104 KDa  5’PTase  Proline rich  5’PTase  Abl Proline rich  1.8 SHIP biochemistry and interactors Three molecules comprise the SHIP family, SHIP, SHIP2 and stem cell, or sSHIP. While SHIP and sSHIP expression are restricted to the hematopoietic and stem cell compartments, respectively, SHIP2 is more ubiquitous and found in most cell types and tissues. SHIPs function by translocating to sites of PIP3 synthesis in cells stimulated in a variety of ways, such as cytokine or immunoreceptor ligation (March, 2002). The SHIP polypeptide is a 145 KDa molecule, 1190 amino acids long, and is composed of three major domains that characterize its function within immune cells. The amino-terminal SH2 domain of SHIP interacts with phosphorylated immuno-tyrosine activating and inhibitory motifs (ITAMs/ITIMs) present in the cytoplasmic tail of many receptors found on immune cells, such as the Fcε RI IgE antibody receptor as well as the Fcγ RIIβ1 IgG antibody receptor (March, 2002; Ono, 1996; Osborne, 1996).  Once  18  phosphorylated, ITAMs or ITIMs associate with SHIP and consumption of PIP3 or IP4 by the central phosphatase domain prevents further downstream cell signaling. In addition, the carboxy-terminus of the molecule mediates multiple interactions between SHIP and other proteins. For example, phosphotyrosines in two NPXY sites are important for SHIP function as they provide binding sites for negative regulator adaptor proteins such as Doks (Lamay, 2000), whereas prolines allow for interactions with SH3 domain containing molecules like Grb (Fig. 4) (Jefferson et al., 1997; Tridandapani et al., 1997). C terminal truncations however, are commonly generated in vivo and these isoforms may perform distinct functions (Rohrschneider LR, 2003). sSHIP and SHIP2 share high homology with SHIP, especially in the phosphatase and C terminal domains, as all three molecules can break down PIP3 as well as interact with adapter molecules (March, 2002). 1.9 The SHIP Knockout Mouse The biological importance of SHIP and the elucidation of its many functions have been exemplified in the SHIP knockout mouse (Helgason, 1998). The ship-/- mouse was developed in 1998 by deleting the first exon of the SHIP gene, located on mouse chromosome 1c5, in the 129/SvJ mouse strain and has subsequently been back-crossed into a C57BL/6 background. The SHIP deletion has no effect on the expression of SHIP2, however sSHIP expression is elevated and more prolonged (Helgason, 1998). Overall, these mice are sickly; they suffer from various maladies including severe pulmonary inflammation, overproduction of granulocytes and macrophages, splenomegaly, extramedullary hematopoiesis, aberrant NK cell development, allograft rejection and osteoporosis (Helgason, 1998; Takeshita, 2002; Wang, 2002). Interestingly, these mice show similar phenotypes to others that have non-functional immune modulators, such as PTEN, SHP-1 and LYN. For example, all share increased proliferation of myeloid and  monocytic  cells,  stem  cell proliferation  and  hyper-responsivity  to  granulocyte/monocyte colony stimulating factor (GMCSF) (Baran, 2003; Harder et al., 2004; Neel et al., 2003; Sly et al., 2007; Xiao et al., 2008b). These  19  phenotypes underscore the importance of regulating PIP3 and its downstream signaling, by a variety of mechanisms. 1.10 The Function of SHIP in Innate Immune Cells Examination of the function of SHIP in the ship-/- mouse has uncovered the pluripotent activity of this molecule in almost every cell type of the immune response (Table 3). These effects are most profound in innate immune cells, where SHIP sets the activation threshold and controls proliferation in response to a variety of stimuli (March, 2002). For example, SHIP has been shown to act as a "gatekeeper" of mast cell degranulation. By negatively regulating PIP3 levels in response to antigen and IgE, calcium flux is restricted and degranulation is modulated (Huber, 1998). SHIP also seems to control the IFNγ response of NK cells, as CD56 bright NK cells from human blood show markedly decreased levels of SHIP and produce more IFNγ upon activation (Trotta, 2004). The function of SHIP in innate immune cells is best characterized in macrophages and monocytes. Because these cells derived from ship-/- mice are hyper-responsive to cytokines and growth factors such as IL-3 and GMCSF, (Kim, 1999) and the mice suffer from severe myeloproliferation (Helgason, 1998), it is clear that SHIPs control over macrophage activation and proliferation is essential in maintaining immune homeostasis. One of the major roles for SHIP in macrophages is to maintain tolerance to LPS. Endotoxin tolerance occurs when macrophages stimulated with low doses of LPS become transiently refractory to high LPS exposure, resulting in a dampened pro-inflammatory response initiated by the cell. This tolerance is thought to protect the host from activated immune cells as well as serve as a protective mechanism from persistent bacterial infections (West, 2002). Interestingly, ship-/- macrophages do not display endotoxin tolerance and hypersecrete pro-inflammatory mediators such as TNFα, IL-6, IL-1β and NO in response to LPS (Rauh, 2004; Sly, 2004) . In addition, SHIP plays an essential role in regulating macrophage phenotype. There are two general categories of macrophages in the immune system, M1 and M2. M1 macrophages are those effector cells we typically  20  associate with an anti-bacterial immune response; they produce ROS/RNS and cytokines central to controlling infection. In contrast, M2 macrophages produce much lower levels of inflammatory mediators, such as nitric oxide (NO), TNFα, IL-12p70 and IL-23 after stimulation by pattern recognition receptor ligation and thus are ineffective in combating pathogens (Gordon and Taylor, 2005; Mantovani et al., 2007). For example, M2 macrophages are impaired in their ability to limit the growth of intracellular Mycobacterium tuberculosis due to reduced NO production and increased iron levels within the phagosome, and they are also associated with murine susceptibility to cutaneous Leishmaniasis (Holscher et al., 2006; Kahnert et al., 2006). However, M2 macrophages do play a vital role in the resolution of immune responses and are essential for promoting tissue healing and repair (Gordon and Taylor, 2005). In macrophages, SHIP represses the generation of an M2 phenotype, presumably by limiting activation signals generated by PIP3 (Rauh, 2005). Thus, SHIP is intimately involved in maintaining the delicate balance of macrophage differentiation, maturation and phenotype that ultimately has a dramatic effect on ensuring an appropriate response by the immune system.  21  Table 3. Regulation of immune cell functions by SHIP Cell Type B Lymphocyte  T Lymphocyte  Mast  Neutrophils  Platelets NK DC MΦ  SHIP Function ⇓ Proliferation ⇓ Chemotaxis ⇓ Activation ⇓ Activation ⇑ Apoptosis Th1/Th2 Bias ⇓ Degranulation ⇓ Cytokine production ⇓ Adherence ⇓Proliferation ⇓TLR2 Activation ⇑ Survival ⇓Activation ⇓Spreading ⇓IFNγ production ⇓Activation ⇓Progenitor number Slows differentiation ⇓ Proliferation ⇑Survival ⇓ H2O2 generation ⇓ NF−kΒ Signaling Skews phenotype to M2 ⇓ TLR mediated LPS response ⇓ Anti-bacterial activity  Reference (Brauweiler et al., 2000a)  (Dong et al., 2006; Tarasenko et al., 2007) (Huber, 2000; Kalesnikoff et al., 2002; Lam et al., 2003) (Gardai, 2002; Hunter et al., 2004; Strassheim et al., 2005) (Giuriato et al., 2003) (Trotta, 2004) (Neill et al., 2007) (Parsa, 2006; Sly et al., 2007)  1.11 The potential role of SHIP during immune responses to Salmonella Studies utilizing the ship-/- mouse have made it clear that SHIP is an indispensable regulator of immune homeostasis, however, it is unknown how SHIP functions during an immune response following infection in vivo. Recent studies suggest that SHIP may direct the outcome of pathogenesis. For example, in vitro, SHIP regulates both the macrophage pro-inflammatory response to the intracellular pathogen, Francisella novicida (Parsa et al., 2006), and phagosome maturation (Kamen et al., 2007).  This data correlates nicely with previously  published work that has shown how other key regulators of immunity, like SHP-1,  22  are central to the outcome of disease (Neel et al., 2003). In addition, SHIP is essential for maintaining endotoxin tolerance in both mice and macrophages (Sly et al., 2004).  Because many bacterial pathogens initially stimulate innate  immune cells like macrophages through LPS and an appropriate inflammatory program is required to control infections, these studies suggest that an aberrant immune response may be mounted against such invaders in ship-/- mice (Ohl and Miller, 2001). In addition, changes in SHIP expression in humans is correlated with various types of inflammatory disorders, such as severe allergies and peritonitis as well as cancers, suggesting that even in the absence of infection, disregulation of SHIP is associated with disease (Jiang et al., 2003; MacDonald and Vonakis, 2002; Muthukuru and Cutler, 2006). 1.12 Rationale Complex immune responses are required to control Salmonella infections and SHIP is an essential regulator in many cell types, such as macrophages, that are central to preventing disease. Furthermore, SHIP dysfunction is associated with human diseases as well as impaired responses to pathogens in vitro. Based on this evidence, the central hypothesis examined in this thesis has been that SHIP is required to control Salmonella infection in vivo. In order to test this hypothesis, susceptibility to typhoid infection in ship-/- mice and the innate immune mechanisms that may be affected in this model were explored. Because SHIP  plays  important  roles  in  regulating  macrophage  behavior,  and  macrophages are central to both innate and adaptive/innate responses to Salmonella, it was further hypothesized that SHIP deficiency would impact the macrophage-dependent cytokine response to Salmonella infection, both in vivo and in vitro. This was tested by monitoring cytokine responses in mice during Salmonella infection (Chapter 3), and of infected BMDM (Chapter 4). During these studies it became clear that the role of SHIP in macrophage heterogeneity could be a defining aspect of the ship-/- mouse's ability to resist Salmonella infection.  23  Throughout these studies of typhoid in the ship-/- mouse, an interesting phenotype was observed in the gut that led to the data presented in chapter 5. Unless pretreated with streptomycin, mice do not get intestinal inflammation after infection with S. Typhimurium. However, this was not the case in ship-/- mice, as they presented with severe inflammation of the ileum after oral infection with Salmonella. This led to the hypothesis that SHIP is required to modulate gut inflammatory  responses  during  enteric  infections.  By  examining  gut  histopathology and inflammatory mediator production in response to Salmonella and Citrobacter rodentium as well as heat-killed bacteria and LPS, it was found that SHIP deficiency predisposes mice to severe inflammation of the ileum in response to infection.  24  CHAPTER 2: MATERIALS AND METHODS  25  2.1 Bacterial culture and preparation 2.1.1 Growth conditions S. Typhimurium SL1344 and Citrobacter rodentium DBS100 were grown overnight in 3 or 5 ml of Luria-Bertani broth (LB) respectively, at 37°C with shaking. To enumerate bacteria from overnight cultures, serial dilutions were prepared in sterile phosphate buffered saline ((PBS+/+)+ 0.901mM CaCl2, HyClone, Mississauga, ON), plated on LB agar supplemented with 100ug/ml streptomycin (Gibco, Burlington, ON) and incubated for 24 hours at 37°C. Approximately 3x109 bacteria were present in each 3 ml overnight culture. 2.1.2 Opsonization For in vitro infections, S. Typhimurium SL1344 were opsonized by washing 100 µl of overnight culture and resuspending in 100 µl of 30% mouse serum in Dulbecco's Modified Eagle Media (DMEM, Hyclone) followed by incubation at 37°C for 25 min. Opsonized bacteria were then diluted 1:10 for infection of bone marrow-derived macrophages (BMDMs). 2.1.3 Bacterial killing To heat-kill bacteria, 100 µl of overnight culture of S. Typhimurium SL1344 were washed and resuspended in 100 µl PBS+/+ followed by incubation at 80°C for 30 min. No heat-killed bacteria were viable after 48 hours incubation on LB agar at 37°C. 2.1.4 Preparation for infection Prior to inoculation, heat-killed bacteria were centrifuged at 8,000 rpm for 4 min in an Eppendorf Mini Spin benchtop centrifuge (F45-12-11 rotor) and diluted 1:10 in 1 ml DMEM for infection of BMDMs.  26  2.2 In vivo infections 2.2.1 Mice Six to eight week-old 129/SvJ x C57BL/6 F2 ship+/+ and ship-/- mice were obtained from the laboratory of Dr. Gerald Krystal at the British Columbia Cancer Research Centre and bred for homozygotes. For mouse genotyping, the primers for SHIP A (sense oligo) 5’-TCTGTGCAGCTCAGTTTCCTCT-3’, SHIP B (antisense oligo) 5’-CGTCCCACCATCCTATGACATAA-3’ and TK Promotor (antisense oligo) 5’-CTGCATCTGCGTGTTCGAATT-3’ were obtained from Sigma Genosys (Oakville, ON, Canada). All in vivo experiments were performed in accordance with the protocols and guidelines provided by the Animal Care Committee at the University of British Columbia. 2.2.2 Salmonella Typhimurium infections Mice were infected orally by gavage with a dose of 1x106 live S. Typhimurium SL1344, or intraperitoneally (IP) with 1x102 S. Typhimurium SL1344 for survival, bacterial load enumeration, cytokine analysis and immunohistochemistry experiments. For heat-killed survival experiments, mice were inoculated either orally by gavage or IP with 1x108 heat-killed S. Typhimurium SL1344. Oral inocula were diluted from overnight cultures in sterile HEPES buffer, pH 8.0 (Gibco), and IP inocula were diluted in sterile Hanks Balanced Salt Solution (HBSS, Sigma, Oakville, ON). Mice were sacrificed immediately when moribund (for survival experiments) and at two or five days post-infection (for bacterial load determination and cytokine analyses). 2.2.3 Citrobacter rodentium infections Mice were infected orally by gavage with 1x108 C. rodentium DBS100, and sacrificed at two or 7 days post-infection for immunohistochemistry or bacterial load determination experiments, respectively. Oral inocula were diluted from overnight cultures in sterile HEPES buffer, pH 8.0 (Gibco).  27  2.3 Enumeration of bacterial load from infected mice Mice were sacrificed two or five days post-infection for oral or IP S. Typhimurium infections, respectively, and colon, small intestine, liver, MLN and spleens were harvested into 1 ml of sterile, cold, PBS+/+. For C. rodentium infections, mice were sacrificed 7 days post-infection and colons were harvested into 1 ml of sterile, cold, PBS+/+. Organs were homogenized in 2ml Eppendorf Saftey-Lock tubes with one 7mm tungsten carbite bead using a Mixer Mill MM 200 (Retsch Technologies, Haan, Germany) and serial dilutions of homogenate were prepared in sterile PBS+/+ for plating. Bacteria were grown on LB Agar + 100µg/ml streptomycin for 24 hours at 37°C and subsequently counted. 2.4 In vivo cytokine analyses For analysis of mouse serum cytokine levels, blood was obtained by cardiac puncture at various time points post-infection (as indicated in legends), incubated at 37°C for 1 hour and then spun at 13,200 rpm in an Eppendorf 5415D benchtop centrifuge for 10 min to separate serum. Serum was aliquoted and frozen at – 80°C and analyzed using the mouse inflammation cytometric bead array (CBA) assay kit (BD Biosciences, Mississauga, ON) as per manufacturer's instructions. Briefly, cytokine levels were analyzed in a multiplex fashion, whereby single samples of mouse serum were incubated with a combination of antibody coated beads  specific  for  IL-12p70,  IL-6,  IL-10,  TNFα  and  IFNγ  and  phycoerythrin (PE) detector solution. Samples were assessed by flow cytometry on a BD Facscalibur Flow Cytometer and cytokine levels then analyzed using the kit associated CBA software (BD Biosciences).  28  Table 4. Antibodies and dyes used in studies. (WB) Western blot, (IHC) Immunohistochemistry, (FACS) Fluorescence activated cell sorting. Antibody YM-1  Species rabbit  SHIP  mouse  Arg-1  mouse  GAPDH  mouse  Biotin F/480  rat  Biotin GR-1  Dilution Use 1:10,000, WB, IHC 1:500 1:10,000 WB 1:10,000, WB, IHC 1:100 1:10,000 WB  Source Stem Cell Technology, Vancouver, BC Santa Cruz Biotechnology, Santa Cruz, CA BD Transduction, Lexington, KY Research Diagnostics, Flanders, NJ ADB Serotec, Hornby, ON  IHC, FACS  rat  1:1000, 1:100 1:100  apc Mac-1  rat  1:100  FACS  PE CD11c  hamster  1:200  FACS  PE CD-3  hamster  1:400  FACS  Biotin B220  rat  1:200  FACS  Mouse-HRP Rabbit-HRP Rat -568 AlexaFluor® Rabbit-488 AlexaFluor® Mouse-468 AlexaFluor® SA-FITC  goat goat goat  1:5000 1:5000 1:200  WB WB IHC  BD Biosciences, Mississauga, ON BD Biosciences, Mississauga, ON BD Biosciences, Mississauga, ON BD Biosciences, Mississauga, ON BD Biosciences, Mississauga, ON Invitrogen, Burlington, ON Invitrogen, Burlington, ON Invitrogen, Burlington, ON  goat  1:200  IHC  Invitrogen, Burlington, ON  IHC  Invitrogen, Burlington, ON  -  1:400  FACS  7AAD  -  1:250  FACS  BD Biosciences, Mississauga, ON BD Biosciences, Mississauga, ON  goat  FACS  2.5 Immunohistochemistry 2.5.1 Preparation of tissues Tissues were removed from mice and immediately fixed in 10% neutral buffered formalin and incubated at 23°C for 24 hours and then transferred into 70% 29  ethanol. Fixed tissues were embedded in paraffin, cut into 5mm sections and either left untreated, or stained for hematoxylin and eosin (H&E) and Masson's trichrome stain using standard techniques by Wax-it Histology Services (Vancouver, BC, Canada). 2.5.2 Immunohistochemistry for M2 macrophages For immunohistochemistry staining, slides with 5mm sections of tissue were deparrafinized and re-hydrated. Antigen retrieval was carried out by digesting tissues in 20mg/ml proteinase K (Sigma) in TE buffer (50mM Tris base, 1mM EDTA, pH 8.0) for 15 min. Tissues were then immunostained with primary antibodies against Ym-1, arginase 1 (Arg1) and F4/80, followed by incubation with secondary antibodies. After staining, cover slips were mounted over tissues using ProLong Gold Antifade reagent (Invitrogen) containing DAPI for DNA staining. Images were obtained using a Zeiss AxioImager microscope equipped with and AxioCam HRm camera operating through AxioVision software (Carl Zeiss Ltd., Toronto, ON, Canada). 2.5.3 Pathology Scoring Tissue sections prepared in H&E stain by Waxit Histology Services were scored for inflammation in the lumen, surface epithelium, mucosa and submucosa using the criteria outlined in appendix 5. 2.6 Western Analysis 2.6.1 Ex-vivo cell preparation Peritoneal macrophages were obtained from infected and uninfected mice by lavage with 10 ml DMEM (HyClone) + 10% FBS (Gibco). Cells were washed 1 time in DMEM, counted, spun at 1000 rpm in a Beckman Coulter Allegra X-12 R benchtop centrifuge at 23°C for 5 min and lysed directly into 1× Laemmli’s Western sample buffer.  30  2.6.2 SDS-PAGE and Western blotting Samples were boiled for 3 min in Laemmli's sample buffer and spun at 13,200 rpm in an Eppendorf 5415D benchtop centrifuge for 30 sec before loading on 10% SDS-polyacrylamide gels for electrophoresis. Proteins were transferred onto nitrocellulose membranes (Millipore, Bedford, MA) using a semi-dry transfer system (BioRad, Mississauga, ON). The membranes were incubated for one hour at 23°C in tris-buffered saline + 0.1% Tween 20 (TBST) + 5% FBS (blocking buffer) and then incubated overnight at 4°C with primary antibodies against Ym1, Arg1, SHIP or GAPDH diluted in blocking buffer. After washing 3 x 10 min in TBST, membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in blocking buffer for 1 hour at 23°C. Membranes were washed again 3 x 10 min in TBST and proteins were detected using a chemiluminesence (ECL) kit according to manufacturer's instructions (Amersham Biosciences, Piscataway, NJ). 2.7 Flow Cytometry 2.7.1 Leukocyte isolation Spleens and MLN from orally infected mice were pooled and harvested into 5 ml DMEM + 1% HEPES Buffer + 0.01mg/ml V. alginolyticus collagenase (Roche Diagnostics, Indianapolis, IN) and then minced using scissors and forceps, followed by incubation at 37°C for 1 hr. Cell suspensions were separated by drawing through an 18 gauge needle 3 times with a 5 ml syringe. Cells were washed in PBS-/- (without added calcium) + 2% FBS + 0.5% NaN3 (FACS buffer), centrifuged at 1200 rpm in a Beckman Coulter Allegra X-12 R benchtop centrifuge at 23°C for 5 min and resuspended in 10 ml FACS buffer for flow cytometry staining. 2.7.2 Leukocyte staining for flow cytometry Approximately 1x106 cells were stained per well in 96 well round bottom tissue culture plates with primary antibodies against CD3, B220, F4/80, Mac-1, Gr-1 31  and CD11c diluted in FACS buffer for 30 min at 4°C. Cells were then washed 2 x in FACS buffer, followed by secondary conjugate staining for 30 min at 4°C. Cells were washed again 2 times and resuspended in 300µl FACS buffer and transferred into FACS tubes for acquisition. Percent positive cells were assessed using a Cell Quest Pro software on a BD Facscalibur Flow Cytometer and analyzed using FlowJo flow cytometry software (Tree Star, Ashland, OR). 2.8 Macrophage Tissue Culture 2.8.1 BMDMs Bone marrow cells were obtained by flushing tibiae and femora from uninfected ship+/+ and ship-/- mice. 5x106 cells were first suspended in 10 ml DMEM supplemented with 10% heat-inactivated FBS, 2mM L-glutamine, 1mM sodium pyruvate, 100U/ml penicillin/100µg/ml streptomycin (Gibco) and 30% conditioned media from L929 cells as a source of macrophage colony stimulating factor (MCSF). M2 inducing media was further supplemented with 2% mouse serum taken from ship+/- mice. After allowing cells to adhere to non-tissue culture treated petri plates for 3 hours at 37°C and 5% CO2, non-adherent cells were transferred to fresh petri plates for differentiation at 37°C, 5% CO2 for 10 days with complete media changes on adherent cells at days four and 7. This procedure results in a population of cells that is 95% positive for the macrophage markers F4/80 and Mac-1 (Sly et al., 2004). 2.8.2 Raw 264.7 macrophages Raw 264.7 macrophages were obtained from American Type Culture Collection cell biology stock centre (Rockville, MD). Cells were grown in culture in DMEM + 10% FBS and skewed to an M2 phenotype 24 hours prior to infection using 100ng/ml recombinant mouse IL-4 (R&D Systems, Minneapolis, MN).  32  2.9 In vitro infections 2.9.1 Preparation of BMDMs Cells were removed from petri plates using non-enzymatic cell dissociation buffer (Gibco) and washed twice in 10 ml DMEM to remove antibiotics. Cells were seeded at a density of 1x105 cells/well in 1 ml of DMEM + 10% FBS, 2mM Lglutamine and 1mM sodium pyruvate (BMDM media). 2.9.2 Preparation of Raw 264.7 macrophages Cells were removed from petri plates using non-enzymatic cell dissociation buffer and washed twice in 10 ml DMEM to remove antibiotics. Cells were seeded at a density of 1x105 cells/well in 1 ml of DMEM + 10% FBS + 100ng/ml recombinant mouse IL-4 (raw media, R&D Systems) in 24 well tissue culture plates 12 hours prior to infection. 2.9.3 S. Typhimurium infections Cells were infected at a multiplicity of infection of 10 with either opsonized S. Typhimurium SL1344 or inoculated with heat-killed S. Typhimurium SL1344 and centrifuged at 1,000rpm in a Beckman GS-6R benchtop centrifuge at 23°C for 5 min to synchronize infection, followed by incubation at 37°C, 5% CO2 for 15 min. To remove extracellular bacteria, cells were washed three times in sterile PBS+/+ and supplied with 1 ml BMDM or raw media + 50µg/ml gentamicin (Sigma) for 2 hours. Cell supernatants then were replaced with 1 ml BMDM or raw media + 10µg/ml gentamicin. LPS-treated control cells were given BMDM media + gentamicin + 100ng/ml S. Typhimurium LPS (Sigma) for the amounts of time indicated.  2.10 Intracellular replication (gentamicin protection) assays  33  BMDMs and Raw 264.7 macrophages were infected with S. Typhimurium SL1344 as above and supernatants were removed at two and 24 hours postinfection. After washing 3 times in PBS+/+, cells were lysed in 250 ml PBS+/+ + 1% TritonX-100 + 0.1% SDS. Lysates were serially diluted and plated on LB Agar + 100µg/ml streptomycin for 24 hours at 37°C and subsequently counted. Fold replication numbers were generated by dividing the bacterial load enumerated from 24 hour lysates by 2 hour lysates. 2.11 Cell death assays BMDMs were infected with live, or inoculated with heat-killed S. Typhimurium SL1344, or treated with LPS and supernatants were removed at 8 and 24 hours post-infection. After washing three times with PBS+/+, cells were scraped using a rubber scraper from the wells into 300 ml FACS buffer. Cells were transferred to 96 well round bottom tissue culture plates and stained for necrosis using 7amino-actinomycin D (7AAD). 7AAD positivity was assessed by flow cytometry using a Cell Quest Pro software on a BD Facscalibur flow cytometer and samples were analyzed using FlowJo flow cytometry software (BD Biosciences). 2.12 Cytokine analyses At designated time points post-infection, 1 ml of supernatant was removed from each well of infected or control BMDMs, aliquoted and frozen at –80°C until assayed for cytokines using mouse TNFα, IL-10, IL-6 and IL-12p70 ELISA kits (BD Biosciences). 2.13 Collagen assays Approximately 0.5 cm or 0.10 g of ilea was removed from mice at two days postoral infection and immediately placed into 500 µl of 0.5M Acetic Acid + 10mg/ml pepsin (Sigma). Samples were incubated overnight at 23°C with rotation followed by the addition of 1 ml Sircol dye (Biocolor, N. Ireland). Samples were incubated for 1 hour at 23°C and spun for 10 minutes at 10,000 rpm in an Eppendorf 5415D benchtop centrifuge followed by the removal of supernatant. Bound dye was 34  resuspended in 1 ml 0.5 M Alkali Solution (Biocolor) and incubated at 23°C for 10 min. Samples were vortexed and 100 µl were transferred to a 96 well clear bottom microplate. Fluorescence was read with a TECAN SpectraFluor Plus plate reader (TECAN, Research Triangle Park, NC) at wavelength of 560nm. 2.14 Statistical analyses For in vivo time of death experiments, logrank statistical analyses for survival data were performed on curves generated in Graph Pad Prism 4.0 (MacKiev Software). For enumeration of bacterial loads from infected mice as well as replication assay, flow cytometry, ELISA and CBA data, statistical analyses were performed using two-tailed, unpaired student’s t tests with a 95% confidence interval in Graph Pad Prism 4.0 (MacKiev Software). For lymphocyte distributions, one-way analysis of variance (ANOVA) with a 95% confidence interval were performed with Bonferroni tests applied post-hoc. On all graphs, error bars represent standard error from the arithmetic mean. Statistical significance based on P values of <0.05, <0.01 and <0.001 are represented on graphs by one, two or three asterisks, respectively.  35  CHAPTER 3: The role of SHIP in S. Typhimurium infections in vivo  36  3.1 Introduction SHIP negatively regulates various hematopoietic cell functions and is critical for balancing pro- and anti-inflammatory responses of both innate and adaptive cells. Negative regulation is particularly important during bacterial infections in order to create the proper interface between innate and adaptive immunity and to protect the host from potent inflammatory mediators. This is especially true during infections with pathogens like Salmonella, where strong innate responses are required for an extended period of time in order to prevent bacterial replication. Because alterations in SHIP function have been implicated in various human diseases as well as interactions with pathogens in cell culture, it is important to examine the role it may play in vivo during the pathogenesis of Salmonella, which has such an impact on global health. To test the hypothesis that SHIP is required to control Salmonella infection in vivo, susceptibility and the innate immune responses of ship+/+and ship-/- mice to S. enterica serovar Typhimurium infection were compared. Survival experiments and enumeration of bacterial loads in intestinal and systemic sites indicated the high degree of susceptibility to Salmonella infections in ship-/- mice. Cytokine analysis of mouse serum-used to profile innate immune responses in the presence or absence of SHIP showed that ship-/- mice produce lower levels of Th1 polarizing cytokines compared to ship+/+ animals at two days postinfection, and time courses revealed the defective nature of the cytokine response throughout the duration of infection. To investigate the possibility that M2 macrophages could be contributing to this cytokine profile, these cells were looked for at sites of Salmonella infection. Indeed, M2 macrophages were the predominant population in vivo; tissue macrophages within the small intestine and peritoneal macrophages from ship-/- mice showed elevated levels of the M2 macrophage markers Ym1 and Arg1 compared to ship+/+ cells. When the distribution of other lymphocyte populations was examined, it was found that in correlation with previously published studies, there are inherent differences between macrophage and B cell populations between ship+/+ and ship-/- mice, however these differences were not exaggerated upon infection. These results 37  indicate that SHIP does indeed play a crucial role in modulating the immune response during Salmonella pathogenesis in vivo and suggests a contribution of the M2 macrophage in this system.  3.2 Results  3.2.1 SHIP controls susceptibility to Salmonella infection in vivo Because endotoxin tolerance is associated with resistance to Salmonella infection (Lehner et al., 2001) and this response is defective in ship-/- mice (Sly et al., 2004), it was hypothesized that these animals would be more susceptible to Gram-negative bacterial infection. To test this, ship+/+ and ship-/- mice were infected orally or IP with S. Typhimurium and their survival was monitored. ship-/mice were significantly more susceptible to oral Salmonella infection compared to ship+/+ mice (P<0.001, Fig. 5 A). Even with a low dose of 1x106 bacteria, ship-/mice died as early as day two post-infection and no animals survived longer than 10 days, while 47% of ship+/+ mice survived to at least 21 days post-infection. This phenotype was not specific to oral Salmonella infection, since ship-/- mice were significantly more susceptible to Salmonella following IP infection with 1x102 bacteria than ship+/+ mice (P=0.0119, Fig. 5 B). Administration of LPS via IP injection is lethal to ship-/- mice within 54 hours (Sly et al., 2004). Therefore, the possibility that death seen at early time points after oral or IP infection in ship-/- mice was due to endotoxic shock from Salmonella LPS in the infection inoculum was examined. To do this, ship+/+ and ship-/- mice were inoculated orally or IP with a dose of 1x106 or 1x102 heat-killed Salmonella, respectively, and survival was monitored.  Results showed that  100% of both ship+/+ and ship-/- mice survived these treatments (Fig. 5). In addition, 100% of mice infected either orally or IP with a high dose of 1x108 heatkilled Salmonella, which more closely mimicked bacterial load levels seen when mice were moribund, survived infection (Figs. 6 and 7). Furthermore, ship-/- mice were not susceptible to sepsis induced by LPS found on replicating Citrobacter 38  rodentium (Fig. 8). While the possibility that LPS may still play some role in mortality in ship-/- mice cannot be completely excluded, these data suggest that while ship-/- mice can not control Salmonella replication, levels of LPS present in infection inocula are not sufficient to cause mortality. Figure 5. SHIP controls susceptibility to Salmonella in vivo. (A) ship+/+and ship-/- mice were infected orally with 1x106 live, or 1x106 and 1x108 heat-killed (HK) S. Typhimurium SL13344 and time of death was assessed over 3 weeks. (B) ship+/+and ship-/- mice were infected IP with 1x102 live, or 1x102 and 1x108 heat-killed (HK) S. Typhimurium SL13344 and time of death was assessed over 2 weeks. For A and B three independent experiments were performed with a total N=12 for both ship+/+and ship-/- mice. A. 100  ship-/ship+/+  75  ship-/- HK ship+/+ HK  50 25 0  0  7 14 Time (days)  21  B. 100  ship-/ship+/+  75  ship-/- HK ship+/+ HK  50 25 0  0  7 14 Time (days)  21  39  3.2.2 Increased susceptibility is associated with high bacterial load in ship-/- mice Higher bacterial burdens in the organs of infected mice corresponded to increased susceptibility to infection. During oral S. Typhimurium infection, the most significant differences were seen on days two and four-post infection. On day two, bacterial loads were significantly higher in all organs except the small intestines (SI) of ship-/- mice compared to ship  +/+  mice (spleen P=0.0245, liver  P=0.0459 and colon P=0.005) and significantly higher in all organs taken on day four (spleen P=0.0828, liver P=0.0238, SI P=0.0013 and colon P=0.0103) (Fig. 6). During IP infections, colony counts were also significantly higher in all organs taken from ship-/- mice compared to ship+/+mice at days two, three, and five postinfection, and all organs except liver, on day four post-infection (Table 5, Fig. 7). Table 5. Statistical Significance of CFU values in ship-/- vs. ship+/+ organs after IP Salmonella infection ship+/+ Colon  ship-/- SI  ship-/- Spleen  ship-/- Liver  Day 2  P=0.0248  P=0.0085  P=0.0136  P=0.0122  Day 3  P=.0069  P=.0020  P=0.0007  P=0.0002  Day 4  P=0.0017  P=0.0040  P=0.0276  NS  Day 5  P=0.005  P=0.0011  P=0.0230  P=0.0068  40  Figure 6. Increased susceptibility to Salmonella is associated with higher bacterial loads in organs of orally infected mice. ship+/+and ship-/- mice were infected orally with 1x106 S. Typhimurium SL1344 and sacrificed at days 1-4 days post-infection (A-D). Colon, SI, liver, and spleen were harvested from the mice, homogenized and plated to enumerate bacterial load. 3 independent experiments were performed with a total N=9 for days 1-3, or N=12 on day 4, for both ship+/+and ship-/- mice.  A.  B. Day 1  Day 2  1010.0 10  ship-/ship+/+  7.5  1010.0  105.0  105.0  102.5  102.5  100.0  Colon  SI  Spleen  100.0  Liver  C.  **  *  *  Spleen  Liver  107.5  Colon  SI  *  **  ship-/ship+/+  D. Day 3 1010.0  Day 4  *  -/-  ship ship+/+  107.5  1010.0  105.0  102.5  102.5  Colon  SI  Spleen  Liver  ship-/ship+/+  107.5  105.0  100.0  *  100.0  Colon  SI  Spleen  Liver  41  Figure 7. Increased susceptibility to Salmonella is associated with higher bacterial loads in organs of IP infected mice. ship+/+and ship-/- mice were infected IP with 1x102 S. Typhimurium SL13344 and sacrificed at 1-5 days postinfection (A-E). Colon, SI, liver and spleen were harvested from the mice, homogenized and plated to enumerate bacterial load. 3 independent experiments were performed with a total N=3 for days 1-4, or N=12 on day 5, for both ship+/+and ship-/- mice.  A.  B. Day 2  Day 1 5  10  ship-/ship+/+  4  10  1010.0  **  *  *  107.5  103  ship-/ship+/+  105.0  102  102.5  101 100  *  Colon  SI  Spleen  100.0  Liver  C.  Colon  SI  Spleen  Liver  D. Day 4  Day 3 1010.0  **  **  ***  ***  107.5  ship-/ship+/+  1010.0  105.0  102.5  102.5  Colon  SI  Spleen  100.0  Liver  **  *  ship-/ship+/+  107.5  105.0  100.0  **  Colon  SI  Spleen  Liver  E. Day 5 1010.0  ***  **  *  **  Colon  SI  Spleen  Liver  107.5  ship-/ship+/+  105.0 102.5 100.0  42  Importantly, I found that when challenged with Citrobacter rodentium, an extracellular attaching and effacing pathogen that does not cause systemic disease (Mundy et al., 2005), there was no difference in colonization of the colon between ship+/+ and ship-/- mice (Fig. 8) and infection in either strain did not lead to morbidity or mortality in mice used in CFU experiments after 7 days. These data suggest that the outcome of infection in ship-/- mice may be dependent on the intracellular or extracellular nature of the pathogen and it highlights the role SHIP may play in preventing systemic infection. Figure 8. ship+/+ and ship-/- mice are equally susceptible to colonization by Citrobacter rodentium. ship+/+and ship-/- mice were infected orally with 1x108 C. rodentium DBS100 and sacrificed at 7 days post-infection. Colons were harvested from the mice, homogenized and plated to enumerate bacterial load. 3 independent experiments were performed with a total N=9 for both ship+/+and ship-/- mice.  1010.0  ship-/ship+/+  107.5 105.0 102.5 100.0 Colon  3.2.3 ship-/- mice produce levels of inflammatory cytokines typical of M2 macrophages during Salmonella infection IL-12 and IFNγ comprise the central axis of Th1 polarizing cytokines that are known to drive the immune response against Salmonella (Eckmann and Kagnoff, 2001; Mastroeni, 2002).  Thus, the association between increased  susceptibility to Salmonella in ship-/- mice and lower concentrations of these cytokines during infection was examined. Mice were infected orally or IP with 1x106 or 1x102 S. Typhimurium SL1344, respectively, and blood was taken from 43  the mice at two days after oral infection or five days after IP infection for cytokine analyses. These were the same animals used to generate bacterial load data shown in figures 6 B and 7 E, and these days were chosen to prevent ship-/- mice from becoming moribund during oral or IP infections. In response to oral infection, ship-/- mice produced significantly lower amounts of IFNγ (P=0.0377), IL-6 (P=0.0259) and IL-10 (P=0.0301) than ship+/+ mice, and production of IL12p70 was also decreased, albeit not significantly (Fig. 9 A). In an oral time course experiment where cytokine levels were examined on days one to four post-infection (Fig. 9 B-E) it was also observed that ship-/- mice produce lower levels of IL-12p70, IFNγ, IL-10 and IL-6 in comparison to ship+/+ mice. During an IP infection, a similar trend was observed on day five post-infection as oral infection cytokines taken on day two post-infection, with the exception of IL-6; ship-/- mice produced significantly higher levels of IL-6 (p=0.0299) but lower levels of IL-12p70 and IFNγ than ship+/+ mice, however these differences were not significant (Fig. 10 A). Tracking the kinetics of cytokine production during an IP infection showed that while ship-/- mice produced lower levels of IL-12p70, IFNγ, IL-10 and IL-6 at early time points, by day three these levels were much higher than those produced in ship+/+ mice. In the case of IL-12p70, IFNγ and IL10, production in ship-/- mice was lower than ship+/+ at day five post-infection. In both oral and IP infections TNFα levels were significantly higher in ship-/- than in ship+/+ mice (P=0.0173 and P=0.0085, respectively) throughout the duration of infection (Fig. 9 A and C, Fig. 10 A and C). Importantly, no significant differences were found between cytokine levels in uninfected ship-/- and ship+/+ mice (Fig. 11) and altered cytokine levels in ship-/- mice correlate with increased bacterial load in organs. Trends toward low IL-12 and IFNγ levels produced in ship-/- mice upon Salmonella  infection  suggest  a  cytokine  profile  characteristic  of  M2  macrophages; therefore, association between increased susceptibility to Salmonella and SHIP deficiency could be due to a lack of M1 macrophages that are important for skewing of Th1 responses required to prevent disease.  44  Figure 9. SHIP deficiency leads to altered levels of inflammatory cytokine production after oral Salmonella infection in vivo. ship+/+and ship-/- mice were infected orally with 1x106 S. Typhimurium SL1344 and sacrificed on days 1-4 post-infection. Blood samples were obtained via cardiac puncture and serum was separated for cytokine analysis. Cytokines were analyzed using the CBA mouse inflammation kit. (A) Cytokine production on day 2 post-infection. (B-F) Kinetics of cytokine production during a 4 day oral infection. 3 independent experiments were performed with a total N=9 for days 1-4, or N=12 on day 2, for both ship+/+and ship-/- mice. A.  B. IL-12  *  3100  *  *  *  450  ship-/ship+/+  2100  ship-/ship+/+  300  1100 700 150  450 200  0  IFN"  TNF!  IL-12  IL-10  0  1  IL-6  2  3  4  5  Time (days)  C.  D. TNFα  IFNγ  300  ship-/ship+/+  4000  ship-/ship+/+  3000  200  2000  100 1000  0  0  1  2  3  4  0  5  0  1  Time (days)  2  3  4  5  Time (days)  E.  F. IL-10  IL-6  600  ship-/ship+/+  450  5000  ship-/ship+/+  4000 3000  300  2000  150 1000  0  0  1  2  3  Time (days)  4  5  0  0  1  2 3 Time (days)  4  5  45  Figure 10. SHIP deficiency leads to altered levels of inflammatory cytokine production after IP Salmonella infection in vivo. ship+/+and ship-/- mice were infected IP with 1x102 S. Typhimurium SL1344 and sacrificed on days 1-5 postinfection. Blood samples were obtained via cardiac puncture and serum was separated for cytokine analysis. Cytokines were analyzed using the CBA mouse inflammation kit. (A) Cytokine production on day 5 post-infection. (B-F) Kinetics of cytokine production during a 5 day IP infection. 3 independent experiments were performed with a total N=9 for days 1-4, or N=12 on day 5, for both ship+/+and ship-/- mice. A. B. IL-12  **  3100  *  175  ship-/ship+/+  2100  ship-/ship+/+  150 125  1100  100 75  100 20  50 25  10  0  0  IL-12 TNF-! IFN-"  IL-10  0  1  2  IL-6  3  4  5  6  Time (days)  C.  D. IFNγ  TNFα 450  2500  ship-/ship  +/+  ship-/ship+/+  2000  300  1500 1000  150  500 0  0  1  2  3  4  5  0  6  0  1  2  Time (days)  3  4  5  6  Time(days)  E.  F. IL-6  IL-10 1750  ship-/ship+/+  1500  3000  ship-/ship+/+  1250 1000  2000  750 500  1000  250 0  0  1  2  3  Time (days)  4  5  6  0  0  1  2 3 4 Time (days)  5  6  46  Figure 11. Cytokine production in uninfected ship-/- and ship+/+ mice. Uninfected ship+/+and ship-/- mice were sacrificed, blood samples were obtained via cardiac puncture, and serum was separated for cytokine analysis. Cytokines were analyzed using the flow cytometry based CBA mouse inflammation kit. 3 independent experiments were performed with a total N=12 for both ship+/+and ship-/- mice.  140  ship-/ship+/+  120 100 80 60 40 20 0  IL-12 TNF-! IFN-"  IL-10  IL-6  3.2.4 M2 macrophage skewing is associated with increased susceptibility to Salmonella in ship-/- mice Previous work has shown that SHIP deficiency skews the macrophage phenotype in vivo towards M2 in the lung and peritoneal cavity (Rauh et al., 2005) and M2 macrophages are ineffective at mounting immune responses against pathogens, especially those requiring Th1 cytokines for clearance (Mantovani et al., 2007; Parsa et al., 2006). Therefore, it was hypothesized that increased susceptibility to Salmonella infection in ship-/- mice could be due, in part, to a lack of M1 effector macrophages. To address whether macrophages in ship-/- mice were M2, the presence of two M2 macrophage markers, Ym1 and Arg1, in tissue sections and peritoneal macrophages isolated from both uninfected mice and those infected orally or IP with S. Typhimurium SL1344, was assessed. Histological sections of the small intestine of orally infected ship-/- mice showed a large amount of Ym1 and Arg1 positive macrophages in the submucosa, whereas infected ship+/+ mice showed few Ym1 or Arg1 positive 47  cells. YM1 and Arg1 are stained in green and mature macrophages were identified with F4/80 in red. M2 macrophages were identified as these two colors merged, in yellow (bottom color panel, Figs. 12 A and B). In addition, peritoneal macrophages from IP infected ship-/- mice showed elevated levels of Ym1 and Arg1 compared to macrophages from uninfected and infected ship+/+ mice (Fig. 12 C). Taken together, these results suggest that macrophages in the gut and peritoneal cavity in ship-/- mice are heavily skewed to an M2 phenotype; thus these sites may be less protected by effector cells during Salmonella infection.  48  Figure 12. M2 macrophage markers are found in the gut and peritoneal cavity of ship-/- mice. (A and B) Sections of small intestine were taken from uninfected (ship+/+ and ship-/-) and orally infected ship+/+and ship-/- mice (ship+/+ SL1344 and ship-/- SL1344) at day 2 post-infection and stained with DAPI, the M2 macrophage markers YM1 or Arg1 (green) or and F4/80 (red) as a macrophage control. All photographs were taken under 40x magnification. (C) Peritoneal macrophages were obtained from uninfected (-SL1344 ) or IP infected (+SL1344) ship+/+and ship-/- mice at 2 days post-infection. Cells were washed, counted and lysed directly into Western Sample Buffer for protein analysis. Both bands are specific for Arg 1 (Rauh et al., 2005). For A-C representative experiments are shown.  49  A.  50  B.  C.  +/+  ship  -/-  ship  +/+  ship  -/-  ship  SHIP YM1 Arg1 GAPDH -SL1344  +SL1344  51  3.2.5 Leukocyte distribution in ship-/- mice Because many other cell types besides macrophages are key in clearing Salmonella infections, and there are known differences in various immune cell populations between uninfected ship+/+ and ship-/- mice (Brauweiler et al., 2000b; Helgason et al., 1998; Sly et al., 2007), it was questioned whether Salmonella infection affected the distribution of T cells, B cells, DCs, macrophages or neutrophils found in the spleens and MLN of ship+/+ vs. ship-/- mice. In comparing cell populations between uninfected ship+/+ and. ship-/- mice, results showed that there were significantly fewer B cells (B220+) in both the MLN (P<0.01) and spleens (P<0.001) of ship-/- than ship+/+ mice (Tables 6 and 7, Fig. 13 B). In contrast, uninfected ship-/- mice had significantly more macrophages (Mac1/F4/80+) in the MLN (P<0.001) than ship+/+ mice and these levels were also elevated in the spleen, albeit not significantly (Tables 6 and 7, Fig. 13 C). These results support previously published data that showed decreased levels of B cells and increased macrophage populations in ship-/- mice (Brauweiler et al., 2000a; Helgason, 1998). Upon infection, levels of T cells (CD3+, P<0.05) and B cells (P<0.05) were significantly elevated in the spleens of ship+/+ mice, while in ship-/mice levels of macrophages in the MLN increased significantly (P<0.001) and dendritic cells (CD11c+) decreased significantly (P<0.05) (Tables 6 and 7, Fig. 13 A-D.) Despite these differences however, the distribution of none of these cells in the spleens and MLN of ship+/+ vs. ship-/- mice were significantly changed upon infection with Salmonella when normalized against uninfected lymphocyte levels (Fig. 13 F). Thus, while inherent differences in lymphocyte populations may contribute to susceptibility to Salmonella infection, these phenotypes are not exaggerated in infected mice.  52  Table 6. Statistical changes in lymphocyte populations in the spleens of uninfected and Salmonella infected ship+/+ and ship-/- mice. Statistical analyses were performed using a one-way ANOVA with Bonferroni post-test. CD3  B220  Mac-1  GR-1  CD11c  P>0.05  P>0.05  P>0.05  F4/80 +SL1344  ship-/ship+/+  +SL1344  ship-/-  -SL1344  ship-/-  +SL1344  P<0.001 -/-  < +/+  P<0.001 -/-  < +/+  P>0.05  P>0.05  P>0.05  P>0.05  P>0.05  ship+/+  P<0.05  P<0.05  P>0.05  P>0.05  P>0.05  -SL1344  ship+/+  -<+  -<+  -SL1344  ship-/-  P>0.05  P<0.001  P>0.05  P>0.05  P>0.05  ship+/+  Table 7. Statistical changes in lymphocyte populations in the MLN of uninfected and Salmonella infected ship-/- and ship+/+ mice. Statistical analyses were performed using a one-way ANOVA with Bonferroni post-test. CD3  B220  Mac-1  GR-1  CD11c  F4/80 +SL1344  ship-/-  P>0.05  P>0.05  P>0.05  P>0.05  P>0.05  P>0.05  P>0.085  P<0.001  P>0.05  P<0.05  ship+/+ +SL1344  ship-/-  -SL1344  ship  -/-  +SL1344  ship+/+  -SL1344  ship+/+  -SL1344  ship-/ship+/+  ->+  ->+  P>0.05  P>0.05  P>0.05  P>0.05  P>0.05  P>0.05  P<0.01  P<0.001  P>0.05  P>0.05  -/-  < +/+  -/-  > +/+  53  Figure 13. Lymphocyte distribution in Salmonella infected ship+/+ and ship-/mice. Mice were left uninfected (-) or were infected orally with 1x106 S. Typhimurium SL1344 (+) and spleens and MLN were harvested at 2 days postinfection. Cell suspensions were prepared from organs and stained for flow cytometry for T cells (A), B cells (B), macrophages (C), DCs (D), and neutrophils (E) followed by acquisition and analysis using a BD Facscalibur and FlowJo software. Graphs represent percent positivity of each cell type, and the final graph (F) was generated by creating a ratio of percent positive ko:wt cells normalized against uninfected mice. For all graphs, 2 independent experiments were performed with N=3 for both ship+/+ and ship-/- mice and organs were pooled for cell staining. 10,000 total cells were counted for each organ and staining. A.  B. B220  CD3  40  ship-/ship+/+  30  50  ship-/ship+/+  40 30  20  20  10 0  10  - Spleen +Spleen  -MLN  0  +MLN  C.  - Spleen +Spleen  -MLN  +MLN  D. Mac-1/F480  CD11c  15  3  ship-/ship  10  2  5  1  0  - Spleen +Spleen  -MLN  ship-/ship+/+  +/+  0  +MLN  E.  - Spleen +Spleen  -MLN  +MLN  F. KO:WT  GR-1 75  ship-/ship+/+  1.75  +SL1344  1.50  -SL1344  1.25  50  1.00 0.75  25  0.50 0.25  0  0.00  - Spleen +Spleen  -MLN  +MLN  CD3  B220 Mac-1 CD11c GR-1 F4/80  54  3.3 Discussion Salmonella species pose a global threat to human health. Research using the S. Typhimurium mouse model of systemic salmonellosis has provided many insights into the behavior of this pathogen and the nature of immune responses required to clear intracellular infection. However, despite this progress, the ultimate cause of mortality in mice remains unknown. One possibility is that negative regulation of immune responses during bacterial infections ultimately decides the fate of the host. For example, the ability to both sense and respond appropriately to LPS is required for resistance to S. Typhimurium infections (Freudenberg et al., 2001; Lehner et al., 2001; Xu and Hsu, 1992). In addition, regulation of pro-inflammatory pathways by enzymes such as PI3K has also been shown to play a critical role in determining the outcome of infections (Fukao and Koyasu, 2003). For example, PI3K-/- mice show increased susceptibility to nematode infection and Gram-negative induced septic peritonitis (Fukao et al., 2002b; Hirsch et al., 2000) but higher resistance to Toxoplasma and Leishmania due to PI3K dependent skewing of a Th1 immune response (Fukao et al., 2002a). SHIP modulates immune homeostasis, endotoxin tolerance, PI3K signaling and macrophage inflammatory responses in vitro, but its role in the immune response to in vivo infection was undefined prior to this study. Because SHIP suppresses PI3K and PI3K has been shown to affect outcomes of various pathogenic infections, it is probable that SHIP is an important mediator in this regulatory cascade. Furthermore, since ship-/- mice do not display endotoxin tolerance (Rauh et al., 2004; Sly et al., 2004) and ship-/- macrophages can not respond to Francisella novicida infection (Parsa et al., 2006), it was hypothesized that SHIP would be required to control Salmonella infection. These data show that SHIP dependent regulation of innate immune responses is critical for the control of intracellular bacterial infections in vivo. Furthermore, these results suggest that an excess of M2 macrophages in ship-/- mice may be one contributing factor to exacerbated Salmonella pathogenesis.  55  Th1 mediated immunity is essential for final clearance of Salmonella infection both in vivo and in vitro (Eckmann and Kagnoff, 2001). For example, neutralization of IFNγ and IL-12 increases murine susceptibility to Salmonella infection, whereas exogenous addition of these cytokines increases host survival, and patients able to clear gastroenteritic Salmonella infection have higher serum levels of these cytokines (Bao et al., 2000; Nauciel and Espinasse-Maes, 1992; Stoycheva and Murdjeva, 2005). Consistent with this, slightly lower levels of IL12p70 and significantly lower levels of IFNγ were produced in ship-/- mice during oral Salmonella infection (Fig. 9 A), and a similar trend was observed during IP infections as well, albeit not significant (Fig. 10 A). Importantly, this phenotype was associated with a significant increase in susceptibility to disease (Fig. 5). In addition, levels of other innate immune cytokines, such as IL-6 and IL10, were altered in infected ship-/- mice. However, the role for these in Salmonella infection is less clear. The fact that IL-6 is upregulated during Salmonella infections in vivo and regulates PMN killing of Salmonella in vitro (Eckmann and Kagnoff, 2001; Nadeau et al., 2002), suggests it plays a protective role against disease. Interestingly, we found IL-6 was lower in ship-/- mice orally infected with Salmonella and higher in IP infected mice, suggesting that IL-6 modulation in ship-/- mice likely plays a critical role in controlling Salmonella in ship-/- mice independently of the other Th1 polarizing cytokines examined and may be heavily dependent on the route of infection. This is supported by experiments that plotted IL-6 production over the duration of oral vs. IP infections where we found that ship-/- mice produce lower levels of IL-6 at early time points during both oral and IP infections, but not later. Therefore, SHIP may play an important role in IL-6 regulation in both oral and IP infections and at distinct times during Salmonella pathogenesis. In contrast to IL-6, it has been suggested that IL-10 may be anti-protective against Salmonella infection (Pie et al., 1996), due to its classical role as an antiinflammatory cytokine that suppresses the functions of macrophages, DCs, NK cells and T cells (Moore et al., 2001). However, more recently it has been shown that adequate IL-10 production is an essential component of the immune  56  response against intracellular pathogens such as Leishmania and Toxoplasma (O'Garra and Vieira, 2007; Trinchieri, 2007). Thus, the lower levels of IL-10 produced by Salmonella infected ship-/- mice may also exacerbate disease. The recruitment and activation of phagocytes within Salmonella infected tissues, is heavily dependent on M1, or classic macrophages; thus these cells are essential in the fight against this intracellular pathogen (Gordon and Taylor, 2005; Mantovani et al., 2007). In contrast, M2 macrophages are incapable of controlling other intracellular pathogens like M. tuberculosis and Leishmania, both of which require strong Th1 immunity for clearance (Holscher et al., 2006; Kahnert et al., 2006). Interestingly, in the case of the ship-/- mouse, there is a skewing of macrophages in the peritoneal cavity and lungs to an M2 phenotype and it is hypothesized that this is due to uncontrolled PI3K signaling in the absence of SHIP, or other src kinases such as Lyn and Hck (Rauh et al., 2005; Xiao et al., 2008a). Based on data showing disregulated Th1 polarizing cytokines in infected ship-/- mice, we suspected that M2 skewing was contributing to increased susceptibility to Salmonella infection in vivo. Consistent with this, peritoneal macrophages from IP infected ship-/- animals showed a strong M2 phenotype (Fig. 12 C). During this type of infection, macrophages within the peritoneal cavity are the first innate immune cells to encounter Salmonella and are responsible for front line defense to prevent further spread to the blood and systemic sites (Mastroeni, 2002). During oral Salmonella infection however, resident tissue macrophages as well as DCs present in the Peyer's patches of the small intestine are the cells that first interact with bacteria colonizing gut tissues (McSorley et al., 2002). Positive staining for Ym1 and Arg1 in these areas (Figs. 12 A and B) further supported that M2 macrophages are indeed poised at critical sites during both oral and IP Salmonella infection in ship-/- mice. Taken together, the results presented in this chapter show that the high susceptibility of ship-/- to Salmonella infection seems to rely heavily on their inability to regulate cytokine responses critical to preventing infection. Furthermore, they suggest that the presence of M2 macrophages at sites of Salmonella infection in both IP and oral models, could be contributing to  57  increased susceptibility, as they are poor producers of antimicrobial molecules as well as Th1 polarizing cytokines, such as IL-12. Other contributing factors that could predispose the ship-/- mouse to Salmonella infection may also be lowered B cell populations, however the relative contribution of these cells during the early time points of infection examined in both our IP and oral model suggests that poor innate defenses are most likely key to governing susceptibility to Salmonella.  58  CHAPTER 4: The role of SHIP in S. Typhimurium infection of macrophages in vitro  59  4.1 Introduction Macrophages are integral in the pathogenesis of Salmonella. While it is known now that Salmonella can survive in multiple cell types, originally unique to its virulence strategies were the multiple mechanisms of surviving within the macrophage and utilizing this cell type as a vehicle to spread throughout the body and cause systemic disease. As such, in vitro modeling of Salmonella pathogenesis has made extensive use of macrophages in order to determine both aspects of bacterial survival and replication as well as macrophagedependent immune responses (Linehan and Holden, 2003). In addition, the macrophage has also been an integral tool in studying the role of SHIP in innate immune responses. While SHIP plays an important role in cells of both the innate and adaptive immune system, its function is of particular interest in macrophages and their relationship with pathogens. This is because SHIP regulates the macrophage pro-inflammatory response to PAMPs, such as LPS and CpG DNA, as well as receptor mediated phagocytosis and phagosome formation (Sly et al., 2007). Importantly, SHIP also can regulate macrophage phenotypes, which are increasingly recognized as important in determining the outcome of tumorogenesis, tissue regeneration and infectious diseases.  In  particular, the response of M2 macrophages is poor against most pathogens, except for protazoans, and in following they are associated with Th2 immunity. In addition, while M2 macrophages are important in wound healing and repair, they have also been implicated in the development of fibrosis during chronic inflammation as well as the development of tumors (Mantovani et al., 2007). As such, they are important contributors to the development of infectious as well as autoimmune diseases and therefore may play an important role in the outcome of Salmonella infections. Because SHIP deficiency has such widespread effects on both the innate and adaptive immune compartments, results using ship-/- mice for infectious disease studies are complicated at best. Despite this, much is known regarding the importance of the macrophage in Salmonella infection and the role of SHIP in macrophage responses. Therefore, it follows that studying the macrophage-  60  dependent immune responses against Salmonella in a ship-/- background is a logical place to begin assessing the relative importance of SHIP in innate responses to Salmonella. The results in this chapter provide insight into how SHIP deficiency alters classical macrophage responses to Salmonella, such as ability to limit intracellular replication, cell death and cytokine production. Importantly, these results highlight the fact that only M2 BMDMs produce lower levels of inflammatory cytokines when infected with Salmonella, which is what is seen in vivo, suggesting that these cells do indeed play an important role in the cytokine environment in the mouse during infection. Furthermore, the fact that SHIP deficiency paired with macrophage phenotype had the most dramatic effect on inflammatory cytokine production, and little effect on macrophage ability to control intracellular replication or the susceptibility of macrophages to Salmonella induced death, supports how critical the cytokine milieu is to preventing infection.  4.2 Results 4.2.1 Intracellular Salmonella replication in macrophages is independent of SHIP expression or macrophage phenotype Classically activated macrophages are the primary reservoirs for Salmonella in vivo and modulate bacterial clearance via the production of cytokines such as IL-12p70 and IFNγ (Linehan and Holden, 2003).  To  investigate the role of the macrophage in the ship-/- response to Salmonella infection, macrophages were derived from bone marrow of ship+/+ and ship-/- mice under M1 or M2 derivation conditions and their responses to Salmonella infection were compared. To assess the capability of M1 vs. M2 macrophages to prevent intracellular Salmonella replication, numbers of viable Salmonella were assessed using a standard 24 hour gentamicin protection assay. Replication was slightly higher in BMDMs from ship+/+ and ship-/- mice derived in the presence of mouse serum, which skews to an M2 phenotype, however these differences were not significant (Fig. 14).  Importantly, there was no significant difference in  intracellular Salmonella replication between M1 derived ship+/+ and ship-/-  61  BMDMs or between M2 derived ship+/+ and ship-/- BMDMs (Fig. 14 A), indicating that SHIP expression does not have a direct effect on internal bacterial numbers. Because Salmonella generally does not replicate to high numbers in BMDM however, the importance of SHIP in the macrophage bacteriostatic capability was examined by using Raw264.7 macrophages (Mastroeni and Maskell, 2006). These cells typically permit greater fold increases of bacteria over a 24 hour period and can be skewed to an M2 phenotype using IL-4 (Mantovani et al., 2007). However, even in Raw cells, still no significant difference was found in Salmonella replication in M1 vs. M2 cells (Fig. 14 B).  62  Figure 14. Changes of S. Typhimurium numbers in M1 vs. M2 macrophages (A) BMDMs were obtained from ship+/+ and ship-/- mice and derived in the presence of FBS alone (M1) or FBS + 2% mouse serum (M2) and infected with S. Typhimurium SL1344. (B) Raw 264.7 macrophages were grown in either DMEM + 10% FBS (M1) or skewed to an M2 phenotype by the addition of 100ng/ml IL-4 (M2) 24 hours prior to infection. For A and B cells were seeded and infected with an MOI of 10 S. Typhimurium SL1344 in a gentamicin protection assay with bacteria enumerated at 2 and 24 hours post-infection. Graphs represent fold replication of intracellular S. Typhimurium by dividing CFUs obtained at 24 hours by those at 2 hours. For both A and B three independent experiments were performed with each treatment being performed in triplicate for a total N=9 for each treatment. A. 1.75  ship-/-  1.50  ship+/+  1.25 1.00 0.75 0.50 0.25 0.00  M1  M2  M1  M2  B. 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00  63  4.2.2 M2 skewing of BMDMs provides a model for macrophage function in oral Salmonella infection in ship-/- mice Differences in cytokine profiles produced by M1 or M2 BMDMs during infection with S. Typhimurium SL1344 were more apparent and dependent upon SHIP genotype. As in orally infected ship-/- mice, Salmonella infected M2 derived ship-/- BMDMs produced significantly lower levels of IL-12p70 (P=0.0201), IL-6 (P=0.017) and IL-10 (P=0.0027) than ship+/+ cells (Fig. 15). Significant differences were not found in TNFα production from Salmonella infected M2 BMDM ship+/+ and ship-/- cells, or IL-6 and IL-10 production from ship+/+ and ship-/cells stimulated with LPS or heat-killed bacteria (Figs. 15, B, C and D). IL-12p70 production by ship+/+ cells was significantly greater than ship-/- with LPS (P=0.0465) and heat-killed bacteria (P=0.0052, Fig. 15 A). Interestingly, lower cytokine production by Salmonella infected ship-/- cells could only be seen using M2 derivation conditions; M1 derived macrophages showed significantly higher levels of IL-12p70 (P=0.0326), TNFα (P=0.0077), IL-6 (P=0.0017) and IL-10 (P=0. 0143) when infected with Salmonella (Fig. 16). Under M1 conditions, ship-/and ship+/+ macrophages stimulated with LPS or heat-killed Salmonella showed no significant differences in production of the four cytokines tested at 8 or 24 hours post-infection (Fig. 16). Taken together, these results suggest that M2 BMDMs are better able to mimic the cytokine production seen in vivo during Salmonella infection.  64  Figure 15. Salmonella infected BMDMs from ship-/- mice derived under M2 inducing conditions show decreased levels of inflammatory cytokines compared to ship+/+ cells. (A-D) BMDMs were obtained from ship+/+and ship-/mice and were polarized to an M2 phenotype by derivation for 10 days in the presence of FBS + 2% mouse serum. Cells were seeded and either left untreated (-), infected with S. Typhimurium SL1344 (SL1344) or heat-killed S. Typhimurium SL1344 (HK) at an MOI of 10, or treated with 100ng/ml S. Typhimurium LPS (LPS), for 8 hours and supernatants were collected. Cytokine analysis was performed using ELISAs. For A-D three independent experiments were performed with each treatment being performed in triplicate for a total N=9 for each treatment. A.  B. TNFα  IL-12 **  *  *  1250  ship-/-  1000  ship+/+  1250 1000  750  750  500  500  250  250  0  -  HK  SL1344  ship-/ship+/+  0  LPS  C.  -  HK  SL1344  LPS  D. IL-10  IL-6  **  400  ship-/ship  300  +/+  *  1750  ship-/ship+/+  1500 1250 1000  200  750 500  100  250  0  -  HK  SL1344  LPS  0 -  HK  SL1344  LPS  65  Figure 16. Salmonella infected BMDMs from ship-/- mice derived under M1 inducing conditions show increased levels of inflammatory cytokines compared to ship+/+ cells. (A-D) BMDMs were obtained from ship+/+and ship-/mice and were polarized to an M1 phenotype by derivation for 10 days in the presence of FBS. Cells were seeded and either left untreated (-), infected with S. Typhimurium SL1344 (SL1344) or heat-killed S. Typhimurium SL1344 (HK) at an MOI of 10, or treated with 100ng/ml S. Typhimurium LPS (LPS), for 8 hours and supernatants were collected. Cytokine analysis was performed using ELISAs. For A-D three independent experiments were performed with each treatment being performed in triplicate for a total N=9 for each treatment. A.  B. IL-12  TNFα  *  1500  **  250  ship-/ship+/+  1250  ship-/ship+/+  200  1000 150  750  100  500  50  250 0 -  HK  SL1344  0  LPS  -  C.  HK  SL1344  LPS  D.  IL-10  IL-6 *  500  ship-/ship+/+  400  **  1250  ship-/ship+/+  1000 750  300  500 200  250  100  10  0  0 -  HK  SL1344  LPS  -  HK  SL1344  LPS  66  4.2.3 SHIP deficiency does not influence cell death in vitro during Salmonella infection SHIP controls cell survival pathways and as well as macrophage phenotype, which can have direct effects on the longevity of a cell (Mantovani et al., 2007). Thus, whether the lower cytokine production by ship-/- BMDM was due to increased cell death in these cells was examined. As figure 17 shows, cell death was not significantly greater in ship-/- than ship+/+ M2 BMDM at either 8 or 24 hours post-infection, suggesting that this is not the reason for lowered cytokine production upon Salmonella infection by ship-/- M2 BMDM. Figure 17. ship-/- BMDM are not more susceptible to death upon infection with Salmonella. BMDMs were obtained from ship+/+and ship-/- mice and were polarized to an M2 phenotype by derivation for 10 days in the presence of FBS + 2% mouse serum. Cells were seeded and either left untreated (-), infected with S. Typhimurium SL1344 (SL1344) or heat-killed S. Typhimurium SL1344 (HK) at an MOI of 10, or treated with 100ng/ml S. Typhimurium LPS (LPS), for 8 hours (A) or 24 hours (B). Cells were collected and stained with the cell death marker 7AAD and analyzed via flow cytometry. 3 independent experiments were performed with each treatment being performed in triplicate for a total N=9 for each treatment. A.  B.  8 Hours  24 Hours  70  ship-/ship+/+  60  70  50  50  40  40  30  30  20  20  10  10  0  -  HK  SL1344  LPS  ship-/ship+/+  60  0  -  HK  SL1344  LPS  67  4.3 Discussion The high susceptibility of ship-/- mice to Salmonella infection is interesting from the standpoint of both Salmonella pathogenesis and SHIP dependent regulation of immune responses. However, the variety of cellular responses that SHIP negatively regulates makes it difficult to assess the importance of this molecule in specific cell types during their responses to Salmonella in vivo. Because macrophages play such a large role in determining the outcome of Salmonella pathogenesis and because SHIP regulates so many macrophage responses, extrapolating data from in vitro Salmonella infections of BMDM can give insight into how these cells may contribute to infection in mice. Importantly, because SHIP skews macrophages to an M2 phenotype and these cells were found to be poised at sites of Salmonella infection, the relative importance of both SHIP genotype in macrophages and cell phenotype in response Salmonella was assessed. M2 macrophages do not produce high levels of bactericidal mediators like reactive nitrogen or oxygen intermediates that play a role in controlling intracellular replication of Salmonella (Mantovani et al., 2007). As expected, cells derived under M2 inducing conditions using mouse serum or IL-4 allowed slightly higher Salmonella replication in a 24 hour period (Fig. 14), however this difference was independent of SHIP genotype in BMDMs. In addition, consistent with other unpublished work from our laboratory, Salmonella did not replicate well in BMDMs from either ship+/+ or ship-/- mice (Fig. 14 A). Interestingly, the cytokine profile produced by ship-/- mice infected with Salmonella was not mimicked by ship-/- macrophages derived under traditional M1 polarizing conditions in vitro. However, M2 polarized  ship-/- BMDMs infected with Salmonella produced a  cytokine profile that most closely paralleled the one seen in orally infected ship-/mice. For example, M2 ship-/- BMDMs produced significantly lower levels of IL12p70, IL-10, and IL-6 upon Salmonella infection. Lower, but not significant, production of TNFα was also a hallmark sign of an M2 phenotype (Fig. 15), however this did not match what was observed in vivo. Importantly, reduction in cytokines was not attributed to increased cell death in ship-/- BMDM (Fig. 17). In  68  contrast, experiments using conventional derivation conditions that are known to induce an M1 phenotype (Rauh et al., 2005) showed that Salmonella infected ship-/- M1 BMDMs produced a cytokine profile that was opposite to the one seen in in vivo oral Salmonella infections, with significantly higher levels of IL-12p20, IL-10 and IL-6 being produced (Fig. 16). Taken together, these results showed that while M2 derived BMDMs from ship-/- mice may not be less effective in preventing intracellular Salmonella replication than ship+/+ cells, they do produce very different cytokine profiles that closely mimic those seen in infected ship-/- mice. In correlation with results showing excess M2 macrophages in the gut and peritoneal cavity during oral Salmonella infections, the in vitro behavior of M2 macrophages may provide important insight into how these cells contribute to Salmonella infection in ship-/mice.  69  CHAPTER 5: The role of SHIP in the intestinal inflammatory response to enteric infection  70  5.1 Introduction Studying both intestinal inflammation and negative regulation of immune responses goes hand in hand. This is because the immune response in the gut must be down-regulated in the presence of commensal microbes that have the potential to stimulate strong immune responses. This regulation is a complex process that relies on balancing low pro-inflammatory mediator production with maintenance of regulatory secretory antibodies, CAMPs, and mucous products as epithelial and immune cells respond to commensal bacteria and their PAMPs. If this balance is lost and especially if there is disregulation of pro-inflammatory and Th1, Th2 or Th17 cytokine responses, inflammatory bowel diseases (IBD), like ulcerative colitis (UC) and Crohn's disease (CD) can result (Kelsall, 2008). The severe mucosal damage inflicted by unchecked inflammation in the intestine during IBD requires wound healing in the form of collagen deposition and epithelial restitution. However, this process in CD patients is also dysregulated and fibrosis, characterized by intestinal wall thickening and uncontrolled ECM deposition, occurs (Rieder et al., 2007). Fibrosis one of the most serious complication of CD, as it leads to the formation of intestinal strictures, which must be removed surgically. Unfortunately, because fibrosis is a reactive process to the chronic inflammation seen in CD, stricture formation is recurrent in most patients, even after surgery (Burke et al., 2007). SHIP has the potential to play a significant role in the development of inflammation and fibrosis in the intestine; however this has not been previously examined. SHIP controls the pro-inflammatory status of macrophages that is strongly implicated in the development of IBD (Kelsall, 2008; Sly et al., 2007). For example, ROS production by macrophages is a requirement for the tissue destruction that characterizes IBD and macrophages of CD patients have high levels of NF−ΚB transcription and downstream production of TNFα (Kelsall, 2008; Kruidenier and Verspaget, 2002). Similarly in ship-/- macrophages, other groups have found high levels of TNFα are produced in response to LPS. In addition, TGFβ, which is one of the most potent pro-fibrotic mediators in the body, regulates macrophage pro-inflammatory responses via SHIP (Sly, 2004).  71  Also, SHIP actively represses the generation of M2 macrophages, and these cells have been implicated in playing a role in fibrosis development (Sangaletti et al., 2003). Therefore, since SHIP regulates many cellular processes required to prevent intestinal inflammation, it is probable that ship-/- mice might develop intestinal pathology in response to commensals or enteric pathogens that mimics IBD. Oral infection of mice with S. Typhimurium provides and excellent model to study human typhoid infection, which is a systemic, febrile disease. However, unlike in humans, where NTS such as Typhimurium cause gastroenteritis, this phenotype is not seen in murine models unless the microbiota is altered with antibiotic treatment prior to infection (Bartel, 2003). However, during the course of experiments examining susceptibility of ship-/- mice to oral S. Typhimurium infection, these mice exhibited severe swelling of the small intestine primarily located in the ileum. The experiments presented in this chapter therefore, were designed to investigate the nature and cause of intestinal inflammation in the ship-/- mouse. Histology and examination of cytokines showed that the increase in size of the ileum was in fact due to excessive inflammation. In addition, signs of fibrosis, such as collagen deposition and the presence of high levels of profibrotic factors, like TGFβ and MCP-1 were also found in the ileum. Importantly, results showed that this phenotype is not specific to Salmonella infection, as Citrobacter rodentium, Helicobacter, as well as heat-killed bacteria and LPS also led to inflammation. 5.2 Results 5.2.1 Salmonella infected ship-/- mice have highly inflamed small intestines The nature of S. Typhimurium infection in mice is a systemic disease, minimal intestinal pathology is seen (and limited to Peyer's patches) and typically the small intestine is not highly colonized by bacteria. However, upon infection of ship-/- mice with oral S. Typhimurium and without pre-treatment of mice with  72  streptomycin, what appeared to be severe swelling of the ileum was observed. This phenotype was accompanied by significant increases in small intestinal weight (Fig. 18 A) as well as high colonization with S. Typhimurium at two days post-infection (Fig. 18 B). Upon examination of small intestinal sections by hematoxylin and eosin Y (H&E) histology, which stains nucleic acids, ribosomes and cellular proteins, it was clear that intestines from ship-/- mice exhibited increased size and weight due to massive infiltration of inflammatory cells and edema (Fig. 18 C). A pathology scoring system that assessed inflammation in the lumen, surface epithelium, mucosa and submucosa of the iliea, showed a more quantitative difference between ship-/- and ship+/+ ilea (Fig. 18 D). The inflammation seen in these H&E slides correlated with a significant increase in the inflammatory cytokines TNFα (P<0.0001) and MCP-1 (P=0.0087) in the small intestine taken at day two post-infection (Fig. 19). Importantly, uninfected ship-/mice and ship+/+ mice showed no inflammation by histology and cytokine levels were lower than the detectable limit (Fig. 18 E).  73  Figure 18. Salmonella infection in ship-/- mice leads to inflammation of the ileum. ship+/+and ship-/- mice were infected orally with 1x106 S. Typhimurium SL1344 and were sacrificed at 2 days post-infection. (A and B) Small intestines were removed from uninfected (-SL1344) and Salmonella infected (+SL1344) mice, weighed and homogenized for bacterial counts. (C and D) Prior to homogenization, 0.25 micron sections of ilea were excised and stained with H&E for pathology and scored. (E) H&E sections of ilea from uninfected mice. For A, B and D, 3 independent experiments were performed with a total N=12 for both ship+/+ and ship-/- mice. For C and E, representative experiments are shown. A.  B. Small Intestine *  ***  1.25  106 ship-/ship+/+  1.00  105 104  0.75  103  0.50  102  0.25  101  0.00 +SL1344  -SL1344  100 ship-/-  ship+/+  C. ship+/+  ship-/-  5x  20x  74  D. 15.0  Lumen Surface Epithelium Mucosa Submucosa  12.5 10.0 7.5 5.0 2.5 0.0  ship-/-  ship+/+ Ileum  E. ship+/+  ship-/-  5x  20x  75  Figure 19. TNFα and MCP-1 are increased in ship-/- small intestines during Salmonella infection. ship+/+and ship-/- mice were infected orally with 1x106 S. Typhimurium SL1344 and were sacrificed at 2 days post-infection. (A and B) Small intestines were homogenized and supernatants were analyzed for proinflammatory cytokines using ELISAs. For A and B, 3 independent experiments were performed with a total N=12 for both ship+/+ and ship-/- mice. A.  B.  MCP-1  TNFα 120  ***  **  1000  100  750  80 60  500  40 250  20 0  ship-/-  ship+/+  0  ship-/-  ship+/+  4.2.2 Fibrosis is a consequence of inflammation induced during Salmonella infection in the small intestine of ship-/- mice Fibrosis is the formation of excess connective tissue in the small intestine and it is a severe complication of IBD. In IBD, fibrosis occurs as a direct result of chronic or recurrent inflammation of the intestine and it is a reactive process-the more inflammation occurs, the more fibrotic tissue develops (Rieder et al., 2007). Because of the high degree of intestinal inflammation caused by S. Typhimurium infection in ship-/- mice and the infiltration of M2 macrophages, which are associated with fibrotic tissue generation, to this area (Chapter 3), it was questioned whether fibrosis was occurring in the ilea of Salmonella infected ship/-  mice. Masson's trichrome histology stain was used to determine collagen  deposition in the ilea of ship+/+ and ship-/- mice orally infected with S. Typhimurium. As Figure 20 A shows, collagen (blue stain) deposition in ship-/mice is widespread in the submucosa as well as muscularis propria, whereas in 76  ship+/+ mice it remains tightly compacted to a thin layer at the muscularis mucosae.  To quantify collagen deposition, sections of ilea were digested in  pepsin and soluble collagens were assessed by a sircol dye based collagen assay. Results of these tests showed a significant increase in collagen levels upon infection of ship-/- mice, whereas in ship+/+ mice levels stayed steady (P<0.001, Fig. 20 B). Several pro-fibrotic mediators are essential for the development of fibrosis in the intestine, including TGFβ and MCP-1. TGFβ is the most potent pro-fibrotic indicator in the intestine, as it regulates induces the production of other profibrotic mediators and cell types, like collagen transforming growth factor (CTGF), insulin-like growth factor (IGF) and Th17 cells, as well as reduces the production of matrix metalloproteases (MMP) which are necessary for collagen breakdown (Rieder et al., 2007). MCP-1 is a critical chemoattractant for the inflammatory cells that are responsible for tissue destruction that precedes fibrosis, and was already found to be elevated in the intestines of ship-/- infected mice (Fig. 19). Because TGFβ is necessary for the induction of fibrosis, its presence was assessed in ilea from Salmonella infected ship+/+ and ship-/- mice. Figure 20 C shows that both latent and active forms of TGFβ are significantly higher (P=0.0001) in the ilea of ship-/- than ship+/+ mice. Both uninfected ship+/+ and ship/-  mice produced levels of TGFβ below the level of detection. Taken together,  these results suggest that despite the short duration of inflammation in the ileum induced by oral Salmonella infection in ship-/- mice, pro-fibrotic mediators are present and collagen deposition leading to fibrosis can occur.  77  Figure 20. Fibrosis is a consequence of small intestinal inflammation in ship-/- ilea during Salmonella infection. ship+/+and ship-/- mice were infected orally with 1x106 S. Typhimurium SL1344 and were sacrificed at 2 days postinfection. (A) Masson's Trichrome stain of ilea sections taken at 2 days postinfection. (B) 0.5 cm of Ileum was removed at 2 days post-infection and pepsin soluble collagen was assessed using a standard collagen assay. Small intestines were removed and homogenized at 2 days post infection and supernatants were analyzed via ELISA for TGFβ (C). In A, a representative experiment is shown. For B and C, 3 independent experiments were performed with a total N=12 for both ship+/+ and ship-/- mice. A.  ship+/+  ship-/-  5x  20x  B. 70 60 50 40 30 20 10 3 2 1 0  *** ship-/ship+/+  -SL1344  +SL1344  78  C.  TGFβ 3000  ***  *** ship-/ship+/+  2000  1000  0  Active  Latent  4.2.3 Development of inflammation and fibrosis in the ileum of ship-/- mice is not specific to Salmonella infection A large contributing factor to the development of IBD is the immune response against microflora that colonize the intestine (Duchmann et al., 1995). In fact, there is a direct correlation between the number of bacteria in the colon, terminal ileum and caecum and the development of CD; these areas are where the commensal population is most dense and where CD manifests in the gastrointestinal tract (D'Haens et al., 1998). For the most part, it did not seem that ship-/- mice were susceptible to inflammation induced by commensal bacteria, since the majority of uninfected mice presented with no intestinal pathology (Fig. 18 E). However, a few uninfected mice were found with enlarged ilea, raising the possibility that the intestinal inflammatory response in ship-/- mice was not specific to Salmonella infection. To assess to what degree other pathogens or products could be affecting intestinal pathology in ship-/- mice, contamination of mice with Helicobacter species as a potential cause of intestinal inflammation in "uninfected" controls was investigated. There are 8 species of Helicobacter that infect the mouse intestinal tract and contamination of commercial and academic colonies is  79  extremely high. colonization  with  Importantly, in many immunocompromised mouse strains, typically  non-pathogenic Helicobacter  sp.  can  induce  inflammatory disease (Whary and Fox, 2006). PCR analysis specific for all 8 Helicobacter species was done by RADIL on pooled stool samples from ship-/and ship+/+ mice. Results showed infection with three different species of Helicobacter, hepaticus, typhilonius and rodentium. Based on correspondence with collaborators at the British Columbia Cancer Research Centre (BCCRC), the introduction of Helicobacter infection in rederived ship-/- mice corresponded with the development of small intestinal inflammation, suggesting that this infection was a probable cause for inflammation seen in my "uninfected" controls. After confirmation that both ship-/- and ship+/+ mice were infected with Helicobacter, it was essential to define if co-infection was a prerequisite for Salmonella-induced intestinal inflammation. Helicobacter-free ship-/- and ship+/+ mice, that were rederived from another mouse colony and housed in a separate facility as well as screened as Helicobacter negative by PCR, were infected with S. Typhimurium orally and intestinal pathology was examined at two days postinfection. As figure 21 shows, Helicobacter-free ship-/- mice also showed severe inflammation as well as collagen deposition in the ileum. Importantly, ship-/- and ship+/+ mice free of Helicobacter and left uninfected did not show intestinal pathology (Fig. 21 D). To investigate whether another enteric pathogen was capable of inducing intestinal inflammation, ship-/- mice were infected with Citrobacter rodentium and intestinal pathology was monitored at seven days post-infection, which is the time it typically takes for this bacterium to colonize the colon (Mundy et al., 2005). While ship-/- mice were not more susceptible to colonization by C. rodentium (Fig. 8) and did not become sick upon infection, they did display severe inflammation and fibrosis of the ilea after 7 days (Fig. 22). Taken together, these results show that both intracellular and extracellular enteric pathogens, as well as commensal species like Helicobacter are not tolerated by the immune response in the small intestine of ship-/- mice.  80  Interestingly, infection with viable bacteria was not a requirement for the induction of intestinal inflammation in ship-/- mice, since infection with both heatkilled S. Typhimurium as well as C. rodentium, resulted in similar intestinal pathology as live infections (Fig. 23). In addition, gavage with LPS from S. Typhimurium was also able to induce inflammation after two days, suggesting that strong PAMPs on the microbe surface are sufficient to initiate the inflammatory response in these mice (Fig. 23). Figure 21. Helicobacter infection is not a prerequisite of Salmonella induced intestinal inflammation. Helicobacter-free ship+/+and ship-/- mice were infected orally with 1x106 S. Typhimurium SL1344 and were sacrificed at 2 days post-infection. 0.25 cm of ilea were excised and pathology was scored based on H&E histology staining (A and B). (C) Masson's trichrome stain for collagen deposition in infected mice. (D and E) H&E and Masson's trichrome histology on ilea from uninfected Helicobacter-free mice. For A, C, D and E representative experiments are shown. For B, 3 independent experiments were performed with a total N=12 for both ship+/+ and ship-/- mice. A.  ship+/+  ship-/-  5x  20x  81  B. 15.0  Lumen Surface Epithelium Mucosa Submucosa  12.5 10.0 7.5 5.0 2.5 0.0  ship-/-  Ileum  ship+/+  C. ship+/+  ship-/-  5x  20x  82  D.  ship+/+  ship-/-  5x  20x  E.  ship+/+  ship-/-  5x  20x  83  Figure 22. Citrobacter rodentium induces intestinal inflammation in ship-/mice. ship+/+ and ship-/- mice were infected orally with 1x108 C. rodentium DBS100 and were sacrificed at 7 days post-infection. 0.25 cm of ileum were excised and pathology was scored based on H&E histology staining (A and B). (C) Masson's trichrome stain for collagen deposition. For A and C, representative experiments are shown. For B, 3 independent experiments were performed with a total N=12 for both ship+/+ and ship-/- mice. A.  ship+/+  ship-/-  5x  20x  B.  17.5  Lumen Surface Epithelium Mucosa Submucosa  15.0 12.5 10.0 7.5 5.0 2.5 0.0  ship-/-  ship+/+ Ileum  84  C. ship+/+  ship-/-  5x  20x  85  Figure 23. Heat-killed bacteria and LPS can induce intestinal inflammation in ship-/- mice. ship+/+ and ship-/- mice were infected orally with 1x106 heat-killed S. Typhimurium SL1344 or 1x108 heat-killed C. rodentium DBS100 and were sacrificed at 2 or 7 days post-infection, respectively. 0.25 cm of ileum were excised from ship-/- mice infected with heat-killed Salmonella (A-C) or Citrobacter (D-F), stained with H&E, pathology scored and Masson's trichrome stained, respectively. (G-H) ship+/+ and ship-/- mice were treated orally with 50mg/kg S. Typhimurium LPS, sacrificed at 2 days post infection and ilea sections were stained for H&E and Masson's trichrome histology, respectively. For A, C, D, F, G and H representative experiments are shown. For B and E, 3 independent experiments were performed with a total N=12 for both ship+/+ and ship-/- mice. ship+/+  A.  ship-/-  5x  20x  B. 15.0  Lumen Surface Epithelium Mucosa Submucosa  12.5 10.0 7.5 5.0 2.5 0.0  ship-/-  ship+/+ Ileum  86  C. ship+/+  ship-/-  ship+/+  ship-/-  5x  20x  D.  5x  20x  87  E.  15.0  Lumen Surface Epithelium Mucosa Submucosa  12.5 10.0 7.5 5.0 2.5 0.0  ship-/-  ship+/+ Ileum  F. ship+/+  ship-/-  5x  20x  88  G.  ship+/+  ship-/-  ship+/+  ship-/-  5x  20x  H.  5x  20x  89  5.3 Discussion IBD results from a disregulation of both inflammatory and wound healing processes in the intestines. While pathology seen in UC is typically restricted to the intestinal mucosa, CD is a transmural disease where inflammation is found throughout the mucosa, submucosa and muscle layers. Fibrosis, characterized by increased collagen deposition and disruption or expansion of the muscle layer of the intestine, is the most severe complication of CD. During fibrosis, overexpansion  of  fibroblasts  and  mesenchymal  cells  together  with  overexpression of adhesion molecules and pro-fibrotic factors, leads to the formation of excess ECM, thickening the gut wall and decreasing elasticity (Rieder et al., 2007). In fact, despite the available treatments for IBD, such as immunomodulatory therapies, antibiotics and probiotics, fibrotic stricutre formation in CD patients still leads to significant mortality (Van Assche et al., 2004) . Susceptibility to IBD is controlled by a number of factors, including the gut microflora, environmental stimuli, genetics and immunity (Bouma and Strober, 2003). Immune responses generated against normal flora in the intestine are highlighted as one of the major causes for IBD development (Elson et al., 2005). In a healthy individual, inflammation induced by commensal bacteria is minimized by the epithelium and immune cells residing in the lamina propria. In addition to the production of CAMPs and bacterial permeability inducing protein (BPI) that directly attack bacterial membranes (Eckmann, 2005), epithelial cells produce mucus, which provides a physical barrier against bacteria and their products from moving to deeper tissues (Podolsky, 1999). Secretory IgA is also an important defense at the epithelial surface against commensal organisms (Macpherson et al., 2000). If the epithelial barrier is breached however, PMNs and macrophages in the lamina propria are responsible for clearing bacteria by traditional innate responses such phagocytosis and lysosomal degradation (Kelsall, 2008). The differentiation of T regulatory cells in this region is also of critical importance in regulating immune responses (Bouma and Strober, 2003). However, in IBD patients, both the epithelial barrier and immunity in the lamina propria is 90  compromised. Overproduction of pro-inflammatory mediators by innate immune cells, such as TNFα and high mobility group box protein 1 (HMGB-1), as well as mast cell proteases, break down epithelial tight junctions and can cause epithelial cell apoptosis that leads to increased intestinal permeability (Bruewer et al., 2003; Jacob et al., 2005; Liu et al., 2006). Uncontrolled positive feedback of IL23, Th1 cytokines, IL-17 and TGFβ produced by innate immune cells in the lamina propria are then responsible for the uncontrolled inflammation and fibrosis development seen in CD (Fantini et al., 2006; Uhlig et al., 2006; Wilson et al., 2007). Few effective animal models are available that mimic human IBD, in part because mice are not susceptible to developing fibrosis in the intestine (Pucilowska et al., 2000). This resistance to inflammatory disease correlates well with the fact that mice do not develop the same intestinal pathology upon infection with enteric bacteria, such as S. Typhimurium, that is seen in humans. However, inflammation and fibrosis can occur upon infection with Salmonella after oral pre-treatment with streptomycin. Chronic infection in this model results in both severe inflammation and fibrosis development in the caecum and colon that mimics pathology and cytokine responses that are seen in CD patients (Grassl et al., 2008). In addition, these data show that inflammation also occured in ship-/- mice upon infection with Salmonella, however antibiotic pretreatment was not required. This phenotype raises the interesting questions of how SHIP controls the inflammatory response in the intestine, and whether ship-/- mice may be used as an effective model to study IBD. SHIP deficiency may affect the development of intestinal inflammation and fibrosis by altering immune defenses of both the epithelial layer and lamina propria. For example, results showed that there is significantly more TNFα in the small intestine of Salmonella infected ship-/- mice, and TNFα contributes to both the breakdown of tight junctions as well as epithelial cell apoptosis in IBD (Bruewer et al., 2003). Degranulation of mast cells and the subsequent release of proteases is also important for increasing the permeability of the epithelial barrier in IBD (Jacob et al., 2005). Because SHIP sets the activation threshold of mast  91  cells and limits degranulation, lack of this regulation in the intestine during infection has the potential to increase inflammation (Huber, 2000). Finally, because ship-/- mice have lower levels of B cells, the production of secretory IgA, which is critical in preventing bacteria from breaching the epithelium, may be diminished (Brauweiler et al., 2000a; Macpherson et al., 2000) In the lamina propria, elevated levels of both TNFα and MCP-1 are required for inflammation (Kelsall, 2008). The fact that both cytokines were significantly higher in ship-/- mice may be a direct effect of the lack of negative regulation on LPS dependent pro-inflammatory signaling in ship-/- macrophages. Furthermore, the combination of the presence of these cytokines with the fact that SHIP negatively regulates PMN recruitment to these mediators may further exacerbate inflammation in the lamina propria (Sly et al., 2007). In ship-/- mice, fibrosis may be occurring quickly because of the hyper-responsiveness of may be dependent on the high density of M2 macrophages in the small intestine during infections. While M2 macrophages are required for normal wound healing processes, they are also found at sites of fibrotic tissue development (Mantovani et al., 2007). Thus, the combination of uncontrolled inflammatory responses and M2 skewing in the ship-/- gut could provide an ideal environment for rapid collagen deposition. Whether ship-/- mice could be used to study IBD will depend on the similarities and differences between the inflammatory phenotype seen in the ship-/- ileum and factors implicated in the development of CD in other mouse models and in humans (Table 8). The primary paradigm for the development of IBD, specifically CD, is the overproduction of Th1 polarizing cytokines, such as IL-12 and IFNγ as well as TNFα, paired with a deficient regulatory T cell response. More recent studies have also shown that these cytokines as well as IL-6 regulate the development of Th17 T cells and the production of IL-17 and IL23, which are present in both CD patients as well as mouse models of IBD (Kelsall, 2008). Fibrosis development is characterized by increased collagen deposition that requires pro-fibrotic factors like TGFβ (Rieder et al., 2007). In the ship-/- mouse however, while TGFβ and MCP-1 levels were high, TNFα was the  92  only pro-inflammatory cytokine seen to be elevated in the intestines. ship-/- mice had no significant increase in IL-6 in the small intestine, which acts in a positive feedback loop in the development of Th17 responses that characterize IBD. Importantly, it is unknown what levels of IL-17 or IL-23 are in the ship-/- intestine, highlighting the fact that more work needs to be done to better characterize intestinal inflammation in this model and compare it to IBD. Nonetheless, the occurrence of inflammation in the intestine of ship-/- mice provides new avenues to explore the importance of SHIP in gut immunity as well as alternative mechanisms that may lead to the development of IBD. Table 8. Similarities and differences between intestinal inflammation seen in the ship-/- mouse compared to IBD. SIMILAR TO IBD High TNFα High MCP-1 High TGFβ  DIFFERENT THAN IBD IL-12 not elevated IFNγ not elevated IL-6 not elevated  Increased collagen deposition  Located in ileum instead of caecum and colon  UNKNOWN IL-17 levels IL-23 levels Presence of regulatory T cells in small intestine Th2 cytokine levels  93  CHAPTER 6: DISCUSSION  94  6.1 Regulation of anti-Salmonella immunity by SHIP The importance of negative regulators during immune responses to pathogens has been highlighted using various mouse models. PI3K, SHP-1, LYN and PTEN+/- mice all have severe impairment of immune homeostasis and are susceptible to various pathogens, such as Mycobacterium tuberculosis, Leishmania, Pseudomonas, as well as cancers. However, in some models, such as SHP-1-/- mice infected with cytomegalovirus, deficient immune modulation can actually have a protective effect against disease (Veillette et al., 2002). Indeed, ship-/- mice have many immune characteristics that could both increase or decrease their susceptibility to pathogens. For example, myeloproliferation and decreased activation thresholds in both innate and adaptive immune cells might prime immune responses, while skewing of macrophage, NK and T cell responses towards an inhibitory phenotype would easily predispose ship-/- mice to infection. Data presented here confirmed that the latter of these scenarios is true; ship-/- mice are highly susceptible to Salmonella infection. Contributing factors in this susceptibility are poor induction of Th1 polarizing cytokines, M2 macrophage skewing at sites of infection, and possibly hyper-responsive immunity in the gut to either the bacteria themselves, or LPS. However, what remains unclear is when SHIP dependent regulation of immune responses is most important during Salmonella infection. Potentially, SHIP deficiency may impact susceptibility to Salmonella either i) during systemic phases of disease; an idea which is supported by the fact that ship-/- mice were not susceptible to Citrobacter rodentium, ii) during systemic responses to LPS or iii) in the gut, where Salmonella clearly induces an overzealous innate response. Regardless of where SHIP plays a more substantial role, it is clear that it, like so many other negative regulators, is an essential component in our defenses against infectious disease.  95  6.2 The role of SHIP in systemic Salmonellosis The simple lack of negative regulation does not necessarily equal increased susceptibility to disease-it seems that the nature of the pathogen and cell responses governed by the regulator are both important factors in determining pathogenesis. This fact is exemplified in the ship-/- mouse, which was found to be highly susceptible to both oral and IP Salmonella infection, yet was as well colonized by Citrobacter rodentium as were ship+/+ mice. While both pathogens cause enteric disease, the main distinguishing features between the two is that in mice, S. Typhimuium is an intracellular pathogen that causes systemic disease, while Citrobacter is not. In addition, being an attaching and effacing pathogen, Citrobacter targets epithelial cells of the colon to establish infection, whereas Salmonella rapidly breaches the epithelial barrier and disseminates to systemic organs within phagocytic cells (Mastroeni and Maskell, 2006; Mundy et al., 2005). Salmonella establishes systemic infection by secreting a variety of effector molecules via its TTSS that manipulate the host cell environment and make it conducive to bacterial growth (Gal-Mor and Finlay, 2006). Once the epithelium is breached, SPI-2 effectors are essential for creating the SCV and allowing Salmonella replication within macrophages and a variety of other cell types. Importantly, macrophages and DCs are the primary cell types that shuttle bacteria from the gut to systemic organs where infection foci are established, such as the liver, and spleen (Mastroeni and Maskell, 2006). In the ship-/- mouse, it was found that there are significantly higher bacterial counts in these sites during infections. SHIP deficiency may allow for higher bacterial numbers in the liver and spleen in two ways. Firstly, ship-/- mice suffer from severe over proliferation of myeloid cells; it is well known that ship-/- mice have higher levels of circulating macrophages and myeloid DC's, and this leads to the pulmonary inflammation that eventually kills the mice (Helgason et al., 1998). Therefore, it is possible that higher CFU counts are found in systemic sites simply because there are more myeloid cells to infect. However, whether these cells have the same phagocytic capacity towards Salmonella, or if they are able to home  96  properly from the gut to systemic sites during infection, remains unknown. Certainly, the phenotype of the macrophage could be an important factor in determining whether more cells would equal greater levels of infection. Secondly, the bacteriostatic capabilities of ship-/- macrophages or other cells that permit intracellular replication might be impaired. From studies presented in chapter 4, it is clear that at least M2 macrophages do not produce cytokines necessary to initiate bacteriostatic responses against Salmonella, such as IL-12 and IL-6. These "adaptive/innate" responses of macrophages are essential for preventing mortality in mice and are also important for the bacteria to establish a persistent, yet non-fatal infection of systemic organs (Fig. 2). The bacteriostatic capability of macrophages in vivo is exemplified by the fact that Salmonella does not replicate to high numbers per cell in systemic sites (Sheppard et al., 2003). In the ship-/- mouse however, it is unknown to what degree and in what cell types Salmonella may be replicating, or whether M2 cells are truly the culprits of the lower levels of IL-12, IL-6 and IFNγ that are produced during infection. However, the fact that M2 cells were found at sites of infection suggests that they may provide a reservoir where unchecked bacterial replication can occur. This is supported by in vitro data showing not significant, but elevated, bacterial replication in M2 vs. M1 macrophages. Furthermore, while it is known that M2 cells are highly phagocytic for cellular debris (Mantovani, 2007), we do not know whether these cells behave similarly towards pathogens or whether Salmonella can replicate to high numbers per M2 cell in vivo. Adaptive immunity is also strictly governed by SHIP in both T and B cells and it is critical in the control and eventual clearance of systemic Salmonellosis. However, the question of whether T and B cells control susceptibility to Salmonella in the ship-/- mouse model is interesting since most ship-/- mice die from infection much earlier than classic adaptive responses are required. Despite this fact however, the lower number of B cells in ship-/- mice could affect systemic Salmonellosis in that B cells, apart from producing antibodies, can act as APC's as well as harbor replicating Salmonella (Mastroeni et al., 2000). In particular, the  97  APC function of B cells during Salmonella infection may be essential for imparting protection from disease. 6.3 The role of SHIP in endotoxin tolerance during systemic Salmonellosis In survival experiments, ship-/- mice were highly susceptible to infection with live Salmonella via both oral and IP infection routes, but did not become ill from injection or gavage with heat-killed bacteria. These experiments were designed to assess the relative importance of LPS present in infection inoculum in causing death. However, they can not answer whether LPS present at systemic sites from replicating bacteria is the real cause of mortality seen in Salmonella infected ship-/- mice. The fact that systemic levels of TNFα, a major inducer of endotoxic shock, were consistently higher in Salmonella infected ship-/mice suggests this may be the case. The highly sensitive phenotype of ship-/macrophages to LPS, at least in vitro, would easily contribute to this cytokine profile during infection and could lead to endotoxin mediated death. Despite these factors however, there is evidence to suggest that LPS sensitivity may not be the cause of increase susceptibility to Salmonella in ship-/mice. For example, other classic mediators of toxic shock, like IL-12 and IFNγ, were significantly lower in ship-/- mice during Salmonella infections. In addition, LPS from Citrobacter rodentium infection was not sufficient to cause death in ship-/- mice, although this is most likely due to the fact that Citrobacter does not migrate to systemic sites. Most importantly however, is the fact that the phenotype of macrophages in ship-/- mice is skewed to an M2 phenotype. The major role of these cells in the body is not to combat bacterial infections, but rather to remodel tissues after destructive immune responses. While results presented in chapter 5 showed that M2 BMDM from both ship+/+ and ship-/- mice can respond to LPS, ship-/- M2 cells did not produce more inflammatory cytokines than ship+/+ cells with this stimulus. This contrasts with the response of M1 ship-/BMDM to LPS, since it has been shown that they hypersecrete pro-inflammatory cytokines upon stimulation (Sly et al., 2004). Therefore, despite the fact that there are many more circulating myeloid cells in the ship-/- mouse, a majority of  98  them are of an M2 phenotype with a dampened pro-inflammatory response so might not contribute to endotoxin sensitivity. Results showing that both ship-/mice as well as ship-/- M2 BMDM produce lower levels of IL-12, IFNγ and IL-6 support this idea. Further biochemical analysis of M2 macrophage responses to LPS in vitro would also shed light on how these cells function during bacterial infections. In addition, it has been found that SHIP is upregulated in response to other TLR ligands, such as CpG DNA but not double stranded RNA, indicating that its function may be to regulate MyD88 dependent signaling in innate immune cells (L. Sly and G. Krystal, submitted). Therefore, in the ship-/- mouse during Salmonella infection there are potentially other bacterial products besides LPS that have the capacity to overstimulate immune cells and mediate death. Again however, it is unknown how these stimuli signal in M2 macrophages and what types of immune responses they are capable of mounting in the presence of these PAMPs. Thus, while M2 cells may play a large role in allowing increased bacterial dissemination and replication in systemic sites, whether they can contribute to shock caused by LPS or other PAMPs remains unknown. Clearly, many more biochemical experiments are needed in vitro on ship-/- M2 macrophages in order to tease out the relative importance of these cells during bacterial infections. 6.4 The role of SHIP in gut immunology The importance of SHIP regulating gut immunology is highlighted by the fact that ship-/- mice develop severe inflammation in the ileum during challenge with either enteric bacteria or LPS. This response is rapid and strong; by two days post-challenge the ilea are filled with inflammatory cells and there is already deposition of collagen in both the muscle layers and submucosa. Most likely, this is due to both a breakdown of integrity in the epithelium and poor immune responses in the lamina propria to invading bacteria or bacterial products. Indeed, SHIP is required to negatively regulate the production of both proinflammatory and pro-fibrotic factors that were found to be elevated in the guts of  99  ship-/- mice, such as TNFα, MCP-1 and TGFβ, and it of course regulates the M2 phenotype of macrophages, which also play a role in the development of fibrosis. However, many questions remain regarding this inflammatory phenotype. For example, why does SHIP play such an important role in regulating immunity in the small intestine but not in the caecum or colon? Why isn't there an overactive immune response to bacterial products from resident microflora in the ship-/- gut? Finally, what impact does gut inflammation have on the development of systemic Salmonellosis? SHIP deficiency alters immune homeostasis because it regulates the distribution of different types of immune cells in the body as well as the strength and duration of their responses to various stimuli. Therefore, how immune cells are distributed in the ship-/- mouse may explain why the small intestine is so susceptible to inflammation yet the rest of the GI tract is not. For example, M2 macrophages were seen in the small intestine during Salmonella infection and maybe these cells either do not differentiate in, or home to, other sites of the gut as readily. Other cell populations, such as regulatory T cells, are extremely important in mediating tolerance in the gut to microbial stimuli (Bouma and Strober, 2003). It is unknown what the distribution of these cells is in the small intestine or colon of ship-/- mice, although it is known that ship-/- mice have overall higher populations of both myeloid suppressor and regulatory T cells in systemic organs (Kashiwada et al., 2006). If this is the case however, while these cells may show "regulatory" phenotypes by cell surface marker or protein expression, whether they are functional in the small intestine has not been determined. Importantly, the distribution of cell types in the small intestine vs. the caecum or colon will dictate the cytokine milieu in the area that ultimately is the cause for inflammation. As discussed in chapter 5, how similar the cytokine profile in ship-/small intestines is to those seen in UC or CD is still largely unknown. LPS in the ship-/- intestine obviously does play a role in the inflammatory response since both heat-killed Salmonella and Citrobacter as well as LPS induced inflammation. However, whether the degree of inflammation seen in the intestine is dependent on the dose of LPS remains unknown. Identifying a low  100  dose of LPS that would lessen the degree of inflammation in ship-/- ilea, or not induce the response altogether, would suggest that the reason microflora do not cause inflammation in the ship-/- mouse is because they do not shed enough LPS to generate a response. Indeed the number of microflora in the small intestine, around 104 bacteria, is many logs lower than the 1014 organisms that reside in the colon (Duchmann et al., 1995). But, this fact again raises the question as to why LPS from microflora in the colon would not generate an inflammatory response in ship-/- mice. Once again, the population of cells in the area and cytokine milieu most likely has a large impact on the outcome of intestinal pathology. Finally, it is important to question the impact intestinal inflammation has on the development of systemic bacterial infections. While in the case of Citrobacter infection, small intestinal inflammation does not seem to affect the ability of bacteria to colonize the colon, Salmonella was found to replicate to high numbers in systemic sites and cause mortality in ship-/- mice. A major virulence strategy for Salmonella is to breach the epithelial barrier in the intestine to gain access to underlying immune cells. In inflamed ilea of the ship-/- mouse, more Salmonella may have access to the lamina propria, since there is extensive breakdown of the epithelial layer. In addition, the massive infiltration of immune cells to the submucosa provides ample opportunity for the bacterium to establish intracellular infection and traffic to systemic sites. Furthermore, the fact that inflammatory cytokine levels, besides TNFα, were not high in inflamed ship-/- ilea may indicate that the innate cells present, such as macrophages, would not be activated against invading Salmonella. As such, the phenotype of these macrophages most likely is a strong determining factor in how efficient Salmonella is at establishing infection. 6.5 Future directions Because SHIP is such a pluripotent regulator of both innate and adaptive immune cell signaling, it is most likely a critical component of gut and systemic immune responses to Salmonella and LPS during infection. How then can such  101  a complex mouse model be dissected to provide a mechanism by which SHIP determines susceptibility to Salmonella? In order to try to answer what the role of SHIP may be during systemic infection, in vivo studies using Salmonella mutants as well as infections of ship/RAG-/- mice would be very effective. For example, SPI-2 deficient Salmonella mutants do not establish infection in systemic organs. Infecting ship-/- mice with these bacteria, and assessing susceptibility to disease as well as bacterial load in various organs, would highlight whether cells involved in spreading Salmonella throughout the body are impaired in the ship-/- mouse. In addition, using individual SPI-2 effector mutants may elucidate specific mechanisms by which SHIP could be targeted by Salmonella during in vivo infections. Furthermore, studies where wild type S. Typhimurium and SPI-2 mutants are tracked via immunofluoresence to see which cell types they may replicate within, and to what degree, would aid in our understanding of how SHIP deficiency effects the spread of systemic bacteria. Combining SHIP deficiency and a lack of T and B cells, as in a ship/RAG-/mouse, would determine whether increased susceptibility to Salmonella infection in the ship-/- mouse is dependent on adaptive immune responses. This would be an interesting model to study the outcome of both systemic replication of Salmonella as well as gut inflammation, since it is known that T and B cells play distinct roles in clearing Salmonella infections from systemic sites as well as the development of IBD. Once it is determined whether functional SHIP is required for adaptive cells to respond to Salmonella infection, it would be best to specifically inhibit the function of SHIP in single immune cell populations. This method has been extremely useful in determining the role of SHIP in regulating T cell responses. For example, T cell specific deletion of SHIP, using a Cre-lox targeting system, showed that T cell development, activation and phenotype are independent of SHIP expression, but Th2 bias requires functional SHIP (Tarasenko et al., 2007). This work highlighted the fact that skewing of T cell populations towards a regulatory phenotype and elevated activation states of T cells in ship-/- mice is not due to a lack of SHIP in the cells as originally thought, but rather due to the cytokine environment most likely created by hyper-  102  responsive innate cells. Especially useful to further this work, would be to target SHIP deletion in macrophages and, if possible in M2 macrophages, and to examine the effects on both gut inflammation and systemic Salmonellosis. Finally, it would be extremely interesting to test the outcome of infectious diseases in a model where SHIP signaling was enhanced. Exploring this possibility has become realistic since a naturally occuring small molecule, called pelorol, can be used to activate SHIP in vivo (Yang et al., 2005).  More  importantly, synthetic analogs of pelorol not only activate SHIP in vivo, but are also effective in preventing sepsis as well as various inflammatory disorders such as arthritis, dextran-sulfate induced colitis and cutaneous inflammation (Ong et al., 2007).  Therefore, would these SHIP activators be able to reduce  susceptibility to Salmonella infection in susceptible mouse strains? If so, SHIP may be targeted as a therapy for systemic infections. Of course, many other questions remain about the ship-/- mouse and how it responds to disease. Since ship-/- mice are highly susceptible to Salmonella but not Citrobacter infection, it would seem that SHIP deficiency may only affect responses to intracellular or systemic pathogens; however, other infection models must be established to more fully answer this question. Furthermore, would there be a difference in response to parasitic vs. bacterial infections, since the M2 macrophage population may skew T helper responses to a Th2 phenotype? Also, what is the mechanism behind the strong inflammatory response seen in the guts of ship-/- mice and is this inflammation useful in studying IBD? The fact that so many questions remain, highlights the importance SHIP has in immunity and the potential to study other negative regulators and their roles not just in signaling, but also in determining the outcome of disease. 6.6 Significance Salmonella infections are a significant threat to human health worldwide. While vaccinations and some antibiotic treatments can be effective in curbing disease, there are still millions of people that suffer severe illness or death from both typhoid and NTS. Research has provided a wealth of insight into the nature  103  of this pathogen, however it is clear that the immune response to Salmonella infection is extremely complicated. Because negative regulation of innate and adaptive responses to Salmonella plays an important role in the outcome of disease, examining how negative regulators like SHIP impact enteric disease can shed light on how better to control these infections. In fact, it is already being proposed that targeting negative regulation of immune responses by SHIP may be an effective means of providing therapy for inflammatory disorders such as arthritis and colitis, suggesting that negative regulator modulatory therapies may also be designed to combat infectious diseases. In a time when multifaceted anti-Salmonella vaccines are difficult to design and there is increased risk of antibiotic resistant Salmonella strains, the need for new infectious disease treatments is essential. In addition, effective therapies that dampen immune responses in the gut are necessary for the treatment of inflammatory bowel diseases (IBD) like Crohn's Disease (CD) and Ulcerative Colitis (UC) that are becoming increasingly common. This work shows that SHIP does play an important role in immune responses to both enteric pathogens as well as regulating gut immune responses, and could be explored as a potential target for both infectious diseases and IBD. 6.7 Concluding remarks The ship-/- mouse is a complex model to use in studying Salmonella, or any infections. Inherently, these mice have severe perturbations of many immune cell types and responses that would render them vulnerable to disease. Therefore, it is not surprising that the ship-/- mouse is highly susceptible to Salmonella infection. However, what is interesting, is that SHIP deficiency does not impact the immune response to all pathogens in the same way and that it clearly has a major role in maintaining tolerance in the gut to microbial products. Furthermore, these studies suggest that macrophage heterogeneity plays an important role in Salmonella pathogenesis. Clearly this work has just begun to probe how SHIP may be involved in immune responses to pathogens, but it underscores the importance of yet  104  another negative regulator in determining the outcome of disease. In addition, it has provided opportunities for future work to be done that may elucidate specific mechanisms by which SHIP regulates susceptibility to infection or how pathogens like Salmonella may have evolved to interact with negative regulators in the immune system. 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Mol Microbiol 39: 248-259.  119  Appendix 1: Thesis Material Included in Publications  Chapters 3 and 4 include material from the following manuscript, published in Infection and Immunity in May, 2008. Jennifer L. Bishop, Laura M. Sly, Gerald Krystal, and B. Brett Finlay (2008). The inositol phosphatase SHIP controls Salmonella Typhimurium infection in vivo. Infect. Immun., 76:2913-22. Chapter 5 includes material from a manuscript in preparation entitled "A role for SHIP in the small intestinal inflammatory response to enteric bacteria".  120  Appendix: 2 Contribution of Others Chapter 3 I designed and performed all experiments in this chapter. Dr. Gerald Krystal kindly provided ship+/- mice used for breeding ship-/- and ship+/+ mice used in experiments. Dr. Laura Sly provided technical assistance for mouse genotyping. Drs. Guntram Grassl and Erin Boyle provided technical assistance for immunofluorescence and immunohistochemistry. Chapter 4 I designed and performed all experiments in this chapter. Dr. Laura Sly provided technical assistance and guidance for the derivation of BMDM under M1 vs. M2 inducing conditions.  121  Appendix 3: Publications Arising from Graduate Work Peer-reviewed articles Bishop, J.L., L.M. Sly, G. Krystal, and B.B. Finlay. 21 April 2008. The inositol phosphatase SHIP controls Salmonella Typhimurium infection in vivo. Infect. Immun. doi:10.1128/IAI.01596-07. Boyle, E.C.*, J.L. Bishop*, G.A. Grassl* and B.B. Finlay (2007). Salmonella: from pathogenesis to therapeutics. J.Bacteriology, 189(5):1489-95. Bishop, J.L.*, E.C. Boyle* and B.B. Finlay (2007). Bacterial cell wall modification as a means of surviving and evading the host innate immune response. Proc. Natl. Acad. Sci., 104(3): 691-2. Coombes, B.K., M.J. Lowden, J.L. Bishop, M.E. Wickham, N.F. Brown, N. Duong, S. Osborne, O. Gal-Mor, B. Brett Finlay (2006). SseL is a novel Salmonella-specific translocated effector integrated into the SsrB-controlled type III secretion system. Infect. Immun., 75(2):574-80. Bishop, J.L. and B.B. Finlay (2006). Friend or Foe? Antimicrobial peptides trigger pathogen virulence. Trends Mol. Med. 12(1):3-6. *These authors contributed equally to this work. Abstracts Bishop, J.L., L.M. Sly, G. Krystal and B.B. Finlay. SHIP and the M2 macrophage control susceptibility to Salmonella Typhimurium infection in vivo (2008). Keystone Symposia on Innate Immune Signaling, Keystone, CO. Guarna, M., N. Glavas, H. Yang, A. Wang, A. Thompson, E. Dullaghan, N. Mookherjee, J.L. Bishop, O. Donini, M. Scott, M. Gold, B.B. Finlay, R. Hancock, J. North (2007). The Macrophage: Homeostasis, Immunoregulation and Disease. Keystone Symposia on Immunology, Copper Mountain, CO. Coombes B.K., M.J. Lowden, J.L. Bishop, M.E. Wickham, N.F. Brown, N. Duong, S. Osborne, O. Gal-Mor, B.B. Finlay (2006). SseL is a novel Salmonellaspecific translocated effector integrated into the SsrB-controlled SPI2 type III secretion system. Interscience conference on Antimicrobial Agents and Chemotherapy, Annual Meeting, San Francisco, CA. 122  Bishop, J.L., L.M. Sly, G. Krystal and B.B. Finlay (2005). Characterizing the role of the inositol phosphatase SHIP-1 in the innate immune response to Salmonella infection. Society for Leukocyte Biology 38th Annual Meeting, Oxford, UK.  123  Appendix 4: Animal Ethical Approvals  124  125  Appendix 5: Pathology Scoring Worksheet Sample ID Tissue Lumen  Empty Necrotic epithelial cells Neutrophils  Scant Moderate Dense Scant Moderate Dense  0 1 2 3 2 3 4  ______ ______ ______ Total ______  Surface Epithelium No pathological changes Regenerative change Desquamation Neutrophils in epithelium Ulceration  0 Mild (<20%) 1 Moderate 2 Severe 3 Patchy(<30%)1 Diffuse 2 1 1  ______ ______ ______ ______ ______ Total ______  Mucosa No pathological changes Crypt abcesses Rare (<15%) Moderate (15-50%) Abundant (>50%) Mucinous plugs Granulation tissue  0 1 2 3 1 1  No pathological changes Monocytes 1 aggregate >1 small aggregate Large aggregate PMNs None Single PMNs Aggregates Edema Mild (<10%) Moderate (10-80%) Severe (>80%)  0 0 1 2 0 1 2 0 1 2  ______ ______ ______ ______ Total ______  Submucosa ______ ______ ______ ______ Total ______  TOTAL SCORE=  126  

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