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Immunomoduatory properties of host defence peptide LL-37 during infection and inflammation in human blood… Yu, Jie 2006

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IMMUNOMODULATORY PROPERTIES OF HOST DEFENCE PEPTIDEJX-37 DURING INFECTION AND INFLAMMATION IN HUMAN BLOOD CELLS by JIEYU B. Med., Medical School, Shandong University, China 2001  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE  in THE F A C U L T Y OF GRADUATE STUDIES  (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH C O L U M B I A November 2006 © JieYu,2006  ABSTRACT The human cathelicidin, L L - 3 7 , is a cationic host defence peptide and serves as an essential component of innate immunity. In addition to its modest antimicrobial activity, L L - 3 7 has been demonstrated to be a multifunctional modulator of innate immune responses, although the mechanism(s) of which have not been,elucidated. The present study demonstrated that L L - 3 7 .could synergistically enhance IL-ip-induced production of cytokines (IL-6, IL-10) chemokines ( M C P - 3 ) LPS-induced  secretion  in primary of  human P B M C s .  pro-inflammatory  In  cytokines,  contrast LL-37  to  the neutralization  dramatically  and of  augmented  LPS-stimulated M C P - 3 production. L L - 3 7 by itself induced transient phosphorylation of IKB-O. and the subsequent nuclear translocation of N F - K B subunits p50 and p65, which could be further enhanced in the presence of IL-1B. Similar effects of L L - 3 7 and I L - i p were also observed oh activation of A k t and C R E B . Therefore, we propose that, in addition to its well-known anti-inflammatory activity, the human host defence peptide L L - 3 7 also plays an important role in boosting the innate immune responses in combination with inflammatory mediator ( I L - i p ) , which provides a new mechanism for L L - 3 7 in modulating the inflammatory responses in innate immunity.  ii  TABLE OF CONTENTS ii  ABSTRACT  iii  T A B L E OF CONTENTS LIST OF TABLES  v vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENTS Chapter 1. GENERAL INTRODUCTION  1  1.1 Innate Immune System 1.1.1 T L R s and I L - 1 R 1.1.2 T L R s / I L - 1 R mediated signalling pathways 1.1.3 Inflammatory responses and mediators 1.1.4 Negative regulation o f T L R s / I L - I R pathways 1.2 Cationic Host Defence Peptides 1.2.1 General features o f h C A P - 1 8 / L L - 3 7 1.2.2 Immunomodulator properties o f L L - 3 7 1.3 Hypothesis and Experimental Goals Chapter 2. MATERIALS AND METHODS 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10  1 1 2 4 5 7 7 9 11 12  Isolation o f Peripheral B l o o d Mononuclear Cells C e l l Culture Stimulants, Reagents and Antibodies Treatment with Various Stimuli Western Immunoblotting Detection o f Cytokines and Chemokines R N A Extraction Quantitative Real-time P C R ( q R T - P C R ) Semi-quantitative R T - P C R Statistical Analysis  12 12 13 14 14 15 15 16 16 17  Chapter 3. HOST DEFENCE PEPTIDE LL-37 AUGMENTS IMMUNE RESPONSES AND MULTIPLE PATHWAYS IN T H E PRESENCE OF I N F L A M M A T O R Y MEDIATOR IL-1(3 3.1 3.2 3.3  Introduction Results Discussion  19 19 21 27  Chapter 4. EFFECTS OF LL-37 ON LPS-TREATED HUMAN MONOCYTIC CELLS  iii  40  4.1 4.2 4.3  Introduction Results Discussion  40 42 44  .'  Chapter 5. GENERAL DISCUSSION AND CONCLUSIONS 5.1 5.2 5.3 5.4 5.5 5.6  Introduction L L - 3 7 Differentially Modulates Response of Innate Immune Effector Cells to Inflammatory Stimuli L L - 3 7 Enhances IL-1 B-induced Intracellular Signalling Events via Multiple Molecules Signalling Transduction Events Induced by L L - 3 7 Receptors Involved in LL-37-induced Chemokine Production Conclusions  REFERENCES  52 52 53 54 55 57 58  59  iv  LIST OF TABLES Table 2.1:  Sequence o f primers (human) used for quantitative real-time P C R  v  LIST OF FIGURES Figure 3.1:  L L - 3 7 induced degradation of IKB-OC, and subsequent nuclear translocation of N F - K B subunit p50 and p65  32  Figure 3.2:  Enhanced M C P - 3 and IL-6 gene expression in response to co-stimulation with L L - 3 7 and IL-1B in human P B M C s 33  Figure 3.3:  Enhanced M C P - 3 , IL-6 and IL-10 protein production in human P B M C s upon co-stimulation with L L - 3 7 and I L - i p  34  Effects o f inhibition of G-protein coupled receptors ( G P C R s ) on the synergistic production of M C P - 3 induced by L L - 3 7 and I L - i p  35  Figure 3.4: Figure 3.5:  Influence of P I 3 K and IKB-OC inhibition on the synergistic production of M C P - 3 induced by L L - 3 7 and I L - l p 36  Figure 3.6:  Effects o f P K A and P K C on the synergistic production o f M C P - 3 induced by L L - 3 7 and I L - i p  37  LL-37-induced IKB-O, phosphorylation, and subsequent translocation of N F - K B subunits (p50 and p65) in the presence of I L - i p  38  LL-37-induced phosphorylation of A k t and C R E B in the presence of IL-ip  39  T N F - a gene expression and protein production on co-stimulation with L L - 3 7 and L P S induced in human P B M C s and T H P - 1 cells  47  L L - 3 7 suppresses LPS-induced secretion of T N F - a as dose-dependent manner  48  IL-6 gene expression and protein production on co-stimulation with L L - 3 7 and L P S induced in human P B M C s  49  M C P - 3 gene expression and protein production on co-stimulation with L L - 3 7 and L P S induced in human P B M C s  50  Protein expression of A 2 0 in LPS-stimulated THP-1 cells with/without LL-37  51  Figure 3.7: Figure 3.8: Figure 4.1: Figure 4.2: Figure 4.3: Figure 4.4: Figure 4.5:  vi  LIST OF ABBREVIATIONS ATCC  American Type Culture Collection  DC  dendritic cell  ECL  enhanced chemiluminescence  EGFR  epidermal growth factor receptor  ELISA  enzyme-linked immunosorbent assay  ERK1/2  extracellular regulated protein kinase  FCS  foetal calf serum  FPRL-1  formyl peptide like receptor-1  G-CSF  granulocyte colony stimulating factor  GM-CSF  granulocyte macrophage- colony stimulating factor  HBD  human beta defensin  HBE  human bronchial epithelial cells  HNP  human neutrophil peptide  IL-  interleukin  LBP  lipopolysaccharide binding protein  LPS  lipopolysaccharide  LTA  lipoteichoic acid  MAPK  mitogen activated protein kinase  MCP-1  macrophage chemotattractant protein 1  MCP-3  monocyte chemotactic protein 3  PBMC  peripheral blood mononuclear cells  PBS  phosphate buffered saline  PMSF  phenyl methyl sulfonyl fluoride  PTx  pertussis toxin  RT-PCR  reverse transcriptase polymerase chain reaction  SDS  sodium dodecyl sulphate  S D S - P A G E S D S polyacrylamide gel electrophoresis TBS  Tris buffered saline  TBST  Tris buffered saline + 0.1% Tween 20  TLR  toll-like receptor  TNF-oc  tumor necrosis factor alpha  viii  ACKNOWLEDGEMENTS I would like to express my sincere thanks to my supervisor Dr. Bob Hancock for giving me the opportunity to start my graduate student life in his brilliant research group. His wisdom, patience and encouragement helped me in all the time of research. I truly appreciate his support and guidance during the years.  I have furthermore to thank my committee members Dr. Michael G o l d and Dr. Ninan Abraham who kept an eye on the progress of my work and always took effort in providing me with valuable discussion and advices.  I am grateful to the many people who provided discussions, ideas and suggestions essential to the planning and execution of this research. I owe a lot to Dr. Dawn Bowdish for her valuable support and for encouraging me so many times. Many amazing thanks have to go to my lab partners and wonderful friends, Y u e x i n L i , Jelena Pistolic and Linda Rehaume. I could not image what my life w i l l be without them. They always pick me up when I was down and push me forth when I needed a nudge. Thanks for all the vivid discussions about experiments and of course foods as well.  M y parents, sister and her family deserve a warm and special acknowledgement for their love and care. A special thank finally goes to my husband L e i for enduring this process with me. Thanks for spending those late nights with me in the lab. Without his loving support and incredible patience I would never have completed my present work.  ix  CHAPTER 1 GENERAL INTRODUCTION 1.1. Innate Immune System Mammals are constantly exposed to a broad range o f microorganisms in the environment, yet healthy individuals rarely become infected due to the protection o f the immune system. Pre-existing innate immunity serves as the first line o f the host defence and acts within minutes of infection, leading to ultimate elimination o f most invading microbes. The innate immune system recognizes microbial threats and initiates inflammatory responses by utilization o f a variety o f pattern recognition receptors (PRRs) expressed on the cell surface, in intracellular compartments, or secreted into the bloodstream and tissue fluids (Medzhitov and Janeway, 1997).  1.1.1. TLRs and IL-1R The Toll-like receptor ( T L R ) family is the first group o f P R R s studied in detail and at least 11 members o f the T L R family have been identified in mammals to date (Medzhitov, 2001; Zhang et al, 2004). T L R s are transmembrane glycoproteins with highly conserved structural domains in the cytoplasmic region known as T I R domains because this domain is also found in the interleukin-1 receptors (IL-IRs). In contrast, the extracellular region o f the T L R s and I L - I R s differs markedly: the extracellular region of T L R s contains leucine-rich repeat ( L R R ) motifs as the  ligand-binding  site,  whereas  the extracellular  region  of  IL-IRs  contains  three  immunoglobulin-like domains specific for IL-1 (Akira and Sato, 2003; A k i r a and Takeda, 2004). Although there is a high degree o f structural similarity among the various T L R s , ligand recognition is very specific. For example, T L R 4 senses Gram-negative bacteria through  1  recognition o f the lipid A moiety o f bacterial lipopolysaccharide ( L P S or endotoxin), T L R 2 recognizes outer components o f Gram-positive bacteria L T A , and T L R 9 is responsible for recognition o f bacterial D N A or its synthetic counterpart, C p G oligodeoxyribonucleotides (Han and Ulevitch, 2005). I L - 1 R is the principal signalling receptor for IL-1 which has two main forms: IL-lct and IL-1 P ( L i u et al., 2003). Unlike IL-1 and other T L R ligands that directly interact with receptors and initiate signal transduction, the T L R 4 ligand L P S first forms a complex with the plasma protein LPS-binding protein ( L B P ) , which is next delivered to C D 14, a receptor protein either free in the plasma or bound to the cell surface (Wang et al., 2003). Subsequently, L P S ' L B P C D H complexes are specifically recognized by T L R 4 and M D - 2 on the membrane of effector cells involved in innate immunity, resulting in the activation of multiple signal transduction events (Ulevitch and Tobias, 1999).  1.1.2. T L R s / I L - l R mediated signalling pathways  Upon recognition o f their cognate ligands, T L R / I L - 1 R activates a set o f proximal signalling pathways that induce the expression o f a variety o f "early response" host defence genes, encoding inflammatory cytokines and chemokines, antimicrobial peptides, acute-phase proteins, cell adhesion molecules, and proteins required for negative feedback to suppress these responses. Activation o f T L R / I L - 1 R recruits the adaptor protein myeloid differentiation protein 88 (MyD88) v i a the cytoplasmic T I R domain, which then activates a cascade involving interleukin-1 receptor associated kinase ( I R A K s ) , tumour-necrosis-factor-receptor-associated factor 6 ( T R A F 6 ) (Hull et al., 2002; M a y and Ghosh, 1998) and a complex containing T A K 1 (transforming-growth-factor-P-activated kinase), T A B 1 ( T A K 1 -binding protein 1) and T A B 2 (Takeda et al., 2003; Viatour et al., 2005). The activation o f T A K 1 in the cytoplasm leads to the phosphorylation o f the I K K complex (inhibitor of nuclear factor-KB (IicB)-kinase complex),  2  which consists of I K K - a , I K K - B and I K K - y . Once activated, the I K K complex phosphorylates IKB proteins (IKB-OC, IKB-B, IKB-£, IKB-^ and B C L - 3 ) , that are subsequently ubiquitinated and degraded via the proteasome pathway (May and Ghosh, 1998; Viatour et al., 2005), which frees nuclear factor-KB ( N F - K B ) to translocate to the nucleus. Phosphorylation o f T A K - 1 also activates M A P kinases, including extracellular signal-regulated kinase 1 ( E r k l ) and Erk2, p38 and c-Jun N-terminal kinase ( I N K ) , leading to gene transcription and induction o f an inflammatory response (Frobose et al., 2006; L u et al., 2006; McDermott and O ' N e i l l , 2002). The expression o f many inflammatory molecules is controlled at the transcriptional level by multiple transcription factors including N F - K B , A P - 1 , and c A M P response element-binding protein ( C R E B ) families. Most predominant is N F - K B that is expressed in a variety o f cell types and is evolutionarily conserved. In mammals, five family members of N F - K B have been identified: R e l B , c-Rel (Rel), p65 ( R e l A ) , 100/p52 (NF-KB2), and 1 0 5 / p 5 0 (NF-KB 1). A l l o f P  P  the N F - K B family members share a ~300-residue N-terminal Rel-homology-domain which is responsible for dimerisation, nuclear translocation and D N A binding (Ghosh et al., 1998; Takeda et al., 2003). M i c e lacking individual N F - K B subunits are very susceptible to microbial infections, indicating that every member o f the N F - K B family plays an important role in Toll-mediated responses (Takeda et al., 2003). These subunits function as homo- and heterodimers and p50/p65 is the most common one while p50/p50 tends to mediate repression (Caamano and Hunter, 2002; L i u and M a l i k , 2006; Moynagh, 2005). In parallel, activation of I N K triggers phosphorylation of transcription factor c-Jun and activation o f E r k l / 2 stimulates phosphorylation o f Elk-1 which in turn controls the synthesis of the transcription factor c-Fos (Karin, 1995). c-Jun and c-Fos form activation protein ( A P ) - l which was found to be activated by a variety o f stimuli, such as T N F - a , IL-1 and L P S (Brenner et al., 1989; Muegge et al., 1989), suggesting that A P - 1 is most likely to  3  be involved in inflammation and the innate immune response. In addition, recent studies demonstrated that p38 also activates phosphorylation o f cAMP-responsive transcription factor C R E B (Park et al., 2005). C R E B is a leucine zipper protein that binds to the c A M P response element ( C R E ) with the consensus sequence, 5 ' - T G A C G T C A - 3 ' (Brindle and Montminy, 1992). Typically, C R E B is phosphorylated at serine 133 by protein kinase A ( P K A ) in response to c A M P , leading to transcriptional expression o f target genes whose promoters contain the C R E sequence (Brindle and Montminy, 1992). There are other signalling pathways that lead to phosphorylation and activation o f C R E B , such as increased intracellular C a  (Brindle and  Montminy, 1992) and the activation o f M A P K , p38 induced by L P S / I L - 1 (Xing et a l , 1996). The activation o f transcription factor complexes, including N F - K B , C R E B , and A P - 1 , is required for LPS-induced transcriptional activation o f multiple inflammatory mediators, including T N F - q , IL-6, IL-8, M C P - 1 , M C P - 3 and anti-inflammatory cytokines IL-10, and transforming growth factor T G F - B (Gupta et al., 1999; L i u and M a l i k , 2006; Platzer et al., 1999; Shaulian and Karin, 2001).  1.1.3. Inflammatory responses and mediators Bacterial  pathogens and their  products  trigger  the inflammatory  response by  transcriptional activation o f inflammatory genes, leading to the release o f a large number o f inflammatory mediators, including cytokines, chemokines, adhesion molecules, reactive oxygen species (ROS), and reactive nitrogen species ( R N S ) ( L i u and M a l i k , 2006). Although these mediators are required for the initiation o f an effective host defence, their uncontrolled and excessive production (especially T N F - a ) by effector cells in the liver, spleen and other sites frequently causes sepsis and even septic shock (Fiuza and SufTredini, 2001; Hancock and Scott, 2000; L i u and M a l i k , 2006; Platzer et al., 1999). In addition, a breakdown in the inflammatory  4  reactions during an acute bacterial infection may also result in a wide range of chronic diseases such as arthritis, inflammatory bowel disease, and asthma (Uthaisangsook et al., 2002). To control the excessive inflammatory process, anti-inflammatory pathways are also simultaneously activated, leading to the release of anti-inflammatory cytokines, including IL-4, IL-10, IL-13, IL-1 receptor antagonist ( I L - 1 R A ) and T G F - B , which serves as counterregulatory mechanisms (Liu and M a l i k , 2006). Most importantly, negative regulation of signalling transduction along the TLR/IL-1R  to  NF-KB  pathway  is  necessary for  balancing the  pro-inflammatory  and  anti-inflammatory responses. It has been demonstrated that the N F - K B pathway is under negative autoregulatory control at multiple levels.  1.1.4. Negative regulation of T L R s / I L - l R pathways The zinc finger protein A 2 0 is encoded by an immediate early response gene T N F A I P - 3 . T N F A I P - 3 is induced by different stimuli including T N F - a , IL-1B, and bacterial L P S in an NF-KB-dependentmanner (Hu et al., 1998; Sarma et al., 1995; Song et al., 1996). A 2 0 is now known to be a deubiquitinating enzyme that removes ubiquitin chains from T R A F 6 through association with T R A F - 6 , leading to the degradation o f T R A F - 6 . B y doing so, A 2 0 terminates LPS-induced I K K activation and N F - K B transcriptional activity in macrophages (Boone et al., 2004).  Recent studies  demonstrated  that  overexpression o f  A20  inhibits  TLR2-  and  TLR4-induced NF-KB-dependent production of IL-8 by interfering with a TLR-mediated signalling pathway (Gon et al., 2004). M i c e with T N F A I P 3 (A20) deficiency exhibited elevated sensitivity to endotoxic shock and severe inflammation when injected with T N F - a due to persistent activation o f N F - K B . These observations provide evidence that A 2 0 might work as a negative regulator in T L R - N F - K B signalling pathways and it may serve as a target for  5  modulating inflammation and protecting against sepsis. (Boone et al., 2004; Lee et al., 2000a; O'Reilly and Moynagh, 2003). In addition, activation of T L R s also induces phosphatidylinositol 3 - O H kinase (PI3K) activation at the early stage of the pathway although a mechanism needs to be further understood (Akira and Takeda, 2004). Typically, potent ligands (such as L P S ) bind to receptors and activate the  receptor  associated PI3K,  leading  to  the  production  of  the  second messenger  phosphatidylinositol (3,4,5) trisphosphate (PIP3), which in turn recruits A k t via its P H domain to the plasma membrane.  These membrane  changes allow  docking  of the  lipid kinases  phosphatidylinositol-dependent kinase 1 and protein kinase B/Akt. After membrane localization, A k t is phosphorylated at Ser-473 and Thr-308 by phosphoinositide-dependent protein kinase-1 ( P D K - 1 ) . The phosphorylated A k t modulates the function o f numerous substrates involved in the regulation of cell survival, cell cycle progression and cellular growth (Guha and Mackman, 2002; Lee et al., 2000b; Park et al., 1997; Scheid, 2000). The P I 3 K - A k t pathway has been shown to negatively regulate N F - K B and the expression of inflammatory genes in macrophages (Fukao and Koyasu, 2003). The activation of the P I 3 K / A k t pathway suppresses both M A P K s and N F - K B cascades in response to L P S in human monocytes, resulting in decreased production of T N F - a (Guha and Mackman, 2002). Consistent with these observations in monocytes, P I 3 K seems to block the p38 activation pathway that is essential for transcriptional activation of both the IL-12 p35 and p40 genes in D C s (Fukao et al., 2002). Gratton and colleagues have shown recently that Akt-dependent  phosphorylation  of M E K K 3 reduces its kinase activity  and inhibits  the  M E K K 3 / 6 - p 3 8 pathway (Gratton et al., 2001) and similar negative regulation effects were also detected in murine macrophages (Park et al., 1997). These data suggest that the P I 3 K / A k t signalling pathway  may  be an endogenous negative  6  regulator  that serves to  balance  pro-inflammatory and anti-inflammatory responses and by doing so, maintains homeostasis and the integrity o f the immune response (Fukao and Koyasu, 2003). Furthermore, Bommhardt et al. demonstrated that overexpression o f A k t decreased sepsis-induced lymphocyte apoptosis, increased production o f the T h l cytokine IFN-y, and improved survival in sepsis in vivo (Bommhardt et a l . , 2004). Thereby, it has been proposed by many investigators that the PI3K-mediated pathway could be a new therapeutic target for certain pro-inflammatory and/or septic diseases (Williams et al., 2006).  1.2. Cationic Host Defence Peptides 1.2.1. General features of hCAP-18/LL-37 Small cationic peptides are abundant i n nature and have been described as 'Nature's antibiotics'  due to their  direct  antimicrobial  activity  against microorganisms  including  Gram-positive bacteria, Gram-negative bacteria, fungi, parasites and viruses, as demonstrated by early work with insects, amphibians and mammalian (Hancock and Diamond, 2000). More recently, it has been demonstrated that these peptides have a myriad o f functions in modulating innate immune responses which have an impact on infections and inflammation and are now often termed 'cationic host defence peptides' (Bowdish et al., 2005). Cationic host defence peptides are evolutionary ancient components of the innate immune system. Most o f peptides are 12-50 amino acids in length with a net positive charge o f +2 to +7 due to an excess o f basic amino acid residues (arginine, lysine and histidine) (Hancock and Chappie, 1999a). Based on their secondary structure, these peptides can be grouped into four classes: P-sheet peptides, unstructured peptides, extended peptides, loop peptides (Hancock and Diamond, 2000). Since the 1970's, more than 500 host defence peptides have been identified in a wide variety o f species, such as plants, amphibians, insects and mammals including humans  7  (Hancock and Lehrer, 1998; Lehrer and Ganz, 2002a). One example o f mammalian cationic host defence peptides are those derived from cathelicidin gene family conserved N-terminal  (Zanetti, 2004b). The cathelicidins are characterized by  cathelin domain and signal sequence and a structurally  highly variable  antimicrobial domain at the C-terminus (Hancock and Chappie, 1999a; Lehrer and Ganz, 2002b). B y homology to the cathelin domain, many cathelicidin sequences have been discovered in various species, including cows, pigs, sheep, horses, mice, guinea pigs and rabbits (Bals and Wilson, 2003b). The C-terminal domain contains the active peptide that can be released from the precursor protein by the action of serine proteinases. To date, the 18-kDa h C A P 1 8 is the only cathelicidin peptide identified in humans (Bals and Wilson, 2003b). L i k e most peptides, h C A P 1 8 is stored as an unprocessed inactive form in the granules of neutrophils. U p o n stimulation inflammatory  cytokines), the N-terminal  (such as by invading microorganisms or  cathelin domain  is cleaved extracellularly  by  neutrophil-derived serine proteinase 3, resulting in the release of the C-terminal domain as a mature  37-amino  acid  peptide,  LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES  (Gudmundsson et al., 1996; Larrick et al., 1995; Lehrer and Ganz, ; Lehrer and Ganz, 2002b; Sorensen et al., 1997). In addition to neutrophils, L L - 3 7 has also been shown to be expressed in monocytes, specific lymphocyte populations (B cells and y5 T-cells), mast cells, keratinocytes during inflammatory disorders, airway epithelium, urinary tract epithelial cells, and a number of tissues and bodily fluids including gastric juices, saliva, semen, sweat, plasma, airway surface liquid and breast milk (Durr et al., 2006; Tjabringa et al., 2003b). Upregulation of L L - 3 7 production is found i n bronchoalveolar lavage from infants with pulmonary infections, in individuals with Cystic Fibrosis lung disease and in psoriatic skin lesions. These indicate that  8  L L - 3 7 assists the immune system in fighting disease (Durr et al., 2006). .  1.2.2. Immunomodulatory properties of LL-37 In addition to its antimicrobial activities, abundant evidence strongly the idea supports that L L - 3 7 functions as a potent modulator o f immune responses. First o f all, L L - 3 7 was demonstrated to trigger cell accumulation at local inflammatory sites by direct chemoattraction of effector cells and induction o f chemokines. L L - 3 7 is able to chemoattract neutrophils, monocytes and T cells through binding to formyl peptide-like receptor 1 ( F P R L - 1 ) , a pertussis toxin-sensitive, G protein-coupled receptor ( G P C R ) (De et al., 2000). In contrast, receptors coupled with G proteins, other than F P L R - 1 , are involved in LL-37-induced chemotaxis o f mast cells (Niyonsaba et al., 2002a). Two other receptors mediated LL-37-induced biological activities are P2X7, a purinergic receptor and epidermal growth factor receptor ( E G F R ) (Elssner et al., 2004; Nagaoka et al., 2006; Tjabringa et al., 2003a; Tokumaru et al., 2005). P2X7 mediates LL-37-induced maturation and release o f IL-1B in LPS-primed monocytes (Elssner et al., 2004). A proposed mechanism for E G F R suggests that exposure o f airway epithelial cells to L L - 3 7 results in activation o f a metalloproteinase ( M P ) , cleavage o f membrane-anchored EGFR-ligands and activation o f the E G F R by these ligands, which leads to activation o f M A P K cascades and transcription o f target genes (Tjabringa et al., 2003a). Consistently, L L - 3 7 can induce phosphorylation o f the M A P K , E R K 1 / 2 and p38 in human peripheral blood-derived monocytes and human bronchial epithelial cell line (HBEs), resulting in activation o f transcription factor Elk-1 and transcription o f target genes that encode chemokines I L - 8 , M C P - 1 , and M C P - 3 (Bowdish et al., 2004b). LL-37-induced activation o f M A P K p38 and E R K 1 / 2 also contributes to IL-18 secretion from human keratinocytes, suggesting the involvement o f L L - 3 7 in innate and  9  adaptive immunity because IL-18 is involved in both T h l and Th2 functions (Niyonsaba et al., 2005) . Davidson et al demonstrated L L - 3 7 could modulate the ability of dendritic cells ( D C s ) to uptake and present antigens (Davidson et al., 2004b). Additionally, the fact that internalization of L L - 3 7 by monocyte-derived dendritic cells ( M D D C s ) can affect cellular adaptive immunity also supports that L L - 3 7 plays a critical role in linking innate and adaptive immunity (Bandholtz et al., 2006) . Recently L L - 3 7 was suggested to be involved in protecting neutrophils from apoptosis via the P2X7 and/or G-protein coupled receptors (Barlow et al., 2006; Nagaoka et al., 2006). Other identified functions of L L - 3 7 include stimulation of mast cell degranulation (Di_Nardo et al., 2003; Niyonsaba et al., 2003), inhibition of tissue protease (Zaiou et al., 2003), promotion of wound healing (Heilborn et al., 2003) and induction of angiogenesis v i a F P R L 1 (Koczulla et al., 2003b). There is much evidence that L L - 3 7 is an effective anti-endotoxin agent according to its pronounced ability to suppress L T A or LPS-induced production of a variety of pro-inflammatory cytokines (including IL-6, IL-1B and T N F - a ) and prevent lethal endotoxemia in an animal models (Dankesreiter et al., 2000; Gough et al., 1996b). Likewise, over-expression of L L - 3 7 in mouse airways can considerably prolong the survival of mice after challenge with a lethal dose of L P S (Bals et al., 1999). L L - 3 7 itself and peptides derived therefrom, offers considerable promise as a treatment for sepsis. Although previous studies suggested neutralization  of  endotoxin by L L - 3 7 is partly due to blockage of L P S binding to L B P and C D 14, further investigations demonstrated that the anti-endotoxin effect of L L - 3 7 was also based on the ability of L L - 3 7 to directly interact with host cells and alter the transcription of a variety  of  inflammatory genes, suggesting that L L - 3 7 plays a pivotal role in modulating innate immune responses (Finlay and Hancock, 2004; Mookherjee et al., 2006; Scott et al., 2002b)  10  1.3. Hypothesis and Experimental Goals The aims o f the studies described in this thesis were to examine the potential roles of L L - 3 7 in modulating T L R / I L - 1 R agonist induced inflammatory responses, and the underlying mechanisms. Since L L - 3 7 has been shown to activate M A P K pathways by direct interaction with effector cells involved in innate immunity, I hypothesized that other cellular signalling pathways which are important i n regulating innate immune responses, such as the N F - K B and P I 3 K / A k t pathways, also contributed to the immunomodulatory properties o f L L - 3 7 . Furthermore, the impacts o f L L - 3 7 on production o f inflammatory mediators at the transcriptional and protein levels were investigated in human mononuclear cells, including the human monocytic THP-1 cell line and human peripheral blood mononuclear cells ( P B M C s ) . Thus, m y studies help to further understand the role o f L L - 3 7 i n innate immune responses to inflammatory stimuli.  11  CHAPTER 2 MATERIALS AND METHODS  2.1. Isolation of Peripheral Blood Mononuclear Cells Primary human P B M C s were isolated as described previously (Mookherjee et al, 2006). Briefly, 100 m l of human venous blood was collected from healthy volunteers in sodium heparin Vacutainer collection tubes ( B D Biosciences, Mississauga, Ontario, Canada) according to University of British Columbia clinical research ethics board approval and guidelines. The blood was mixed, at a 1:1 ratio, with R P M I 1640 medium (supplemented with 10% (v/v) F C S , 2 m M L-glutamine, and 1 m M sodium pyruvate) and separated by centrifugation over a Ficoll-Paque® Plus (Amersham Biosciences, Piscataway, N J , U S A ) . P B M C were isolated from the buffy coat and the cell concentration determined by trypan blue exclusion. P B M C ( l x l O /ml) were seeded 6  into tissue culture dishes (Falcon; Becton Dickinson) at 37°C in 5 % CO2 and rested for 1-2 hr before addition o f various treatments.  2.2. Cell Culture The human monocyte-like cell line, THP-1 (25) was obtained from American type culture collection, (ATCC® TIB-202) and were grown in suspension in RPMI-1640 media (Gibco®, Invitrogen™ L i f e technologies, Burlington, O N ) , supplemented with 10% (v/v) heat-inactivated F B S , 2 m M L-glutamine, and 1 m M sodium pyruvate (all from Invitrogen Life Technologies), at 37°C in a humidified 5 % (v/v) CO2 incubator up to a maximum of six passages. Before stimulations  with various treatments the cells were differentiated  into adherent  macrophage-like cells by treatment with phorbol 12-myristate 13-acetate ( P M A ; Sigma-Aldrich  12  Canada, Oakville O N ) as described previously (26). A l l experiments using human THP-1 cells or P B M C s involved at least three independent biological replicates.  2.3. Stimulants, Reagents and Antibodies The  cationic  human  host  defence  peptide,  LL-37  ( L L G D F F R K S K E K I G K E F K R I V Q R I K D F L R N L V P R T E S ) was synthesized by F-moc chemistry at The Nucleic Acid/Protein Synthesis Unit at University o f British Columbia, and was resuspended i n endotoxin-free water. The concentration (w/v) o f the peptide in solution was determined by amino acid analysis. Peptide W K Y M V M was a gift from Dr. C . Dahlgren (University o f Goteborg, Goteborg, Sweden). Recombinant human IL-1 (3 was purchased from Research  Diagnostics  (Flanders, N J , U S A ) . Pharmacological  inhibitors  pertussis toxin,  wortmannin were purchased from Calbiochem, Merch Biosciences (Nottingham, U K ) and Biomol (Plymouth Meeting, P A , U S A ) respectively. A n inhibitors o f IKB-CC ( B A Y 11-7085) was purchased from B i o m o l . Inhibitors o f P I 3 K (LY294002, Wortmannin), P K C (GF109203x) and P K A (6-22 Amide) were purchased from C e l l Signalling Technology, Inc., (Mississauga, O N , Canada). A n t i - A 2 0 mouse monoclonal antibody was purchased from Imgenex Corporation (San Diego, C A , U S A ) . The mouse monoclonal antibody recognized phospho-IicB-a, and rabbit anti-total-lKB-a, anti-pl05/50, anti-phospho-Akt, anti-total-Akt and anti-phospho-CREB were purchased from  Cell  Signalling  Technology  Inc. HRP-conjugated goat  anti-rabbit and  anti-mouse IgG antibodies were purchased from Cell Signalling Technologies and Amersham Biosciences (Piscataway, N J , U S A ) respectively. L P S from P. aeruginosa strain HI03 was purified free o f proteins and lipids using the Darveau-Hancock method as previously described (Darveau and Hancock, 1983). A l l reagents were tested for endotoxin by L A L assay and reconstituted i n endotoxin-free water.  13  2.4. Treatment with Various Stimuli Human P B M C or T H P - 1 cells were stimulated with L L - 3 7 (20 ug/ml), L P S (100 ng/ml), or recombinant human I L - l p (10 ng/ml) for the time period indicated in the results section. Alternatively, the cells were pre-treated with the various mentioned inhibitors for 1 hr before treatment with the various stimulants. For studies demonstrating synergistic mechanism of the peptide L L - 3 7 with inflammatory mediator IL-1B, both the stimulants were added simultaneously.  2.5. Western Immunoblotting THP-1 cells (3 x 10 ) and human P B M C 6  (5 x 10 ) seeded into 60-mm petri dishes 6  ( V W R International) were stimulated with I L - i p , L L - 3 7 , L P S or endotoxin-free water as a vehicle control for 30 or 60 m i n at 37°C, 5% CO2. Cells were subsequently detached by gentle scraping, followed by washing and re-suspending the cells in ice-cold P B S containing 1 m M sodium vanadate. Nuclear and cytoplasm extracts were isolated using N E - P E R Nuclear and Cytoplasmic Extraction Reagents K i t (Pierce, Rockford, I L , U S A or Cramlington, U K ) according to the manufacturer's instructions. The extracts were stored at - 8 0 ° C until further use. Protein concentrations o f the lysates were determined by B C A assay (Pierce). Nuclear extracts (7.5 pg) and cytoplasmic extracts (15 u.g) were resolved on a 10% S D S - P A G E , followed by subsequent transfer to Immuno-blot P V D F membranes (Bio-Rad).  The membranes were probed with  specific antibodies at 1/1000 dilution i n T B S T (20 m M Tris p H 7.4, 150 m M N a C l , and 0.1% Tween 20) containing 5 % skimmed milk powder ( T B S T / m i l k ) or 5 % B S A ( T B S T / B S A ) , followed by incubation with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies as required. The membranes were developed using chemiluminescence peroxidase  14  substrate (Sigma-Aldrich), according to manufacturer's instructions. The blots were re-probed with an a n t i - G A P D H antibody to determine equal amount o f protein were loaded.  2.6. Detection of Cytokines and Chemokines Human P B M C were seeded at 8 x 10 / m l in complete R P M I - 1 6 4 0 media. Cells were 5  stimulated for 24 hrs with L L - 3 7 , indicated cytokines and endotoxin-free water was used in parallel as vehicle controls. Following stimulation, the tissue culture supernatants were centrifuged at 2500 x g for 5 m i n to obtain cell-free samples. Supernatants were aliquoted and stored at -20°C prior to assay for various cytokines. The concentrations o f T N F - a , IL-8, M C P - 3 , IL-10  and IL-6 in the supernatants were measured using capture E L I S A  as per the  manufacturer's suggestion ( M C P - 3 E L I S A : R & D Minneapolis, M N ; IL-10 and IL-6 E L I S A : ebioscience San Diego, C A ; IL-8 E L I S A : BioSource International, Camarillo, C A ) .  2.7. RNA Extraction Fresh P B M C s were isolated and placed in 60-mm tissue culture dishes at 5 x 10 cells/dish in complete R P M I 1640, and incubated for 1-2 hrs at 37°C i n 5 % C 0 . 2  6  Cells then  were incubated with treatments for indicated hours. After stimulation, suspended cells were collected into 15 m l flacon tubes and spin at 1000 rpm for 3 minutes. C e l l pellets and attached cells were washed with ice-cold P B S . Total R N A was isolated using RNeasy M i n i kit (Qiagen Inc., Canada) and treated with RNase-Free DNase as per the manufacturer's instructions to remove contaminating genomic D N A . The R N A was eluted in RNase-free water (Ambion Inc., Austin, T X , U S A ) . R N A s e inhibitor (Ambion) was added to the R N A sample to prevent R N A degradation. R N A quality and quantity was assessed using a spectrometer and visualization by electrophoresis on a 1% agarose gel.  15  2.8. Quantitative Real-time PCR (qRT-PCR) Gene expression was analyzed by quantitative real time P C R using Superscript III Platinum Two-Step q R T - P C R kit with S Y B R Green (Invitrogen Life Technologies), as the manufacturer's  instructions, in the A B I Prism 7000 sequence detection system (Applied  Biosystems). Briefly, 50 ng o f total R N A was reverse transcribed in a 20-pi reaction volume for 50 min at 42°C, and the reaction was terminated by incubating for 5 m i n at 85°C and then digested for 30 min at 37°C with RNase H . The P C R was conducted in a 12.5-ul reaction volume containing 2.5 p i o f 1/5 diluted c D N A template. A melting curve was performed to ensure that any product detected was specific to the desired amplicon. Fold changes were calculated after normalization to endogenous G A P D H and using the comparative Ct method (Pfaffl, 2001). The primers used for q R T - P C R are summarized in Table 2.1.  2.9. Semi-quantitative RT-PCR Semi-quantitative R T - P C R was also performed to measure the level o f gene transcription induced by L P S and/or L L - 3 7 . Total R N A was isolated as described above and 1 pg o f total R N A samples were incubated with 1 pi oligo(dT) (500 pg/ml) and 1 p i mixed d N T P stock at 1 m M in a 12-pl volume with diethyl pyrocarbonate-treated water at 65°C for 5 min in a fhermocycler. Briefly, 4 p i 5x first-strand buffer, 2 p i 0.1 M D T T , and 1 pi R N a s e O U T recombinant ribonuclease inhibitor (40 U/pl) were added and incubated at 42°C for 2 min, followed by the addition o f 1 p i (200 U ) o f Superscript I I  Tm  (Invitrogen, Burlington, Ontario,  Canada). Each P C R was performed with a thermal cycler by using 25-35 cycles consisting of 30 s o f denaturation at 94°C, 30 s o f annealing at 55°C and 40 s of extension at 72°C. The number of cycles o f P C R was optimized to lie in the linear phase of the reaction for each primer and set  16  of R N A samples. A housekeeping gene, G A P D H , was amplified in each experiment to evaluate extraction procedure and to estimate the amount o f R N A .  2.10. Statistical Analysis Student's t-test was performed in order to determine the statistical significance, with p < 0.05 being considered statistically significant. Values shown are expressed as mean ± standard deviation or standard error as indicated in the result section and figure legends.  17  Table 2.1.  Sequence of primers (human) used for quantitative real-time PCR.  Gene  Forward primer (5'-3')  Reverse Primer (5'-3')  GAPDH  GTCGCTGTTGAAGTCAGAGG  GAAACTGTGGCGTGATGG  IL-6  AATTCGGTACATCCTCGACGG  GGTTGTTTTCTGCCAGTGCC  MCP-3  TGTCCTTTCTCAGAGTGGTTCT  TGCTTCCATAGGGACATCATA  18  CHAPTER 3 HOST DEFENCE PEPTIDE LL-37 AUGMENTS IMMUNE RESPONSES AND MULTIPLE PATHWAYS IN THE PRESENCE OF INFLAMMATORY MEDIATOR IL-ip  3.1. Introduction Cationic host defence peptides are evolutionarily-ancient components of the innate immune system (Peschel and Sahl, 2006). While some can directly k i l l microorganisms (Zasloff, 2002), recent studies have demonstrated that these peptides have immunomodulatory functions in that they can selectively boost innate immunity and/or link innate and adaptive immunity (Bowdish et al., 2005; Yang et al., 1999). The two major families o f host defence peptides in mammals are the cathelicidins and defensins. Cathelicidins are defined by a highly conserved N-terminal  cathelin pro-domain and a structurally-variable  antimicrobial domain at the  C-terminus (Hancock and Chappie, 1999b; Lehrer and Ganz, 2002a), and they have been identified in various species, including cows, pigs, sheep, horses, mice, guinea pigs and rabbits (Bals and Wilson, 2003a). The only known human cathelicidin is the 37-amino acid peptide L L - 3 7 (Gudmundsson et al., 1996; Larrick et al., 1995). This peptide is produced by various cell types including neutrophils, lung epithelial cells, keratinocytes, monocytes and mast cells (Agerberth et al., 2000; Bals et al., 1998; D i Nardo et al., 2003; Frohm et al., 1997; Gudmundsson et al., 1996). It is stored intracellularly as an inactive pro-peptide, h C A P 1 8 , which upon stimulation and secretion is extracellularly cleaved from the N-terminal cathelin domain by  19  proteinase 3, leading to the generation of active L L - 3 7 (Sorensen et al., 2001). Recent evidence indicates that L L - 3 7 can function as a potent modulator of innate immune responses. It can trigger mast cell degranulation, and stimulate the production of chemokines, as well as acting as a direct chemoattractant for neutrophils, monocytes and T cells, resulting in the recruitment of effector cells to local sites of infection (De_Yang et al., 2000; Scott et al., 2002a). In addition, L L - 3 7 can suppress endotoxin-induced inflammatory responses by delicately balancing gene transcription involved in homeostasis, inflammation and sepsis (Mookherjee et al., 2006; Scott et al., 2002a), prolong neutrophil life span by suppressing neutrophil apoptosis (Barlow et al., 2006) and influence the polarity of dendritic cells in the adaptive immune response (Davidson et al., 2004a). Several of these activities have been demonstrated in vivo, including the ability of L L - 3 7 to protect against endotoxic shock in mice and rats (Fukumoto et al., 2005; Scott et al., 2002a). Previous studies have suggested that certain immune responses of effector cells can be augmented by L L - 3 7 in the presence of stimuli such as G M - C S F and IL-1 p that would naturally be present in local inflammatory site during the course of an infection (Bowdish et al., 2004a; Mookherjee et al., 2006). However, the mechanism(s) whereby L L - 3 7 stimulates biological responses or enhances responses in synergy with other immune effectors have not yet been fully elucidated. This study focused on these physiologically relevant questions in human primary cells. L L - 3 7 is known to interact with and/or transactivate a variety of receptors (De_Yang et al., 2000; Elssner et al., 2004; L a u et al., 2005; Niyonsaba et al., 2002b; Tjabringa et al., 2003a; Tjabringa et al., 2006); for example the direct chemoattractant activity of L L - 3 7 is due, at least in part, to activation of the G-protein coupled receptor F P R L - 1 ( D e Y a n g et al., 2000), although this  20  receptor is not involved in mediating other L L - 3 7 activities (Bowdish et al., 2004a). Cellular events important in the biological effects of L L - 3 7 include the triggering of certain signaling pathways. L L - 3 7 was shown to induce phosphorylation of the mitogen-activated protein ( M A P ) kinases, E R K 1 / 2 and p38 i n human peripheral blood monocytes, a bronchial epithelial cell line and skin mast cells (Bowdish et al., 2004a; Chen et al., 2006). Recent evidence indicates that L L - 3 7 may also utilize phosphoinositide 3-kinases (PI3K) in the inhibition of neutrophil apoptosis (Barlow et al., 2006), and that it might directly interfere with the pathway along the Toll-like receptor ( T L R ) - 4 to N F - K B in inhibiting endotoxin-stimulated  pro-inflammatory  cytokine expression (Mookherjee et al., 2006). However, the mechanism(s) by which L L - 3 7 modulates the N F - K B pathway and its interaction with other signaling molecules that play a pivotal role in innate immune responses remain unclear. In this study we have investigated the complex mode of action o f L L - 3 7 in combination with the inflammatory mediator I L - i p . The results suggest that multiple signalling pathways are activated in human P B M C s , leading to the activation o f pivotal transcriptional elements.  3.2. Results LL-37 by itself stimulated the activation of IKB-O/NFKB  pathway in THP-1 cells  A s it has been demonstrated that L L - 3 7 is able to induce up-regulation of multiple NF-KB-controlled gene expression such as IL-8, IL-6, M C P - 1 (Bowdish et al. 2004), the ability of L L - 3 7 to modulate iKB-a/NF-KB-mediated signal transduction was firstly assessed in human monocytic T H P - 1 cells. Cells were stimulated with L L - 3 7 (20 u.g/ml) at different time points. L L - 3 7 induced a transient degradation of IKB-OC with kinetics (Fig. 3.1^4) similar to that of L P S (100 ng/ml) (Fig. 3.15). A s demonstrated with Western blots, total IKB-OC decreased transiently  21  within 30 min in human T H P - 1 cells, and was restored back after 60 min of stimulation with L L - 3 7 . To further investigate the impact of the transient phosphorylation of IKB-O. induced by L L - 3 7 , subsequent nuclear translocation of N F - K B subunit p50 was assessed by Western blots. Nuclear extracts of human monocytic cell line, THP-1 cells consistently revealed that 20 pg/ml o f L L - 3 7 induced nuclear translocation o f N F - K B subunits p65 and p50 after 30min of stimulation (Fig. 3.1C). These results together demonstrated that L L - 3 7 had a transient effect on the phosphorylation of IKB-CC leading to a subsequent increase in the nuclear translocation of N F - K B subunit p50 in human mononuclear cells.  Synergistic induction of IL-6 and MCP-3 transcription was induced by LL-37 and IL-1/3 in human PBMCs. L L - 3 7 has been previously demonstrated to be a robust anti-endotoxin agent due to its ability to selectively modulate L P S - and lipoteichoic acid (LTA)- induced pro-inflammatory cytokine production in human mononuclear cells (Mookherjee et al., 2006; Scott et al., 2002a). In contrast, we previously demonstrated that L L - 3 7 substantially enhanced the production of IL-6 and IL-8 in synergy with I L - i p (but only slightly influencing T N F - a ) (Mookherjee et al., 2006). Since I L - i p is an inflammatory cytokine involved in a variety of acute and chronic diseases, and similar to the T L R agonists L P S and L T A activates a common T L R / I L - 1 R signalling pathway leading to i K B - a phosphorylation and N F - K B nuclear translocation, the effects of L L - 3 7 on IL-ip-mediated immune responses were further investigated. The effect of L L - 3 7 on IL-ip-mediated M C P - 3 and IL-6 gene expression was analyzed by qRT-PCR. Primary human P B M C s were treated with I L - l p (10 ng/ml) and/or L L - 3 7 (20 pg/ml) for 8 and 12 hr. Treatment with I L - i p or L L - 3 7 alone elicited modest (2- to 7-fold) up-regulation  22  of M C P - 3 expression compared to that in untreated cells (Fig.3.2A). In contrast, co-stimulation of the cells with IL-1B and L L - 3 7 resulted in a substantially augmented M C P - 3 gene expression (to 12- to 27-fold) when compared with either IL-1 (3 or L L - 3 7 alone (Fig.3.2A). Similarly, while IL-1B  or L L - 3 7 itself in P B M C s from most donors caused up-regulation of IL-6 m R N A  accumulation, treatment with the two stimulants together caused substantial increases in the expression of IL-6 at 8hr (19-fold) and 12 hr (198-fold) (Fig 3.2B). It should be noted that the level of IL-6 gene expression by L L - 3 7 alone appeared to be donor dependent (from 1 to 20 fold increased compared to untreated controls in P B M C preparations from 5 individuals after 12 hr). These results confirmed that the host defence peptide L L - 3 7 synergistically augmented the transcriptional responses induced by the inflammatory mediator I L - 1 B .  Synergistic effects of LL-37 and IL-1B on cytokine and chemokine production were investigated. To further characterize the above described responses at a protein level, human P B M C were stimulated with L L - 3 7 (20 ug/ml) in the presence or absence of IL-1B (10 ng/ml) for 6, 12, 18 and 24 hr. The amount of the chemokine M C P - 3 , and cytokines IL-6 and IL-10 secreted by the stimulated P B M C s were assessed in the tissue culture supernatants by E L I S A . L L - 3 7 by itself induced very modest secretion of M C P - 3  (between 2 and 7 fold increase compared to  unstimulated cells), whereas IL-1B induced less than a 2-fold increase in M C P - 3 production. In contrast, stimulation of P B M C s with IL-1 (3 and L L - 3 7 in combination resulted in a substantial (100-fold) increase in the production of M C P - 3 after 18 hr of stimulation (Fig. 3.3A). Similarly, addition of L L - 3 7 together with IL-1 (3, led to the induction of a substantial (~10-fold) increase in the release of IL-6 after 12 hr of treatment (Fig. 3.3B) and an 8-fold increase in IL-10, a known  23  anti-inflammatory cytokine, after 24 hours incubation (Fig. 3.3C). These cytokines were detected by E L I S A at very modest levels upon stimulation with either L L - 3 7 or I X - i p alone. These results together confirm and extend the observation that L L - 3 7 enhances immune responses induced by IL-1B, an inflammatory mediator present during infection and inflammation.  Synergistic production of MCP-3 was suppressed by the presence inhibitors of GPCRs, PI3K or IKB-OC  The immunomodulatory effects of L L - 3 7 have been proposed to be dependent upon signaling through a number of receptors potentially relevant in this system, including G-protein coupled receptors ( G P C R s ) such as the chemokine receptor F P R L - 1 , specific transactivated receptors such as E G F R , and undetermined high and low affinity receptors (De_Yang et al., 2000; Yang et al., 2001). To assess the possible significance of G P C R , human P B M C s were pre-incubated with pertussis toxin (100 ng/ml), which is known to inhibit the activation of G P C R s , for 1 hr prior to the addition of L L - 3 7 (20 pg/ml) and/or I L - i p (10 ng/ml). The amount of M C P - 3 released into the tissue culture supernatant was subsequently determined by capture E L I S A after 24 hr of incubation. In the presence of pertussis toxin, the production of M C P - 3 induced by the combination of I L - l p and L L - 3 7 was significantly suppressed (Fig. 3.3). In contrast, treatment of cells with the FPRL-1-specific agonist W K Y M V m (Le et al., 1999) i n the presence of I L - i p , resulted in the synergistic induction of M C P - 3 (Fig. 3.3), and this effect was abrogated by pre-treatment with pertussis toxin. These results together were consistent with the possibility that GPCR(s) were required for the synergistic production of M C P - 3 induced by IL-1 p and L L - 3 7 . Phosphoinositide 3-kinases (PI3K) have been implicated in the effects of L L - 3 7 on inhibiting neutrophil apoptosis. P I 3 K is downstream of G P C R s (Toker and Cantley, 1997) and has also  24  been proposed to act as an early intermediary of M A P K activation in response to inflammatory stimuli (Yart et al., 2002). In addition it has been previously demonstrated that L L - 3 7 induces the activation of the M A P kinases, E R K 1 / 2 and p38, in peripheral blood derived monocytes (Bowdish et al., 2004a). Therefore the significance of P I 3 K in M C P - 3 induction by L L - 3 7 in conjunction with I X - i p , was tested by pre-incubating P B M C with LY294002 or the alternative P I 3 K inhibitor wortmannin (Fig 3.4A) for 1 hr, prior to stimulation with L L - 3 7 and/or I L - i p for 24 hr. Pretreatment with either inhibitor significantly inhibited (> 95%), the augmented release of M C P - 3 induced by the combination of L L - 3 7 and I L - i p . In addition, it has been demonstrated that the expression of M C P - 3 is regulated, during innate immune responses in part by the transcription factor N F - K B (Hoffmann et al., 1996). Primary human P B M C were pre-incubated with IKB-CC inhibitor (Bay 11-7085) for 1 hr prior to treatment with L L - 3 7 (20 pg/ml) in the presence or absence of I X - i p (10 ng/ml) for 24 hr, and the level of secreted M C P - 3 in the tissue culture supernatant were monitored by capture E L I S A . The inhibitor B A Y 11-7085, which selectively inhibits phosphorylation of IKB-CC and subsequent nuclear translocation of transcription factor N F - K B , significantly inhibited the synergistic production of M C P - 3 induced by I L - l p and L L - 3 7 by greater than 9 5 % (Fig. 3.5B). Protein kinase C ( P K C ) and protein kinase A ( P K A ) are also known to be downstream of G P C R s . Therefore, the P K C inhibitor GF109203x, and P K A inhibitor 6-22 amide were used to assess the role o f these two kinases in the induction of M C P - 3 by the peptide in the presence of I L - i p . Neither P K A (Fig. 3.6A) nor P K C (Fig. 3.6B) inhibitors were able to suppress the production of M C P - 3 induced by co-stimulation with I L - i p and L L - 3 7 , indicating that P K C and P K A are unlikely to be key mediators in the synergistic induction of M C P - 3 by L L - 3 7 and I L - i p .  25  LL-37, in combination with IL-1B, stimulated the activation of multiple signal pathways To provide a more detailed understanding of the mechanism(s) influencing the observed synergy between host defence peptide L L - 3 7 and the inflammatory mediator IL-1B in human primary cells, the activation of relevant proteins in the elucidated pathways was assessed. The activation of transcription factor N F - K B is a major downstream target of IL-1B (Mercurio and Manning, 1999). In resting cells, N F - K B (particularly p65/p50) is bound to inhibitory Ikappa-B (IKB) proteins termed IKBS and is sequestered in the cytoplasm. Stimulation and subsequent phosphorylation of iKB-proteins lead to the degradation of the complex, which allows the liberated N F - K B subunits to translocate to the nucleus and enhance the transcription of several target genes, including those encoding for cytokines, chemokines, and adhesion molecules (Li and Stark, 2002; Regnier et al., 1997). A s IL-6 and M C P - 3 are partially controlled by N F - K B , the involvement of IKB-CC and N F - K B subunits was also investigated in human P B M C s . L L - 3 7 by itself induced transient phosphorylation of IKB-CC after 30 min of stimulation. IL-1 (3 by itself led to more sustained phosphorylation of IKB-OC, but the peptide in the presence of IL-1 p enhanced this particularly at the 30 min time point (Fig. 3.7A). Moreover, nuclear translocation of N F - K B subunits p50 and p65 was assessed by Western blots. We found that increased levels of p65 and p50 were detected in the nuclear extract of P B M C stimulated with L L - 3 7 alone after 30-60 min dependent on donor variation (Fig. 3.7B). Consistent with the activation of IKB-OC, the combination of IL-1B and L L - 3 7 led to enhanced nuclear translocation of p65 and p50 at the same time points (Fig. 3.7B). Since the serine/threonine protein kinase A k t is known to be activated by phosphorylation through a PI3K-dependent pathway, the influence of stimulation with L L - 3 7 and/or IL-1 p on  26  cellular A k t phosphorylation was analyzed by immunoblotting using an antibody directed at phosphorylated S e r  473  of Akt. The results demonstrated that the phosphorylation of Akt was  modestly increased after stimulation with L L - 3 7 (20 ug/ml) or I L - i p (10 ng/ml) alone for 30 min; however treatment with these two stimuli together induced an increase in phosphorylation of Akt after 30 min o f stimulation o f human P B M C (Fig. 3.8A). It has been established that L L - 3 7 induces activation of the E R K - 1 M A P K  pathway  (Bowdish et al, 2004), and it is also known that E R K - 1 phosphorylates and promotes nuclear translocation of the transcription factor C R E B ( X i n g et al., 1996). Therefore the effect of the peptide on C R E B activity was assessed by immunoblotting using an antibody directed against I  Ser  -phosphorylated C R E B . Primary human P B M C s were stimulated with L L - 3 7 and/or IL-1 P  for 60 min. Both L L - 3 7 and I L - i p by themselves increased the amount of phosphorylated C R E B , but this was substantially enhanced by the combination of L L - 3 7 and IL-1 P (Fig. 3.8B).  3.3. Discussion The innate immune system is a multicomponent host defence system the functioning of which is balanced by complex interactions between its various effector and regulatory molecules. The  sole  human  cathelicidin  immunomodulatory  molecule  LL-37 that  has  enhances  been  increasingly  certain  described  inflammatory  as  a  robust  responses (e.g.  the  upregulation o f certain chemokines while generally suppressing the production of certain pro-inflammatory  cytokines such as TNFoc. It has been shown that L L - 3 7 expression is  up-regulated during infection and inflammation, e.g. in bronchoalveolar lavage of infants with pulmonary infections, in individuals with cystic fibrosis and in psoriatic skin lesions (Chen and Fang, 2004; Ong et al., 2002a; Schaller-Bals et al., 2002). In addition, L L - 3 7 can also be locally  27  induced at sites o f inflammation within epithelial cells (Bals, 2000). This led us to speculate that raised level of L L - 3 7 could enhance, sustain or even amplify certain immune responses in presence o f other endogenous signals. The most interesting o f these would appear to be IL-1B since it can induce L L - 3 7 expression in vitro in keratinocytes (Erdag and Morgan, 2002), while L L - 3 7 is known to promote IL-1 processing through transactivation of the receptor P2X7 (26). Previous data (17) indicted that L L - 3 7 had quite different effects on L P S - and IL-lB-induced pro-inflammatory cytokine responses, suppressing the L P S mediated T N F a , IL-6 and IL-8 production but substantially enhancing the production of IL-6 and IL-8 (while only slightly influencing T N F a ) . Here we have confirmed and extended this initial observation and demonstrate that L L - 3 7 interacts in a mechanistically complex fashion with the inflammatory mediator IL-1 B, to modulate the production of important effectors o f inflammation. A variety o f transcription factors have been separately implicated previously in the biological activities of L L - 3 7 , e.g. L L - 3 7 induces activation of the MAP-kinases Erk-1/2 and p38 and transcription factor Elk-1 that control the transcription and secretion o f IL-8 in primary human monocytes (Bowdish et al., 2004a); L L - 3 7 indirectly stimulates of E G F R and downstream transcription factor STAT-3 in keratinocytes (Tokumaru et al., 2005); and L L - 3 7 appears to act in part through the P I 3 K pathway in inhibiting neutrophil apoptosis (18). This is the first study that demonstrated that multiple transcription factors were involved in an immunomodulatory function of L L - 3 7 , including the pivotal transcription factor N F - K B that is involved in the inflammatory and immune responses. L L - 3 7 by itself was able to transiently activate IicB-a and initiate nuclear translocation o f N F - K B subunits in primary human P B M C s . There is recent evidence suggesting that the N F - K B p50 subunit specifically associates with endogenous IKB-C, in a manner that is essential for the transcription o f IL-6 (Yamamoto et al., 2004). In addition to N F - K B transcription  28  factor binding sites ( T F B S ) , the 5'-flanking sequence upstream of the IL-6 gene contains several response elements for the transcription factors A P - 1 , C R E B and C / E B P indicating that its transcription may require the activity of a complex of transcription factors (Baccam et al., 2003). Similarly, a transcription factor complex containing N F - K B , C R E B and A P - 1 is essential for the production of C C family chemokines such as IL-8, M C P - 1 , and M C P - 3 ( L i et al., 2002; Zhang et al., 2005), as well as the anti-inflammatory cytokine, IL-10 (Agrawal et al., 2003; Dillon et al., 2004; Martin et al., 2005; Moore et al., 2001; Platzer et al., 1999). Alterations in the relative amounts or activities of transcription factors has generally been thought to control the regulation of gene expression at the transcriptional level (Spiegelman and Heinrich, 2004). This leads us to speculate that a complex of multiple transcriptional elements such as N F - K B , C R E B and E l k - 1 , etc., could be essential for the delicate balancing o f immunomodulatory activities by L L - 3 7 . The outcome of action o f this presumed complex would be the selective up-regulation of several key components involved in innate immunity. Recent reports indicate that L L - 3 7 enhances the release of IL-8 from primary neutrophils and monocytes after stimulation with IL-1 P or G M - C S F , respectively (Barlow et al., 2006; Bowdish et a l , 2004a; Bowdish et al., 2004b). L L - 3 7 also augments the ability ofthe T L R / I L - 1 R agonist, IL-1B in human P B M C to induce production of specific cytokines (Mookherjee et al., 2006). Similarly, this report conclusively demonstrated that L L - 3 7 could synergistically enhance specific immune responses, in the presence of the inflammatory mediator IL-1 p. Under inflammatory conditions, effector cells o f the innate immune system are exposed to a broad range of cytokines and inflammatory mediators in vivo. This study showed that IL-1 B and L L - 3 7 induced synergistic production of IL-10, M C P - 3 and IL-6, although it did not induce significant production of TNF-ot (Mookherjee et al., 2006), indicating that the synergistic effect between  29  L L - 3 7 and IL-1 (3 is selective with respect to the inflammatory mediators induced. Therefore we speculate that .the ability o f the host defence peptide L L - 3 7 to boost certain immune responses during the initial course o f infection can be reinforced and/or by inflammatory mediator IL-1 (3, and thus have a significant impact on the efficient balancing o f inflammation by L L - 3 7 , leading to an effective clearance of infection in the host. To date, a number o f putative binding and transactivated receptors, including G P C R s , have been described as influencing the activity of L L - 3 7 (De_Yang et al., 2000). This report further demonstrated the involvement of GPCR(s) in the IL-IB-mediated enhancement of LL-37-induced M C P - 3 production in P B M C . To gain insight into the intracellular transduction events involved in this synergistic enhancement of immunomodulation activity, the underlying mechanism(s) o f the synergistic effect between IL-1 p and L L - 3 7 was further investigated in this study. P I 3 K inhibitors significantly attenuated M C P - 3 production induced by IL-1 P and L L - 3 7 and the presence o f IL-1 B reinforced LL-37-induced activation of multiple molecules, including IKB-OC, N F - K B subunit p50 and p65, A k t and C R E B . Therefore, one possible interpretations for the observation that the modest responses induced by the host defence peptide were enhanced in the presence of the inflammatory mediator IL-1 P, may be due to the reinforcement by IL-1 P of the multiple transcriptional elements involved in the biological activity o f L L - 3 7 , and vice versa. In addition, IL-1B may itself induce other signaling pathways that either supplement or feed into the activation of the transcription factor complex(es) mediating L L - 3 7 responses, thereby stabilizing or enhancing the transcriptional events and thus leading to selective immune responses. In conclusion, this report demonstrates that the biological activity of the human cathelicidin host defence peptide L L - 3 7 is mediated by multiple signaling pathways in human  30  PBMC.  The peptide by itself induced transient activation of IKB-CC/NF-KB, and modest  phosphorylation of A k t and C R E B , which were further augmented in the presence of IL-1 p. The enhanced activation of LL-37-induced transcription factors, in the presence ofthe inflammatory mediator IL-1 P, apparently led to the augmented, synergistic production of IL-6, M C P - 3 and IL-10. This report has provided meaningful insights into the mechanism(s) of the host defence peptide L L - 3 7  in selectively augmenting certain immune responses in the presence of  inflammatory mediators, such as IL-1 P, that would be naturally present at the site of infection, and thereby are likely involved in the delicate balancing of inflammation for the effective functioning o f the immune system.  31  Figure 3.1 A. 50. Total-lKBoc 37" GAPDH  25  0  0.5  6h  +LL-37  B. 50Total-lKBa 37~ GAPDH  250  0.5  1  6h  +LPS  go-  p65  er  p50  50" Ctrl  IL-ip  LL-37  30min  Fig. 3.1: LL-37-induced degradation of NF-KB  Ctrl  IL-ip  LL-37  60min  IKB-CC,  and subsequent nuclear translocation of  subunit p50 and p65. THP-1 cells were stimulated with LL-37 (20 ug/ml), IL-1 B (10  ng/ml) for the indicated time periods. Whole cell lysates of THP-1 cells stimulated with A . LL-37 and B. LPS for 30 min to 6 hr analyzed for total IKB-O. by Western blots. Immunoblotting using anti-GAPDH (housekeeping gene) was performed to allow correction for protein loading in the Western blot analysis. C, Nuclear extracts of THP-1 cells stimulated with LL-37 or IL-1B for 30 and 60 min analyzed for the nuclear translocation of  NF-KB  subunit p50 and p65 by  Western blots using anti-p 105/50 and anti-p65 antibodies. One gel, representative of two independent experiments, is shown for each panel.  32  Figure 3.2 D  A. MCP-3  8 hr  Untreated  12 hr  Fig. 3.2: E n h a n c e d M C P - 3 a n d IL-6 gene expression i n response to co-stimulation w i t h  LL-37 a n d IL-ip i n h u m a n P B M C s . Primary human PBMCs were treated with LL-37 (20 pg/ml) and/or IL-1 P (10 ng/ml) for the indicated time period. Transcriptional induction of A, MCP-3 and B, IL-6 was analyzed by qRT-PCR. Fold changes (Y-axis) for each gene were normalized to G A P D H and are relative to the gene expression in unstimulated cells (normalized to 1 using the comparative Ct method). Typical results of an experiment with cells from one donor, representative of three separate donors, are shown. The biological variability between the three different donors at the 8 hr time point was between ± 2.4 folds for LL-37, ± 2.7 folds for IL-1 P and ± 20 folds for LL37+IL-1P - induced MCP-3 gene expression; between ± 0 . 1 7 folds for LL-37, ± 0 . 1 9 folds for IL-lp and ± 108 folds for LL-37+IL-1 p-induced IL-6 gene expression. Similarly the biological variability between the three different donors at the 24 hr time point was between ± 122 folds for LL-37, ± 4 folds for IL-ip and ± 2 1 9 folds for LL37+IL-1P - induced MCP-3 gene expression; between ± 6.5 folds for LL-37, ± 7.7 folds for IL-1 P and ± 5 9 folds for LL-37+IL-1 p-induced IL-6 gene expression.  33  Figure 3.3 A. =5 600 E  J  400  ^  200  c  0  t r l  hr  •  6  •  12 hr  • •  18 hr 24 hr  LL-37  IL-ip  ILip+LL-37  LL-37  IL-ip  ILlp+LL-37  B. 1.5 SOD  s  1 4-  so  Ctrl  c.  1  150 120  5  90  o I  -J  60 30 n  Ctrl  LL-37  IL-ip  ILip+LL-37  Fig. 3.3: Enhanced M C P - 3 , IL-6 and IL-10 protein production in human PBMCs upon co-stimulation with L L - 3 7 and IL-ip. Human P B M C were incubated with LL-37 (20 pg/ml) and/or IL-lp (10 ng/ml) for 6, 12, 18 and 24 hr. Tissue culture supernatants were analyzed by capture ELISA for the amount of secreted A , MCP-3 and B, IL-6. C, Tissue culture supernatants were analyzed for IL-10 by capture ELISA after PBMCs were stimulated for 24 hr by LL-37 and/or IL-1 p. Results are representative of three independent experiments with different donors. Values are the mean ± SE for replicate samples from same cells.  34  Figure 3.4 150  ^ 120  • -PTx • +PTx  90 60 30  0  Ctrl  IL-ip  LL-37  IL-ip  WKYMVm IL-ip  +LL-37  + WKYMVm  Fig. 3.4: Effects of inhibition of G-protein coupled receptors (GPCR) on the synergistic production of MCP-3 induced by  LL-37 and IL-1 p. Human P B M C were pre-treated with the  inhibitor of G P C R , pertussis toxin (100 ng/ml), for 1 hr. Subsequently the cells were stimulated with L L - 3 7 (20 ug/ml) and I L - i p (10 ng/ml) for 24 hr. M C P - 3 production in the culture supernatant was monitored by capture E L I S A . The agonist of G-protein coupled receptor F P R L - 1 , peptide W K Y M V m (5 u M ) , was used as a positive control. Results are representative of three independent experiments. Values are the mean ± S E .  35  Figure 3.5 A. 1200 ] Untreated cells  1000  I | + wortmannin  800  •  600  +LY294002  400  £  200 0  C  m  B.  1000  •-Bay11-7085  800  • + B a y l 1-7085  600 400  \  200 0  Ctrl  Fig. 3.5: Influence of PI3K and  IL-lp  LL-37  IKB-CC  IL-ip+LL-37  inhibition on the synergistic production of MCP-3  induced by LL-37 and IL-1 p. Human PBMCs were pre-treated with A, PI3K inhibitor LY294002 (10 uM) or wortmannin (1 pM), or B,  IKB-CC  inhibitor Bayl 1-7085 for 1 hr.  Subsequently the cells were stimulated with LL-37 (20 pg/ml) and IL-1 (3 (10 ng/ml) for 24 hr. MCP-3 production in the tissue supernatant was monitored by capture ELISA. Results are representative of three independent experiments. Values are the mean ± SE.  36  Figure 3.6 800 600  •  - P K A inhibitor  M  +PKA inhibitor  •  - P K C inhibitor  400 200 OJD  a.  • CM  U  0 1200 1000 800 600 400 200  0  Ctrl  LL-37  IL-1 (3  IL-ip+LL-37  Fig. 3.6: Effects of PKA and PKC on the synergistic production of MCP-3 induced by LL-37 and IL-ip. Human P B M C s were pre-treated with A, P K A inhibitor 6-22 Amide (10 n M ) or B, P K C inhibitor GF109203x (10 n M ) . Subsequently the cells were stimulated with L L - 3 7 (20 ug/ml) and I L - i p (10 ng/ml) for 24 hours. M C P - 3 production in the tissue supernatant was monitored by capture E L I S A . Results are representative o f three independent experiments. Values are the mean ± S E .  37  Figure 3.7 A.  Ctrl  IL-lp  LL-37  IL-ip+LL-37  30min  Donor 1  60Ctrl  IL-IP  LL-37  D o n o r  ' IL-ip+LL-37  2  ' Ctrl  60min  IL-lp  LL-37  ' IL-ip+LL-37  30min  Fig. 3.7: LL-37-induced IKB-CX phosphorylation, and subsequent translocation of N F - K B subunits (p50 and p65) in the presence of IL-ip. Human P B M C were stimulated with L L - 3 7 (20 ug/ml) and/or IL-1B (10 ng/ml). A, Cytoplasmic extracts were analyzed by immunoblotting after 30 min o f stimulation for phosphorylation o f IKB-CC (upper panel). Immunoblotting using a n t i - G A P D H (housekeeping gene) was performed to allow correction for protein loading in the Western blot analysis. B, Nuclear extracts of cells stimulated for 30 or 60 min were analyzed for nuclear translocation o f N F - K B subunits p50 and p65 by Western blots using anti-pl05/50 and anti-p65 antibodies. One gel, representative o f three independent experiments, is shown for each panel.  38  Figure 3.8 A. 75kDp-Akt (60 kD) 50kD—I 75kD total-Akt (60kD) 50kDCtrl  IL-ip  LL-37  IL-ip+LL-37  30min 50kl> p-CREB (43kD) 37kD—\ 50kD—f total-CREB (43 kD) 37kD  Ctrl  IL-IP  LL-37  IL-ip+LL-37  60min  Fig. 3.8: LL-37-induced phosphorylation of A k t and C R E B in the presence of I L - i p . Human P B M C were stimulated with L L - 3 7 (20 pg/ml) and/or I L - l p  (10 ng/ml). A,  Cytoplasmic cell extracts were analyzed after 30 min of stimulation by immunoblotting for phosphorylation o f A k t (upper panel), or total A k t (middle panel); B, Nuclear extracts were analyzed for phosphorylation of C R E B (lower panel), or anti-total C R E B (housekeeping gene) to allow correction for protein loading in the Western blot analysis. One gel, representative o f three independent experiments, is shown for each panel.  39  CHAPTER 4 EFFECTS OF LL-37 ON LPS-STIMULATED HUMAN MONOCYTIC CELLS  4.1. Introduction The presence o f infection in the bloodstream, known as sepsis, causes nearly 200,000 deaths per year i n North A m e r i c a (Sommers, 2003). Although sepsis can occur i n response to a wide range o f pathogens, it is most commonly caused by the release o f a Gram-negative outer membrane component, endotoxin (lipopolysaccharide, L P S ) (Alexander and Rietschel, 2001). To initiate the signalling cascades, L P S first interacts with LPS-binding protein ( L B P ) which facilitates its binding to the receptor, C D 14 ( L i u and M a l i k , 2006; Wright, 1995). This complex is specifically recognized by T L R 4 and M D - 2 expressed on surface o f macrophages and other cells involved in the innate immunity, leading to the activation o f intracellular signalling events including I K K / I K B / N F - K B and the E R K , J N K and p38 mitogen-activated protein kinases. Initiation o f these signalling pathways eventually leads to the transcription o f a vast number of genes encoding multiple inflammatory mediators such as TNF-ct, I L - 1 , I L - 6 , IL-10, IL-8, and T G F - B (Scherle et al., 1998; Ulevitch and Tobias, 1999; van der P o l l , 2001). These biological mediators have beneficial effects for combating infections and protective immune responses in infectious diseases. O n the other hand, uncontrolled and excessive acute immune response can cause sepsis during early stage o f bacteria infections. During sepsis, the generation of inflammatory mediators can trigger amplified cascade responses and overwhelming secretion o f  40  pro-inflammatory cytokines, especially T N F - a , by macrophages in the liver, spleen, and other organs. Such a systemic secretion of T N F - a results in systemic inflammation and host damage that lead to septic shock and death (Fiuza and Suffredini, 2001; Hehlgans and Pfeffer, 2005; Tracey et al., 1986; Zanotti et al., 2002). In addition to bacterial infections, traditional  antibiotics  stimulate the release of  endotoxin and thus contribute to sepsis (Hancock, 1999; Prins et a l , 1995a; Prins et al., 1995b). Therefore, it is necessary to develop novel strategies to overcome Gram-negative sepsis, especially given the disappointing results obtained from other therapies such as neutralization antibodies for T N F - a and L P S (Glauser et al., 1994; Hancock, 2001). Strikingly, it was reported that in sepsis mice models, cationic host defence peptides display abilities to prevent lethal endotoxaemia by dramatically suppressing T N F - a production. Therefore, these peptides have attracted considerable attention to develop potential therapeutic drugs for the treatment of sepsis in terms of their anti-endotoxin activity (Bowdish et al, 2005; Hancock and Scott, 2000; Scott et al., 2002a). There is much evidence that L L - 3 7 is an effective anti-endotoxin agent due to its pronounced ability to suppress LPS-induced production of pro-inflammatory cytokines including T N F - a , IL-6, and IL-1 and prevent lethal endotoxemia in the animals (Cirioni et al., 2006; Dankesreiter et a l , 2000; Fukumoto et al., 2005; Glauser et al., 1994; Gough et al., 1996b). Likewise, over-expression of L L - 3 7 in mouse airway could considerably prolong survival period of mouse model after challenge with lethal dose of L P S (Bals et al,  1999). Despite growing  interests in the anti-endotoxin property of L L - 3 7 , little is known about the mechanism(s) by which L L - 3 7 modulates inflammatory reaction in response to L P S . One acceptable explanation is that L L - 3 7 could competitively block the formation of L P S • L B P complex owing to high  41  affinity of L L - 3 7 binding to L P S (Scott et al, 2000). Recent microarray studies performed in R A W 2 6 4 . 7 cells and human THP-1 cells demonstrated that anti-endotoxin effect of L L - 3 7 is alternatively due to the capability of L L - 3 7 to selectively regulate pro- and anti-inflammatory gene expression induced by L P S (Mookherjee et al, 2006; Scott et al, 2002a). In this study, the impacts of L L - 3 7 on modulating LPS-induced inflammatory responses were further examined in human monocytic cells.  4.2. Results LL-37 selectively regulates transcription and secretion of LPS-induced inflammatory molecules in PBMCs and THP-1 cells Cationic host defence peptides have been shown to block many LPS-induced responses and are being considered as candidates for the treatment of sepsis (Hancock, 2001). Therefore, the effects of L L - 3 7 in modulating LPS-stimulated human monocytic cells ( P B M C s and THP-1) were investigated. Fresh human P B M C s or the human monocytic T H P - 1 cell line were treated with endotoxin-free water as vehicle control, or L P S (100 ng/ml) with or without L L - 3 7 (20 pg/ml) for the indicated times and total R N A was isolated to perform quantitative R T - P C R (qRT-PCR) to generate gene expression patterns. We showed that exposure to L L - 3 7 dramatically inhibited LPS-induced production of m R N A for the pro-inflammatory cytokine T N F - a by 9 5 % in both P B M C s and THP-1 cells (Fig. 4.1 A ) , which is consistent with the involvement of L L - 3 7 in suppressing T N F - a secretion (Fig. 4.IB). I further showed that the inhibitory effect of L L - 3 7 on T N F - a production occurred in a dose-dependent manner and 1 pg/ml of L L - 3 7 was able to inhibit 50% of T N F - a released into the tissue culture supernatant, suggesting that a physiological concentration of L L - 3 7 exhibited anti-endotoxin activity (Fig 4.2).  42  In contrast to previous observations that L L - 3 7 can significantly increase IL-lB-induced IL-6 gene transcription  and protein  secretion from P B M C s ,  LPS-mediated IL-6  release was  dramatically decreased by exposure to L L - 3 7 although it has been demonstrated that IL-1 p utilizes the common T L R / I L - 1 R signal pathway with L P S (Fig. 4.3A). Consistently, IL-6 m R N A accumulation was substantially reduced (-93%) when L L - 3 7 (20 ug/ml)  and L P S were  simultaneously added to cells (Fig. 4.3B). Moreover, as a representative o f chemokines, M C P - 3 gene expression and production were also examined in P B M C s treated with L L - 3 7 and/or L P S . Interestingly, stimulation by L L - 3 7 and L P S synergistically enhanced M C P - 3 secretion in a time-dependent manner when compared with either L P S or L L - 3 7 alone, while L P S alone induced detectable but small amounts of M C P - 3 production (Fig 4.4B). Similar synergy between L P S and L L - 3 7 was also detected in gene transcription levels after 8-12 hours (Fig 4.4A). These results confirmed that L L - 3 7 is able to selectively modulate LPS-induced inflammatory responses, thereby balancing the pro- and anti-inflammatory responses and avoiding potential harmful inflammation.  Expression of NF-kB negative regulator A20 in THP-1 cells in presence of LPS and LL-37 D N A microarray studies demonstrated that L L - 3 7 selectively regulates LPS-induced inflammatory gene transcription in THP-1 cells (Mookherjee, et al. 2006). It was showed that LL-37  significantly  downregulated  pro-inflammatory  cytokine  genes,  whereas  certain  anti-inflammatory genes that encode negative regulators of the T L R 4 - t o - N F - K B pathway were only slightly reduced in the presence of L L - 3 7 . These genes included TNFAIP3 protein 3) encoding A 2 0 and the NF-KB-inhibitor, NF-KBIA  (TNF-a-inducible  encoding (IKB-OC) (Mookherjee et al.,  2006). Therefore, the protein expression of A 2 0 was examined in T H P - 1 cells stimulated with L P S and/or L L - 3 7 . Whole cell lysates from LPS-treated THP-1 cells were collected at different  43  time points (0 hr, 2 hr, 4hr and 18 hr). It was found that following the initial induction, the expression o f protein A 2 0 remained stable for up to 18 hr after L P S stimulation (Fig 4.5A). Interestingly, when cells were co-stimulated with L L - 3 7 and L P S for either 2 or 18 hr, L L - 3 7 only reduced 30-50% expression o f A 2 0 induced by L P S (Fig. 4.5B and C ) compared with unstimulated control cells, indicating that L L - 3 7 did not completely inhibit LPS-stimulated A 2 0 synthesis, despite the fact that A 2 0 protein expression is known to be controlled by N F - K B . This observation is not consistent with expression of NF-KB-controlled pro-inflammatory cytokines. A s A 2 0 terminates LPS-mediated N F - K B activation by negative feedback pathway (Boone et al., 2004; Lipniacki et al., 2004; Wertz et al., 2004), I speculate that the effects o f L L - 3 7 on maintaining intracellular expression o f A 2 0 would provide a possible mechanism, by which L L - 3 7 selectively modulates LPS-induced inflammatory responses.  4.3. Discussion L L - 3 7 , a natural human cationic host defence peptide, has been demonstrated to be a potent anti-endotoxin agent (Bowdish et al., 2005; Ciornei et al., 2005). Previous microarray studies  indicated that  LL-37  selectively alters transcription  o f LPS-induced pro- and  anti-inflammatory genes downstream o f T L R - 4 (Mookherjee et al., 2006; Scott et al., 2002a). Here, my results provide further evidence indicating the involvement o f L L - 3 7 in modulating LPS-initiated inflammatory responses i n human monocytic cells. Moreover, L L - 3 7 exhibits the ability to retain a N F - K B negative regulation pathway in presence o f L P S . In mouse model experiment, it was demonstrated that administration o f L L - 3 7 can protect against sepsis induced by Escherichia coli L P S (Scott et al. 2002). Similarly, the effects of L L - 3 7 in neutralizing LPS-induced pro-inflammatory cytokine production in murine macrophage cell  44  line R A W 264.7 (Scott et al, 2002b) are consistent with the results presented here that treatment of L L - 3 7 dramatically suppressed LPS-stimulated T N F - a and IL-6 gene transcription and protein secretion in human P B M C s and THP-1 cells. In addition, this inhibition of T N F - a production is dose-dependent, with significant inhibition (-60%) at 1 pg/ml of L L - 3 7 , a physiological concentration found in healthy individuals (Murakami et al., 2002). Moreover, the concentration of L L - 3 7 has been reported to increase during infection and inflammation, for example, in bronchoalveolar lavage from infants with pulmonary infections and in individuals with Cystic Fibrosis lung disease at up to 30 pg/ml and 15 pg/ml respectively (Chen et al, 2004; Chernish and Aaron, 2003; Schaller-Bals et al, 2002; Starner et al., 2005), and at levels of - 1.5 mg/ml in psoriatic skin lesions (Frohm et al., 1997; Ong et al., 2002b). Although previous studies showed that the anti-endotoxin activity of L L - 3 7 is attributed in part to blockage o f L P S - L B P binding, there is emerging evidence suggesting that L L - 3 7 directly interacts with effector cells and balances LPS-induced pro- and anti-inflammatory responses (Braff et al, 2005). Active internalization of L L - 3 7 into cells was detected in A549 epithelial cells, T H P - 1 cells and D C s (Bandholtz et al, 2006; L a u et al, 2005; Rosenfeld et  al,  2006). Our lab observed that L L - 3 7 can still reduce T N F - a production even when added to the murine cell line and human T H P - 1 cells an hour after L P S addition (Gough et al, Mookherjee et al,  2006; Scott et al,  1996a;  2002a). The present study described for the first time  synergy between L L - 3 7 and L P S in production of the chemokine M C P - 3 , which definitely cannot be explained solely by extracellular neutralization of L P S . A previous study by Elssner et al. showed that L L - 3 7 increases maturation and release of IL-1 B in LPS-primed monocytes via the P2X7 receptor (Elssner et al,  2004). The mechanisms by which L L - 3 7 synergistically  enhance LPS-induced chemokine production remain to be further characterized.  45  To further understand the role of L L - 3 7 in modulating LPS-induced inflammatory responses, the expression of the N F - K B pathway negative regulator A 2 0 was examined in the presence of L P S and L L - 3 7 . Recent reports have shown that A 2 0 is inducible by T N F - a and L P S in macrophages (O'Reilly and Moynagh, 2003; Wertz et al,  2004) and serves as a negative  feedback regulator to terminate TLR-induced N F - K B activity, and pro-inflammatory cytokine secretion as well as blocking LPS-induced septic shock in animal models (Boone et al., 2004; Lee et al., 2000a). In parallel, A 2 0 can terminate the TLR-induced I F N regulatory factor 3 (IRF-3) signalling pathway (Saitoh et al, 2005). Thus, A 2 0 is considered to be an important regulator of innate immune responses (Heyninck and Beyaert, 2005) and can be a likely target for modulating inflammatory responses and thus sepsis (Boone et al,  2004). Here, Western blotting results  demonstrated that the presence of L L - 3 7 did not significantly  inhibit LPS-induced A 2 0  expression in T H P - 1 cells at either early 2 hr or later 18 hr time points. Therefore, it is reasonable to propose that the involvement of L L - 3 7 in the maintenance of this N F - K B negative autoregulation feedback loop is partially responsible for regulating LPS-induced inflammatory responses. In conclusion, I demonstrated that L L - 3 7 significantly suppressed T N F - a and IL-6 secretion, whereas it synergistically increased M C P - 3 production in LPS-stimulated P B M C s . Further, L L - 3 7 has no effect on A 2 0 expression level. These findings suggest that L L - 3 7 regulate LPS-induced inflammatory responses by selectively modulating production of inflammatory mediators (e.g. cytokines and chemokines).  46  Figure 4.1 TNF-a  B 800 600 400 200  Ctrl  LPS  LL-37  LPS+LL-37  ED  Ctrl  LPS  LL-37  Ctrl  a.  100  2500 2000 1500 1000 500 0  LPS+LL-37  rfr-i  LPS  LL-37  LPS+LL-37  LL-37  LPS+LL-37  m Ctrl  LPS  Figure 4.1: TNF-a gene expression and protein production on co-stimulation with LL-37 and LPS induced in human PBMC and THP-1 cells. Cells were treated with L L - 3 7 (20 ug/ml) and/or L P S (100 ng/ml) for 4 hrs. Transcriptional induction of A, T N F - a from P B M C s (upper panel) and T H P - 1 cells (lower panel) was analyzed by q R T - P C R and semi-qRT-PCR respectively. Fold changes (Y-axis) o f each gene was normalized to G A P D H and are relative to the gene expression i n un-stimulated cells (normalized to 1) using the comparative Ct method. Result of experiment from one donor representative o f four is shown. B, T N F - a production was analyzed by E L I S A from P B M C s (upper panel) and THP-1 cells (lower panel). Results are representative of three independent experiments (± standard error) from three different donors.  47  Figure 4.2  8  ta. I  z  H  0 LPS  -  LL-37  -  lOOng/ml lOOng/ml lug/ml  lOOng/ml lOOng/ml 5ug/ml  lOug/ml  lug/ml  5ug/ml  10ug/ml  Figure 4.2 LL-37 suppresses LPS-induced secretion of TNF-a as dose-dependent manner. THP-1 cells were stimulated with 100 ng/ml L P S in the presence o f increasing concentrations o f L L - 3 7 (x-axis) for 4 h. The concentration o f the pro-inflammatory cytokine T N F - a (y-axis) was monitored in the tissue culture supernatant by E L I S A . The results are an average (±SD) o f three independent experiments.  48  Figure 4.3 IL-6 12000 10000 8000 C L  6000  SO  4000 2000 0  Ctrl  LL-37  LPS  LPS+LL-37  LPS  LPS+LL-37  B. 1000 o S  100  a JS  10  Ctrl  LL-37  Figure 4.3: IL-6 gene expression and protein production on co-stimulation with LL-37 and LPS induced in human PBMCs. IL-6 production was analyzed by E L I S A from P B M C s treated for 24 hr (upper panel). Results are representative o f three independent experiments ( ± standard error) from three different donors. P B M C s were treated with L L - 3 7 (20 pg/ml) and/or L P S (100 ng/ml) for 8 hr and total R N A was isolated (lower panel). Transcriptional induction was analyzed by q R T - P C R . F o l d changes (log) (Y-axis) o f each gene was normalized to G A P D H and are relative to the gene expression in un-stimulated cells (normalized to 1) using the comparative Ct method. Results o f experiment from one donor representative o f four are shown.  49  Figure 4.4 MCP-3 1000  8P 100  2  s  10  o  1000 800 ox;  600  fill •  400  n a  12hrs  •  18hrs  •  24hrs  20h  12h  8h  B.  6hr  &.  s  200 0  Ctrl  LL-37  LPS  LPS+LL-37  Figure 4.4: MCP-3 gene expression and protein production on co-stimulation with LL-37 and LPS induced in human PBMCs. A. P B M C s were treated with L L - 3 7 (20 ug/ml) and/or L P S (100 ng/ml) for 8, 12, and 20 hr and total R N A was isolated. Transcriptional induction was analyzed by q R T - P C R . F o l d changes (log) (Y-axis) of each gene was normalized to G A P D H and are relative to the gene expression i n un-stimulated cells (normalized to 1) using the comparative Ct method. Result o f experiment from one donor representative o f four is shown. B. M C P - 3 production was analyzed by E L I S A from P B M C s treated for 6hr, 12hr, 18hr, and 24 hr. Results are representative o f three independent experiments (± standard error) from three different donors.  50  Figure 4.5  Fig 4.5: Protein expression of A20 i n L P S - s t i m u l a t e d THP-1 cells w i t h or without L L - 3 7 . A. THP-1 cells were incubate with L P S (lOOng/ml) for the indicated times and endotoxin-free water treatment was used as a vehicle control. Cell lysate collected from stimulated cells were used to determine A 2 0 protein expression using monoclonal anti-A20 antibody. B. Protein lysates collected from T H P - 1 cells incubated with L P S (lOOng/ml) in presence or absence of L L - 3 7 (20 pg/ml) for either 2 hours or 18 hours. G A P D H was quantified to allow correction for protein loading. The expression of A 2 0 was quantified by densitometry. G A P D H was quantified to allow correction for protein loading.  51  CHAPTER 5 GENERAL DISCUSSION AND CONCLUSIONS 5.1 Introduction The human cathelicidin peptide L L - 3 7 is a potent modulator o f the immune system, which contributes to its ability to augment innate host defence against acute infection. B y participating in the recruitment o f leukocytes, such as neutrophils, monocytes, mast cells, and T cells (De et al., 2000), and inducing the degranulation o f mast cells (Niyonsaba et al., 2002a), L L - 3 7 acts as a mediator o f positive amplification loops o f innate immune responses (Zanetti, 2004a). Furthermore, L L - 3 7 has the ability to alter transcriptional responses o f inflammatory genes in the murine macrophage cell line R A W 264.7, and the human epithelial cell line A549 (Scott et al., 2002a), and to selectively modulate LPS-stimulated inflammatory responses in human monocytic cell line T H P - 1 , which might mediate its anti-endotoxin activity (Mookherjee et al., 2006). Activation o f M A P K s , E R K 1 / 2 and p38 mediated by L L - 3 7 has been demonstrated to stimulate IL-8 production in peripheral blood derived monocytes and airway lung epithelial cells and to induce IL-18 secretion in keratinocytes (Bowdish et al., 2004a; Niyonsaba et al., 2005; Tjabringa et al., 2003a). Recently, L L - 3 7 has been shown to suppress neutrophil apoptosis, whereas it induces apoptosis i n primary airway epithelial cells (Barlow et al., 2006; Lau et al., 2006; Nagaoka et al., 2006). In this thesis, the role of L L - 3 7 in infection and inflammation was further explored and the signal transduction events underlying LL-37-induced activities were investigated in human peripheral blood mononuclear cells and the human monocytic cell line THP-1.  52  5.2 LL-37 Differentially Modulates Responses of Innate Immune Effector Cells to Inflammatory Stimuli It has been demonstrated that L L - 3 7 is a potent anti-endotoxin agent as a result of its abilities to inhibit LPS-induced T N F - a production in vitro and to protect animal models from sepsis in vivo (Bowdish et a l , 2005; Cirioni et al., 2006; Mookherjee et al., 2006; Scott et a l , 2002a). In addition, L L - 3 7 has also been shown to promote IL-1 (3 processing and release from LPS-primed monocytes (Elssner et al., 2004). In the present study, we have demonstrated that L L - 3 7 synergistically enhanced LPS-stimulated M C P - 3 production in P B M C s in contrast to the inhibition o f pro-inflammatory cytokine secretion. This is inconsistent with the hypothesis that L L - 3 7 simply specifically binds to and neutralizes the extracellular existing L P S , and instead supports the proposed mechanism that L L - 3 7 may selectively modulate and balance pro- and anti-inflammatory responses during endotoxin challenge. During the course o f infection or inflammation, effector cells w i l l be exposed to a broad range o f inflammatory cytokines and other mediators in vivo. I L - i p is a cytokine which is produced by macrophages and epithelial cells, and activates common signal transduction downstream o f receptor engagement, leading to the induction o f various inflammatory responses. Therefore, co-incubation with IL-1 P and L L - 3 7 should help to analyze the impacts o f L L - 3 7 on IL-ip-induced signalling pathways and consequent outcomes. Interestingly, L L - 3 7 and IL-1 P act in synergy to promote the production of IL-6, IL-10 and M C P - 3 , but not T N F - a in primary human blood cells (Bowdish D M , unpublished data). In addition, recent reports demonstrated that L L - 3 7 enhanced the release of IL-8 from primary neutrophils and monocytes after co-stimulation with IL-1 P or G M - C S F , respectively (Barlow et al., 2006; Bowdish et al., 2004a). These observations suggest that L L - 3 7 is not simply endotoxin neutralizing in nature, but might modulate innate immune responses in a  53  stimulus-specific manner by more complicated mechanisms. The presence o f an inflammatory milieu provides a beneficial environment for L L - 3 7 to initiate immune responses at early stages of infection, which might affect the later phases o f inflammation significantly.  5.3 LL-37 Enhances IL-ip-induced Intracellular Signalling Events via Multiple Molecules L L - 3 7 has been reported to directly activate transcription factor Elk-1 that controls transcription and secretion o f IL-8 in primary human monocytes (Bowdish et al., 2004a). Keratinocyte migration induced by L L - 3 7 is mediated by indirect stimulation o f E G F R and downstream transcription factor STAT-3 (Tokumaru et al., 2005). M y findings further suggest that L L - 3 7 regulates activation o f multiple transcription factors, which in turn control expression of downstream genes. The impact o f L L - 3 7 on N F - K B subunit p50 and p65 was first examined since N F - K B plays a pivotal role in inflammatory and immune responses. We demonstrated here that exposure to L L - 3 7 activated p50 and p65 nuclear translocation in T H P - 1 cells and P B M C s . Recent evidence suggests that p50 specifically associates with endogenous IKB-C^ and is recruited to the N F - K B binding site o f the IL-6 promoter, leading to transcription o f IL-6 (Yamamoto et al., 2004). In p50' cells, production o f IL-6 in response to stimulation by L P S and IL-1 p was A  severely impaired, indicating the significance o f p50 in IL-6 expression (Yamamoto et al., 2004). In addition to N F - K B binding site, the 5'-flanking sequence upstream o f the IL-6 gene contains several response elements to the transcription factors A P - 1 , C R E B and C / E B P (Baccam et al., 2003). Similarly, activation o f a distinct set o f transcription factors including N F - K B , C R E B and AP-1 is required by transcriptional activation o f C C family chemokines such as IL-8, M C P - 1 , and M C P - 3 ( L i et al., 2002; Zhang et al., 2005) and the anti-inflammatory cytokine IL-10  54  (Agrawal et al., 2003; D i l l o n et al., 2004; Martin et al., 2005). We have demonstrated here that L L - 3 7 was able to modestly stimulate C R E B phosphorylation to a similar extent to that induced by IL-1 p. L L - 3 7 augmented p50/p65 and C R E B activation in P B M C s stimulated with I L - i p , which likely contributed to increased M C P - 3 and IL-6 m R N A levels and protein secretion. Although IL-6 has been long assigned as a pro-inflammatory cytokine, it is now defined as a resolution factor that balances pro- and anti-inflammatory  outcomes to modulate the  immunological response due to its pro- and anti-inflammatory characteristics (Jones, 2005). M C P - 3 belongs to C - C chemokine family and is active on mononuclear phagocytes, T cells, N K cells, basophils, eosinophils and D C s (Allavena et al., 1994; Dahinden et al., 1994; Loetscher et al., 1994; Sozzani et al., 1995; Taub et al., 1995). In addition, among the genes that are up-regulated directly by L L - 3 7 are anti-inflammatory  cytokines (IL-10, IL-19), chemokines  (Gro-a, M C P - 1 ) and M E K 6 , an activator of M A P K (Mookherjee, unpublished data). Taken together, it is tempting to speculate that the immunomodulatory abilities of L L - 3 7 might be directed in part by its ability to modulate activation of these inflammatory molecules. Future studies should focus on understanding if L L - 3 7 can affect the interactions of different transcription factors, such as N F - K B and C R E B .  5.4 Signalling Transduction Events Induced by L L - 3 7 LL-37-induced intracellular transduction events that are upstream of transcription factors have not been well characterized. Bowdish et al showed that L L - 3 7 induces IL-8 production in part by activating M A P kinase, E R K 1 / 2 and p38 and transcription factor Elk-1 in primary human monocytes and H B E cells (Bowdish et al., 2004a). Recent studies suggested that activation of PI3K, not M A P kinases, is involved in inhibiting neutrophil apoptosis via activation of the  55  effector caspase-3, and alters the balance of Bcl-2 family proteins (Barlow et al., 2006). Additionally, L L - 3 7 appears to be involved in the regulation of endothelial cells by inducing the rise in intracellular C a  2 +  concentration and consequent activation of the P L C - y / P K C / N F - K B and  P I 3 K / A k t cascades (Koczulla et al., 2003a). Signalling analysis showed that pretreatment with Bay 11-7085 (IKB-CC inhibitor), PD98058 ( E R K 1 / 2 inhibitor), SB203589 (p38 inhibitor) and LY294002 (PI3K inhibitor) significantly attenuated LL-37-induced IL-8 production (Mookherjee, unpublished data). These results are partially consistent with previous finding that L L - 3 7 activates M A P K to induce secretion of IL-8 (Bowdish et al., 2004a), and for the first time provides evidence that IKB-GC and P I 3 K also contribute to the release of IL-8. Consistently, Western blotting results demonstrated that L L - 3 7 stimulated transient activation of IKB-CC and slight phosphorylation of A k t in primary human P B M C s . The effect of other cationic peptides on activation of IKB-OC has also been studied in different cell types. Polymyxin B activates some early steps of the complex process of D C maturation through the transient activation of iKB-a/NF-KB pathways. Conversely, the mechanism of L L - 3 7 is clearly distinct from the one described for P R - 3 9 , a porcine cathelicidin peptide, which appears to affect angiogenesis by inhibiting degradation of IKBCC and NF-KB-dependent gene expression in cultured cells and in two different mice models. Therefore, the impacts of L L - 3 7 on the IKB-OC/NF-KB pathway are cell type dependent and function specific. Moreover, we showed that LL-37-induced activation of IKB-CC and A k t is enhanced in the presence of IL-1 p. Studies using P I 3 K inhibitors further confirmed the role of P I 3 K / A k t pathway in the synergistic production of M C P - 3 induced by combination of L L - 3 7 and IL-1B in P B M C s . These results imply that L L - 3 7 enhances activation of multiple steps (Akt, IKB-CC, p50, and C R E B ) along IL-1 B-initiated signalling pathway, which may result in amplification of IL-1 P-induced immune responses. Since L P S and IL-1 p activate a  56  common signalling pathway that results in the activation of N F - K B , M A P K S and others, future research may focus on the effect of L L - 3 7 in regulation of LPS-mediated inflammatory signals.  5.5 Receptors Involved in L L - 3 7 - i n d u c e d Chemokine Production To date, a number of identified and undefined receptors have been described to mediate LL-37-induced activates on different cells such as monocytes, neutrophils, T cells, mast cells, epithelial cells and keratinocytes (De et al., 2000; L a u et al., 2005; Nagaoka et al., 2006; Niyonsaba et al., 2002a; Tjabringa et al., 2003a; Tokumaru et al., 2005). However, the only characterized receptor demonstrated to directly mediate interaction between L L - 3 7 and effecter cells is the formyl-peptide receptor-like 1 ( F P R L 1 ) which belongs to the seven transmembrane domain G P C R families. Through F P R L - 1 , L L - 3 7 functions as a chemoattractant for neutrophils, eosinophils, mast cells, monocytes, and T cells (De et al., 2000; Yang et al., 2001). In addition, LL-37-induced wound healing of human airway epithelial cells and angiogenic activity was suggested to be mediated via F P R L - 1 (Kurosaka et al., 2005; Shaykhiev et al., 2005). Furthermore, modulation of D C differentiation was shown to involve activation of G P C R s distinct from F P R L - 1 (Davidson et al., 2004a), while G P C R s other than F P R L - 1 were shown to mediate LL-37-induced anti-apoptosis of human primary neutrophils (Barlow et al., 2006). Human neutrophil peptides (defensins) stimulate IL-8 production from A 5 4 9 lung epithelial cells via P2Y6, a Gi-protein coupled nucleotide receptor (Khine et al., 2006). In the present study, I showed that production of M C P - 3 induced by combination of IL-1 B and L L - 3 7 was inhibited by G-protein inhibitor pertussis toxin, suggesting involvement of G-protein coupled receptors although it has not yet been demonstrated whether specific formyl-peptide receptors involved (such as F P R L - 1 ) .  57  5.6 Conclusions In summary, this thesis demonstrated that the host defence peptide L L - 3 7 was able to augment immune responses i n effector cells during the inflammation and infection. In addition, the mechanisms underlying  LL-37-induced activities have been studied, indicating the  involvement o f L L - 3 7 in modulating a variety o f intracellular signalling events in immune cells. 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