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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 J I E Y U B. Med., Medical School, Shandong University, China 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH C O L U M B I A November 2006 © J ieYu,2006 A B S T R A C T The human cathelicidin, LL -37 , is a cationic host defence peptide and serves as an essential component of innate immunity. In addition to its modest antimicrobial activity, LL -37 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 LL-37 .could synergistically enhance IL- ip- induced production of cytokines (IL-6, IL-10) and chemokines (MCP-3) in primary human P B M C s . In contrast to the neutralization of LPS-induced secretion of pro-inflammatory cytokines, LL -37 dramatically augmented LPS-stimulated M C P - 3 production. LL -37 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 LL -37 and I L - i p were also observed oh activation of Akt and C R E B . Therefore, we propose that, in addition to its well-known anti-inflammatory activity, the human host defence peptide LL -37 also plays an important role in boosting the innate immune responses in combination with inflammatory mediator ( IL- ip) , which provides a new mechanism for LL-37 in modulating the inflammatory responses in innate immunity. i i T A B L E O F C O N T E N T S ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i LIST OF ABBREVIATIONS v i i ACKNOWLEDGEMENTS ix Chapter 1. GENERAL INTRODUCTION 1 1.1 Innate Immune System 1 1.1.1 T L R s and IL-1R 1 1.1.2 TLRs / IL -1R mediated signalling pathways 2 1.1.3 Inflammatory responses and mediators 4 1.1.4 Negative regulation of T L R s / I L - I R pathways 5 1.2 Cationic Host Defence Peptides 7 1.2.1 General features of hCAP-18 /LL-37 7 1.2.2 Immunomodulator properties of LL-37 9 1.3 Hypothesis and Experimental Goals 11 Chapter 2. MATERIALS AND METHODS 12 2.1 Isolation of Peripheral B lood Mononuclear Cells 12 2.2 Cel l Culture 12 2.3 Stimulants, Reagents and Antibodies 13 2.4 Treatment with Various Stimuli 14 2.5 Western Immunoblotting 14 2.6 Detection of Cytokines and Chemokines 15 2.7 R N A Extraction 15 2.8 Quantitative Real-time P C R (qRT-PCR) 16 2.9 Semi-quantitative R T - P C R 16 2.10 Statistical Analysis 17 Chapter 3. HOST DEFENCE PEPTIDE LL-37 AUGMENTS IMMUNE RESPONSES AND MULTIPLE PATHWAYS IN THE PRESENCE OF INFLAMMATORY MEDIATOR IL-1(3 19 3.1 Introduction 19 3.2 Results 21 3.3 Discussion 27 Chapter 4. EFFECTS OF LL-37 ON LPS-TREATED HUMAN MONOCYTIC CELLS 40 i i i 4.1 Introduction 40 4.2 Results .' 42 4.3 Discussion 44 Chapter 5. GENERAL DISCUSSION AND CONCLUSIONS 52 5.1 Introduction 52 5.2 LL -37 Differentially Modulates Response of Innate Immune Effector Cells to Inflammatory Stimuli 53 5.3 LL-37 Enhances IL-1 B-induced Intracellular Signalling Events via Mult iple Molecules 54 5.4 Signalling Transduction Events Induced by LL -37 55 5.5 Receptors Involved in LL-37-induced Chemokine Production 57 5.6 Conclusions 58 REFERENCES 59 iv LIST OF TABLES Table 2.1: Sequence of primers (human) used for quantitative real-time P C R v LIST OF FIGURES Figure 3.1: LL -37 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 LL -37 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 LL-37 and I L - i p 34 Figure 3.4: Effects o f inhibition of G-protein coupled receptors (GPCRs) on the synergistic production of M C P - 3 induced by LL -37 and I L - i p 35 Figure 3.5: Influence of P I3K 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 LL -37 and I L - i p 37 Figure 3.7: 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 Figure 3.8: LL-37-induced phosphorylation of Akt and C R E B in the presence of I L - i p 39 Figure 4.1: T N F - a gene expression and protein production on co-stimulation with LL -37 and L P S induced in human P B M C s and THP-1 cells 47 Figure 4.2: LL -37 suppresses LPS-induced secretion of T N F - a as dose-dependent manner 48 Figure 4.3: IL-6 gene expression and protein production on co-stimulation with LL -37 and L P S induced in human P B M C s 49 Figure 4.4: 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 Figure 4.5: Protein expression of A20 in LPS-stimulated THP-1 cells with/without LL -37 51 vi LIST OF ABBREVIATIONS A T C C American Type Culture Collection D C dendritic cell E C L enhanced chemiluminescence E G F R epidermal growth factor receptor E L I S A enzyme-linked immunosorbent assay E R K 1 / 2 extracellular regulated protein kinase F C S foetal calf serum FPRL-1 formyl peptide like receptor-1 G - C S F granulocyte colony stimulating factor G M - C S F granulocyte macrophage- colony stimulating factor H B D human beta defensin H B E human bronchial epithelial cells H N P human neutrophil peptide IL- interleukin L B P lipopolysaccharide binding protein L P S lipopolysaccharide LTA lipoteichoic acid M A P K mitogen activated protein kinase M C P - 1 macrophage chemotattractant protein 1 M C P - 3 monocyte chemotactic protein 3 P B M C peripheral blood mononuclear cells P B S phosphate buffered saline P M S F 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 SDS polyacrylamide gel electrophoresis T B S Tris buffered saline T B S T Tris buffered saline + 0.1% Tween 20 T L R toll-like receptor TNF-oc tumor necrosis factor alpha vi i i 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 Gold 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, Yuexin L i , Jelena Pistolic and Linda Rehaume. I could not image what my life wi 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 viv id 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 Le 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 of microorganisms in the environment, yet healthy individuals rarely become infected due to the protection of the immune system. Pre-existing innate immunity serves as the first line of the host defence and acts within minutes of infection, leading to ultimate elimination of most invading microbes. The innate immune system recognizes microbial threats and initiates inflammatory responses by utilization of a variety of 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-l ike receptor (TLR) family is the first group of PRRs studied in detail and at least 11 members of 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 TIR domains because this domain is also found in the interleukin-1 receptors (IL-IRs). In contrast, the extracellular region of the T L R s and IL- IRs differs markedly: the extracellular region of T L R s contains leucine-rich repeat (LRR) 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; Ak i ra and Takeda, 2004). Although there is a high degree of 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 of the l ipid A moiety of bacterial lipopolysaccharide (LPS or endotoxin), T L R 2 recognizes outer components of Gram-positive bacteria L T A , and T L R 9 is responsible for recognition of bacterial D N A or its synthetic counterpart, C p G oligodeoxyribonucleotides (Han and Ulevitch, 2005). IL-1R is the principal signalling receptor for IL-1 which has two main forms: IL-lct and IL-1 P (L iu et al., 2003). Unl ike 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 (LBP) , 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 of their cognate ligands, T L R / I L - 1 R activates a set of proximal signalling pathways that induce the expression of a variety of "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 of T L R / I L - 1 R recruits the adaptor protein myeloid differentiation protein 88 (MyD88) via the cytoplasmic TIR domain, which then activates a cascade involving interleukin-1 receptor associated kinase ( IRAKs) , tumour-necrosis-factor-receptor-associated factor 6 (TRAF6) (Hul l et al., 2002; May and Ghosh, 1998) and a complex containing TAK1 (transforming-growth-factor-P-activated kinase), TAB1 (TAK1 -binding protein 1) and TAB2 (Takeda et al., 2003; Viatour et al., 2005). The activation o f T A K 1 in the cytoplasm leads to the phosphorylation of the I K K complex (inhibitor of nuclear factor-KB (IicB)-kinase complex), 2 which consists of I K K - a , IKK - B and IKK-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 (NF-KB) to translocate to the nucleus. Phosphorylation o f TAK-1 also activates M A P kinases, including extracellular signal-regulated kinase 1 (Erk l ) and Erk2, p38 and c-Jun N-terminal kinase ( INK) , leading to gene transcription and induction o f an inflammatory response (Frobose et al., 2006; L u et al., 2006; McDermott and O'Nei 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 of cell types and is evolutionarily conserved. In mammals, five family members of N F - K B have been identified: Re lB , c-Rel (Rel), p65 (RelA), P 100/p52 (NF-KB2), and P 105/p50 (NF-KB 1). A l l o f 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 ice 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 Mal ik , 2006; Moynagh, 2005). In parallel, activation of I N K triggers phosphorylation of transcription factor c-Jun and activation o f E rk 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 (AP) - 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 AP-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 of 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 (CRE) 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 of 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 of C R E B , such as increased intracellular Ca (Brindle and Montminy, 1992) and the activation of M A P K , p38 induced by LPS/ IL-1 (Xing et a l , 1996). The activation of transcription factor complexes, including N F - K B , C R E B , and A P - 1 , is required for LPS-induced transcriptional activation of 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 TGF-B (Gupta et al. , 1999; L i u and Mal ik , 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 of inflammatory mediators, including cytokines, chemokines, adhesion molecules, reactive oxygen species (ROS), and reactive nitrogen species (RNS) (Liu and Mal ik , 2006). Although these mediators are required for the initiation of 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 Mal ik , 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 ( IL-1RA) and T G F - B , which serves as counterregulatory mechanisms (Liu and Mal ik , 2006). Most importantly, negative regulation of signalling transduction along the TLR/ IL -1R to N F - K B 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 A20 is encoded by an immediate early response gene TNFAIP-3 . TNFAIP-3 is induced by different stimuli including T N F - a , IL-1B, and bacterial LPS in an NF-KB-dependentmanner (Hu et al., 1998; Sarma et al., 1995; Song et al., 1996). A20 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 of T R A F - 6 . By doing so, A20 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 of A20 inhibits T L R 2 - and TLR4-induced NF-KB-dependent production of IL-8 by interfering with a TLR-mediated signalling pathway (Gon et al. , 2004). M ice with TNFAIP3 (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'Reil ly and Moynagh, 2003). In addition, activation of T L R s also induces phosphatidylinositol 3-OH kinase (PI3K) activation at the early stage of the pathway although a mechanism needs to be further understood (Akira and Takeda, 2004). Typical ly, potent ligands (such as LPS) 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 Ak 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, Akt is phosphorylated at Ser-473 and Thr-308 by phosphoinositide-dependent protein kinase-1 (PDK-1) . The phosphorylated Ak 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 PI3K-Akt 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 PI3K/Akt 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, PI3K 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 MEKK3/6 -p38 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 PI3K/Akt signalling pathway may be an endogenous negative regulator that serves to balance 6 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 of Akt decreased sepsis-induced lymphocyte apoptosis, increased production of the T h l cytokine IFN-y, and improved survival in sepsis in vivo (Bommhardt et al., 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 (Wil l iams et al., 2006). 1.2. Cationic Host Defence Peptides 1.2.1. General features of hCAP-18/LL-37 Small cationic peptides are abundant in 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 of 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 of peptides are 12-50 amino acids in length with a net positive charge of +2 to +7 due to an excess of 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 of 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 (Zanetti, 2004b). The cathelicidins are characterized by highly conserved N-terminal cathelin domain and signal sequence and a structurally variable antimicrobial domain at the C-terminus (Hancock and Chappie, 1999a; Lehrer and Ganz, 2002b). By 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 hCAP18 is the only cathelicidin peptide identified in humans (Bals and Wilson, 2003b). L ike most peptides, hCAP18 is stored as an unprocessed inactive form in the granules of neutrophils. Upon stimulation (such as by invading microorganisms or inflammatory cytokines), the N-terminal 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, 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 (Gudmundsson et al., 1996; Larrick et al., 1995; Lehrer and Ganz, ; Lehrer and Ganz, 2002b; Sorensen et al., 1997). In addition to neutrophils, LL -37 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 mi lk (Durr et al., 2006; Tjabringa et al., 2003b). Upregulation of LL-37 production is found in bronchoalveolar lavage from infants with pulmonary infections, in individuals with Cystic Fibrosis lung disease and in psoriatic skin lesions. These indicate that 8 LL-37 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 LL-37 functions as a potent modulator of immune responses. First of al l , LL-37 was demonstrated to trigger cell accumulation at local inflammatory sites by direct chemoattraction of effector cells and induction of chemokines. LL-37 is able to chemoattract neutrophils, monocytes and T cells through binding to formyl peptide-like receptor 1 (FPRL-1) , a pertussis toxin-sensitive, G protein-coupled receptor (GPCR) (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 of 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 (EGFR) (Elssner et al., 2004; Nagaoka et al., 2006; Tjabringa et al., 2003a; Tokumaru et al., 2005). P2X7 mediates LL-37-induced maturation and release of IL-1B in LPS-primed monocytes (Elssner et al., 2004). A proposed mechanism for E G F R suggests that exposure of airway epithelial cells to LL-37 results in activation of a metalloproteinase (MP), cleavage of membrane-anchored EGFR-l igands and activation of the E G F R by these ligands, which leads to activation of M A P K cascades and transcription of target genes (Tjabringa et al., 2003a). Consistently, LL -37 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 of transcription factor Elk-1 and transcription of target genes that encode chemokines IL-8, M C P - 1 , and M C P - 3 (Bowdish et al., 2004b). LL-37-induced activation of M A P K p38 and E R K 1 / 2 also contributes to IL-18 secretion from human keratinocytes, suggesting the involvement of LL -37 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 LL -37 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 LL-37 by monocyte-derived dendritic cells (MDDCs) can affect cellular adaptive immunity also supports that LL -37 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 LL -37 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 ia FPRL1 (Koczulla et al., 2003b). There is much evidence that LL -37 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 TNF-a ) and prevent lethal endotoxemia in an animal models (Dankesreiter et al., 2000; Gough et al., 1996b). Likewise, over-expression of LL-37 in mouse airways can considerably prolong the survival of mice after challenge with a lethal dose of L P S (Bals et al., 1999). LL -37 itself and peptides derived therefrom, offers considerable promise as a treatment for sepsis. Although previous studies suggested neutralization of endotoxin by LL -37 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 LL -37 was also based on the ability of LL -37 to directly interact with host cells and alter the transcription of a variety of inflammatory genes, suggesting that LL-37 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 of the studies described in this thesis were to examine the potential roles of LL-37 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 in regulating innate immune responses, such as the N F - K B and PI3K/Akt pathways, also contributed to the immunomodulatory properties of LL -37 . Furthermore, the impacts of 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 (PBMCs) . Thus, my studies help to further understand the role of LL -37 in 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 ml of human venous blood was collected from healthy volunteers in sodium heparin Vacutainer collection tubes (BD Biosciences, Mississauga, Ontario, Canada) according to University of Brit ish 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 6 /ml) were seeded into tissue culture dishes (Falcon; Becton Dickinson) at 37°C in 5% CO2 and rested for 1-2 hr before addition of 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™ Li fe technologies, Burlington, ON) , 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, Oakvil le ON) 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 Brit ish Columbia, and was resuspended in endotoxin-free water. The concentration (w/v) of 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 of 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 of IKB-CC ( B A Y 11-7085) was purchased from Biomol . Inhibitors of PI3K (LY294002, Wortmannin), P K C (GF109203x) and P K A (6-22 Amide) were purchased from Cel l Signalling Technology, Inc., (Mississauga, O N , Canada). Ant i -A20 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 Cel l Signalling Technology Inc. HRP-conjugated goat anti-rabbit and anti-mouse IgG antibodies were purchased from Cel l Signalling Technologies and Amersham Biosciences (Piscataway, N J , U S A ) respectively. L P S from P. aeruginosa strain HI03 was purified free of 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 in endotoxin-free water. 13 2.4. Treatment with Various Stimuli Human P B M C or THP-1 cells were stimulated with LL -37 (20 ug/ml), LPS (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 LL -37 with inflammatory mediator IL-1B, both the stimulants were added simultaneously. 2.5. Western Immunoblotting THP-1 cells (3 x 106) and human P B M C (5 x 106) seeded into 60-mm petri dishes ( V W R International) were stimulated with I L - i p , LL -37 , L P S or endotoxin-free water as a vehicle control for 30 or 60 min 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, IL, U S A or Cramlington, U K ) according to the manufacturer's instructions. The extracts were stored at -80°C until further use. Protein concentrations of 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 in 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 (TBST/mi lk) or 5% B S A (TBST/BSA) , 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 an t i -GAPDH antibody to determine equal amount of protein were loaded. 2.6. Detection of Cytokines and Chemokines Human P B M C were seeded at 8 x 10 5 /ml in complete RPMI-1640 media. Cells were stimulated for 24 hrs with LL -37 , indicated cytokines and endotoxin-free water was used in parallel as vehicle controls. Fol lowing stimulation, the tissue culture supernatants were centrifuged at 2500 x g for 5 min to obtain cell-free samples. Supernatants were aliquoted and stored at -20°C prior to assay for various cytokines. The concentrations of 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 (MCP-3 E L I S A : R & D Minneapolis, M N ; IL-10 and IL-6 EL ISA : ebioscience San Diego, C A ; IL-8 E L I S A : BioSource International, Camaril lo, 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 6 cells/dish in complete R P M I 1640, and incubated for 1-2 hrs at 37°C in 5% C 0 2 . Cells then were incubated with treatments for indicated hours. After stimulation, suspended cells were collected into 15 ml flacon tubes and spin at 1000 rpm for 3 minutes. Ce l l pellets and attached cells were washed with ice-cold P B S . Total R N A was isolated using RNeasy M in 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 Li fe Technologies), as the manufacturer's instructions, in the A B I Prism 7000 sequence detection system (Applied Biosystems). Brief ly, 50 ng of 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 min 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 pi of 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 of gene transcription induced by L P S and/or LL -37 . Total R N A was isolated as described above and 1 pg of total R N A samples were incubated with 1 pi oligo(dT) (500 pg/ml) and 1 pi mixed dNTP stock at 1 m M in a 12-pl volume with diethyl pyrocarbonate-treated water at 65°C for 5 min in a fhermocycler. Brief ly, 4 p i 5x first-strand buffer, 2 pi 0.1 M D T T , and 1 pi RNaseOUT recombinant ribonuclease inhibitor (40 U/pl) were added and incubated at 42°C for 2 min, followed by the addition of 1 pi (200 U) of Superscript I I T m (Invitrogen, Burlington, Ontario, Canada). Each P C R was performed with a thermal cycler by using 25-35 cycles consisting of 30 s of denaturation at 94°C, 30 s of annealing at 55°C and 40 s of extension at 72°C. The number of cycles of 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 of 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') G A P D H G T C G C T G T T G A A G T C A G A G G G A A A C T G T G G C G T G A T G G IL-6 A A T T C G G T A C A T C C T C G A C G G G G T T G T T T T C T G C C A G T G C C M C P - 3 T G T C C T T T C T C A G A G T G G T T C T T G C T T C C A T A G G G A C A T C A T A 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 l ink innate and adaptive immunity (Bowdish et al., 2005; Yang et al., 1999). The two major families of 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 LL -37 (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, hCAP18, which upon stimulation and secretion is extracellularly cleaved from the N-terminal cathelin domain by 19 proteinase 3, leading to the generation of active LL-37 (Sorensen et al., 2001). Recent evidence indicates that LL -37 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, LL -37 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 LL -37 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 LL -37 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. LL -37 is known to interact with and/or transactivate a variety of receptors (De_Yang et al., 2000; Elssner et al. , 2004; Lau et al., 2005; Niyonsaba et al., 2002b; Tjabringa et al., 2003a; Tjabringa et al., 2006); for example the direct chemoattractant activity of LL -37 is due, at least in part, to activation of the G-protein coupled receptor FPRL-1 ( D e Y a n g et al., 2000), although this 20 receptor is not involved in mediating other LL -37 activities (Bowdish et al., 2004a). Cellular events important in the biological effects of LL -37 include the triggering of certain signaling pathways. LL -37 was shown to induce phosphorylation of the mitogen-activated protein (MAP) kinases, E R K 1 / 2 and p38 in 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 LL-37 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-l ike receptor (TLR)-4 to N F - K B in inhibiting endotoxin-stimulated pro-inflammatory cytokine expression (Mookherjee et al., 2006). However, the mechanism(s) by which LL-37 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 As it has been demonstrated that LL-37 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 LL-37 to modulate iKB-a/NF-KB-mediated signal transduction was firstly assessed in human monocytic THP-1 cells. Cells were stimulated with LL -37 (20 u.g/ml) at different time points. LL -37 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). As demonstrated with Western blots, total IKB-OC decreased transiently 21 within 30 min in human THP-1 cells, and was restored back after 60 min of stimulation with LL-37 . To further investigate the impact of the transient phosphorylation of IKB-O. induced by LL-37 , 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 LL -37 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 LL -37 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. LL-37 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 LL -37 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 IL - 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 LTA 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 LL-37 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 LL-37 (20 pg/ml) for 8 and 12 hr. Treatment with I L - i p or LL-37 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 LL-37 resulted in a substantially augmented M C P - 3 gene expression (to 12- to 27-fold) when compared with either IL-1 (3 or LL-37 alone (Fig.3.2A). Similarly, while IL-1B or LL -37 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 LL -37 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 LL -37 synergistically augmented the transcriptional responses induced by the inflammatory mediator IL -1B. 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 LL -37 (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 . LL-37 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 LL-37 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 LL -37 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 IX - i p alone. These results together confirm and extend the observation that LL -37 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 LL -37 have been proposed to be dependent upon signaling through a number of receptors potentially relevant in this system, including G-protein coupled receptors (GPCRs) 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 LL -37 was significantly suppressed (Fig. 3.3). In contrast, treatment of cells with the FPRL-1-speci f ic agonist W K Y M V m (Le et al. , 1999) in 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 LL-37. Phosphoinositide 3-kinases (PI3K) have been implicated in the effects of LL-37 on inhibiting neutrophil apoptosis. P I3K 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 LL-37 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 I3K in M C P - 3 induction by LL-37 in conjunction with I X - i p , was tested by pre-incubating P B M C with LY294002 or the alternative PI3K inhibitor wortmannin (Fig 3.4A) for 1 hr, prior to stimulation with LL -37 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 LL-37 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 LL-37 (20 pg/ml) in the presence or absence of IX - ip (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 LL-37 by greater than 95% (Fig. 3.5B). Protein kinase C (PKC) and protein kinase A (PKA) 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 of 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 LL -37 and IL - ip . 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 LL-37 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). As 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 . LL-37 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 LL -37 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 LL -37 led to enhanced nuclear translocation of p65 and p50 at the same time points (Fig. 3.7B). Since the serine/threonine protein kinase Akt is known to be activated by phosphorylation through a PI3K-dependent pathway, the influence of stimulation with LL -37 and/or IL-1 p on 26 cellular Akt phosphorylation was analyzed by immunoblotting using an antibody directed at phosphorylated Se r 4 7 3 of Akt . The results demonstrated that the phosphorylation of Akt was modestly increased after stimulation with LL-37 (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 LL -37 induces activation of the E R K - 1 M A P K pathway (Bowdish et al, 2004), and it is also known that ERK-1 phosphorylates and promotes nuclear translocation of the transcription factor C R E B (Xing 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 LL -37 and/or IL-1 P for 60 min. Both LL -37 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 LL -37 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 LL -37 has been increasingly described as a robust immunomodulatory molecule that enhances certain inflammatory 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 LL -37 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, LL -37 can also be locally 27 induced at sites of inflammation within epithelial cells (Bals, 2000). This led us to speculate that raised level of LL -37 could enhance, sustain or even amplify certain immune responses in presence of other endogenous signals. The most interesting of 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 LL -37 is known to promote IL-1 processing through transactivation of the receptor P2X7 (26). Previous data (17) indicted that LL-37 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 LL -37 interacts in a mechanistically complex fashion with the inflammatory mediator IL-1 B, to modulate the production of important effectors of inflammation. A variety of transcription factors have been separately implicated previously in the biological activities of LL -37 , e.g. LL -37 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); LL-37 indirectly stimulates of E G F R and downstream transcription factor STAT-3 in keratinocytes (Tokumaru et al., 2005); and LL -37 appears to act in part through the PI3K pathway in inhibiting neutrophil apoptosis (18). This is the first study that demonstrated that multiple transcription factors were involved in an immunomodulatory function of LL -37 , including the pivotal transcription factor N F - K B that is involved in the inflammatory and immune responses. LL -37 by itself was able to transiently activate I icB-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 of IL-6 (Yamamoto et al., 2004). In addition to N F - K B transcription 28 factor binding sites (TFBS) , 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 AP-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; Di l lon 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 Elk -1 , etc., could be essential for the delicate balancing of immunomodulatory activities by LL-37 . The outcome of action of this presumed complex would be the selective up-regulation of several key components involved in innate immunity. Recent reports indicate that LL-37 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). LL-37 also augments the ability ofthe TLR/ IL -1R 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 of 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 LL-37 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 LL-37 and IL-1 (3 is selective with respect to the inflammatory mediators induced. Therefore we speculate that .the ability of the host defence peptide LL-37 to boost certain immune responses during the initial course of infection can be reinforced and/or by inflammatory mediator IL-1 (3, and thus have a significant impact on the efficient balancing of inflammation by LL -37 , leading to an effective clearance of infection in the host. To date, a number of putative binding and transactivated receptors, including G P C R s , have been described as influencing the activity of LL -37 (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) of the synergistic effect between IL-1 p and LL -37 was further investigated in this study. PI3K inhibitors significantly attenuated M C P - 3 production induced by IL-1 P and LL-37 and the presence of IL-1 B reinforced LL-37-induced activation of multiple molecules, including IKB-OC, N F - K B subunit p50 and p65, Akt 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 of LL -37 , 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 LL -37 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 LL -37 is mediated by multiple signaling pathways in human 30 P B M C . The peptide by itself induced transient activation of IKB-CC /NF-KB , and modest phosphorylation of Ak 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 l ikely involved in the delicate balancing of inflammation for the effective functioning of the immune system. 31 Figure 3.1 A . 50. 37" 25 Total-lKBoc GAPDH 0 0.5 B. 50-37~ 25-6 h +LL-37 Total-lKBa 0 0.5 1 GAPDH 6 h +LPS go-er 50" p65 p50 Ctrl IL-ip LL-37 Ctrl IL-ip LL-37 30min 60min Fig. 3.1: LL-37-induced degradation of IKB-CC , and subsequent nuclear translocation of N F - K B 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 N F - K B 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 A . M C P - 3 D Untreated 8 hr 12 hr Fig. 3.2: Enhanced M C P - 3 and IL-6 gene expression in response to co-stimulation with LL-37 and IL-ip in human 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 ±0.17 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. 3 3 Figure 3.3 A . =5 600 E J 400 ^ 200 0 c t r l B. 1.5 SOD 1 4-s so Ctrl c. 150 1 120 5 90 o I 60 -J 30 n LL-37 LL-37 • 6 hr • 12 hr • 18 hr • 24 hr IL-ip ILip+LL-37 IL-ip ILlp+LL-37 Ctrl LL-37 IL-ip ILip+LL-37 Fig. 3.3: Enhanced MCP-3, IL-6 and IL-10 protein production in human PBMCs upon co-stimulation with L L - 3 7 and IL-ip. Human PBMC 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 90 60 30 0 • -PTx • +PTx 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 LL-37 (20 ug/ml) and I L - i p (10 ng/ml) for 24 hr. MCP-3 production in the culture supernatant was monitored by capture E L I S A . The agonist of G-protein coupled receptor FPRL-1 , peptide W K Y M V m (5 uM), was used as a positive control. Results are representative of three independent experiments. Values are the mean ± SE. 35 Figure 3.5 A. £ C m 1200 1000 800 600 400 200 0 1000 800 600 400 \ 200 0 ] Untreated cells I | + wortmannin • +LY294002 B . • -Bay11-7085 • + B a y l 1-7085 Ctrl LL-37 IL-lp IL-ip+LL-37 Fig. 3.5: Influence of PI3K and IKB-CC 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 OJD a . • CM U 800 600 400 200 0 1200 1000 800 600 400 200 0 • - P K A inhibitor M +PKA inhibitor • - P K C inhibitor 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 nM) or B, P K C inhibitor GF109203x (10 nM). Subsequently the cells were stimulated with LL-37 (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 of 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 D o n o r 2 6 0 - ' ' ' Ctrl IL-IP LL-37 IL-ip+LL-37 Ctrl IL-lp LL-37 IL-ip+LL-37 60min 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 LL-37 (20 ug/ml) and/or IL-1B (10 ng/ml). A, Cytoplasmic extracts were analyzed by immunoblotting after 30 min of stimulation for phosphorylation of IKB-CC (upper panel). Immunoblotting using an t i -GAPDH (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 of N F - K B subunits p50 and p65 by Western blots using anti-pl05/50 and anti-p65 antibodies. One gel, representative of three independent experiments, is shown for each panel. 38 A. 75kD-50kD—I 75kD 50kD-50kl> 37kD—\ 50kD—f 37kD Figure 3.8 p-Akt (60 kD) total-Akt (60kD) Ctrl IL-ip LL-37 IL-ip+LL-37 30min p-CREB (43kD) total-CREB (43 kD) Ctrl IL-IP LL-37 IL-ip+LL-37 60min Fig. 3.8: LL-37-induced phosphorylation of Akt and C R E B in the presence of IL - ip . Human P B M C were stimulated with LL-37 (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 of Akt (upper panel), or total Ak 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 of 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 of infection in the bloodstream, known as sepsis, causes nearly 200,000 deaths per year in North America (Sommers, 2003). Although sepsis can occur in response to a wide range of pathogens, it is most commonly caused by the release of a Gram-negative outer membrane component, endotoxin (lipopolysaccharide, LPS) (Alexander and Rietschel, 2001). To initiate the signalling cascades, L P S first interacts with LPS-binding protein (LBP) which facilitates its binding to the receptor, C D 14 (L iu and Mal ik , 2006; Wright, 1995). This complex is specifically recognized by T L R 4 and M D - 2 expressed on surface of macrophages and other cells involved in the innate immunity, leading to the activation of 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 of these signalling pathways eventually leads to the transcription of a vast number of genes encoding multiple inflammatory mediators such as TNF-ct, IL-1, IL-6, IL-10, IL-8, and TGF-B (Scherle et al., 1998; Ulevitch and Tobias, 1999; van der Po l l , 2001). These biological mediators have beneficial effects for combating infections and protective immune responses in infectious diseases. On the other hand, uncontrolled and excessive acute immune response can cause sepsis during early stage of bacteria infections. During sepsis, the generation of inflammatory mediators can trigger amplified cascade responses and overwhelming secretion of 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 LL-37 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 LL -37 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 LL -37 , little is known about the mechanism(s) by which LL -37 modulates inflammatory reaction in response to L P S . One acceptable explanation is that LL-37 could competitively block the formation of L P S • L B P complex owing to high 41 affinity of LL -37 binding to L P S (Scott et al, 2000). Recent microarray studies performed in RAW264.7 cells and human THP-1 cells demonstrated that anti-endotoxin effect of LL-37 is alternatively due to the capability of LL -37 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 LL -37 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 LL -37 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 THP-1 cell line were treated with endotoxin-free water as vehicle control, or L P S (100 ng/ml) with or without LL-37 (20 pg/ml) for the indicated times and total R N A was isolated to perform quantitative RT-PCR (qRT-PCR) to generate gene expression patterns. We showed that exposure to LL-37 dramatically inhibited LPS-induced production of m R N A for the pro-inflammatory cytokine T N F - a by 95% in both P B M C s and THP-1 cells (Fig. 4.1 A ) , which is consistent with the involvement of LL -37 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 LL -37 was able to inhibit 50% of T N F - a released into the tissue culture supernatant, suggesting that a physiological concentration of LL -37 exhibited anti-endotoxin activity (Fig 4.2). 42 In contrast to previous observations that LL-37 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 LL-37 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 LL -37 (20 ug/ml) and L P S were simultaneously added to cells (Fig. 4.3B). Moreover, as a representative of chemokines, M C P - 3 gene expression and production were also examined in P B M C s treated with LL-37 and/or LPS. Interestingly, stimulation by LL -37 and L P S synergistically enhanced M C P - 3 secretion in a time-dependent manner when compared with either L P S or LL -37 alone, while LPS alone induced detectable but small amounts of M C P - 3 production (Fig 4.4B). Similar synergy between L P S and LL -37 was also detected in gene transcription levels after 8-12 hours (Fig 4.4A). These results confirmed that LL -37 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 LL -37 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 TLR4- to -NF - K B pathway were only slightly reduced in the presence of LL -37 . These genes included TNFAIP3 (TNF-a-inducible protein 3) encoding A 2 0 and the NF-KB- inhibitor, NF-KBIA encoding (IKB-OC) (Mookherjee et al., 2006). Therefore, the protein expression of A20 was examined in THP-1 cells stimulated with L P S and/or LL -37 . 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 of protein A20 remained stable for up to 18 hr after L P S stimulation (Fig 4.5A). Interestingly, when cells were co-stimulated with LL -37 and L P S for either 2 or 18 hr, LL-37 only reduced 30-50% expression of A20 induced by L P S (Fig. 4.5B and C) compared with unstimulated control cells, indicating that LL -37 did not completely inhibit LPS-stimulated A20 synthesis, despite the fact that A20 protein expression is known to be controlled by N F - K B . This observation is not consistent with expression of NF-KB-control led pro-inflammatory cytokines. As A20 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 of LL-37 on maintaining intracellular expression of A20 would provide a possible mechanism, by which LL-37 selectively modulates LPS-induced inflammatory responses. 4.3. Discussion LL-37 , 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 of LPS-induced pro- and anti-inflammatory genes downstream of T L R - 4 (Mookherjee et al., 2006; Scott et al., 2002a). Here, my results provide further evidence indicating the involvement of LL-37 in modulating LPS-initiated inflammatory responses in human monocytic cells. Moreover, LL -37 exhibits the ability to retain a N F - K B negative regulation pathway in presence of L P S . In mouse model experiment, it was demonstrated that administration of LL -37 can protect against sepsis induced by Escherichia coli L P S (Scott et al. 2002). Similarly, the effects of LL-37 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 LL -37 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 LL-37 , a physiological concentration found in healthy individuals (Murakami et al., 2002). Moreover, the concentration of LL -37 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 LL -37 is attributed in part to blockage o f L P S - L B P binding, there is emerging evidence suggesting that LL-37 directly interacts with effector cells and balances LPS-induced pro- and anti-inflammatory responses (Braff et al, 2005). Active internalization of LL -37 into cells was detected in A549 epithelial cells, THP-1 cells and D C s (Bandholtz et al, 2006; Lau et al, 2005; Rosenfeld et al, 2006). Our lab observed that LL -37 can still reduce T N F - a production even when added to the murine cell line and human THP-1 cells an hour after L P S addition (Gough et al, 1996a; Mookherjee et al, 2006; Scott et al, 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 LL -37 increases maturation and release of IL-1 B in LPS-pr imed monocytes via the P2X7 receptor (Elssner et al, 2004). The mechanisms by which LL -37 synergistically enhance LPS-induced chemokine production remain to be further characterized. 45 To further understand the role of LL -37 in modulating LPS-induced inflammatory responses, the expression of the N F - K B pathway negative regulator A20 was examined in the presence of L P S and LL -37 . Recent reports have shown that A20 is inducible by T N F - a and L P S in macrophages (O'Rei l ly 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, A20 can terminate the TLR-induced IFN regulatory factor 3 (IRF-3) signalling pathway (Saitoh et al, 2005). Thus, A20 is considered to be an important regulator of innate immune responses (Heyninck and Beyaert, 2005) and can be a l ikely target for modulating inflammatory responses and thus sepsis (Boone et al, 2004). Here, Western blotting results demonstrated that the presence of LL -37 did not significantly inhibit LPS-induced A20 expression in THP-1 cells at either early 2 hr or later 18 hr time points. Therefore, it is reasonable to propose that the involvement of LL -37 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 LL-37 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, LL -37 has no effect on A20 expression level. These findings suggest that LL-37 regulate LPS-induced inflammatory responses by selectively modulating production of inflammatory mediators (e.g. cytokines and chemokines). 46 Figure 4.1 T N F - a 100 Ctrl LPS LL-37 LPS+LL-37 B ED a. Ctrl LPS LL-37 LPS+LL-37 800 600 400 200 rfr-i 2500 2000 1500 1000 500 0 m Ctrl LPS LL-37 LPS+LL-37 Ctrl LPS LL-37 LPS+LL-37 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 LL-37 (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 THP-1 cells (lower panel) was analyzed by qRT-PCR 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 in un-stimulated cells (normalized to 1) using the comparative Ct method. Result of experiment from one donor representative of 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 I ta. z H 0 LPS -LL-37 -lOOng/ml lOOng/ml lOOng/ml lOOng/ml -lug/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 of increasing concentrations of LL-37 (x-axis) for 4 h. The concentration of 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) of three independent experiments. 48 Figure 4.3 IL-6 C L SO B. o S a JS 12000 10000 8000 6000 4000 2000 0 1000 100 10 Ctrl Ctrl LL-37 LPS LL-37 LPS LPS+LL-37 LPS+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 LL -37 (20 pg/ml) and/or LPS (100 ng/ml) for 8 hr and total R N A was isolated (lower panel). Transcriptional induction was analyzed by qRT-PCR. Fold 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 of experiment from one donor representative of four are shown. 49 Figure 4.4 8P 2 s o M C P - 3 1000 100 10 B. 8h 12h 20h ox; &. fill • s 1000 800 600 400 200 0 n 6hr a 12hrs • 18hrs • 24hrs 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 LL -37 (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 . Fold changes (log) (Y-axis) of 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. Result of experiment from one donor representative of four is shown. B. MCP-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 of three independent experiments (± standard error) from three different donors. 50 Figure 4.5 Fig 4.5: Protein expression of A20 in LPS-st imulated THP -1 cells wi th 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. Cel l lysate collected from stimulated cells were used to determine A20 protein expression using monoclonal anti-A20 antibody. B. Protein lysates collected from THP-1 cells incubated with L P S (lOOng/ml) in presence or absence of LL-37 (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 A20 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 LL-37 is a potent modulator of the immune system, which contributes to its ability to augment innate host defence against acute infection. By participating in the recruitment of leukocytes, such as neutrophils, monocytes, mast cells, and T cells (De et al., 2000), and inducing the degranulation of mast cells (Niyonsaba et al., 2002a), LL-37 acts as a mediator of positive amplification loops of innate immune responses (Zanetti, 2004a). Furthermore, LL -37 has the ability to alter transcriptional responses of 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 of M A P K s , E R K 1 / 2 and p38 mediated by LL-37 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, LL -37 has been shown to suppress neutrophil apoptosis, whereas it induces apoptosis in primary airway epithelial cells (Barlow et al., 2006; Lau et al., 2006; Nagaoka et al., 2006). In this thesis, the role of LL -37 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 LL-37 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; Cir ioni 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 LL-37 synergistically enhanced LPS-stimulated M C P - 3 production in P B M C s in contrast to the inhibition of pro-inflammatory cytokine secretion. This is inconsistent with the hypothesis that LL-37 simply specifically binds to and neutralizes the extracellular existing L P S , and instead supports the proposed mechanism that LL -37 may selectively modulate and balance pro- and anti-inflammatory responses during endotoxin challenge. During the course of infection or inflammation, effector cells w i l l be exposed to a broad range of 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 of receptor engagement, leading to the induction of various inflammatory responses. Therefore, co-incubation with IL-1 P and LL-37 should help to analyze the impacts of LL -37 on IL- ip- induced signalling pathways and consequent outcomes. Interestingly, LL -37 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 LL -37 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 LL-37 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 of an inflammatory milieu provides a beneficial environment for LL -37 to initiate immune responses at early stages of infection, which might affect the later phases of inflammation significantly. 5.3 LL-37 Enhances IL-ip-induced Intracellular Signalling Events via Multiple Molecules LL-37 has been reported to directly activate transcription factor Elk-1 that controls transcription and secretion of IL-8 in primary human monocytes (Bowdish et al., 2004a). Keratinocyte migration induced by LL -37 is mediated by indirect stimulation of E G F R and downstream transcription factor STAT-3 (Tokumaru et al., 2005). M y findings further suggest that LL-37 regulates activation of multiple transcription factors, which in turn control expression of downstream genes. The impact of LL -37 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 LL -37 activated p50 and p65 nuclear translocation in THP-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 of the IL-6 promoter, leading to transcription of IL-6 (Yamamoto et al., 2004). In p50'A cells, production of IL-6 in response to stimulation by L P S and IL-1 p was severely impaired, indicating the significance of p50 in IL-6 expression (Yamamoto et al., 2004). In addition to N F - K B binding site, the 5'-flanking sequence upstream of 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 of a distinct set of transcription factors including N F - K B , C R E B and AP-1 is required by transcriptional activation 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) and the anti-inflammatory cytokine IL-10 54 (Agrawal et al., 2003; Di l lon et al., 2004; Martin et al., 2005). We have demonstrated here that LL-37 was able to modestly stimulate C R E B phosphorylation to a similar extent to that induced by IL-1 p. LL -37 augmented p50/p65 and C R E B activation in P B M C s stimulated with IL - 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 LL -37 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 LL -37 might be directed in part by its ability to modulate activation of these inflammatory molecules. Future studies should focus on understanding i f LL -37 can affect the interactions of different transcription factors, such as N F - K B and C R E B . 5.4 Signall ing 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 LL -37 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, LL -37 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 PLC-y /PKC / N F - K B and PI3K/Akt cascades (Koczul la et al., 2003a). Signalling analysis showed that pretreatment with Bay 11-7085 (IKB-CC inhibitor), PD98058 (ERK1/2 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 LL-37 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 PI3K also contribute to the release of IL-8. Consistently, Western blotting results demonstrated that LL-37 stimulated transient activation of IKB-CC and slight phosphorylation of Akt 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 LL-37 is clearly distinct from the one described for PR-39, 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 LL -37 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 Ak t is enhanced in the presence of IL-1 p. Studies using PI3K inhibitors further confirmed the role of P I3K/Akt pathway in the synergistic production of M C P - 3 induced by combination of LL -37 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 LL -37 in regulation of LPS-mediated inflammatory signals. 5.5 Receptors Involved in LL-37- induced 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; Lau 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 LL-37 and effecter cells is the formyl-peptide receptor-like 1 (FPRL1) which belongs to the seven transmembrane domain G P C R families. Through F P R L - 1 , LL -37 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 FPRL-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 FPRL-1 (Davidson et al., 2004a), while G P C R s other than FPRL-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 A549 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 LL -37 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 FPRL-1 ) . 57 5.6 Conclusions In summary, this thesis demonstrated that the host defence peptide LL -37 was able to augment immune responses in effector cells during the inflammation and infection. In addition, the mechanisms underlying LL-37-induced activities have been studied, indicating the involvement of LL -37 in modulating a variety of intracellular signalling events in immune cells. 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