Transcript Case_4x
CASE 4 THE IMMUNE RESPONSE Ashley Wang Path417 March 2016 The Case 10-year-old Ronnie McDonald has developed abdominal cramps, bloody diarrhea and a low grade fever. His parents take him to see the family doctor. The doctor asks about what Ronnie may have eaten in the past week and his parents recall that last weekend at a neighbor’s barbecue they were concerned that the hamburgers may not have been cooked thoroughly and Ronnie had eaten two burgers. The doctor performs a physical examination noting no rebound tenderness just some mild periumbilical tenderness. He asks the parents to collect a stool sample for the Microbiology Laboratory and to take Ronnie to the local lab for some routine bloodwork. The Cause Foodborne illness-causing organisms, include but are not limited to Organism Onset Time Signs & Symptoms Duration Food Source Salmonella 6 – 48 hrs Diarrhea, fever, abdominal cramps, vomiting 4 – 7 days Eggs, poultry, meat… Shigella 4 – 7 days Abdominal cramps, fever, and diarrhea. Stools may contain blood and mucous 24 – 48 hrs Raw produce, uncooked foods, contaminated water… Although the onset time is not provided, Ronnie is more likely to be infected with Salmonella based on the epidemiology. Q1: Host Response What elements of the innate and adaptive (humoural and cellular) immune response are involved in this infection. Basic Anatomy of The GI Tract Gastrointestinal Barrier A single layer of intestinal epithelial cells (IEC) that provides a physical and chemical barrier to the gut IEC express TLR5 on basolateral side, low levels of TLR2 and TLR4 on apical side, and NOD1/NOD2 inside the cells Tight junctions between these cells to block the entry of pathogen and commensals Lamina propria Houses a variety of lymphocytes, including T cells, B cells, dendritic cells, macrophages, and neutrophils Contains Peyer’s patches (PP), which are aggregated lymphoid follicles M cells are specialized cells that transport bacteria via transcytosis Broz, P., et. al, 2012 Fig. 1 The Gastrointestinal Immune System Schematic representation of the structure of immune system in the gastrointestinal tract and the different ways Salmonella can take to invade the intestinal mucosa. Initial Barrier Goblet cells Produce mucins that are essential for the formation of a thick mucous layer, which covers the surface of the gut epithelium and provides protection Paneth cells Secret antimicrobial peptides that break down bacterial cell membrane Some antimicrobial peptides are expressed constitutively (i.e. α- and βdefensins, CRS peptides, and lysozyme) Many antimicrobial peptides are induced in response to invading pathogens (i.e. cathelicidins, and angiogenin 4) Other epithelial cells can produce antimicrobial peptides as well Initial Encounter Pattern Recognition Receptors (PRRs) detects pathogen-associated molecular patterns (PAMPs). The first PRRs to detect the presence of Salmonella via PAMPs are the following Toll-like receptors (TLRs)… TLRs present on the outer membrane of the cell TLR1, 2, 4, 5, 6, 10 TLRs present in intracellular vesicles TLR3, 7, 8, 9, 11, 13 What PAMPs are been detected? LPS (TLR4) Bacterial lipoproteins (TLR1/2/6) Flagellin FliC (TLR5) (NLRC4) Bacterial DNA (TLR9), RNA, and more TLR Signaling Process Ligand binding between TLRs and PAMPs TLRs interact with adaptors MyD88 and TRIF Induction of signaling cascades Activation of transcriptional factors NFkB and IRF3 Production of inflammatory cytokines (next slide) followed by a type I IFN response Detection by TLRs TLRs detect extracellular Salmonella TLR signaling Induces increased secretion of antimicrobial peptides RegIIIβand RegIIIγ, which aids in the clearance of intestinal pathogens Mice lacking MyD88 fails to produce RegIIIγ Up-regulates expression of proinflammatory cytokines (i.e. IL-23) Promotes the production of IL-8, IL10, pro-IL1β, pro-IL18, and more Broz, P., et. al, 2012 NLR Signaling Process Ligand binding between NLRs and PAMPs Ligand binding between NLRs and PAMPs Induction of various signaling cascades Induction of the assembly of inflammasome, a signaling complex NOD1 and NOD2 interact with RIP2 kinase Adaptor protein ASC brings proCaspase-1 to inflammasome RIP2 kinase activates transcriptional factors NFkB Activation of pro-Caspase-1 via dimerization and autoproteolytic cleavage Production of inflammatory cytokines Secretion of mature cytokines via activated protease-mediated cleaving of IL-1β and IL-18 Detection by NLRs NLRs detect intracellular Salmonella NOD-like Receptors Mice lacking NOD1/2 or RIP2 showed decreased levels in inflammatory cytokines and increased colonization in mucosa NLRC4/NLPR3 inflammasomes detect T3SS (virulence factor) and activate Caspase-1, inducing pyroptosis, a proinflammatory cell-death program Unlike apoptosis, pyroptosis intensifies the inflammatory response via subsequent release of pro-inflammatory cytokines and cellular contents, once again making pathogens vulnerable towards extracellular immune defenses. Broz, P., et. al, 2012 Inflammation of Gut Mucosa Secretion of cytokines induced by PRRs IL-18 and IL-23 Induce the inflammatory response via paracrine signaling mechanisms T cells release increased amount of IFNγ under the influence of IL-18 IL-23 induces the release of IL-22 and IL-17, leading to increased production of mucins and antimicrobial peptides Innate Lymphoid Cells (i.e. NK cells) may also produce IL-22 IL-17 promotes release of CXC chemokines, resulting in an influx of neutrophils into the mucosa Broz, P., et. al, Recruitment of Neutrophils As an intracellular pathogen, Salmonellae can be found extracellularly after transcytosis or host cell lysis (pyroptosis). Infiltrating neutrophils IL-17-/- mice demonstrate reduced amount of neutrophils recruited to the intestinal mucosa Caspase-1 deficient mice, lacking secretion of IL-1β, produce less CXC chemokines than wild-type mice Play an important role in the elimination of extracellular Salmonellae in the gut Promote diarrhea and inflammation due to intestinal tissue damage Broz, P., et. al, Macrophages & Dendritic Cells Once crossed the epithelial cell barrier, Salmonella face other immune defences in Gut-Associated Lymphoid Tissue GALT… Macrophages and Dendritic Cells (DCs) Eliminate pathogens via phagocytosis Activate other immune cells, mainly the adaptive immune system, directly or via producing pro-inflammatory cytokines Effector SipB in macrophages can activate Caspase 1, producing IL-1β and IL18 Modified from LaRock, D. L., et. al, 2015 Antigen-Presenting Cells (APCs) During Salmonella infection, professional APCs – Macrophages and DCs… Crucial to the activation of T cells Process and present antigen (Ag) to T cells Upregulate the expression of the following molecules Costimulatory surface molecules: CD40, CD80, and CD86 Cytokines: IFNγ, TNF-α, IL-12, and IL-18 Dendritic Cells Essential activators of naïve T cells with diverse subpopulations Present in the subepithelial area of PP Where initial encounter with pathogen takes place The primary APCs during early stages of infection Express low levels of lysosomal proteases Take longer than microphages to degrade bacterial proteins Environment surrounding mature DC is more supportive of Ag processing and presentation than elimination of bacteria Stronger induction of IFN-γ production by CD4+ T cells Contribute more to Th1-type responses than macrophages Immature Ag-capturing cells (Phagocytic) Undergo morphological and functional changes after bacterial uptake Mature APCs Macrophages Research suggests macrophages contribute more to the production of proinflammatory chemokines and cytokines than DCs Chemokines: CCl5, CCL20, CXCL10 Cytokines: IL-18, TNF-α Macrophage-depleted mice can generate CD8+ T cells against Salmonella-specific antigens More essential in inducing proinflammatory responses rather than carrying out APC function May be more efficient in the stimulation of effector T cells rather than naïve T cells Natural Killer (NK) Cells NK cells are activated by macrophages, leading to Secretion of IFN-γ Degranulation Decreased number of live bacteria inside macrophages Interaction between NK cells and infected macrophages Contact-dependent IL-2 and/or IL-15 required to prime NK cells to produce IFN-γ Infected macrophages secrete IL-12 and IL-18, which activate NK cells Lapaque, N., et. al, 2009 Types of T Lymphocytes Express heterodimeric receptors (TCRs) on surface TCRs interact with Ag Most of TCRs consist of one α- and one β- chain One group expresses CD4 coreceptors One group expresses CD8 coreceptors A minority of TCRs consist of one γ- and δ- chain Abundant in epithelial tissues, especially in the gut mucosa Less diverse than αβT cells MHC presentation of Ags is not required for activation May have a role in both innate and adaptive immune responses Both αβ T cells and γδ T cells are detected after Salmonella infections; however the role of γδ T cells is not well understood. Wolfert, M. A. and Boons, G., 2013 Modified from Wolfert, M. A. and Boons, G., 2013 Activation of CD4+ T Cell TCRs interact with bacterial peptides presented on MHC-II molecules Peptides obtained from endocytic or phagocytic vacuoles CD4+ T cells recognize FliC, an abundant subunit protein found in the flagella Salmonella invasion protein SipC is also an immune target CD4+ T Cells Express CD4 co-receptors on surface Responsible for production of cytokines Induce B-cell class switching and affinity maturation Stimulate activation and growth of functional cytotoxic T cells Mediates macrophage activation IFN-γ Production Essential for clearance of infection CD4+ T-cell-deficient mice produce 100-fold less IFN-γ than wild-type mice, failing to clear an infection with an attenuated strain Reduced IFN-γ or CD4+ T cells lead to reactivation of latent Salmonella infection Wolfert, M. A. and Boons, G., 2013 Activation of CD8+ T Cell TCRs interact with endogenous antigens presented on MHC-I molecules CD8+ cells are activated, becoming cytotoxic T cells Recognize peptides derived from GroEL More research is required to identify additional Ags targeted by T cells CD8+ T Cells Express CD8 co-receptors on surface Involved in secretion of cytokines Involved in bacterial clearance Mice lacking MHC-I have higher bacterial loads during primary infection than wild-type mice TCR-deficient mice develop more severe infection than MHC-II knockouts Re-expose intracellular pathogen to phagocytes Granulysin A protein found in cytotoxic T cell (CTL) granules CD4+ T-cell-deficient mice produce 100-fold less IFN-y than wild-type mice, failing to clear an Modified from Wolfert, M. A. and Boons, G., 2013 Activation of B Cells B cells that present the same antigenic peptides on MHC-II molecules as the ones on activated TH cells can become activated via cytokines. Antibody-secreting plasma cells Memory B Cells B Cells Antibody production Rely on help from T-cell for antibody isotype switching O-antigen polysaccharide chains on LPS are the main targets Also generated against unknown antigenic proteins Mediate the priming of T cells and the generation of memory T-cells Influence immune response based on the cytokines produced (IFN-y: Th1-type response; IL-4: Th2- type response) Activated B cells function as APCs to prime naïve T cells (important in the spleen) Secretory IgA (sIgA) Present in mucosal tissues Reduces adherence of pathogen to cells, but not required for protective immunity Mice that cannot generate Salmonella-specific sIgA are able to mount a same protective immune response as wild-type mice in Typhimurium infection General Overview of Intestinal Immunity General Overview of Intestinal Immunity Salmonella in the gut lumen encounter initial barriers (i.e. antimicrobial peptides and sIgA) and cross the epithelium via M cells Bacteria express FliC protein, which is recognized by TLR5, initiating an inflammatory response along with production of cytokines Cytokines lead to an influx of macrophages, neutrophils, and DCs DC or Macrophage-mediated phagocytosis of bacteria Pyroptosis Bacteria persist within phagocyte after its uptake Inhibited T-cell activation and increased expression of MHC and co-stimulatory molecules on DCs Degradation of bacteria within neutrophils or macrophages DCs in the PP present processed Ags to naïve T cells and enhance activation of T cells via cytokine production (TNF-α and IL-12) Activated TH cells produce cytokines that activate B cells, which proliferate into antibody-secreting plasma cells and memory B cells Q2: Host Damage What damage ensues to the host from the immune response? The Microbiota Gut Microbiota Functions Required for the proper development of immune system Has important homeostatic immune & metabolic functions Affects the proliferation and survival of epithelial cells Provides protection against pathogens Dysbiosis Inflammation from host immune response may result in disequilibria between the host and microbiota, aka dysbiosis Increased risk for neoplatic transformation in host Zitvogel et al., 2015 Inflammation and Colorectal Cancer Inflammation contributes to tumour development Premalignant cells may activate proliferation and anti-apoptotic mechanisms Activated inflammatory cells produce ROS, which promotes DNA damage and mutation Teric et al., 2010 Tumour-Promoting Cytokines Teric et al., 2010 Intestinal inflammation caused by microbial pathogens can lead to tissue injury, which may promote tumour if inflammation becomes chronic Inflammation may also lead to septic shock, organ failure, and/or chronic inflammatory diseases Q3: Bacterial Evasion How do the bacteria attempt to evade these host response elements. Overview of Immune Evasion Pyroptosis of APC Cummings, L. A. et. al, 2009 Inhibition of Ag Processing and Presentation Inhibition of T Cell Activation Intracellular Replication Caspase-1-Induced Pyroptosis From death comes life… Evasion of NLRC4 (Reminder: NLRC4 recognizes FliC and T3SS) SPI2 T3SS rod protein, SsaI, changes amino acid to avoid being detected by NLRC4 Presence of SPI2 T3SS results in suppressed expression of flagellin NLRC4 can still detect pathogen, however at a delayed time post-infection With expression of FliC, NLRC4 is triggered within 6h; 17h without FliC Leads to intracellular replication of bacteria in macrophages via suppression of pyroptosis Miao, E.A. and Rajan, J.V., 2011 Antigen Processing & Presentation Salmonella alter DC functions, compromising activation of T cells SPI-2 genes encode virulence proteins (i.e. SpiC) that are secreted into the cytoplasm to prevent Ag processing by avoiding phagosome-lysosome fusion These virulence proteins target P13K activity, preventing bacterial degradation Salmonella can reduce expression of bacterial antigens such as flagellin and LPS in MLNs and spleen, preventing their presentation to T cells Bueno, S.M. et. al, 2007 Intracellular Survival Avoid neutrophil-mediated killing by invading, surviving, and replicating inside epithelial cells, macrophages, and DCs Salmonellae rely on bacterial membrane remodeling via regulatory proteins to enhance intracellular survival Spi-2 type III secretion system secrete effectors such as SifA to change the composition of the Salmonella-containing vacuole, preventing phagosome acidification (avoid NO- and NADPH oxidase-mediated killing) Modify lipid A and other components of LPS via mechanisms such as deacylation and palmitylation to reduce recognition by TLR2 and 4 PgtE, an outer membrane expressed on Salmonella, cleaves αantimicrobial peptides, promoting its resistance to innate immune response While inside phagocytes, Salmonella can spread to other parts of the body (i.e. liver and spleen) for colonization Q4: Outcome Is the bacteria completely removed, does the patient recover fully and is there immunity to future infections with these candidate infectious agents? Salmonella persistence Most people are able to have the bacteria completely cleared, but some become carriers of Salmonella Salmonella infections can survive activation of the innate and adaptive immune responses, and remain in the host “immune status-quo” is reached, meaning an equilibrium is established between the immune system and bacteria Mostly found in the mesenteric lymph nodes, sometimes in the spleen and liver During persistent infections, pathogens ensure survival by Undergoing antigenic variation and antigenic imitation Inhibition synthesis of host proteins Inactivation of humoural immune components Avoiding immune recognition by hiding Immune System in Chronic Infections Macrophages Research suggests that Salmonella becomes dormant after entering macrophages ~80% of the bacteria are found within MOMA-2 expressing macrophages in mesenteric lymph nodes (MLN) Dendritic Cells Can transport Salmonella from the GI tract to MLN; transportation is mainly accomplished by CD103+ CD11B+ DCs Decreased intracellular proliferation of Salmonella within DC affects antigen presentation, reducing T-cell responses, allowing for persistent infection A subset of DCs carrying pathogen can migrate from the lamina propria into the intestinal lumen, leading to shedding of live bacteria in stools (transmission) Adaptive Immunity in Chronic Infections Early resistance phase (acute infection) High Th1 (pro-inflammatory) and low Th2 (anti-inflammatory) responses Maintenance of “Immune Status-quo” A lower Th1 response; however production IFN-y is still required as reactivation of acute infection is observed in mice injected with anti-IFN-y antibodies Regulatory T cells suppress Th1 response Microbiota Needed to reduce bacterial load in the gut and stools during chronic infections Infected individuals may shed bacteria from a year up to lifetime Protection Against Future Infections Strong, persistent B- and T-cell memory is established IFN-y-secreting memory T-Cell populations are detected in vaccinated subjects Memory B cells are crucial in fighting future infections as well because humoural protection is positively correlated with CD4+ T cell IFN-y production Circulating antibodies also help protect against future infections B cells are crucial in the development of Th1 memory cells References – Q1 & Q4 Broz, P., Ohlson, M. B., & Monack, D. M. (2012). Innate immune response to salmonella typhimurium, a model enteric pathogen. Gut Microbes, 3(2), 62-70. Bueno, S. M., Riquelme, S., Riedel, C. A., & Kalergis, A. M. (2012). Mechanisms used by virulent salmonella to impair dendritic cell function and evade adaptive immunity. Immunology, 137(1), 28-36. Cummings, L. A., Deatherage, B. L., & Cookson, B. T. (2009). Adaptive immune responses during salmonella infection. EcoSal Plus, 3(2), 10.1128/ecosalplus.8.8.11. Griffin, A. J., & McSorley, S. J. (2011). Development of protective immunity to salmonella, a mucosal pathogen with a systemic agenda. Mucosal Immunology, 4(4), 371-382. Kupz, A., Scott, T. A., Belz, G. T., Andrews, D. M., Greyer, M., Lew, A. M., et al. (2013). Contribution of Thy1+ NK cells to protective IFN-gamma production during salmonella typhimurium infections. Proceedings of the National Academy of Sciences of the United States of America, 110(6), 2252-2257. 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J., & Podolsky, D. K. (2007). Unravelling the pathogenesis of inflammatory bowel disease. Nature, 448(7152), 427-434. Xavier, R. J., & Podolsky, D. K. (2007). Unravelling the pathogenesis of inflammatory bowel disease. Nature, 448(7152), 427-434. References – Q2 Rakoff-Nahoum, S., and R. Medzhitov. "Role of toll-like receptors in tissue repair and tumorigenesis." Biochemistry (Moscow) 73.5 (2008): 555-561. Uronis, Joshua M., et al. "Modulation of the intestinal microbiota alters colitisassociated colorectal cancer susceptibility." PloS one 4.6 (2009): e6026. Terzić, Janoš, et al. "Inflammation and colon cancer." Gastroenterology 138.6 (2010): 2101-2114. Plottel, Claudia S., and Martin J. Blaser. "Microbiome and malignancy." Cell host & microbe 10.4 (2011): 324-335. Huber, Samuel, et al. "IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine." Nature 491.7423 (2012): 259-263. Zitvogel, Laurence, et al. 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