lect 39 - immune-mediated GI diseases_SM1.pdf

Recall: Physical Barriers of G.I. Tract • Epithelial structures • For example, villi (gut) and cilia (lung) • Function specialized to that surface (e.g., adsorption, sweeping removal) • Lymphoid structures • • • • Specialized for each mucosal surface In submucosal layer under epithelium (e.g., lam...

Recall: Physical Barriers of G.I. Tract • Epithelial structures • For example, villi (gut) and cilia (lung) • Function specialized to that surface (e.g., adsorption, sweeping removal) • Lymphoid structures • • • • Specialized for each mucosal surface In submucosal layer under epithelium (e.g., lamina propria of gut) Organized, but not encapsulated like LN Contains T and B lymphocytes, innate lymphoid cells, DCs, and macs • Function of lymphoid structures • Generate specialized adaptive immune response • Regulate local immune responses as needed in that tissue • Adaptive lymphocytes and DCs generated in these structures and in regional draining LNs will traffic back to the particular mucosal surface (i.e., homing) after entering the circulation Figure 14-1 – Cellular and Molecular Immunology, 10th ed., 2022 Recall: Cellular and Chemical Barriers of G.I. Mucosa • Specialized epithelial cells – Arise from stem cells at base of epithelial layer – Absorptive epithelial cells – absorb nutrients and secrete cytokines – Goblet cells – secrete mucus – Microfold or M cells – antigen transport – Paneth cells – secrete antimicrobial peptides • Specialized molecules – Poly-Ig receptor – Mucins – Defensins and other antimicrobial peptides Figure 14-1 – Cellular and Molecular Immunology, 10th ed., 2022 Gut Mucosal Immunity • M cells sit on top of the “dome” of the Peyer’s patch, have fenestrations that facilitate uptake of antigens, and use transcytosis and clathrin-mediated endocytosis to transport antigens to APCs (Note: they do NOT process Ag). • Some pathogens exploit M cells (e.g., Salmonella typhimurium). Once engulfed, they kill the M cell, leaving a gap in the epithelium. Note: in addition to Peyer’s patches, there are 100-150 mesenteric lymph nodes surrounding the gut! Immune-Mediated GI Disease The normal gut has a predominance of anti-inflammatory cytokines: • IL-10 and TGF-beta • Produced by DCs and Tregs • Promote sIgA &Treg production Inflamed GI tract changes to a predominance of pro-inflammatory cytokines – IFN-γ, IL-17, and IL-13. Tolerance in the GI Mucosa • Twice as many Tregs in intestine compared with other tissues • Induced in the gut in response to Ag encountered locally (so induced rather than natural Tregs) by: – Production of retinoic acid and TGF-β by specialized DCs (CD103+CD11b+) – Presence of metabolites from GI commensal bacteria, esp. short chain fatty acids (e.g., butyrate) • Tregs then inhibit differentiation of Th1 and Th2 by production of IL-10 and TGF-β • Other specialized DCs (CD103-CD11b+) are more immunogenic and induce differentiation of Th17 cells which promote inflammation Figure 14-8 – Cellular and Molecular Immunology, 10th ed., 2022 CELIAC DISEASE (CD) Celiac Disease • Celiac disease – Also called gluten-induced enteropathy – Inflammatory disease of small bowel caused by immune response to gliaden (major protein component in gluten) • Classic presentation: – A 6-year-old Caucasian male child with a history of progressive chronic (non-bloody) diarrhea and poor growth without fever or other evidence of (viral) infection • Risk factor: – Genetics – strongly associated with HLA-DQ2 and HLA-DQ8 – Strong evidence these MHC alleles present altered gluten peptides to mucosal CD4 T cells, activating them to secrete proinflammatory cytokines (next slide • Treatment – avoidance of gluten with a gluten-free diet https://www.precisionnutrition.com/wpcontent/uploads/2009/10/gliadin-glutenin-gluten.gif Symptoms of Celiac Disease • Primary symptoms due to chronic inflammation in sm. intestine: ‒ Villous atrophy (see following slide). This leads to…. ‒ Malabsorption – leading to…. ‒ Various nutritional deficiencies https://theceliacscene.com/wpcontent/uploads/2015/05/osteoporosis.jpg • Secondary symptoms – extraintestinal presentations generally related to nutritional deficiencies ‒ Anemia ‒ Low bone density ‒ Dermatitis herpetiformis – IgA against intestinal transglutaminase 2 (TG2) enzyme in intestine crossreacts with TG3 in basement membrane of skin • Due to subtility and non-specificity of symptoms, pts often initially diagnosed with “failure to thrive” with diagnosis of celiac disease not made until 3rd or 4th decade of life https://www.mdpi.com/2072-6643/10/5/602/htm Pathophysiology of Celiac Disease • Transglutaminase 2 (TG2) in intestine modifies gliadin (deamination), and APCs process this gliadin • During Ag presentation, it seems there is preferred CD4 T cell recognition of these altered gliadin peptides presented on HLA-DQ2+ or –DQ8+ APCs, activating the CD4 T cells • Immune-mediated damage from: ‒ Activated CD4 cells produce IFN-γ, promoting CMI (CD4 Th1, CD8, macs, NK cells) and tissue damage ‒ CD8 T cells, predominantly intraepithelial lymphs, IELs) and NK cells kill intestinal epithelial cells by direct cytotoxicity ‒ TG2 becomes “recognizable” as an autoAg with TG2-specific IgA and IgG made (role in pathogenesis unclear) Figure 19-13, Cellular and Molecular Immunology, ed. Abbas, Lichtman, and Pillai, 10th ed., 2022 Roles of IELs and Cytokines in Celiac Disease • IFN-γ stimulates GI epithelium release IL-15, another pro-inflammatory cytokine • IL-15 – enhances cytotoxicity of CD8 CTL and of NK cells • IL-15 – also has an anti-apoptotic effect on lymphs, so cells accumulate in the lamina propria • Increases epithelial damage due to release of perforin and granzyme • IL-21 is produced by activated CD4 Th cells which enhances CD8 T cell survival, proliferation, production of IFN-γ, and granzyme/perforin-dependent cytotoxicity • IL-21 and IL-15 block the suppressive effects of Tregs • BOTTOM LINE – sets up cycle of immune activation and tissue damage in celiac disease • See supplemental slides This anti-apoptotic effect on IELs also increases risk of malignant transformation to lymphoma in celiac patients. Diagnostic Testing for Celiac Disease • HLA phenotyping ‒ 95% of celiac patients have susceptibility HLA genotype DQ2 (5X risk) and DQ8 • Duodenal biopsy ‒ Villous atrophy, elongated/hyperplastic crypts, plasma cells, T cells, IELs • Antibodies to transglutaminase 2a and gliadin ‒ IgA and IgG responses – role in pathogenesis not defined, but these Ab responses are important for diagnosis • Response to gluten free diet ‒ Improvement of clinical manifestations Histopathological Findings Celiac disease diagnosis is confirmed with duodenal biopsies that demonstrate: • Blunting and flattening of mucosal surface • Absent or broad and short intestinal villi • Elongated crypts (hyperplastic) • Lamina propia infiltrated by mononuclear cells • Increased IELs Lesions often regress w/ gluten-free diet LP = lamina propria Treatments for Celiac Disease • • • • Removal of gluten from the diet (and other sources!) Agents that inactivate TG2a auto-antibodies ** Agents that inhibit IL-15 ** Agents that block gliadin peptide-specific T cell response ** **experimental therapies INFLAMMATORY BOWEL DISEASE (IBD) Inflammatory Bowel Disease or IBD • Heterogenous group of diseases of small or large bowel • Hypothesis – result of dysregulated immune response to GI microbiome • Two main types ‒ Crohn’s disease – most frequently affects terminal ileum, but can affect any part of GI tract ‒ Ulcerative colitis – restricted to colonic mucosa ‒ Similar presentations, but unique immunologic and clinical patterns https://www.online-sciences.com/the-living-organisms/the-function-of-the-small-intestine-in-the-human-digestive-system/ Dysregulated Immunity in IBD • Inflammation in GI mucosa controlled by: • Both mechanisms disrupted in CD and UC • Reduced immune suppression and... • Increased active inflammation (proinflammatory) –IL-10 and TGF-β production DCs and Tregs resident in GIT –Killing of effector cells by apoptosis ATG16L1 and IRGM gene mutations are risk factors for IBD and impair autophagy in Paneth cells leading to decreased defensins and antimicrobial peptides. Risk Factors for IBD • Environmental factors ‒ Smoking – alters cytokine profile and free O2 radical generation ‒ Socioeconomic factors – IBD more prevalent in developed countries; one hypothesis – hygiene theory where less frequent helminth infections → lack of Th2 stimulation → more proinflammatory cytokines → IBD • Bacterial factors ‒ 50% reduction in biodiversity of commensals, e.g., Lactobacillus, Bacteroides ‒ Increased presence of pathogens – possibly due to defect in defensins, leading to ↑ bacterial invasion ‒ Abs against bacterial components detected ‒ Lymphs reactive to fecal Ag found in IBD pts FYI: No IBD in germ-free conditions (animal models) Clinical Presentation of IBD • Crohn’s disease – – – – – – – – – – Affects any part of GI tract, most often terminal ileum Discontinuous inflammation = cobblestone Full thickness lesion = “garden hose” appearance May cause fistulae with bladder, intestine, perineum Anal skin tags Fever Abdominal pain Diarrhea (+/- blood) Fatigue NOD2* is predisposing factor (rel. risk = 38) • Ulcerative colitis – – – – – Affects the colon and rectum only, superficial Bloody diarrhea Abdominal pain Fever Weight loss Differentiating IBDs Crohn’s Disease Ulcerative Colitis Possible etiology Disordered response to GI bact Autoimmune Location Any part of GIT, Skip lesions, rectal Continuous colon/rectal lesions sparing Gross morphology Transmural, cobblestone mucosa, thickening, string sign, fistulas Mucosal/submucosal inflammation, pseudopolyps Microscopic morphology Granulomas, lymphs (Th1) Crypt abscesses, ulcers (Th2) Complications Stricture, fistula, perianal disease, malabsorption, cancer Sclerosing cholangitis, toxic megacolon, cancer Intestinal manifestation Diarrhea +/- blood Bloody diarrhea Treatment Corticosteroids, infliximab ASA preps, 6-mercaptopurine, infliximab, colectomy Imaging and Testing in IBD • In Crohn’s disease, double contrast barium swallow + gas shows: ‒ Narrowing of intestines = “string sign” (white arrow) ‒ Cobblestoning (red star, I think!) ‒ Presence of fistulas • Endoscopic exam is sufficient to diagnose UC and investigate extent of the lesions • Histopathology – UC = superficial inflammation (lymphs, macs, PC) – Crohn’s = granulomas in deep tissue • Other tests: – Pericytoplasmic anti-neutrophil cytoplasmic antibody or P-ANCA – present in 70% of UC patients – Anti-outer membrane protein C (OmpC) and anti-Saccaromyces cerevisiae (ASCA) antibodies – Crohn’s – These tests differentiate 95% of IBD patients, and the remaining 5% are termed “indiscriminate colitis” * Histopathology of IBD https://basicmedicalkey.com/wpcontent/uploads/2016/06/B978070204293 5000269_f13-01-9780702042935.jpg https://www.webpathology.com/slides13/slides/Colon_Gross3_Crohn_Cobblestone Mucosa_resized.jpg https://www.researchgate.net/figure/Endoscopicand-histological-features-of-Crohns-disease-CDand-ulcerative-colitis-UC_fig3_304661756 Cytokine Profiles in IBD Ulcerative Colitis: • Increased IL-13 • Increased IL-5 • Low IL-4 Crohn’s Disease: • Increased IFN-γ • Low IL-4 INFLAMMATORY BOWEL DISEASE (IBD): CROHN’S DISEASE (CD) Pathophysiology of Crohn’s Disease Possible triggers • An environmental agent may cause initial inflammation • IL-2 or IL-10 deficiency → reduces tolerance • Mutation in NOD2 – 15% of patients with Crohn’s have a homozygous mutation CARD15, gene encoding NOD2 ‒ NOD2 is a PRR that binds MDP, a peptidoglycan component present in cell walls of Gram-pos. and Gram-neg. bacteria ‒ In people with normal NOD2 function, bacteria bind TLR2 which activates NF-kB and removes pathogen, and the pathway is turned off. ‒ In people with defective NOD2 signaling, pathogenic bacteria AND normal microflora induce dysregulated, hyper-responsive state, resulting in excessive Th1/Th17 (IL-1, 6, 12, TNF, IFN-γ, IL-17) inflammation (a “gain of function mutation” although controversial roles). ‒ NOD2 mutations also inhibit alpha-defensin production by Paneth Cells. Figure 19-12, Cellular and Molecular Immunology, ed. Abbas, Lichtman, and Pillai, 10th ed., 2022 A Side Bar: Peptidoglycan in Bacterial Cell Walls M = N-acetyl muramic acid G = N-acetyl glucosamine Recall: NOD2 Localization We touched on NOD2 very briefly in Lecture 4 (Innate Immune Receptors) when we talked about cytosolic sensors and the inflammasome. Recall: TLR2 and NOD2 Function Immunity, Vol. 22, 661–667, June 2005 Review NOD2 and Crohn’s Disease: Loss or Gain of Function? Defective NOD2 in Crohn’s Disease Mucosal Response in Healthy and CD Patients • MDP is sensed in bacteria. • Normally, NOD2 signaling is turned on which, in turn, downregulates cytokine production. • In Crohn’s patients where NOD2 is defective, MDPinduced TLR signaling through NF-κB persists, causing excess Th1 and Th17 inflammation. • Result: an excess IL-1, IL-6, IL-12, IL-17, IFN-γ, and TNF. • NOD2 mutations inhibit alpha-defensin production In the Driver’s Seat of CD: IL-1 and IL-17 INFLAMMATORY BOWEL DISEASE (IBD): ULCERATIVE COLITIS (UC) Pathophysiology of Ulcerative Colitis • Not driven by Th1 cytokines like Crohn’s disease. • Ulcerative colitis or UC driven by NKT cells stimulated by IL-13 – What are NKT cells again??? o Constitute ~0.1% of all peripheral blood T cells o Share properties of both T cells and natural killer cells o Have an alpha-beta T cell receptor like T cells o Express surface marker proteins similar to NK cells o Recognize self and foreign lipids/glycolipids presented by CD1d (not MHC-peptide complexes) o When stimulated by glycolipid antigen binding, they produce large quantities of proinflammatory cytokines, including IFN-γ, GM-CSF, IL-2, IL-13, IL-17, and TNF • These proinflammatory cytokines injure epithelial cells and contribute to bowel ulceration. • NKT cells may be important in UC, diabetes, atherosclerosis, cancer, and asthma. NKT Cells and IL-13 in Ulcerative Colitis NKT cell production of IL-13 is key factor in destruction of GI epithelium in ulcerative colitis. https://www.nature.com/articles/nri1132 https://www.sciencedirect.com/science/article/pii/B9780124158474000811 Treating IBD Historical treatments for IBD • Crohn’s Disease – Mild anti-inflammatory agent (such as mesalamine) +/- short-term CCS (prednisone) or azathioprine – Anti-TNF immunotherapy – Consider nutritional deficiencies (iron, B12, protein, vitamins/micronutrients) – Monitor for cancer of GI tract (5-6x increased risk) • Ulcerative Colitis: – Similar approach to treatment as in sub-bullets 1 and 2 above – Nutritional deficiencies less common because it is a LI, not SI, lesion; still monitor for iron deficiency anemia due to blood loss – Monitor for toxic megacolon (rare – inflammation of neural plexus in bowel wall) – Monitor for colonic carcinoma (20-30x increased risk). Newer immunotherapies • Anti-IL-12/23 antibody (binds a shared peptide) – inhibits differentiation of Th1 and Th17 cells by neutralizing il-12 and IL-23, respectively • Vedolizumab – antibody against α4β7 integrin, so it inhibits trafficking of gut-homing lymphocytes Immunotherapies for IBD A 2018 review of the currently approved and investigational immunotherapies for IBD – https://pubmed.ncbi.nlm.nih.gov/29624476/ Summary • Review normal physical and chemical barriers that help to protect the GI tract • Normally, IL-10, TGF-beta and Tregs promote anti-inflammatory, tolerogenic environment in the gut • Celiac disease: – Lack of gluten tolerance – Non-bloody diarrhea, poor growth due to villous atrophy and malabsorption – HLA-DQ2 and HLA-DQ8 activated by APCs presenting TG2-processed gliadin protein – Activated T cells produce IFN-γ, IL-15, and IL-21 which enhances cytotoxic function of CD8 CTLs (recruited and IELs) and NK cells and inhibits differentiation of induced Tregs – Net result – cell-mediated destruction of intestinal epithelium and loss of adsorptive villi – TG2a and gliadin autoantibodies aid in diagnosis, but no clear role in disease pathogenesis to date • Crohn’s disease: – Terminal ileum: cobblestoning, string sign, garden hose, fistulae, diarrhea +/- blood – NOD2 ‘gain of function’ mutations causing activation of Th1 and Th17 CD4 T cells which produce IL-1, IL-6, IL-12, IL17, IFN-γ, and TNF – all proinflammatory in nature, increasing tissue destruction • Ulcerative colitis: – Colon and rectum: superficial inflammation and ulceration, bloody diarrhea – IL-13 stimulation of NKT cells – Cytokines: IFN-γ, TNF, IL-2, IL-13, and IL-17 – Greatly increased risk of colon cancer