Lymphocyte Function I and II 2024 PDF

Immune Cell Function in Innate Immunity OBJECTIVES: Define steps and interactions in neutrophil diapedesis Mechanisms for killing of bacteria by neutrophils and macrophages Describe how immune cells cause pathology to the host Differentiate between M1 and M2 macrophage functions The...

Immune Cell Function in Innate Immunity OBJECTIVES: Define steps and interactions in neutrophil diapedesis Mechanisms for killing of bacteria by neutrophils and macrophages Describe how immune cells cause pathology to the host Differentiate between M1 and M2 macrophage functions The Leukocytes: Meet the ‘_O’Phils’ Somebody please feed ‘Phil! Primary Granules Secondary Tertiary Pham Nature Reviews Immunology 6, 541–550 (July 2006) | doi:10.1038/nri1841  Granule Exocytosis Triggered by chemokines (IL-8), cytokines (TNF) and chemoattractants (C5a, N-fMLP peptides) ie; Primarily through G-Protein Coupled Receptors Specific (secondary) and azurophil granules both fuse with the membrane of phagocytic vacuoles upon neutrophil stimulation, and release their contents into the vacuole. Secretory granules are also easily accessible for extracellular release, by fusing with the plasma membrane. Hierarchy of granule mobilization: Secretory > gelatinase > specific > azurophil Consequently, azurophil granule contents are dedicated primarily towards killing of phagocytosed microbes, while secretory granules act primarily as a reservoir for mobilization of adhesion molecules to the neutrophil surface. Major Secretory Granule Proteins CR1 (aka CD35, C3bR, C4bR): Receptor for particles opsonized with C3b) CR3 (aka CD11b, Mac1) Receptor for fibrinogen, ICAM1, and C3b) FPR: Receptor for N-formylated peptides (G protein coupled receptor CD14: Receptor for LPS CD16: Low affinity receptor for IgG 5  Important Concepts Neutrophils are first line of defence against invading pathogens Short life span; large numbers enter and exit the circulation daily Neutrophil function is dependent on granule proteins, which are mobilized in an ordered fashion Most rapidly mobilized required for chemotaxis, neutrophil adhesion, and opsonization of bacteria; slowly mobilized promotes killing of bacteria 6 Road Rash and Innate Immunity https://www.bicycling.com/training/a20043941/11-of-the-worst-road-rash-pictures-weve-seen/  LFA1- lymphocyte function associated antigen 1 ICAM2 Neutrophil Rolling http://www.dnatube.com/video/4140/Leukocyte-Rolling-Adhesion  Key Points Interstitial macrophages encounter bacteria and release cytokines; TNF and IL1 TNF and IL1 activate endothelial cells, to produce P- selectin, ICAMs, and chemokines (IL8) Neutrophils begin to roll on P-selectins, and engage IL8, leading to activation of LFA1, engagement of ICAM2 and stable adhesion. Extravasation through interaction between LFA1, MAC1 and PECAM with endothelial ICAM1 and PECAM. After extravasation, neutrophils adhere to fibronectin/fibrinogen in ECM, phagocytose bacteria, and elaborate more chemokines (IL8) 9 Activation of NADPH Oxidase by Chemokines A reminder that chemokines activate NADPH oxidase through G-Protein coupled receptors 10 The NADPH Oxidase/Myeloperodixase Azurophil System MPO Cl- Br- I- MPO + H2O2 + Halide O2 O2 Toxic Agents Tyrosine oxidation Oxidation OCl- Chlorination + I- NADPH NADP MPO + H2O2 + Cl- HOCl Cytochrome b558 complex OH Cl2 Disorders of Oxygen-Dependent Antimicrobial Systems Myeloperoxidase deficiency: Relatively common disorder, affecting 1:2000 individuals. Most patients with MPO deficiency not at increased risk of infection!? Bactericidal activity is normal, but candidacidal (yeast) activity may be moderately to severely impaired. Phagocytosis is normal or increased. Deactivation of the respiratory burst is delayed, and H2O2 production is greatly enhanced. H2O2 is itself bactericidal, and may compensate for the loss of toxic substance formation by the MPO-halide-hydrogen peroxide complex. Granulomatous disorders do not occur as a result of the defect. Presumably, as a result of increased H2O2 production, patients with MPO deficiency do not have difficulty in metabolizing chemoattractants and other mediators of inflammation.  Chronic Granulomatous Disease Description: Inherited X-linked and autosomal recessive disorder affecting 1:250,000 to 1:500,000 patients. CGD patients suffer from recurrent infections, and in response to inflammation, develop severe chronic inflammation, leading to granuloma formation. S. aureus most common cause of infection in CGD patients. Cause: Failure to produce cytochrome b558 due to defect in gp91phox (60% of patients) and p47phox (30%) components of NADPH oxidase; therefore cannot generate superoxide anion. Assumption: Susceptibility to infection due to inability to generate reactive oxygen species, including H2O2, HOCl, free radicals etc.  Azurophile Granule Contents Matrix: Acid mucopolysaccharide: Negatively charged polysaccharide which is primarily responsible for uptake of the azure stain. Many of the granule proteins are cationic, and bind tightly to acid mucopolysaccharide. Microbicidal Enzymes: Myeloperoxidase; Lysozyme; Phospholipase A2 Acid hydrolases: b-glucuronidase N-acetyl- b-glucosaminidase Phosphatases Neutral Proteinases: Serprocidins: serine protease homologues with microbicidal activity ( Elastase; Cathepsin G; Proteinase 3; Azurocidin); Gelatinase; Collagenase Antimicrobial Peptides: Bactericidal/permeability increasing protein; Defensins (comprise 30-50% of granule protein; small 4 kDa cationic peptides)  Antimicrobial Serprocidins Cathepsin G: Stimulates acute phase response antimicrobial protease (Gram positive); neutrophils from mice defective in cathepsin G have reduced ability to kill S. aureus. Elastase: Neutrophils derived from mice defective in elastase production show reduced killing of Gram-negative bacteria. Elastase probably degrades outer membrane proteins (Porin), and inactivates secreted virulence factors.  Wegeners Granulomatosis Multifocal lung nodules in Fibrinoid necrosis in renal biopsy Wegeners Granulomatosis ANCA (anti-neutrophil cytoplasmic antibodies) induce a necrotizing vasculitis by provoking a respiratory burst and degranulation of leukocytes (neutrophils and monocytes), leading to endothelial injury. Requires an initiating inflammatory event, leading to production of antibodies against previously shielded granule proteins (ie; Proteinase 3; MPO) Binding of antibodies to neutrophil surface promotes constitutive activation and adhesion to endothelium, followed by degranulation; AUTOAMPLIFICATION LOOP.  Wegeners Granulomatosis ANCA-associated vasculitides (AAV) are a group of rare, primary, systemic necrotizing small-vessel vasculitides. Granulomatosis with polyangiitis and microscopic polyangiitis account for 80–90% of all AAV. Exposure to silica dust, farming and chronic nasal Staphylococcus aureus carriage are associated with increased risk of developing AAV. When a diagnosis of AAV is suspected, as in patients with multisystem organ dysfunction or those with features such as chronic recurrent rhinosinusitis, cavitated lung nodules, palpable purpura or acute kidney injury, then appropriate further investigations are needed, including ANCA testing. Granulomatosis with polyangiitis; 2-13 per million  Dual Function of BPI Bactericidial Permeability Increasing Factor: Cationic 50-55 kDa protein with selective cytotoxicity towards Gram negative bacteria. Present exclusively in azurophil granules, but also detected on surface of PMN’s. Exhibits high affinity towards LPS (endotoxin); inserts into outer membrane and disrupts permeability barrier, causing cell death. Synergistic activity with defensins and phospholipase A2; sufficient concentration in inflammatory exudates to exert microbicidal activity. Role in Moderation of Inflammation: BPI is closely related to acute phase LPS binding protein (LBP), which potentiates proinflammatory activity of LPS by presenting it to CD14, and initiating signaling cascade. LPS in complex with BPI is incapable of initiating signaling cascade. Therefore, BPI has a potent activity in down-regulation of LPS signaling at sites of inflammation. Summary of Neutrophil Function 1. Granule contents play important role both in eradication of pathogens, and in directing neutrophil migration towards site of infection. 2. Bactericidal proteins (defensins, phospholipase, BPI, protease) act within phagolysosomes, but are also released into inflammatory exudates at biologically active concentrations. 3. Unregulated neutrophil degranulation has significant pathologic affects. 4. The oxidative burst NADPH oxidase has a key role in promoting the neutrophil’s antimicrobial activity. 5. Both oxidative and proteolytic mechanisms are important in eradication of microbial pathogens, but also capable of inflicting damage on host tissues. Oh, What a Tangled Web We Weave.... Neutrophil Extracellular Traps (NETS) Science 303:1532-35; March 2004  NET COMPOSITION Microbial virulence factors are degraded by NET proteins NET fibres are bactericidal  NET Formation: From ROS to chromatin decondensation: NATURE REVIEWS | IMMUNOLOGY VOLUME 18 | FEBRUARY 2018 NET Formation: From ROS to chromatin decondensation: In a nutshell: ROS generated by NADPH oxidase stimulates MPO to trigger activation of NE (neutrophil elastase; serprocidin) and its transfer from azurophilic granules to the nucleus In the nucleus, NE proteolytically processes histones which disrupts chromatin packaging MPO and NE bind chromatin and synergize to decondense chromatin, independently of proteolytic activity. Abbas et al: Basic Immunology 4th edition Chapter 02: Innate Immunity Macrophages 26 Liver Anatomy Macrophage Function: Liver Kupffer cells J. Exp. Med. 2016 213(7):1141-1145 28  Neutrophils Form NETS That Persist in Liver Vasculature and Cause Local Necrosis in MRSA Sepsis Paul Kubes et al; 2015 Staining of eDNA, Neutrophil Elastase and Histone in Liver Sinusoids NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7673 29  NETs Cause Damage to the Vasculature S. aureus cells that enter the circulation are rapidly cleared by Kupffer cells This leads to necrosis, that is dependent on neutrophil recruitment, activation, and NET release NET structures are anchored to the vascular walls by vonWillebrand Factor Neutrophil elastase, which normally would be inhibited by plasma proteins, remains active when immobilized on NET structures, and causes local necrosis 30  Comparison of Macrophages and Neutrophils Immunological Reviews 2015 Vol 264:182-203 Several Months Macrophage Neutrophil 10-12 hours  Macrophage Bactericidal Functions NADPH Oxidase/Cyt b558 Nitric oxide synthase* Lysosomal hydrolases (protease, lipases, glycanases) Vacuolar ATPase (phagosome acidification)* NRAMP1 (iron and manganese efflux)* ATP7A Ctr1 and Znt1 (import of copper and zinc)* Pathogens 2015, 4, 826-868; doi:10.3390/pathogens4040826 Ronald S. Flannagan 1,Bryan Heit 1,2 andDavid E. Heinrichs 1,2,* 32 A Reminder: Pro-inflammatory role of macrophages 33 F1000Prime Reports 2014, 6:13 Inflammatory M1 and Anti- inflammatory M2 Macrophages 34 Microbial Biofilms Promotes Differentiation of M2 Macrophage Hanke & Kielian; Front. Cell. Infect. Micrbobiol. May 2012 35  Macrophage and Fibroblast Interactions in Biomaterial-Mediated Fibrosis Biomaterials (hip implants, pacemakers..) become coated with extracellular matrix, predisposing to microbial biofilm formation and M2 macrophage polarization Adv Healthcare Materials, Volume: 8, Issue: 4, 2019, DOI: (10.1002/adhm.201801451)  The catheterized bladder environment induces dysregulation of macrophage polarization exacerbating bacterial UTI doi: https://doi.org/10.1101/2024.07.16.603761 Mice with catheterized or naïve bladders were challenged with E. coli or E. faecalis Catheterization promotes tissue damage and fibrin (green) deposition. Fibrin accumulation promotes microbial biofilm formation and M2-Mϕ polarization (C) C57BL/6 female mice bladders were either naïve (N), catheterized without infection (C), or infected in absence (uUTI) or presence (CAUTI) of a catheter with either E. coli UTI89 or E. faecalis OG1RF for 24 hours. At 24 hours, bladders were stained with DAPI for cell nuclei (blue) and antibodies to detect Fg/fibrin (green), E. coli or E. faecalis (red), and Mϕs (anti-F4/80; magenta). White boxes at 20x represent zoomed-in areas of 100x magnification and the white broken line separates the lumen (L) from the urothelium surface (U) and the lamina propria (LP). Orange dotted rectangle depicts uropathogen-Fg biofilms. 37  M1 Macrophage Classically activated macrophages: Produce pro-inflammatory cytokines (IL-1, TNF, IL-18), mediate pathogen and tumor resistance, strong microbicidal properties, but also contribute to tissue destruction Differentiation stimulated by IFN, LPS, GM-CSF In addition to pro-inflammatory cytokines, also produce high levels of iNOS (inducible nitric oxide synthase); contributes to oxidative killing mechanisms Important for eradication of intracellular pathogens; M. tuberculosis and L. monocytogenes Can also promote auto-immune disease; Inflammatory Bowel Disease, Rheumatoid Arthritis, Multiple Sclerosis 38  M2 Macrophage Alternatively activated macrophage: Stimuli such as IL-4, IL-10, TGF-β, helminth (parasitic) infections trigger M2 macrophage differentiation M2 macrophages have anti-inflammatory signature; produce IL-10, decoy receptors, receptor antagonists, etc., to attenuate inflammation Produce arginase; degrades arginine which is needed as a substrate for iNOS Contribute to tissue remodeling, wound healing, and response to helminth (parasitic worms) infections 39