BIO420H Chapter 3 Fall 2024 Virology & Immunology PDF

Department of Biological Sciences Virology & Immunology BIO420H Fall 2024 Dr. Al i ne Hamade  Part I: Basic Immunology Chapter 3 Innate Immunity I. Functions of the innate immunity IV. Complement system II. Anatomic...

Department of Biological Sciences Virology & Immunology BIO420H Fall 2024 Dr. Al i ne Hamade  Part I: Basic Immunology Chapter 3 Innate Immunity I. Functions of the innate immunity IV. Complement system II. Anatomic Barriers V. Pattern recognition by immune cells III. Initial Chemical Defense VI. Induced innate response 2 I. Overview of the innate immunity Innate Immunity  Chapter 3 I. Functions of the innate immunity Features ✓ It is the initial response to microbes that serves to prevent, control, or eliminate infection by many pathogens ✓ It eliminates damaged cells and initiates the process of tissue repair. ✓ Its responses stimulate adaptive immune responses and can influence the nature of the adaptive responses to make them optimally effective against different types of microbes. ✓ Inflammation is also the major reaction to damaged or dead cells and to accumulations of abnormal substances in cells and tissues. I. Functions of the innate immunity Summary ✓ Infection starts when a pathogen reaches one of the host’s anatomic barriers. Some innate immune mechanisms start acting immediately. ✓ These immediate defenses include several classes of preformed soluble molecules that are present in extracellular fluid, blood, and epithelial secretions and that can either kill the pathogen or weaken its effect. ✓ Antimicrobial peptides such as the defensins lyse bacterial cell membranes directly ✓ A system of plasma proteins known as the complement system targets pathogens both for lysis and for phagocytosis by cells of the innate immune system such as macrophages. ✓ If these fail, innate immune cells become activated by pattern recognition receptors (PRRs) that detect molecules called pathogen-associated molecular patterns (PAMPs) that are typical of microbes. I. Functions of the innate immunity Summary ✓ The activated innate cells can engage various effector mechanisms to eliminate the infection. ✓ Neither the soluble nor the cellular components of innate immunity generate long-term protective immunological memory. ✓ Only if an infectious organism breaches these first two lines of defense will mechanisms be engaged to induce an adaptive immune response—the third phase of the response to a pathogen. ✓ This leads to the expansion of antigen-specific lymphocytes that target the pathogen specifically ✓ and to the formation of memory cells that provide long- lasting specific immunity. I. Functions of the innate immunity need to know: infection +immunity Infectious diseases The agents that cause disease fall into five groups: viruses, bacteria, fungi protozoa, and helminths (worms) (parasites) Pathogens can be found in various compartments of the body, where they must be combated by different host defense mechanisms. I. Functions of the innate immunity need to know details? Isolation of CD3-CD56+ human cells and cell lines. no ✓ After infection with obligate intracellular pathogens and gram negative organisms, monocytes elaborate a number of cytokines, such as IL-12, IL-1b, TNF-a, and IL-15, each of which can activate NK cells. ✓ NK cells, in turn, produce IFN-g which is critical for elimination of these pathogens by monocytes ✓ NK cells produce the chemokine MIP-1a in response to co-stimulation by a specific combination of monocyte-derived cytokines. I. Functions of the innate immunity need to know: just mechanism Infectious diseases Infectious diseases differ in their symptoms and outcome depending on where the causal pathogen replicates within the body—the intracellular or the extracellular compartment—and what damage it does to the tissues Pathogens that live intracellularly frequently cause disease by damaging or killing the cells they infect. Obligate intracellular pathogens, such as viruses, must invade host cells to replicate. Facultative intracellular pathogens, such as mycobacteria, can replicate either intracellularly or outside the cell. Endotoxins are membrane compounds of Gram-negative bacteria Exotoxins are peptides that are mostly secreted by Gram-positive bacteria  Chapter 3 II. Anatomic barriers Epithelia: Physico-chemical barriers Epithelia comprise the skin and the linings of the body’s tubular structures— the respiratory, urogenital, and gastrointestinal tracts. Epithelial cells are held together by tight junctions, which effectively form a seal against the external environment. The internal epithelia are known as mucosal epithelia because they secrete a viscous fluid called mucus, which contains many glycoproteins called mucins. Mucous membranes: ✓ Normal flora (competition for nutrients) ✓ Mucus (entrapment) ✓ Cilia (propulsion) Skin and Epithelial barriers: ✓ Mechanical barriers ✓ Antimicrobial peptides II. Anatomic barriers Epithelia: Physico-chemical barriers need to know table  Chapter 3 II. Anatomic barriers Epithelia: Physico-chemical barriers The epidermis has multiple layers of keratinocytes in different stages of differentiation arising from the basal layer of stem cells. Differentiated keratinocytes in the stratum spinosum produce β-defensins and cathelicidins, which are incorporated into secretory organelles called lamellar bodies (yellow) and secreted into the intercellular space to form a waterproof lipid layer (the stratum corneum) containing antimicrobial activity. need to know  Chapter 3 II. Anatomic barriers Epithelia: Physico-chemical barriers The lung, the airways are lined by ciliated epithelium. Beating of the cilia moves a continuous stream of mucus (green) secreted by goblet cells outward, trapping and ejecting potential pathogens. Type II pneumocytes in the lung alveoli \also produce and secrete antimicrobial defensins.  Chapter 3 II. Anatomic barriers Epithelia: Physico-chemical barriers Type II pneumocytes in the lung alveoli \also produce and secrete antimicrobial defensins. RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus. need to know just how to analyze  Chapter 3 II. Anatomic barriers Epithelia: Physico-chemical barriers The intestine : Paneth cells—specialized cells deep in the epithelial crypts— produce several kinds of antimicrobial proteins: α-defensins (cryptdins) and the antimicrobial lectin RegIII. Most healthy epithelial surfaces are also associated with a large population of normally nonpathogenic bacteria, known as commensal bacteria or the microbiota. The microbiota can also make antimicrobial substances, such as the lactic acid produced by vaginal lactobacilli, some strains of which also produce antimicrobial peptides (bacteriocins). II. Anatomic barriers Stages of infection ✓ The infectious agent must first adhere to the epithelial cells and then cross the epithelium. ✓ A local immune response may prevent the infection from becoming established. ✓ If not, it helps to contain the infection and also delivers the infectious agent, carried in lymph and inside dendritic cells, to local lymph nodes. ✓ This initiates the adaptive immune response and eventual clearance of the infection.  Chapter 3 II. Anatomic barriers Gut-Associated lymphoid tissue (GALT) need to know ✓ GALT lies throughout the digestive system, especially intestines. ✓ Both GALT and mesenteric lymph nodes are sites where the immune response is started. ✓ Lymphatic circulation through the tissue is connected to the mesenteric lymph nodes.  Chapter 3 II. Anatomic barriers Gut-Associated lymphoid tissue (GALT) need to know ✓ lamina propria: a thin layer of connective tissue underneath the mucous and epithelial membranes in different tissues. ✓ M cells: Specialized cells that feed antigens to macrophages and DCs of Peyer’s Patches. ✓ T and B cells determine if the antigens whether or not the antigen requires an immune response => Oral immune Tolerance ✓ B cells mature into plasma cells and start secreting IgA.  Chapter 3 II. Anatomic barriers Gut-Associated lymphoid tissue (GALT) B cells mature into plasma cells and start secreting IgA. need to know? Abx: antibiotic-treated group The Antibacterial Lectin RegIIIg Promotes the Spatial Segregation of Microbiota and Host in the Intestine (SCIENCE VOL 334 14 OCTOBER 2011_ II. Anatomic barriers Lymphoid Tissues (a) Antigen might enter through the microfold (M) cells (b) after transfer to local dendritic cells (DC), might then be presented directly to T cells in the Peyer’s patch. (c) Alternatively, antigen or antigen-loaded DC from the Peyer’s patch might gain access to draining lymph. (d) with subsequent T-cell recognition in the mesenteric lymph nodes. (e) A similar process of antigen or antigen-presenting cell dissemination to mesenteric lymph nodes might occur if antigen enters through the epithelium covering the lamina propria. (f) In this case, there is also the possibility that enterocytes might act as local antigen presenting cells.  Chapter 3 II. Anatomic barriers Lymphoid Tissues (g) In all cases, the antigen-responsive CD4 + T cells leave the mesenteric lymph nodes in the efferent lymph (h) and after entering the bloodstream (i) and interact with T cells in peripheral lymphoid tissues. SED, subepithelial dome. TDA, thymus-dependent area.  Chapter 3 III. Initial chemical defenses Categories A. Physiological compounds: Salts, Fatty acids, digestive enzymes B. Antimicrobial Enzymes : Lysozyme, peroxidases, secretory Phospholipase A2. C. Antimicrobial Peptides (AMP) : Defensins, Cathelicidins, Histatins D. Other Proteins : Lactoferrin, Lectin, CRP E. Complement system Proteins F. Cytokines  Chapter 3 III. Initial chemical defenses Physiological compounds ✓ Acidic pH in the stomach, vagina, and skin. ✓ Digestive enzymes (pepsin), bile salts, fatty acids present in the upper gastrointestinal tract.  Chapter 3 III. Initial chemical defenses Antimicrobial Enzymes ✓ Secreted in tears, saliva, specialized cells in the small intestine, and by phagocytes. ✓ Lysozyme: glycosidase that breaks the chemical bonds in the peptidoglycan of the bacterial cell wall. (secreted in tears and saliva, by phagocytes and Paneth cells) ✓ Phospholipase A2: A highly basic enzyme that can enter the bacterial cell wall to hydrolyze phospholipids in the cell membrane. ✓ Peroxidases: to maintain the cellular redox homeostasis. Lysozyme is more effective in acting against Gram-positive bacteria, in which the peptidoglycan cell wall is exposed, than against Gram-negative bacteria, which have an outer layer of LPS covering the peptidoglycan layer.  Chapter 3 III. Initial chemical defenses Antimicrobial Enzymes PLA2-IIA binding to bacteria is charge-dependent. (against Gram-Positive Bacteria) (A). PLA2-IIA is highly positively charged and requires, for optimal binding, negatively charged structures [such as wall teichoic acid (WTA)] on the cell wall of Gram- positive bacteria. (B). Similar to binding, penetration is charge-dependent, taking advantage of the abundant negatively charged teichoic acids that span the entire cell wall (C). Membrane phospholipid degradation is independent of charge but is dependent on calcium. Calcium ions bind to PLA2 and facilitate its interaction with the phospholipid substrate, enabling the enzymatic hydrolysis LTA, lipoteichoic acid.  Chapter 3 III. Initial chemical defenses need to know Selectivity of Antimicrobial Peptides  Chapter 3 III. Initial chemical defenses ELECTRIC FIELD THEN PORE Antimicrobial Peptides (AMP) ✓ Defensin: α-defensins (neutrophils, macrophages, and Paneth cells) and β-defensins (most leukocytes and epithelial cells) are short cationic peptides with a broad- spectrum activity against gram - and gram + bacteria.  Chapter 3 III. Initial chemical defenses Antimicrobial Peptides (AMP) ✓ Cathelicidins: made constitutively by neutrophils and macrophages, and made by keratinocytes in the skin and epithelial cells in the lungs and intestine in response to infection. C-CARPET  Chapter 3 Candida albicans III. Initial chemical defenses Antimicrobial Peptides (AMP) ✓ Histatins: Produced in the oral cavity by the salivary glands. Anti- fungal short, histidine-rich, cationic peptides. oxygen scavenger (L-cysteine) + L- cysteine  Chapter 3 III. Initial chemical defenses Other Proteins need to know ✓ Lactoferrin: (transferrin) iron-binding glycoproteins found primarily in mucosal secretions (saliva, tears, intestine), also present in neutrophilic granules, with anti-bacterial, anti-viral, and anti-fungal activities. ✓ C-Reactive Protein: Acute-phase inflammatory protein secreted routinely by the liver and occasionally by innate immune cells to help in the unspecific activation of complement system.  Chapter 3 III. Initial chemical defenses Other Proteins ✓ Lectin: carbohydrate-binding proteins secreted mainly by Paneth cells that bind to bacterial peptidoglycans and exerts direct bactericidal activity. A) Agglutination - Upon finding membrane carbohydrates on the surface of microorganisms, lectins bind to them, agglutinating the cells together, reducing motility and facilitating the activity of antimicrobial drugs. B) Inhibition of adhesion and biofilm formation – Lectins prevent the sites responsible for attaching microorganism to a surface from playing their role, inhibiting adhesion. C) Synergism with drugs – It is suggested that lectins act by delivering the drug close to the surface of the microorganism, thus facilitating its entry into the cell.  Chapter 3 IV. Complement system Components ✓ A collection of soluble proteins present in blood and other body Fluids. ✓ cooperates with both the innate and the adaptive immune systems to eliminate blood and tissue pathogens. ✓ Discovered by Jules Bordet (1890s) as a heat-labile substance in normal plasma whose activity could ‘complement’ the bactericidal activity of immune sera ✓ Composed of more than 30 different plasma proteins synthesized mainly by the liver. ✓ Circulate in their inactive form in absence of infection ✓ Particular complement proteins interact with each other to form several different pathways of complement activation  Chapter 3 IV. Complement system Functions 1. Lysis of microbes, allografts, and tumors. 2. Opsonization (enhancement of the phagocytosis). 3. Generation of inflammation mediators to attract neutrophils 4. Clearance of Immune complexes and apoptotic cells.  Chapter 3 IV. Complement system Overview (1) The complement pathways are initiated by proteins that bind to pathogens, either directly or via an antibody or other pathogen- specific protein. After a conformational change, (2) enzymatic mediators activate other enzymes that generate the central proteins of the complement cascade, the C3 and C5 convertases, which cleave C3 and C5, releasing active components that mediate all functions of complement, including (3) opsonization, (4) inflammation (5) the generation of the membrane attack complex (MAC).  Chapter 3 IV. Complement system Overview ✓ Effector complement proteins can label an antibody- antigen complex for phagocytosis (opsonins), Initiate inflammation (anaphylatoxins), or bind to a pathogen and nucleate the formation of the MAC. ✓ Often, these effectors act through (6) complement receptors on phagocytic cells, granulocytes, or erythrocytes. (7) Regulatory proteins limit the effects of complement by promoting their degradation or preventing their binding to host cells.  Chapter 3 IV. Complement system Functions MBL: Mannose Binding Lectin GlcNAc: N Acetyl Glucosamine  Chapter 3 IV. Complement system Pathways Classical Pathway: ✓ Initiated by the formation of Ag-Ab complex Alternative Pathway: ✓ Antibody-independent ✓ Part of the innate immunity ✓ Initiated by foreign cell surfaces Lectin Pathway: ✓ Antibody-independent ✓ Initiated by Host proteins bound to microbes  Chapter 3 IV. Complement system Pathways  Chapter 3 IV. Complement system Pathways  Chapter 3 IV. Complement system Classical Pathway of Complement Activation Formation of the membrane attack complex (MAC).  Chapter 3 IV. Complement system Alternative Pathway of Complement Activation  Chapter 3 IV. Complement system Lectin Pathway of Complement Activation Once attached to the carbohydrates, they bind the MASP family serine proteases, which cleave C2 and C4 with the formation of a lectin-pathway C3 convertase.  Chapter 3 IV. Complement system Opsonization and inflammatory response ✓ C3aR and C5aR receptors are members of the G-protein-coupled receptor family. ✓ Binding of the anaphylatoxins to these receptors stimulates the release of proinflammatory mediators from macrophages, neutrophils, basophils, eosinophils, and mast cells need to know  Chapter 3 IV. Complement system Opsonization and inflammatory response Opsonization of microbial cells by complement components and antibodies. Phagocytosis is mediated by many different complement receptors on the surface of macrophages and neutrophils, including CR1, CR3, CR4. Phagocytes, using their Fc receptors, also bind to antigens opsonized by antibody binding.  Chapter 3 V. Pattern recognition by innate immune cells Pattern-Receptor Concept Pathogen-associated molecular patterns (PAMPs): repeating patterns expressed by most microorganisms Damage-associated molecular patterns (DAMPs): Endogenous molecules derived from damaged/dying cells. Receptors that recognize such features are Pattern Recognition Receptors (PRRs) PRRs are expressed on macrophages, neutrophils, and dendritic cells  Chapter 3 V. Pattern recognition by innate immune cells Pathogen-Associated Molecular Patterns need to know? PAMPs were introduced by Janeway (1989) Conserved molecular structures produced by microbes and recognized as foreign by the receptors of the innate immune system Bacterial lipopolysaccharide (endotoxin, Bacterial flagellin, Lipoproteins and peptidoglycan, Mannose residues, Fungal glucans, nucleic acid variants associated with viruses and bacteria. GPI: Glycosylphosphatidylinositol LTA: lipoteichoic acid Zymosan : Carbohydrate-rich complex in cell wall  Chapter 3 V. Pattern recognition by innate immune cells need to know? Damaged-Associated Molecular Patterns NETosis: Neutrophil Extracellular Traps Released following necrotic and late apoptotic tumor death. Calcium-binding molecules Purines metabolites Uric Acid, Heparin Sulfate, Crystals in kidneys, Nuclear proteins (histones, …) Mitochondria and mitochondrial components… HMGB1 : nonhistone chromatin-associated protein HSP: heat shock protein (regulation of cell cycle, apoptosis, signal transduction)  NETosis NETs are composed of microbicidal components that include : - DNA - Histones - Granule proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE).  Chapter 3 V. Pattern recognition by innate immune cells need to know all details? or just the receptors and what they bind to? Pattern Recognition Receptors Proteins capable of recognizing molecules found in pathogens (PAMPs), or released by damaged cells (DAMPs). The PRRs are divided into four families: 1. Toll-like receptors (TLR) IRF: Interferon Regulatory Factor 2. Nucleotide-binding oligomerization domain-like receptors (NLR) 3. C-type lectin receptors (CLR) 4. RIG-1 like receptors (RLR)  Chapter 3 V. Pattern recognition by innate immune cells need to know pathway? Pattern Recognition Receptors The PRRs 1. C-type lectin receptors (CLR) 2. RIG-1 like receptors (RLR) 3. Nucleotide-binding oligomerization domain-like receptors (NLR)  Chapter 3 V. Pattern recognition by innate immune cells Pattern Recognition Receptors Pattern recognition receptors are linked to intracellular need to know? signal transduction pathways activating various cellular responses, e.g. production of pro-inflammatory molecules and molecules that destroy microbes. Membrane-Associated Receptors: Toll-Like Receptors (TLRs). Cytoplasmic Receptors: Nucleotide-binding small molecule (Dipeptide)derived from oligomerization domain proteins (NODs), MDA, RIG the bacterial cell wall peptidoglycan. Endosomal receptors: TLRs Secreted Receptors: C-Reactive Protein (CRP),… DAP: Diaminopimelic : is an amino acid found in the peptidoglycan layer of bacterial cell walls  Chapter 3 VI. Induced Innate response Cytokines ❖ Cytokines: small secreted proteins (~25 kDa) released by cells have a specific effect on the interactions and communications between cells. ✓ Lymphokines ✓ Chemokines ✓ Monokines ✓ Tumor necrosis factors (TNF) ✓ Interleukines ✓ Interferons (IFN) ❖ Cytokines may have autocrine, Paracrine, and endocrine effect.  Chapter 3 VI. Induced Innate response Cytokines : General properties Synergism More than one cytokine can act together to produce an effect that is more profound than the sum of their individual effects. Pleiotropy Redundancy A single cytokine can act on More than one cytokine can act on the several target cells to produce same target to produce similar effects. Antagonism multiple effect A cytokine can block the effect or reduce the influence of another cytokine on target cells.  Chapter 3 VI. Induced Innate response need to know all the IL? Cytokines : General properties Cytokine attributes of (a) Pleiotropy, redundancy, synergism, antagonism, and (b) Cascade induction.  Chapter 3 VI. Induced Innate response each cytokine has many target cells and secreted by many types of immune cells not to be memorized Cytokines : General properties  Chapter 3 VI. Induced Innate response Cytokines ❖ Cytokines exist in broad families that are structurally related but exhibit diverse function: what should we know: name of class ✓ Interleukin [IL-1] superfamily: secreted very early in the immune response by dendritic cells and monocytes or macrophages. viral, parasitic, or bacterial antigens by innate immune receptors. (Proinflammatory) ✓ Hematopoietin [IL-6] superfamily ✓ TNF/TNF receptors superfamily ✓ Interferons (IFN) Type I interferons are composed of Interferons alpha, and interferon beta Type II interferon, otherwise known as interferon-gamma, is produced by activated T and NK cells and is released as a dimer  Chapter 3 VI. Induced Innate response (CXC) : Cys-X-Cys at their C-terminal CXCL: Ligand Cytokines Chemokines A family of chemoattractant cytokines with a role in cell migration from blood into tissue and vice versa, and in the induction of cell movement by chemotaxis. There are 47 chemokines and 19 receptors Chemokines are named according to their amino acid composition Chemokines may also be grouped according to their function: inflammatory, homeostatic, angiogenic (blood vessel formation) or angiostatic (inhibition of blood vessel formation)  Chapter 3 VI. Induced Innate response Cytokines Interferons ❖ A family of secretory proteins induced in response to specific extracellular stimuli through stimulation of toll- like receptors ❖ Acting in paracrine or autocrine modes ❖ First characterized as substances that will inhibit virus replication ❖ Have effects on many immune components ❖ Interferon (IFN)-α and -β are produced by virus- infected cells ❖ IFN-γ is produced as a primary response of T lymphocytes to mitogenic stimulation.  Chapter 3 VI. Induced Innate response Inflammation Chemotaxis: Attraction of WBCs to the inflammation area in response to the release of chemicals from the injured cells and immune cells (histamine and others ). The leukocyte recruited first from the blood into sites of inflammation is the neutrophil Blood Monocytes become increasingly prominent over time. They differentiate into macrophages and DCells in the tissue  Chapter 3 VI. Induced Innate response Inflammation Leucocytes migration into tissues: 1. Secretion of pro-inflammatory cytokines by tissue macrophages and damaged cells. 2. Adhesion of circulating leukocytes to the endothelial lining of capillaries and post- capillary venules. 3. Paracellular transmigration or diapedesis. Diapedesis: Process in which neutrophils (Mainly) enzymatically digest a portion of the capillary membrane, allowing them to leave the vessel and enter inflamed tissue.  Chapter 3 VI. Induced Innate response Inflammation Inflammation: accumulation of leukocytes, plasma proteins, and fluid derived from the blood at an extravascular tissue site of infection or injury Hyperemia: Increased blood flow to an area (site of inflammation).  Chapter 3 VI. Induced Innate response need to know all details Inflammation Cytokines released by macrophages and DCs TNF shock: The excessive release of TNF-α results in the dilation of blood vessels and increased permeability of capillaries, causing a drop in blood pressure and inadequate blood flow to organs and tissues. This can lead to organ dysfunction and failure, as well as other symptoms such as fever, rapid heart rate, respiratory distress… VI. Induced Innate response Inflammation Cytokines released by macrophages and DCs  Chapter 3 VI. Induced Innate response Inflammation Cytokines released by macrophages and DCs OSM: Oncostatin M, which is a cytokine belonging to the interleukin-6 (IL-6) family. ACTH: Adrenocorticotropic Hormone LIF: Leukemia Inhibitory Factor is a cytokine that belongs to the interleukin-6 (IL-6) family.  Chapter 3 VI. Induced Innate response Fever Pyrexia: Fever Fever can be caused by infections, noninfectious disorders, or neurogenic disorders Patterns: 1. Intermittent: temperature returns to normal at least once every 24 hours. 2. Remittent: temperature doesn't return to normal, although it varies a few degrees in either direction 3. Sustained (continuous): temperature remains above normal with minimal variations.  Chapter 3 VI. Induced Innate response Fever Pyrexia: Fever Stages: 1. Prodromal Stage: nonspecific symptoms (headache, fatigue,..) 2. 2nd Stage (Chill): Vasoconstriction, piloerection, shivering.. 3. Third Stage or Flush: Cutaneous vasodilation, skin rush 4. Defervescence: T°C returns to normal, sweating, ….  Chapter 3 VI. Induced Innate response Phagocytosis  need to know for exam? Paper Analysis - MAPKs such as ERK and p38 regulate Nox-dependent NETosis. - The role of JNK/SAPK in typical Nox-dependent NETosis signaling is not established - PMA is used often as an agonist to induce Nox-dependent NETosis. - It activates protein kinase C (PKC), which then activates Nox for producing ROS Lipopolysaccharide (LPS)- Escherichia coli Gram-negative bacteria - SP600125 is a commonly used JNK inhibitor (PMA) :phorbol myristate acetate ; Nox-dependent NETosis Blocking TLR4 signaling with TAK242 need to know for exam? LPS-TLR4-JNK axis regulates Nox-dependent suicidal NETosis. PMA and LPS regulate ROS production in neutrophils via PKC and JNK, respectively. Blocking TLR4 signaling with TAK242 and JNK activation with SP600125 or TCSJNK6o during LPS-mediated NETosis results in the suppression of ROS production and Nox-dependent NETosis, and increased survival of neutrophils. Nox inhibitor DPI suppresses both PMA- and LPS-mediated NETosis.