Innate immunity

Immunology . classification of immunology. terminology of immunology. detail description of innate immunity. classification of innate immunity. Barrier of innate immune system, immune responses , cellular innate response receptors and signaling , PAMPS and DAMPS, Toll like receptors, Phagocytosis , cellular components of innate immunity, innate lymphoid cells, complement pathways, Mannose binding lecthin pathway, alternative pathway, inflammations, process of inflammations, fever mechanism , factor affecting innate systems, defects in innate immunity, deficiency in innate immunity, disease therapy

1. Innate Immunity Hari Sharan Makaju M.Sc. Clinical Biochemistry

2. Basic immunology Definitions Immune system cells , tissues, and molecules that mediate resistance to infections Immunology study of structure and function of the immune system Immunity resistance of a host to pathogens and their toxic effects Many body cells and tissues are involved in the implementation of immunity (Not just lymphocytes and other immune cells ) Immune response collective and coordinated response to the introduction of foreign substances in an individual mediated by the cells and molecules of the immune system

3. Role of the immune system Defense against microbes Defense against the growth of tumor cells kills the growth of tumor cells Homeostasis destruction of abnormal or dead cells (e.g. dead red or white blood cells, antigen-antibody complex)

4. Immune System Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Immune System Adaptive immunity Delayed response to specific antigen B-lymphocytes (humoral immunity) T-lymphocytes (cell-mediated immunity) Plasma cells (synthesize and release antibodies) Physiologic responses (e.g., inflammation, fever) Chemicals (e.g., interferon, complement ) Cells (e.g., macrophages, NK cells) Skin and mucosal Membranes & barriers (prevent entry) Immediate response to wide array of substances Innate immunity Nonspecific internal defenses

5. Innate immunity ( we are born with this capability ) F irst line of immune response Based on genetic make-up Relies on already formed components Rapid response: within minutes of infection Not specific same molecules / cells respond to a range of pathogens Has no memory same response after repeated exposure

6. Innate immunity Type of innate immunity Explanation Examples Species immunity Innate immunity towards a microbe exhibited by all members of a given species frogs are resistant to Bacillus anthracis ; while toads are susceptible. Racial immunity innate immunity confined to a particular race; may be absent in other communities Negroes of America are more susceptible to tuberculosis than the whites. Individual immunity Antimicrobial defense mechanisms that are confined to a particular individual; may not be exhibited by others. One exception is identical twins Innate immunity is constitutive and can be considered at Species, Racial and Individual level.

7. Innate - Nonspecific Defenses 1 st line of defense Physical barriers: Skin and mucosal barriers - keep hazardous materials outside the body 2 nd line of defense Phagocytes: neutrophils and macrophages: engulf pathogens and cell debris Immunological Surveillance: natural killer cells (NK cells) destroy abnormal cells. Interferons : Chemical messengers that coordinate the defenses against viral infections. Antiviral proteins do not kill viruses but block replication in cell Complement : Complement action of antibodies to destroy pathogens Inflammation : Triggers a complex inflammatory response limiting the spread of infection Fever : A high body temperature which increases body metabolism, and accelerates body defenses

8. Protective barriers of the body. The barriers of the body represent the first line of defense and prevent or retard the entry cells and molecules into the body

9. Barriers of the innate immune system Types Function Physical / anatomical barriers Tight junctions In the epidermal skin layer: water-proof and blocks UV Subcutaneous glands In the dermis: contain fatty acids maintaining a pH of 3-5 Mucous membranes Traps microbes Mechanical removal Mucus In the respiratory tract: microorganism trapping and attachment prevention by the mucus Cilia Cilia propels the mucus and trapped microbes towards the sites of removal Cough and sneeze reflex Respiratory tract: removal of microorganisms from the body Vomiting and diarrhea GI tract: removal of toxins and pathogens from the GI tract Flushing of body fluids Systemic: fluids such as tears, urine, saliva and sweat also flush microbes from the body

10. Types Function Physiological barriers Temperature Normal body temperature slows down the growth of some pathogens pH S tomach , skin and vaginal pH inhibit microbial growth Chemical barriers Lysozymes Bactericidal enzyme secreted by the cells, found in tears & at the mucosal surfaces Lactoperoxidase Mucosal secretion that stimulates cells to produce toxic radicals Cryptidins and α-defensins Base of crypt cells in the small intestine: damage cell membranes β-defensins Produced within the skin, respiratory tract: damage cell membranes Surfactant proteins A and D Present in lungs: function as opsonins, enhancing the phagocytic activity of cells. Barriers of the innate immune system

11. Innate immune response Pathogens surviving their transit into the tissues below the epithelial layers are then targeted by the innate immune system’s second line of defense, an array of cells expressing membrane receptors that recognize microbial components and activate a variety of cellular defense mechanisms against the invaders Some white blood cell types are activated to rapidly engulf and destroy extracellular microbes through the process of phagocytosis.

12. Innate immune response Other receptors induce the production of proteins and other substances that have a variety of beneficial effects including direct antimicrobial activity, as well as the recruitment of fluid, cells, and molecules to sites of infection. This influx causes swelling and other physiological changes that collectively are called Inflammation.

13. Cellular Innate Response Receptors and Signaling Several families of cellular pattern recognition receptors (PRRs ) have essential roles in Detecting the presence of a pathogen and activating innate immune responses that combat the infection . PRRs are capable of distinguishing between self tissues and a specific class of microbes by recognizing highly conserved PAMPs . PRRs identify two classes of molecules:  Pathogen-associated molecular patterns  (PAMPs), which are associated with microbial  pathogens , and  Damage-associated molecular patterns  (DAMPs), which are associated with components of host's cells that are released during cell damage or death . The PRRs are divided into four families: Toll-like receptors ( TLR ) Nucleotide-binding oligomerization domain-like receptors ( NLR ) C-type lectin receptors ( CLR ) RIG-1 like receptors ( RLR )

14. C-type lectin receptors ( CLR ) Toll-like receptors ( TLR ) Nucleotide-binding oligomerization domain-like receptors ( NLR ) RIG-1 like receptors ( RLR)

15. Cellular Innate Response Receptors and Signaling Many cell types in the body express PRRs, including all types of myeloid white blood cells and subsets of three types of lymphocytes (B cells, T cells, and NK cells). PRRs are also expressed by some other cell types, especially those commonly exposed to infectious agents; examples include epithelial cells of the skin and mucosal and glandular tissues, vascular endothelial cells that line the blood vessels, and fibroblasts and other stromal support cells in various tissues .

16. Toll-like receptors (TLRs) were the first family of PRRs TLRs are membrane proteins that share a common structural element in their extracellular region called leucine-rich repeats (LRRs) ; multiple LRRs make up the horseshoe shaped extracellular ligand-binding domain of the TLR polypeptide chain When TLRs bind their PAMP or DAMP ligands via their extracellular LRR domains, they are induced to dimerize, either as a homodimer (e.g., TLR3/3) or as a heterodimer (e.g., TLR2/1) TLR signaling results in change in the transcription factors that regulate a multitude of genes, including those encoding important proinflammatory cytokines.

17. Toll like receptors(TLRs) - classical examples of pattern recognition receptors. There are 13 types of Toll like receptors (TLR 1 to 13). Important ones are- TLR-2 binds to bacterial peptidoglycan TLR-3 binds to dsRNA of viruses TLR-4 binds to LPS of Gram negative bacteria TLR-5 binds to flagella o f bacteria TLR-7 & 8 bind to ssRNA of viruses TLR-9 binds to bacterial DNA TLR2, TLR3 and TLR7 binds to Coronavirus When triggered by binding to a PAMP on an infectious organism, TLRs mediate the generation of defensive responses that include transcriptional activation, synthesis, and secretion of cytokines (immune chemicals secreted by immune cells) to promote inflammation, and the attraction of macrophages, neutrophils, natural killer (NK) cells, and dendritic cells to the site of infection.

18. Endosomal TLR signaling through the MyD88 and TRIF adaptors Plasma membrane TLR signaling through the MyD88 adaptor.

19. Phagocytes express membrane-bound PRRs on their cell surface, which often function in concert with the secreted PRRs. When these cell surface PRRs bind PAMPs, they initiate phagocytosis, release of toxic oxidants, and delivery of pathogens to lysosomes filled with microbicidal products. In macrophages, pathogen-derived proteins are also processed into peptides and presented by major-histocompatibility-complex (MHC) molecules on the cell surface to engage and instruct antigen-specific T lymphocytes. PRRs linked to phagocytosis

20. - Inflammatory factors – released by mast cells, etc. - Vasodilation – capillaries become permeable - Margination – WBCs slow down & align on the vessel wall Diapedesis – blood cells leave vessels & enter the CT Chemotaxis – blood cells follow a chemical gradient (move toward the source ie ., bacteria) How do phagocytes invade the area of infection or injury?

21. Phagocytes: engulf bacteria Originally WBCs – they migrate into connective tissue The “ clean-up crew ” : phagocytose debris and digest via lysosomes Neutrophils enter first then macrophages (derived from monocytes) Eosinophils involved with parasitic infections and antigen-antibody complexes

22. Cellular components of Innate immunity NK cells: Class of lymphocytes that kill virus infected cells and tumor cells . Mast cells: Present lining the respiratory and other mucosa. Activated by microbial products binding to toll like receptors or by IgE antibody dependent mechanism. They release abundant cytoplasmic granules rich in histamine, prostaglandins & cytokines that initiate inflammation and proteolytic enzymes that can kill bacteria Dendritic cells: Respond to microbes by producing numerous cytokines that initiate inflammation. Serve as vehicle in transporting the antigen(s) from the skin and mucosal site to lymph nodes where they present the antigen(s ) to T cells - bridge between innate and acquired immunity .

23. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Venule Eicosanoids Histamine Basophil Arteriole Basophil and mast cell: Proinflammatory chemical-secreting cells Heparin Vasodilation Increases capillary permeability Capillary Anticoagulant Increases inflamation Basophils open up vessels & increase blood flow

24. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. R ecognizes unhealthy cell (usually expressing abnormal proteins or viral proteins – uses perforins (make a hole in the membrane) and granzymes (initiate apoptosis – programmed cell death via gene expression Apoptosis Unhealthy or unwanted cell NK cell Granzymes enter pore, causing apoptosis of cell Perforin forms a transmembrane pore Perforin and granzyme NK cell: Apoptosis-initiating cells NK Cell

25. Innate lymphoid cells  M ost recently discovered family of  innate immune  cells, derived from  common lymphoid progenitors  (CLPs). ILCs are primarily tissue resident cells, found in both   lymphoid  (immune associated), and non- lymphoid tissues, and rarely in the peripheral blood . In response to pathogenic tissue damage, ILCs contribute to immunity via the secretion of  signalling molecules , and the regulation of both innate and adaptive immune cells.

26. Innate lymphoid cells  They are particularly abundant at mucosal surfaces, playing a key role in mucosal immunity and  homeostasis . Based on the difference in developmental pathways, phenotype, and signaling molecules produced , In 2013, ILCs were divided into three groups: 1, 2 and 3, however , after further investigation, now appreciate five distinct subsets within these groups :   NK cells , ILC1s ,  ILC2s ,  ILC3s , Lymphoid Tissue inducer ( Lti )cells.

27.  Classification of ILCs into three groups on the basis of their functional characteristics. Transcription factors: GATA-binding protein 3 ( GATA3) Retinoic acid receptor-related orphan receptor- α ( ROR α) Aryl hydrocarbon receptor ( AHR) Eomesodermin (EOMES ) T bet

28. Complement pathways I n the innate immune system , complement can be activated in two ways: via the alternative pathway via the mannose -binding lectin (MBL) pathway Alternate complement pathway is activated in response to bacterial endotoxin. Mannose binding Lectin pathway is stimulated by mannose carbohydrate residues on bacterial surface. Biological function; Lysis of the target microbes (by forming pores on the microbial surfaces) Stimulate inflammation (by secreting inflammatory mediators) Promotion of phagocytosis ( opsonization ) Clearance of immune complexes from circulation.

29. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. C C Elimination of immune complexes Antigen Antibody Complement Erythrocyte Complement (C) cross-links immune (antigen-antibody) complexes to erythrocyte and transports to liver and spleen . Complement proteins create MAC to lyse cell. Complement activates and attracts various cells of innate immunity. Complement (C) binds to pathogen; acts as opsonin Macrophage Pathogen Complement Inflammation Opsonization Cytolysis Complement MAC protein Pathogen Inflammation Macrophage Neutrophil Basophil Mast cell Complement have a number of functions (below) to defend against pathogens Complement pathways

30. Alternative pathway initiated by cell-surface constituents that are recognized as foreign to the host, such as LPS Various enzymes cleave C3 to C3a and C3b Unstable C3b fragment readily attaches t o receptors on cell surfaces C3b binds Factor B . Factor B in the complex is cleaved by Factor D to produce an unstable C3 convertase. C3bBb binds Factor P to produce stabilized C3 convertase, C3bBbP. Additional C3b fragments join the complex to make C3bBbP3b, also known as C5 convertase . C5 convertase cleaves C5 into C5a and C5b . C5b inserts into the cell membrane and is the necessary step leading to formation of the membrane attack complex ( MAC) and cell lysis .

31. Mannose -binding lectin pathway Lectin s are proteins that bind to specific carbohydrates. Activated by binding of mannose-binding lectin (MBL) to mannose-containing residues of glycoproteins on certain microbes (e.g ., Listeria spp. , Salmonell spp . , Candida albicans ) . MBL is an acute phase protein , one of a series of serum proteins whose levels can rise rapidly in response to infection, inflammation, or other forms of stress . MBL, once bound to appropriate mannose-containing residues, can interact with MBL-activated serine protease (MASP ). Activation of MASP leads to subsequent activation of components C2, C4, and C3

32. Innate Immunity: Inflammation When the outer barriers of the innate immune system—skin and other epithelial layers—are damaged , the resulting innate responses to infection or tissue injury can induce a complex cascade of events known as the inflammatory response . Inflammation may be : Acute (short-term effects contributing to combating infection, followed by healing)—for example, in response to local tissue Chronic (long term, not resolved), contributing to conditions such as arthritis , inflammatory bowel disease, cardiovascular disease, and type 2 diabetes

33. The innate immune system contributes to inflammation by activating the alternative and lectin-binding complement pathways, attracting and activating phagocytic cells that secrete cytokines and chemokines, activating NK cells, altering vascular permeability, and increasing body temperature

34. 1 4 3 2 Formation of exudate and “ washing ” of infected area Exudate Increase in fluid uptake by lymphatic capillaries Delivery of plasma proteins Diapedesis Chemotaxis Chemical gradient Injured tissue Bacteria Release of inflamatory and chemotactic factors Mast cells Neutrophil CAMs Lymphatic capillary Lymph Basophil Recruitment of immune cells • Margination • Diapedesis • Chemotaxis Vascular changes include • Vasodilation of arterioles • Increase in capillary permeability • Display of CAMs Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Margination Inflammation

35. Innate Immunity: Fever Fever is a protective defense mechanism of body. The thermoregulatory center in the hypothalamus is sensitive to microbes and their products. Increasing body temperature increases the circulation of blood and flushing of tissue that help to eliminate toxin through urine and sweat. Events of fever Results from release of pyrogens such as interleukin 1, interferons toxins from infectious agents, drug reactions toxins, brain tumors Pyrogens released and circulate through the body target hypothalamus and cause release of prostaglandin E 2 raises temperature set point of hypothalamus

36. Innate Immunity: Fever Benefits of fever Inhibits reproduction of bacteria and viruses Promotes interferon activity Increases activity of adaptive immunity Accelerates tissue repair Increases CAMs on endothelium of capillaries in lymph nodes additional immune cells migrating out of blood Risks of a high fever significant above 100 degrees F High fevers potentially dangerous above 103 in children Changes in metabolic pathways and denaturation of proteins Possible seizures, irreversible brain damage at greater than 106 , death above 109

37. Factors influencing innate immunity Age Very old or very young more susceptible to infectious disease Hormone Endocrine disorders such as Diabetes Mellitus, hypothyroidism and adrenal dysfunctions – enhanced susceptibility to infection Nutrition Immune response is reduced in malnutrition patient

38. Defects in innate immune system Cystic fibrosis- gene defect in ion-gated chloride channel, leading to accumulation of abnormally thick secretion in air ways. Congenital achlorhydria - inability of gastric mucosal cell to produce HCl , Chronic Granulomatous Disease- congenital defect in NADP phagosome oxidase (NADP phox ) production, leading to poor generation of ROSs and RNS by phagocytes. Congenital deficiency of C8 Compliment - Defective MAC formation Factor I deficiency- uncontrolled complement activation, leading to complement protein depletion .

39. Deficiency in Innate immunity Toll-like Receptor (TLR) Deficiencies: typical presentation of TLR deficiencies is susceptibility to infection with either bacteria or viruses.  MyD88 Deficiency M yD88 deficiency was initially described in nine children suffering from recurrent and severe pus-forming or pyogenic bacterial infections. These children were susceptible to invasive infections with S. pneumoniae, S. aureus, and P. aeruginosa, but had normal resistance to other common bacteria, viruses, fungi, and parasites. The defect in the children with this disorder displayed autosomal recessive inheritance.

40. IRAK4 Deficiency Patients with IRAK4 deficiency have recurrent severe infections (cellulitis, arthritis, meningitis, osteomyelitis, organ abscesses and sepsis) mainly caused by S. aureus, S. pneumoniae (pneumococcus) and Pseudomonas aeruginosa. No patients had severe viral, fungal or parasitic infections

41. UNC93B1 Deficiency and TLR3 Mutations Deficiency of UNC93B1 or TLR3 leads to susceptibility to encephalitis caused by herpes simplex (HSV-1) (the virus that causes cold sores) due to decreased production of interferons in the central nervous system. mutations - severely disable innate responses that are critical to protection against CNS infection by HSV

42. NEMO deficiency syndrome NEMO deficiency syndrome is a complex disease caused by genetic mutations in the X-linked NEMO gene NEMO syndrome was originally described as an association between ectodermal dysplasia (ED) and susceptibility to infections. Patients with ED have thickened skin, conical teeth, absence of sweat glands, and thin, sparse h air

43. Human Natural Killer Cell Deficiencies Human NK cell deficiencies have been divided into two categories: Quantitative defects: with decreased numbers of NK cells in the peripheral blood Qualitative defects: with normal numbers of NK cells with abnormal function Two genetic causes of NK cell deficiency have been identified including an autosomal recessive CD16 functional defect autosomal dominant GATA2 mutation causing classic NK deficiency . Defects in Interferon-γ (IFN-γ) and Interleukin-12 (IL-12) Signaling IFN-γ/IL-12 pathway deficiencies are rare genetic disorders characterized by susceptibility to mycobacteria and salmonella infections.

44. Excessive or chronic innate and inflammatory responses results variety of conditions Most dangerous of these conditions is sepsis , a systemic response to infection that includes fever , elevated heartbeat and breathing rate, low blood pressure, and compromised organ function due to circulatory defects . Several hundred thousand cases of sepsis occur annually in the United States , with mortality rates ranging from 20% to 50% but sepsis can lead to septic shock — circulatory and respiratory collapse that has a 90% mortality rate.

45. The systemic inflammatory response triggered by septicemia can lead to circulatory and respiratory failure, resulting in septic shock and death . As high levels of circulating TNF-α and IL-1β are highly correlated with morbidity, considerable effort is being invested in developing treatments that block the adverse effects of these normally beneficial molecules.

46. Disease Therapy: Use of INF- α in HCV infection treatment Use of IL-13-PE (IL-13 immunotoxin) for treatment of Pancreatic Adenocarcinoma Use of aerosolized IL-2 for treatment of pulmonary metastatic liver cancer

47. Barriers – epithelium, secretions , fluid flow Cells : phagocytes (neutrophils, macrophages, eosinophils), NK cells, Innate lymphoid cells Chemical signals – interferons, complement proteins, inflammatory mediators, pyrogens for fever Summary of Innate - Nonspecific Processes

48. Thank you