Chapter 2

1. Chapter 2 Innate Immunity – Early Defense Against Infection

2. Innate Immunity • Always present, ready to recognize and eliminate microbes and host cells damaged by microbes • Doesn’t react against nonmicrobial substances • Powerful early defense mechanism – can control infection before adaptive immunity can be activated • Cross-talk within innate and adaptive immunity

3. Innate vs. Adaptive Immunity

4. Recognition of Microbes • Cells of innate immunity recognize structures that are shared by various classes of microbes and are not present on host cells • if bind to host cell, other molecules will prevent activation • Phagocytes recognize lipopolysaccharide (LPS or endotoxin) on microbes • have receptors for LPS on phogocytes • Recognize molecular patterns with pattern recognition receptors – pathogen-associated molecular patterns • terminal mannose structures which are different than mammalian cells • dsRNA of viruses and unmethylated CpG nucleotides of bacterial DNA

5. Recognition of Microbes • Recognize things on microbes that make them infective and able to survive • Adaptive immune system recognizes Ag whether microbial or nonmicrobial – very specific • Receptors of innate immune system are encoded in the germline • not by somatic recombination as are T and B cell receptors (Chapter 4) • Innate recognizes <1000 microbial patterns thru the many different receptors on each cell, need to start over at every exposure – no memory

6. Recognition of Microbes • Adaptive immunity recognizes over 109 Ag, can get stronger with each exposure because of memory and adaptive changes (Chapter 7) • Innate immunity doesn’t react with the host • inherent specificity to microbes • Even as microbes evolve they are unable to evade the innate system because the microbe cannot alter the things that the innate recognizes as these are important for microbe infectivity • can evade the adaptive immune response because the things that the microbe changes and adaptive recognizes are not an essential component for survival or infectivity

7. Cellular Receptors for Microbes • Found on dendritic cells, phagocytes and other types – lymphocytes, epithelium and endothelial cells • In separate areas where microbes are found • cell surface • endoplasmic reticulum • move to endosomes where microbes end up after ingestion • cytoplasm • Several receptors identified that are specific for different microbes – molecular patterns

8. Toll-Like Receptors (TLR) • Similar to receptors found in fruit flies that protect flies from infection • Specific for different components of microbes • Found on cellular surface or in endosomes

9. TLRs Activity Against Microbes • TLR-2 – bacterial lipoglycans • TLR-3, -7 and -8 – viral nucleic acids (dsRNA) • TLR-4 – bacterial LPS • TLR-5 – flagellin in bacterial flagella • TLR-9 – unmethylated CG-rich nucleotides of bacteria • Binding of these receptors by their ligands activate transcription factors that change gene expression for cytokines, enzymes and other antimicrobial proteins

10. 2 Most Important Transcription Factors • NF-B (nuclear factor- B) – turns on various cytokines and endothelial adhesion molecules • IRF-3 (interferon response factor) – stimulate production of type 1 interferon, a cytokine that blocks viral replication in additional cells

11. Other Receptors • Surface receptors recognize peptides with N-formyl-methionine that is found on bacterial proteins • Receptors for terminal mannose residues • Cytoplasmic receptors – viral nucleic acid or bacterial peptides • May recognize cytokines produced in response to microbes • chemokines – stimulate migration of cells to microbes • interferon  (IFN ) – enhances phagocytes to kill ingested microbes • Others still for Ab and complement proteins on microbes

12. Components of Innate • Epithelia which act as barrier to infection • Cells in circulation and tissues • Several plasma proteins • Different but complementary roles in blocking entry of microbes

13. Epithelial Barrier • Common portals of entry • skin, GI tract, respiratory tract – continuous epithelia • Act as physical and chemical barrier • Enter by physical contact, ingestion or breathing • Epithelial cells produce peptide antibiotic that kill bacteria • Also have epithelial T-cells and B-1 cells in the peritoneal cavity to make Ab

14. Specialized Lymphocytes • Intraepithelial lymphocytes – T-cell with limited Ag receptor expression • not as diverse as adaptive immune T-cells • unique proteins make up the receptor -  chains rather than the  proteins in other T-cell receptors (highly diverse) • can recognize microbial lipids and other substances • poorly understand specificity and function • B-1 cells in peritoneal cavity – limited diversity in making only Ab that is of the IgM nature – recognizes carbohydrates of bacteria • More in Chapters 4 and 5

15. Cells of the Innate Immunity • Phagocytes • monocytes/macrophage (tissue monocyte) • neutrophils (polymorphonuclear cell, PMN) • Recruited to site of infection and ingest and kill bacteria

16. Neutrophils • Most abundant and increases when infection is present • 4000-10,000/L • >20,000/L in infection • A cytokine called colony-stimulating factor is secreted by many cells in response to infection, acts in BM • stimulates proliferation and maturation • First on site of bacterial and fungal infection • Eats bacteria in circulation and enters extravascular tissues • Die after a few hours

17. Monocytes Macrophage • Less abundant than PMN – 500-1000/L • Ingests microbes in blood; enter extravascular tissue to become macrophage • 2 stages of same cell line • Survival is longer than PMN • Also resident macrophages in connective tissue/all organs – functions like newly recruited mononuclear phagocyte

18. Movement of WBC • Consists of a multistep process • Neutrophils and macrophages leave circulation by binding to endothelial adhesion molecules and in response to cytokines (chemoattractants) produced by resident macrophages on the encounter with microbes • Tumor necrosis factor (TNF) and interleukin-1 (IL-1) act on endothelium of small vessels, stimulate the rapid expression of adhesion molecules – E-selectin and P-selectin on epithelial cells which bind to carbohydrates on PMN or monocyte – weak interaction • WBC express integrins that have a low-affinity state on unactivated cells but when activated, the presence of TNF and IL-1 increases the affinity of WBC integrins which cause WBC to stop movement, spread out and migrate thru endothelial wall

19. Extravasation

20. Inner World of the Cell

21. Chemokines • Chemoattractant cytokines • Stimulate the increase in affinity of integrins on WBC for their ligands on the endothelium which are influenced by TNF and IL-1 • When attached tightly, the cell undergoes cytoskeletal changes and migrate thru the wall and follow the gradient to the infection site

22. Inflammation • Allows for the accumulation of leukocytes, causes vascular dilation and increased vascular permeability • This helps to get the cells to the area of where the microbe is located • Deficiencies in integrins and selectin ligands lead to defective leukocyte recruitment • increase susceptibility to infection

23. Recognition Receptors • Neutrophils and macrophages have several different types of receptors to recognize structures and patterns • Toll-like receptors • receptors for mannose, • N-formylmethionine containing peptides • Macrophages have cytokine receptors INF (interferon) – from innate and adaptive immune response; activates microbiocidal function of phagocytes • Also respond to complement proteins or Ab on surface of bacteria – called opsonization

24. Receptors and Responses

25. Phagocyte Killing • Phagocyte will ingest the microbe and form a phagosome around microbe, fuse with the lysosome to form the phagolysosome • Phagocytosis activates several enzymes such as • phagocyte oxidase – converts O2 to superoxide anion and free radicals (called reactive oxygen intermediates, ROI) which are toxic to microbes • inducible nitric oxide synthase which converts Arg to NO which is microbiocidal • lysosomal proteases that breakdown microbial proteins • Doesn’t harm the phagocyte, but if released into the tissues, it will cause tissue damage • why inflammation causes tissue damage

26. Killing of Microbes

27. Immune Deficiency • Chronic granulomatous disease • phagocyte oxidase deficiency • can’t clear intracellular infections so they are walled off into a “coat” of macrophage and lymphocytes called a granuloma

28. Dendritic Cells • Responds to microbes • Produces cytokines to recruit lymphocytes • Initiate adaptive immune response • Bridge between innate and adaptive immune response

29. Natural Killer Cell • Respond to intracellular microbes by killing the infected or stressed cell and producing the macrophage activating cytokine – INF • ~10% total lymphocytes in blood and peripheral lymphoid tissues • Contain cytoplasmic granules and surface markers but no immunoglobulins or T-cell receptors • Recognize altered host cells • virally infected host cells or phagocytes with viruses or intracellular bacteria, cell with severe DNA damage and tumor cells • Activation releases granules toward infected cell, proteins enter cell and initiate apoptosis • similar activities of CTL to kill infected cells (Chapter 6)

30. NK Cell vs Lymphocyte

31. NK Cells

32. NK and Host Cells • Can recognize normal host cells but have inhibitory receptors to prevent activation of NK cell • fine balance between engagement of activating and inhibitory receptors

33. Activating Receptors • Well defined activating receptor on NK cell is NKG2D – recognizes molecules that resemble MHC I • expressed in response to many signals from stressed cells • Receptor for specific for IgG Ab bound to cells • results in killing of Ab-coated cells – Ab-dependent cellular cytotoxicity (ADCC) – Chapter 8

34. ITAMS • Immunoreceptor tyrosine-based activation motifs • Part of the cytoplasmic tail of receptors that activate NK cells • when activated, initiates a cascade that results in alteration of gene expression leading to cytotoxic granule exocytosis and production of IFN-

35. Major Inhibitory Receptor Families • Inhibitory receptors are specific for MHC I – expressed on all nucleated cells • Killer-cell immunoglobulin-like receptor (KIR) • share similarity with Ig molecules • Consist of CD94 and lectin subunit NKG2 • Both have cytoplasmic domain – immunoreceptor tyrosine-based inhibitory motifs (ITIM) • become phosphorylated on a Tyr when bind MHC-I; activates Tyr phosphatase to remove PO4; block NK activation when the activating receptor is activated

36. Inhibitory Receptor Response

37. Functions of NK Cells • Viruses that prevent MHC-I expression can’t get killed by cytotoxic T-cells but without MHC-I the inhibitor is inactive and host cell is killed by NK cell (Chapter 6) • IL-15, type-1 interferons and IL-12 (dendritic cell and macrophage derived) enhance NK cell activity • IL-12 and IFN- to make macrophage better • IL-15 functions in development and maturation of NK cells

38. Other Lymphocytes • Have some features of T- and B-cells but not the diversity seen in regular T- and B-cells •  T-cells • NK-T cells – recognize microbial lipids bound to CD1 (related to MHC-1) • B-1 cells – produce IgM Ab in the peritoneal cavity and mucosal tissues • Marginal zone B cells – present at edges of lymphoid follicles in spleen and other organs • involved in rapid Ab response to blood-bourne polysaccharide-rich microbes

39. Complement System (Chapter 8) • Circulating and membrane-associated proteins used in the defense of microbes • Series of proteolytic enzymes that are cleaved to the active form • Complement fixation is an enzymatic cascade

40. 3 Pathways • Alternative pathway – triggered by some complement proteins being activated on microbe surfaces; host cells have inhibitory proteins to prevent activation; Innate immune system • Classical pathway – triggered by Ab binding to microbe or other Ag; humoral arm of Adaptive immune system • Lectin pathway – when plasma protein mannose binding lectin recognizes mannose on microbe proteins; initiates the classical pathway but from different starting point; innate system because no Ab involved

41. Complement • Activated complement proteins cleave other complement proteins • rapidly amplified • All pathways initiate differently but end up in a common pathway • central component is C3 which is cleaved to C3b and C3a • C3b binds microbe and initiates downstream events • perform the same effector function

42. 3 Functions • C3b coats microbes which lead to phagocytosis by recognition of C3b receptors on phagocyte • Some of the remaining cleavage products (C5a and C3a) serve as powerful chemoattractants to neutrophils and monocytes; promote inflammation at site of C’ fixation • Make multimeric protein complexes in microbe membrane that leads to death • osmotic lysis or apoptotic death • disturb permeability of membrane

43. Cytokines of Innate Immunity • Macrophages, dendritic cells and other cells secrete cytokines that mediate cellular reactions of innate immunity • Communication between leukocytes; and between leukocytes and other cells

44. Cytokines • “Interleukins” – made by leukocytes to act on other leukocytes, definition is too narrow as we are finding other sources and activities • Innate immunity cytokines principle source is macrophage and dendritic cells after recognition of microbes • stimulated by binding of LPS or dsRNA to receptor and TLRs • Adaptive immunity cytokines come from the T-helper cells (Chapter 5) • Produced in very small amounts, bind high affinity receptors on target cells • Function as autocrine (on cell that made it); paracrine (on nearby cells); if enough cells activated - endocrine

45. Functions of Cytokines • Serve various functions in host defense • TNF, IL-1 and chemokines recruit blood neutrophils and monocytes to sites of infection • TNF in high concentrations cause thrombosis, decrease blood pressure by decreasing myocardial contractility and vascular dilatation and increased leakiness • see in septic shock with gram negative bacteria; caused by TNF in response to LPS, disseminated intravascular coagulation and metabolic disorders • Dendritic cells and marcophage produce IL-12 in response to LPS and phagocytosed microbes; activates NK cells • IFN from the NK cells to activate macrophages • IFN is part of both systems; also from T cells • Type 1 IFN’s are produced in macrophages, fibroblasts and infected cells that prevent viral replication; used to treat some viral infections such as hepatitis (IFN-)

46.   = Must Know   

47. Other Plasma Proteins • Plasma mannose-binding lectin (MBL) – recognizes microbial carbohydrates, coat microbes for phagocytosis (activate complement by Lectin pathway) • C-Reactive Protein (CRP) – binds phosphorylcholine of microbes for phagocytosis by macrophages with CRP receptors • Many of these proteins • increases rapidly after infection • protective phase is called Acute Phase Response

48. Innate Responses • Different innate responses to different microbes • extracellular bacteria and fungi • phagocytes • complement and/or • acute phase proteins • intracellular bacteria and viruses • phagocytes, dendritic cell and NK cells with cytokines providing the communication between leukocytes

49. Evasion of Innate Immunity by Microbes • Intracellular bacteria that infect phagocytes are usually resistant to destruction because in the cytoplasm • L. monocytogenes – evades ROI and NO by making protein that allows for its escape from phagocytic vessel • Mycobacterium have a lipid that inhibits fusion of vesicles containing bacteria to the lysosomes • Other bacterial cell walls inhibit complement proteins

50. Mechanisms of Envasion