Innate Immunity

1. Innate Immunity S. Barbour Suggested Reading: Janeway Chapter 2 pp. 37-55, 75-96 Janeway Chapter 6 pp. 228-231 Office hours by arrangement: Please contact me by email (sbarbour@vcu.edu) to schedule an appointment.

2. Characteristics of the Innate Immune System • Localized vs Systemic Response • Humoral vs Cell-Mediated Response • Non-specific Response • Rapid Response of Pre-existing Molecules and Cells Although the innate immune system is non-specific, it is selective and can distinguish self from non-self.

3. Innate Immune Mechanisms • Complement • Phagocytosis • Inflammatory Response • Acute Phase Response • Natural Killer Cells • Fever

4. Outcomes of Innate Immunity • Chemotaxis • Opsonization / Phagocytosis • Induction of Cytokines • Inflammatory Response • Acute Phase Response • Adaptive Immunity • Induction of Adaptive Immunity

5. Synergism Between Innate and Adaptive Immunity

6. Innate Immune Recognition

7. Characteristics of Immune Receptors Note: Innate receptors are SELECTIVE, although not SPECIFIC

8. Pathogen-Associated Molecular Patterns (PAMPS) Microbial Cell Mammalian Cell Note an additional difference that spares mammalian cells: mannose and fucose are often masked by sialic acids.

9. Pattern Recognition Receptors (PRR) • MB Lectin (opsonization / phagocytosis) • Collectins (opsonization / phagocytosis) • Macrophage Mannose Receptor (opsonization / phagocytosis) • Scavenger Receptor (opsonization / phagocytosis) • F-met-leu-phe (fmlp) Receptor (chemotaxis) • Toll-like Receptors (TLR) (induction of cytokines and adaptive immunity)

10. TLR Structures } Leucine-rich repeats Variable, Selectivity Cysteine-rich region Conserved, Signaling Toll / IL-1 receptor domain (TIR)

11. TLRs and their Ligands

12. Microbial Recognition by TLRs • A given microbe has various ligands and can activate multiple TLRs • Some TLRs can heterodimerize; different heterodimers generate different responses • Responses from each TLR pair are integrated to determine the ultimate cellular response

13. TLR 4 is best characterized and will be used as an example of TLR signaling / function

14. Recognition of LPS

15. TLR Signaling Proteins adaptor signaling protein serine-threonine kinase (IRAK) kinase complex regulatory protein transcription factor -pathway is believed to be common to other TLRs -note: MyD88-independent pathways exist as well

16. TLR 4 Signaling Mechanism MyD88 recruits & activates IRAK (SIIK) Phosphorylated TRAF6 activates Ikk complex Ligated TLR4 dimerizes and binds MyD88 Interaction occurs via TIR domains MyD88-IRAK interaction occurs via death domains Ikk complex contains 2 serine-threonine kinases

17. TLR 4 Signaling Mechanism II Ikk phosphorylates IkB. This targets IkB for ubiquitination and destruction Liberated NFKB translocates to nucleus and regulates gene expression NFkB Ikk complex remnants of IkB NFkB IkB target gene

18. Outcomes of TLR Signaling • Induction of Cytokines • Inflammatory Response • Acute Phase Response • Th1 differentiation • Induction of Adaptive Immunity • Upregulation of costimulatory molecules • Maturation of antigen presenting cells (DC)

19. Cytokines Induced Through TLR4/ Activation of Macrophages (IL-8) Activated macrophages produce cytokines that recruit & activate other leukocytes. Many of these cytokines are associated with inflammation, one of the earliest outcomes of the innate immune response.

20. Characteristics of Inflammation • Pain • Redness • Swelling • Loss of function

21. The Process of Inflammation • Increased vascular permeability and blood flow • Exudation of plasma fluid • Leukocyte recruitment and extravasation Inflammation is a protective response intended to isolate infected tissues and prevent the spread of disease. Although inflammation can be “inconvenient”, it is actually one of the initial responses to injury or infection.

22. The Inflammatory Response 4 steps BLOOD and CR3 bacteria TISSUE -Two steps of adhesion (selectin then integrin) allow tight binding to endothelial cells -Integrin-mediated diapedesis allows access to tissue -Chemokines attract leukocytes to migrate to infected area of the tissue -Leukocytes attack bacteria in tissue, resolve infection and / or induce adaptive immunity

23. First Step: Weak Adhesion Through Selectins blood leukocyte tissue endothelial cell

24. Selectin Details -selectins are members of the class of carbohydrate-binding proteins called lectins -cytokines and chemokines induce selectin expression on endothelial cells -initial selectin (P-selectin) is recruited from Weibel-Palade bodies in endothelial cells -leukocytes express glycoproteins that contain fucose sugars -low affinity binding interactions between fucosylated proteins on leukocytes and selectins on endothelial cells allow leukocytes to roll along the endothelium

25. Second Step: Tight Adhesion through Integrins BLOOD and CR3 TISSUE bacteria

26. Integrin Details -integrins are a family of adhesion proteins expressed on leukocytes -integrins mediate the high affinity (tight) binding that arrests leukocytes on the endothelium -chemokines induce changes in integrin conformation on leukocytes -this “activates” integrins and promotes higher affinity interactions (“inside out signaling”)

27. Integrins and Cell Adhesion Molecules (CAMs) -integrins on leukocytes bind cell adhesion proteins (especially ICAM-1) on endothelial cells -resting endothelium expresses low levels of ICAM -chemokines and TNFa induce ICAM expression and thereby promote tight adhesion

28. Third Step: Diapedesis BLOOD and CR3, CR4 TISSUE bacteria -in diapedesis, leukocytes migrate between adjacent endothelial cells -this process is mediated by another integrin, PECAM (CD31) that is expressed both on endothelial cells and leukocytes

29. Fourth Step: Migration BLOOD and CR3 TISSUE bacteria -chemokines recruit leukocytes to infected area of tissue -activated leukocytes ingest and kill bacteria and produce other cytokines that recruit additional leukocytes/ induce adaptive immunity

30. Recruitment of Leukocytes by Chemokines • Chemoattractant proteins for monocytes, neutrophils, and lymphocytes • Induce these cells to move up concentration gradient; this results in recruitment • Bind receptors that have 7 transmembrane domains and signal through G proteins • Two families of chemokines: • CC chemokines have adjacent cysteines in N- terminus • CXC chemokines have C- any aa- C in N-terminus • Chemokine receptor families • CCR1-9 • CXCR1-6 • Some chemokines are both chemoattractants and activators Chemokines also act on the endothelium inducing changes in the conformation of cell adhesion proteins that result in high affinity interactions with leukocytes.

31. Chemokine Receptor Signaling I • Large G protein is also called “heterotrimeric” G protein • This name is derived from its composition: a, b, and g subunits • b and g are so tightly associated that they essentially function as a single subunit • (bg subunit)

32. Chemokine Receptor Signaling II -before chemokine binds, G protein is heterotrimer (a,b,g subunits associated) -after chemokine binding, receptor binds G protein promoting exchange of GDP for GTP and dissociation of a from bg subunit -both subunits have biological activity, bind effectors to promote signaling & cell response

33. Recruitment of Leukocytes by Chemokines • Chemoattractant proteins for monocytes, neutrophils, and lymphocytes • Induce these cells to move up concentration gradient; this results in recruitment • Bind receptors that have 7 transmembrane domains and signal through G proteins • Two families of chemokines: • CC chemokines have adjacent cysteines in N- terminus • CXC chemokines have C- any aa- C in N-terminus • Chemokine receptor families • CCR1-9 • CXCR1-6 • Some chemokines are both chemoattractants and activators Chemokine signaling also acts on integrins inducing changes in the conformation of these proteins that result in high affinity interactions with endothelial cells.

34. CXC Chemokines

35. CC Chemokines Note: bacterial products (f-met-leu-phe) and complement proteins (C5a) are also classified as chemokines.

36. Localized Inflammatory Responses are Protective > 90% of all infections are resolved by local inflammatory responses

37. Systemic Inflammatory Responses Can be Fatal (Septic Shock)

38. Acute Phase Response TLR signaling induces IL-6 IL-6 acts on hepatocytes and induces production of acute phase proteins Acute phase proteins are important components of innate immunity: Some are opsonins and promote phagocytosis (CRP, MBL (MBP), collectins) Some promote blood clots (fibrinogen) in small vessels, block spread of infection Some are complement activators (MBL) Together, these events augment other aspects of innate immunity and increase efficiency of the response

39. Acute Phase Proteins and Phagocytosis -C-reactive protein (CRP) and mannose-binding lectin (MBL) are opsonins, proteins that promote phagocytosis of pathogens -CRP recognizes phosphorylcholine; MBL recognizes mannose sugars -although both of these are expressed on mammalian cells, they are not recognized by CRP or MBL -this is an example of the selective nature of the innate immune response

40. Phagocytosis lysosomes bacteria phagosome phagocyte phagolysosome bacterial remnants

41. Induction of Adaptive Immunity

42. TLR4 signaling induces DC migration to draining lymph node LPS binds immature DC through TLR4 (dendritic cell) -TLR4 signaling upregulates CD80, CD86 -Mature DC activates naïve T cells and thereby induces adaptive immunity T cell area Dendritic Cell (DC) Maturation

43. LPS Activated Dendritic Cells Induce Th1 Response bacteria or other microbes phagocytic receptor antigen processing TLR peptide + MHC TLR signaling Th1 Cell Naïve T Cell costimulatory molecule -ligand is captured by phagocytic receptor,antigen processing and presentation occur -TLR binds microbial ligand, TLR signaling induces costimulatory molecules and Th1 cytokines IL12 IL18 DC -microbial cell induces Th1 response

44. Innate Immune Responses Against Viruses • Interferon-a (IFN-a) • Interferon-b (IFN-b) • Natural Killer (NK) Cells

45. Induction of Type I Interferons (IFN-a, IFN-b) - Type I interferons are induced by viral nucleic acids -Type I interferons are produced by a variety of virally infected cells, including fibroblasts and monocytes---potentially through binding of double-stranded (viral) RNA by TLR3 -Receptors for Type I interferons are widely distributed, most cell types can respond

46. Roles of Type I Interferons in Anti-viral Immunity • Activation of oligoadenylate synthase • Phosphorylation of eIF-2 by PKR/ inhibition of protein synthesis • Induction of Class I MHC protein expression • Activation of Natural Killer (NK) Cells

47. Natural Killer Cells I • large granular lymphocytes • produce large amounts of interferon gamma/ promote inflammation • highly cytolytic • antibody-dependent cellular cytotoxicity (ADCC) • direct recognition of “non-self” on virally infected cells • type I interferons enhance cytolysis

48. Natural Killer Cells II • express FcgRIII; antibody binding triggers cytolysis (ADCC) • directly recognize ligands on virally infected cells • kill virally infected cells • ligands / receptors are poorly characterized • may also be involved in anti-tumor immunity • also recognize ligands on uninfected cells • uninfected cells are spared • ligands = MHC Class I proteins • receptors = Killer Inhibitory Receptors (KIRs)

49. NK and T Cells in Anti-Viral Immunity NK cells are crucial early in the response. Viral infections are prevalent in individuals lacking NK cells.

50. Fever elevated temperatures impede both viral and bacterial replication