Key to resistance to virus/bacteria infection and perhaps cancer disease

1. Adaptive Immunity Innate Immunity What is Innate Immunity? Key to resistance to virus/bacteria infection and perhaps cancer disease Progression lies within the host immune system: The innate immune system is the earliest response to microbial entry and injury Major function of the innate immune system involves the recognition of pathogen molecules by cellular sensors which activates the production of host defense molecules: These help stop pathogen replication and stimulate T cell responses.

2. No Adaptive Immunity No Innate Immunity How important is Innate Immunity? Very!!!!! X Mice defective in key innate immune response pathways die of infection following exposure to small amounts of virus. The host cannot make sufficient antibody and T cells May take up to a week……too late…..

3. What’s the difference between the innate and adaptive immune response?

4. Immune System: Innate V Adaptive immunity.

5. How Does the Innate Immune Response help adaptive immunity? The production of cytokines referred to as the INTERFERONS, are very important at boosting the production of more cytokines that exert anti-viral activity and stimulate T-cell responses.

6. Infected Cells Produce Interferon/Cytokines. Interferons made (by PRR’s) Phagocytes (DC’s) eat dying infected cell and PAMPS activate PRR’s in phagocyte. More cytokines are made. Antigen presentation occurs- Adaptive immunity Interferons protect other cells, recruit phagocytes

7. Viruses (HSV-1) triggers innate immune gene activation: fibroblast WT STING-/- HSV1

8. Cellular Sensors have Evolved to Detect Pathogens: How? PAMP- pathogen associated molecule pattern Viruses- RNA genome, DNA genome. Bacteria- LPS, flagella, DNA genome. DAMP- damage associated molecular pattern Uric acid, ROS. PRR- Pathogen Recognition Receptor Toll-Like Receptors RLR Inflammasome pathway (AIM2) STING Pathway.

9. Viruses have either DNA or RNA genomes. DNA Viruses Adenovirus Herpes Simplex Virus Vaccinia Virus RNA Viruses Newcastle disease virus/Measles/ Mumps. Vesicular Stomatitis Virus/Rabies. Reovirus. Influenza virus HIV is a lentivirus (retrovirus family) that has a genome that exists in both RNA and DNA forms, depending on the stage of its life cycle.

10. Virus/RNA/DNA Signaling Isaacs and Lindemann; 1958: Found ‘interference’ factors were secreted from cells in response to flu infection. These ‘interferons’ exerted anti-viral activity. How are the interferons activated? dsDNA/dsRNA is a very good activator of interferon. dsRNA and DNA Search for dsRNA/DNA activated proteins The Interferon’s Type I α/β 148 aa Interferon genes

11. Isaacs and Lindenmann

12. B paracrine Type I Interferon / induces gene expression INFAR Apoptosis or protection? Jak1, Tyk2/ STAT 1, 2,IRF9 Trail PKR IRF-1 IRF-7 RNAseL PML ISRE Ikk autocrine GAS IkB JNK-2 NF-kB IRF-3 TBK-1 Type II, Interferon   genes  gene Host Primary Response Other genes T- lymphocyte A Viral induction of interferon ?

13. N P M G L 5’ 3’ 11 Kbp Vesicular Stomatitis Virus- VSV Virus: Vesicular Stomatitis Virus (VSV). Negative-stranded RNA Contains only 5 genes Usually harmless to mice and humans Generates 5 subgenomic mRNAs Lytically infects many types of cells Mock VSV Type I IFN + VSV

14. Cellular Sensors have Evolved to Detect Pathogens: How? PAMP- pathogen associated molecule pattern Viruses- RNA genome, DNA genome. Bacteria- LPS, flagella, DNA genome. DAMP- damage associated molecular pattern Uric acid, ROS. PRR- Pathogen Recognition Receptor Toll-Like Receptors RLR Inflammasome pathway (AIM2) STING Pathway.

15. How Did we Find Cellular PAMPs Insects evolved over 400 million years ago Maybe 10 million species Insect immunity relies on three major mechanisms Phenoloxidase Pathway; synthesizes melanin at injury site which may effect invading microorganisms Phagocytosis; cellular arm Humoral response best characterized TOLL IMD Responses to bacteria and fungi are well characterized: Almost nothing is known about viruses (ARBOVIRUSES)

17. Toll Pathway Discovered in 1985 by Christiane Nusslein-Volhard: Drosophila- Toll = weird, loss affected drosophila development. In 1996 Jules Hoffman showed Toll to have role in immune response in flies in response to fungal infection and bacteria. Pattern Recognition Receptors (PRR) that recognize pathogen associated molecular patterns (PAMPs). Members of the Interleukin I receptor superfamily which have a Toll-IL-1-receptor (TIR) domain. Nomura and colleagues showed that Toll homologues existed in humans. Janaway and Medzhitov showed that TLR4 could be activated with antibody to induce innate immune genes. Bruce Buetler proved that TLR4 was receptor for ‘endotoxin’ produced from gram-negative bacteria – Lipopolysaccharide (LPS). Hoffman and Beutler won Nobel prize in 2011 for their work.

18. Gram-positive bacteria Gram-negative bacteria Toll Receptor PGLC ? dMyd88 imd Toll Pathway IMD Pathway Tube dFADD Pelle DREDD TAK1 NF-Kb (cactus/DIF) NF-Kb (Relish) Anti-fungal/bacteria Anti-bacteria Insect Cells

19. Virus dsRNA IL-1 LPS MALP-2 Flagellin CpG IL-1R TLR3 TLR4 TLR2 TLR6 TLR5 TLR7 TLR9 TIR DD TIRAP MyD88 TICAM MyD88 PAMPs (Pathogen Associated Molecular Patterns Trigger Host Defense NFB/AP1 Gene Induction

20. Crystal Structure

21. Which tissues express TLR’s? Mostly macrophages/monocytes TLR1- MyD88 macrophages TLR2- MyD88 macrophages/myeloid DC TR3- TRIF DC’s, B lymphocytes TLR4- MyD88/TRIF macrophages/myeloid DC, intestinal epithelium. TLR5- MyD88 macrophages/myeloid DC, intestinal epithelium. TLR6- MyD88 macrophages, B lymphocytes TLR7- MyD88 macrophages, B lymphocytes, pDC’s TLR9- MyD88 macrophages TLR9- MyD88 macrophages, pDC’s, B-lymphocytes. HIV infects many of these cell types

22. HIV-mediated TLR Signaling in pDC’s

23. Is innate Signaling Involved in Cytokine Production during Acute HIV Infection? cytokines Probably- but difficult to test experimentally, Is STING involved in suppressing HIV Infection during latency?

24. Inhibitory Cytokines Involved in HIV-1 Infection

25. Stimulatory Cytokines Involved in HIV-1 Infection

26. What is known about HIV and the Toll-Like Receptor Pathway HIV may infect dendritic cells and trigger TLR 7 or other sensors. Type I IFN is produced to help fight infection. However, the virus can also upregulate TRAIL (TNF-related apoptic ligand) on the DC’s. This can bind to TRAIL receptors on CD4 cells and induce CD4 depletion! So, Yes, the TLR pathway is engaged (TL7), but it’s influence on AIDS has yet to be clarified Atfield and Gale, Nature Immunology Vol 16, June 2015

27. TLR’s in host defense Activation of the TLRs leads to up regulation of 100’s of genes. Required for immune responses to pathogens- not essential in many cases. Over activation can lead to inflammation…. Autoimmunity? Pathogens/necrosis? Role in inflammatory bowel disease- Therapeutic intervention? CD destruction and opportunistic disease. TB, AND OTHER BACTERIA.. BUT, development of knock out mice indicated that animals lacking TLR3 or 9 Still made IFN in response to viral infection….. THUS, other sensors must exist in addition to the TLR pathway.

28. Discovery of the RIG- Pathway, 2004: Fujita 1 200 300 400 500 600 700 800 900 MDA-5 1 1025 RIG-I 1 925 CARD/ Death-like DEAD BOX RNA Helicase LGP2e 1 678

29. RIG-I and MDA5 Evolved to Detect Viral RNA Species

30. What is known about HIV and RIG-I and MDA5? TLR 7 is more important in pDC’s than the RIG-I like pathway. pDC’S ARE high level type I IFN producers Other cells could make interferon/cytokines in response to HIV infection and be RIG-I/MDA5 specific. Little data exists in vivo to implicate MDA5/RIG-I pawthay… so far! But purified HIV RNA can activate signaling.

31. Pattern Recognitions Receptors

32. Identification of a New Cytosolic DNA innate immune Signaling Pathway and Regulator – STING. Overexpression of STING activates IFN. STING is localized to the ER (translocon). STING is expressed in Dendritic cells, macrophages, endothelial cells, epithelial cells. STING: STimulator of INterferon Genes 1 379 Transmembrane regions. Peripheral leukocyte Small Intestine Skeletal muscle Placenta Kidney Spleen Thymus Liver Lung Brain Heart Colon 2.4 - hSTING 1.35 - lung β-actin Ishikawa and Barber Nature 2008

33. Viruses (HSV-1) triggers innate immune gene activation: fibroblast WT STING-/- HSV1

34. c-GMP-AMP (cGAMP) synthase -cGAS DNA + ATP, GTP 1 mAb21 domain 522 2’-5’- cyclic dinucleotides NTase core Side and top views of cGASMab21 in complex with dsDNA (brown), GTP and ATP (ruby stick models). DNA binds along the platform between spine and Zn thumb. b, Close-up view of the DNA binding site with selected annotated residues. DNA is bound mainly via the minor groove. A notable exception is the Zn thumb near the major groove. c, Schematic representation of DNA–cGAS contacts. Civril et al., Nature, 2013.

35. STING is a sensor for cyclic dinucleotides 2011 Shang et al., Nat Struc Mol Biol, 2012 Cyclic GMP-AMP synthase is a cytosolic DNA sensor… Sun et al., Science 2013

36. Is STING Signaling Involved in Cytokine Production during Acute HIV Infection? cytokines Probably- but difficult to test experimentally, Is STING involved in suppressing HIV Infection during latency?

37. HIV: Does it activate STING Signaling?

38. Innate Immunity activated here?

39. Does HIV Trigger STING activity and Innate Immune Signaling? STING signaling triggered here?

40. What is known about cGAS/STING and Sensing HIV Infection? Cyclic GMP-AMP Synthase Is an Innate Immune Sensor of HIV and Other Retroviruses. Gao et al., SCIENCE, 2013. The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells. Lahaye et al., IMMUNITY, 2013. Cytosolic RNA:DNA hybrids activate the cGAS-STING axis. Mankan et al., EMBO J, 2013. Nucleic acid recognition orchestrates the anti-viral response to retroviruses. Stavrou et al., CELL HOST MICROBE, 2015. PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1. Yoh et al., CELL 2015. Viruses transfer the antiviral second messenger cGAMP between cells. Bridgeman et al., SCIENCE, 2015. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA. Herzner et al.,NATURE IMMUNOLOGY 2015.

41. Why doesn’t STING or other innate immune pathways clear HIV infection? Maybe they do in some instances…. Maybe cytokine production can facilitate HIV replication…. Masybe the STING or other innate immune signaling pathways are suppressed by HIV? Latency? Re-emergence? T-cell depletion? papers

42. Atfield and Gale, Nature Immunology, 2015.

43. How Can Our Understanding of Innate Immunity Help Prevent HIV/AIDS? Understanding whether HIV inhibits these pathway may enable the design of drugs that block this virus/host interaction. Such drugs may prevent efficient HIV replication. Understanding these pathways helps us design new ways to stimulate the immune system: Adjuvants, Vaccines. RIG-I/MDA5 agonists (polyIC), TLR agonists (imiquimod); STING agonists (cyclic- dinucleotides). Opportunistic infections are a key problem, so understanding how the innate immune system is regulated by microbes may help us combat these diseases. HHV8, EBV, HPV, bacteria (tuberculosis), fungi. STING agonists exert potent anti-tumor ability and so may be helpful against AIDS related malignant disease.

44. NOD-like Receptors TLR’s recognize PAMPS (LPS etc) Nucleotide-binding oligomerization domain-NOD receptor family (NLR) are also important for recognizing bacteria- results in an inflammatory response. Mediated by the induction of IL-1beta, IL6 and TNF alpha. NLR family has approx 22 members. Primarily expressed in immune cells, lymphocytes and APC’s, Macrophage, DC’s also in epithelial cells and mesothelial cells. They have a variety of domains- CARD, PYD etc. Three major activation targets are not IFN but NF-kB, MAPKs and caspase-1. NOD family recognize NF-kB and MAPKs, NALP (NACHT-LRR-PYD)- inflammasome. NODs compliment the TLR’s- for effective immunity.

45. NOD-like Receptors II-Recognition of ligands Individual NLRs have been shown to be important against specific pathogens for example, Nod1 and Nod2 recognize peptidoglycan (PGN) moieties found in bacterial cell wall that are secreted by the bacteria. However, a direct interaction between a putative ligand and its corresponding NLR has not been shown for most Nods- perhaps intermediary host factors exist? NOD1/NOD2 recognize peptidoglycan (PGN), major component of bacterial cell wall activates NF-kB and MAPK pathways. NLRC5 (NOD27) regulates antiviral innate and adaptive immunity through the induction of inflammatory cytokines- NF-kB. Unknown ligand. Secretion system in bacteria makes pores in host cell and introduces virulence factors that activate NLRs (secretion system III and IV).

46. PRR’S-NOD-like receptors (NLR’s) and inflammasomes. CARD inflammasomes FIIND LRR LRR NAD NACHT NACHT PYD Pyrin dom CARD NODs NALPs IPAF/NAIP Nucleotide-binding oligomerization domain-NOD Epithelial cells- bacterial muropeptides- NF-kB PAMPS (PGN, cytosolic DNA), ROS, K+ efflux: LRR- ligand recognition, PYD-PYD association and oligomerization of NACHT domain into high molecular weight complexes. Recruits ASC (apoptosis-associated speck-like protein containing a CARD), then caspase-1. Targets substrates IL-1 beta and IL-18- active IL-1R and IL-18R- MyD88 pathway. Activates inflammatory responses acts as an autocrine adjuvant to upregulate co-stimulatory molecules?.

47. Pattern Recognitions Receptors

48. NOD aggregation and inflammasome activation

49. Recognition of RNA and RNA Viruses by RIG-Like-Receptors (RLR’s).

50. NOD-like Receptors III-Inflammasomes NLRP1 MDP NLRC4 virulence factors NLRP3- DAMPs- directly or indirectly Non NLR- AIM III Recognize DAMPs directly or indirectly All activate caspase I in response to a wide variety of bacteria TLR’s and NODs co-operate to fight infection. Recognize bacteria that escape TLR’s, that invade intracellulary that are engulfed.