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What is Innate Immunity? Key to resistance to virus/bacteria infection and perhaps cancer disease Progression lies within the host immune system: Innate Immunity Adaptive Immunity 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. How important is Innate Immunity? Very!!!!! No Innate Immunity X No Adaptive Immunity 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….. What’s the difference between the innate and adaptive immune response? Differences between innate and adaptive immunity Innate Immunity Adaptive Immunity Action Time Early (hours) Late (Days) Cell Types Macrophages, Dendritic cells, Neutrophils B, T Lymphocytes Receptors Fixed in genome e.g., Toll-like receptor Gene rearrangement necessary e.g., B cell receptor, T cell receptor Recognition Conserved molecular patterns e.g., LPS Wide variety of molecular structure (proteins, peptides) ~1,000,000,000,000,000,000 Evolution Evolutionarily conserved (plants, Only vertebrates (jawed fish-human) animals) Immune System: Innate V Adaptive immunity. 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. Infected Cells Produce Interferon/Cytokines. Interferons made (by PRR’s) Interferons protect other cells, recruit phagocytes Phagocytes (DC’s) eat dying infected cell and PAMPS activate PRR’s in phagocyte. More cytokines are made. Antigen presentation occurs- Adaptive immunity Viruses (HSV-1) triggers innate immune gene activation: fibroblast WT HSV1 - STING-/+ - + g34.5 deleted-HSV Cxcl10 LOC667370 Ifit3 Ifit3 Ccl5 Rsad2 Oasl1 Oasl1 Gbp2 Cxcl10 Irf1 Irf1 Oasl2 Gbp3 Gbp3 LOC100038882 Samd9l Tyki Mx2 Cd274 Ifi205 Irf1 Irf1 Irgm1 Gbp6 Trim21 Ifit2 Il33 Irgm1 Ifit2 Cxcl9 Ifih1 OTTMUSG000000 Ifi205 Mx1 EG240327 Ifi47 Gbp1 LOC100048346 Usp18 Usp18 Igtp Iigp2 Parp14 Sting+/+ Sting -/- Color key -4.1 0 4.1 Value 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. 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. 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 Interferon genes Type I α/β 148 aa Isaacs and Lindenmann B paracrine Type I Interferon / induces gene expression A INFAR Jak1, Tyk2/ STAT 1, 2, IRF9 Viral induction of interferon ISRE Ikk autocrine GAS IkB ? NF-kB JNK-2 IRF-3 Apoptosis or protection? Trail PKR IRF-1 IRF-7 RNAseL PML TBK-1 Type II, Interferon genes gene Host Primary Response Other genes T- lymphocyte 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 3’ N P M G L 5’ 11 Kbp Mock VSV Type I IFN + VSV 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. 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; Phagocytosis; Humoral response synthesizes melanin at injury site which may effect invading microorganisms cellular arm best characterized TOLL Responses to bacteria and fungi are well characterized: Almost nothing is known about viruses (ARBOVIRUSES) IMD 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. Insect Cells Gram-positive bacteria Gram-negative bacteria Toll Receptor PGLC ? dMyd88 Toll Pathway imd Tube dFADD Pelle TAK1 NF-Kb (cactus/DIF) Anti-fungal/bacteria DREDD NF-Kb (Relish) Anti-bacteria IMD Pathway PAMPs (Pathogen Associated Molecular Patterns IL-1 Virus dsRNA LPS MALP-2 Flagellin IL-1R TLR3 TLR4 TLR2 TLR6 TLR5 CpG TLR7 TLR9 TIR DD Trigger Host Defense NFB/AP1 Gene Induction TIRAP MyD88 TICAM MyD88 Crystal Structure Which tissues express TLR’s? Mostly macrophages/monocytes TLR1- MyD88 TLR2- MyD88 TR3- TRIF TLR4- MyD88/TRIF TLR5- MyD88 TLR6- MyD88 TLR7- MyD88 TLR9- MyD88 TLR9- MyD88 macrophages macrophages/myeloid DC DC’s, B lymphocytes macrophages/myeloid DC, intestinal epithelium. macrophages/myeloid DC, intestinal epithelium. macrophages, B lymphocytes macrophages, B lymphocytes, pDC’s macrophages macrophages, pDC’s, B-lymphocytes. HIV infects many of these cell types HIV-mediated TLR Signaling in pDC’s 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? Inhibitory Cytokines Involved in HIV-1 Infection Inhibitory Cytokine IFN-α IL-10 Produced by Leukocytes and dendritic cells[9] Monocytes, macrophages, T cells, and B cells[21] Cell Targeted Mode of Action T cells, monocytes, macrophages Inhibits replication of HIV-1 by suppressing reverse transcriptase.[9] Macrophages[21] Inhibits replication of HIV-1 in the early stages of infection. Inhibition is associated with its ability to down-modulate production of IL-6 and TNF-α[21]. IL-13 Dendritic cells and T cells Macrophages[21] Inhibits HIV-1 infection and production by down-modulating CCR5 expression on macrophages, blocks revers transcription, and suppresses HIV-1 replication at the post-transcriptional level.[9] IL-16 T cells, mast cells, eosinophils[9] CD4+ T cells[9] IL-16 is a natural ligand for the CD4 receptor, so it inhibits HIV-1 entry into CD4+ T cells^^1,[9] Stimulatory Cytokines Involved in HIV-1 Infection Stimulatory Cytokine TNF-α M-CSF IL-1 IL-6 IL-12 Produced by Monocytes, macrophages,T cells, B cells, NK cells, and neutrophils [9] Fibroblasts and endothelial cells[21] Monocytes, macrophages, and neutrophils[9] T cells, B cells, and macrophages[21] Macrophages and dendritic cells[9] Cell Targeted Mode of Action Monocytes and macrophages Powerful activator of transcription factor NF-κB.[21][22] [9]Activation of NF-κB is followed by nuclear translocation and binding to HIV long-terminal repeat (LTR), which leads to initiation or increases in viral transcription[22]. Macrophages[21] Stimulates increased surface expression of CD4 and CCR5 receptors. Results in greater HIV-1 entry and replication.[21] Monocytes and macrophages[9] Upregulates HIV-1 viral replication in infected monocytes and macrophages. Stimulates HIV-1 expression in U1 latently-infected cells[9] Monocytes and macrophages[9] Synergizes with TNF-α to stimulate HIV expression in latently infected cell lines. Potentiates TNF-α-induced HIV-1 production and transcription of NF-κB.[9] T cells[9] Stimulates HIV-1 replication in peripheral blood mononuclear cells (PBMC), CD4+ T cells, and T cell lines. It also triggers IFN-γ production. [9] 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 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 diseaseTherapeutic 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. Discovery of the RIG- Pathway, 2004: Fujita 1 MDA-5 1 RIG-I 1 200 1 400 500 600 700 800 900 1025 925 CARD/ Death-like LGP2e 300 DEAD BOX RNA Helicase 678 RIG-I and MDA5 Evolved to Detect Viral RNA Species 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. Pattern Recognitions Receptors 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: STING is expressed in Dendritic cells, macrophages, endothelial cells, epithelial cells. STimulator of INterferon Genes 1 379 Transmembrane regions. 2.4 - hSTING 1.35 - lung β-actin Ishikawa and Barber Nature 2008 Viruses (HSV-1) triggers innate immune gene activation: fibroblast WT HSV1 - STING-/+ - + g34.5 deleted-HSV Cxcl10 LOC667370 Ifit3 Ifit3 Ccl5 Rsad2 Oasl1 Oasl1 Gbp2 Cxcl10 Irf1 Irf1 Oasl2 Gbp3 Gbp3 LOC100038882 Samd9l Tyki Mx2 Cd274 Ifi205 Irf1 Irf1 Irgm1 Gbp6 Trim21 Ifit2 Il33 Irgm1 Ifit2 Cxcl9 Ifih1 OTTMUSG000000 Ifi205 Mx1 EG240327 Ifi47 Gbp1 LOC100048346 Usp18 Usp18 Igtp Iigp2 Parp14 Sting+/+ Sting -/- Color key -4.1 0 4.1 Value c-GMP-AMP (cGAMP) synthase -cGAS 1 mAb21 domain 522 DNA + ATP, GTP 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. 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 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? HIV: Does it activate STING Signaling? Innate Immunity activated here? Does HIV Trigger STING activity and Innate Immune Signaling? STING signaling triggered here? 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 cGASSTING 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. 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? papers Latency? Re-emergence? T-cell depletion? Atfield and Gale, Nature Immunology, 2015. 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 (cyclicdinucleotides). 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. 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. 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). PRR’S-NOD-like receptors (NLR’s) and inflammasomes. CARD inflammasomes FIIND LRR LRR NAD NACHT NACHT PYD Pyrin dom NALPs CARD IPAF/NAIP NODs 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?. Pattern Recognitions Receptors NOD aggregation and inflammasome activation Recognition of RNA and RNA Viruses by RIG-Like-Receptors (RLR’s). 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. Recognition of DNA Viruses [DNA Pathogens] Spaetzle Toll Virus Toll 3 TIR TIR dMyD88 Pelle MyD88 IRAK Tube IKK Cactus TRAF6 IKK Dif Tab1 IKK Tak1 Tab2 Dorsal P38, JNK IB N FB IRF3 Host Defense Genes / Development TBK-1 Type I IFNs-dependent innate immunity Virus infection nucleic acids IFN IFNR JAK-STAT signaling Sensor Signaling Anti-viral genes Type I IFN (IFNa, IFNb) Appropriate induction of IFN Trigger anti-viral responses Regulate adaptive immunity Inappropriate induction of IFN Autoimmune disease PAMPS Recognized by the TLRs and their Adaptors