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.

Adaptive ImmunityInnate Immunity

What is Innate Immunity?

Very!!!!!

Mice defective in key innate immune response pathwaysdie of infection following exposure to small amounts of virus.

The host cannot make sufficient antibody and T cellsMay take up to a week……too late…..

No Adaptive ImmunityNo Innate Immunity X

How important is Innate Immunity?

Differences between innate and adaptive immunity

  Innate Immunity Adaptive Immunity

Action Time Early (hours) Late (Days)

Cell TypesMacrophages, Dendritic cells, Neutrophils

B, T Lymphocytes

ReceptorsFixed in genomee.g., Toll-like receptor

Gene rearrangement necessary e.g., B cell receptor, T cell receptor

RecognitionConserved molecular patterns e.g., LPS

Wide variety of molecular structure (proteins, peptides) ~1,000,000,000,000,000,000

EvolutionEvolutionarily conserved (plants, animals)

Only vertebrates (jawed fish-human)

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

Immune System: Innate V Adaptive immunity.

The production of cytokines referred to as the INTERFERONS, are very important atboosting the production of more cytokines that exert anti-viral activity and stimulate

T-cell responses.

How Does the Innate Immune Response help adaptive immunity?

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

Infected Cells Produce Interferon/Cytokines.

WT STING-/-WT STING-/-

HSV1HSV1

Viruses (HSV-1) triggers innate immune gene activationViruses (HSV-1) triggers innate immune gene activation: : fibroblastfibroblast

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.

Cellular Sensors have Evolved to Detect Pathogens: How?

DNA Viruses

RNA Viruses

AdenovirusHerpes Simplex Virus

Vaccinia Virus

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 bothRNA and DNA forms, depending on the stage of its life cycle.

Viruses have either DNA or RNA genomes.

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

Interferon genes

Search for dsRNA/DNA activated proteins

Virus/RNA/DNA Signaling

Isaacs and Lindenmann

Type I Interferon / induces gene expression

paracrine

autocrine

ISRE

Jak1, Tyk2/STAT 1, 2, IRF9

INFAR

Type II, Interferon

T- lymphocyte

GAS

A

Viral induction of interferon

genes gene

Other genes

Host Primary Response

IkkIkB

IRF-3

JNK-2NF-kB

TBK-1

TrailPKRIRF-1IRF-7RNAseLPML

Apoptosis or protection?

B

?

Virus: Vesicular Stomatitis Virus (VSV).Negative-stranded RNAContains only 5 genesUsually harmless to mice and humansGenerates 5 subgenomic mRNAsLytically infects many types of cells

N P M G L

11 Kbp

3’ 5’

Mock VSV Type I IFN + VSV

Vesicular Stomatitis Virus- VSV

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.

Cellular Sensors have Evolved to Detect Pathogens: How?

Insects evolved over 400 million years agoMaybe 10 million species

Insect immunity relies on three major mechanisms

Phenoloxidase Pathway;Phenoloxidase Pathway; synthesizes melanin at injury site

which may effect invading microorganisms

Phagocytosis;Phagocytosis;cellular arm

Humoral responseHumoral responsebest characterized

TOLL IMDTOLL IMD

Responses to bacteria and fungi are well characterized:Almost nothing is known about viruses (ARBOVIRUSES)

How Did we Find Cellular PAMPs

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.

Toll Pathway

TAK1

PGLC ?

Gram-negative bacteria

imd

dFADD

DREDD

Anti-bacteria

NF-Kb(Relish)

IMDPathway

Toll Receptor

Gram-positive bacteria

Tube

Pelle

Anti-fungal/bacteria

NF-Kb(cactus/DIF)

Toll Pathway

dMyd88

Insect Cells

VirusdsRNA

TLR3IL-1R

IL-1

TIR

MyD88

Trigger Host Defense NFB/AP1 Gene Induction

MyD88

DD

TICAM

TIRAP

LPS MALP-2 Flagellin CpG

TLR4 TLR2 TLR6 TLR5 TLR7 TLR9

PAMPs (Pathogen Associated Molecular Patterns

Crystal Structure

Mostly macrophages/monocytes

TLR1- MyD88 macrophagesTLR2- MyD88 macrophages/myeloid DCTR3- TRIF DC’s, B lymphocytesTLR4- MyD88/TRIF macrophages/myeloid DC, intestinal epithelium.TLR5- MyD88 macrophages/myeloid DC, intestinal epithelium.TLR6- MyD88 macrophages, B lymphocytesTLR7- MyD88 macrophages, B lymphocytes, pDC’sTLR9- MyD88 macrophagesTLR9- MyD88 macrophages, pDC’s, B-lymphocytes.

Which tissues express TLR’s?

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?

Probably- but difficult to test experimentally,

Is STING involved in suppressing HIV Infection during latency?

cytokines

Inhibitory Cytokine Produced by Cell Targeted Mode of Action

IFN-α Leukocytes and dendritic cells[9] T cells, monocytes, macrophagesInhibits replication of HIV-1 by suppressing reverse transcriptase.[9]

IL-10Monocytes, macrophages, T cells, and B cells[21]

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]

Inhibitory Cytokines Involved in HIV-1 Infection

Stimulatory Cytokine Produced by Cell Targeted Mode of Action

TNF-αMonocytes, macrophages,T cells, B cells, NK cells, and neutrophils [9]

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].

M-CSF Fibroblasts and endothelial cells[21] Macrophages[21]

Stimulates increased surface expression of CD4 and CCR5 receptors. Results in greater HIV-1 entry and replication.[21]

IL-1Monocytes, macrophages, and neutrophils[9]

Monocytes and macrophages[9]

Upregulates HIV-1 viral replication in infected monocytes and macrophages. Stimulates HIV-1 expression in U1 latently-infected cells[9]

IL-6 T cells, B cells, and macrophages[21] 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]

IL-12 Macrophages and dendritic cells[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]

Stimulatory Cytokines Involved in HIV-1 Infection

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

What is known about HIV and the Toll-Like Receptor Pathway

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 9Still made IFN in response to viral infection…..

THUS, other sensors must exist in addition to the TLR pathway.

TLR’s in host defense

MDA-5

RIG-I

LGP2e

CARD/Death-like

RNAHelicase

DEADBOX

1 200 300 400 500 600 700 800 900

9251

1 1025

6781

Discovery of the RIG- Pathway, 2004: Fujita

RIG-I and MDA5 Evolved to Detect Viral RNA Species

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 infectionand 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.

What is known about HIV and RIG-I and MDA5?

Pattern Recognitions ReceptorsPattern Recognitions Receptors

                            STING:STING:          STimulator of  INterferon GenesSTimulator of  INterferon Genes

TransmembraneTransmembraneregions.regions.

Identification of a New Cytosolic DNA innate immune Signaling Identification of a New Cytosolic DNA innate immune Signaling Pathway and Regulator – STING.Pathway and Regulator – STING.

11 379379

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Overexpression of STING activates IFN.Overexpression of STING activates IFN.

STING is localized to the ER (translocon).STING is localized to the ER (translocon).

STING is expressed in Dendritic cells, STING is expressed in Dendritic cells, macrophages, endothelial cells, epithelial cells.macrophages, endothelial cells, epithelial cells.

Ishikawa and Barber Nature 2008

WT STING-/-WT STING-/-

HSV1HSV1

Viruses (HSV-1) triggers innate immune gene Viruses (HSV-1) triggers innate immune gene activationactivation: fibroblast: fibroblast

Side and top views of cGASSide and top views of cGASMab21Mab21 in complex with dsDNA (brown), GTP and ATP (ruby stick models). DNA binds along the platform in complex with dsDNA (brown), GTP and ATP (ruby stick models). DNA binds along the platform between spine and Zn thumb. between spine and Zn thumb. bb, Close-up view of the DNA binding site with selected annotated residues. DNA is bound mainly via the , 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. minor groove. A notable exception is the Zn thumb near the major groove. cc, Schematic representation of DNA–cGAS contacts, Schematic representation of DNA–cGAS contacts ..

mAb21 domainmAb21 domainDNADNA

++ATP, GTPATP, GTP

2’-5’- cyclic dinucleotides2’-5’- cyclic dinucleotides

NTase coreNTase core

c-GMP-AMP (cGAMP) synthase -cGASc-GMP-AMP (cGAMP) synthase -cGAS

11 522522

Civril et al., Civril et al., NaturNature, 2013.e, 2013.

20112011

Shang et al., Nat Struc Mol Biol, 2012Shang et al., Nat Struc Mol Biol, 2012

Cyclic GMP-AMP synthase is a cytosolic DNA sensor… Cyclic GMP-AMP synthase is a cytosolic DNA sensor… Sun et al., Science 2013Sun et al., Science 2013

STING is a sensor for cyclic dinucleotidesSTING is a sensor for cyclic dinucleotides

Is STING Signaling Involved in Cytokine Production during Acute HIV Infection?

Probably- but difficult to test experimentally,

Is STING involved in suppressing HIV Infection during latency?

cytokines

HIV: Does it activate STING Signaling?

Innate Immunity activated here?

STING signaling triggered here?

Does HIV Trigger STING activity and Innate Immune Signaling?

Cyclic GMP-AMP Synthase Is an Innate Immune Sensor of HIV and Other Retroviruses. Gao et al., SCIENCE, 2013.

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.

Nucleic acid recognition orchestrates the anti-viral response to retroviruses. Stavrou et al., CELL HOST MICROBE, 2015.

Cytosolic RNA:DNA hybrids activate the cGAS-STING axis. Mankan et al., EMBO J, 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., Lahaye et al., IMMUNITY,IMMUNITY, 2013. 2013.

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 Viruses transfer the antiviral second messenger cGAMP between cells. cells. Bridgeman et alBridgeman et al., ., SCIENCESCIENCE, , 20152015..

What is known about cGAS/STING and Sensing HIV Infection?

Why doesn’t STING or other innate immune pathways clear HIV infection?

papers

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?

Atfield and Gale, Nature Immunology, 2015.Atfield and Gale, Nature Immunology, 2015.

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 mayhelp 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.

How Can Our Understanding of Innate Immunity Help Prevent HIV/AIDS?

TLR’s recognize PAMPS (LPS etc)

Nucleotide-binding oligomerization domain-NOD receptor family (NLR) are alsoimportant 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

Individual NLRs have been shown to be important against specific pathogensfor example, Nod1 and Nod2 recognize peptidoglycan (PGN) moieties found inbacterial cell wall that are secreted by the bacteria.

However, a direct interaction between a putative ligand and its corresponding NLRhas 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 virulencefactors that activate NLRs (secretion system III and IV).

NOD-like Receptors II-Recognition of ligands

NALPs IPAF/NAIP NODs

PYD

NACHT

NAD

LRR

FIIND

CARD

CARD

LRR

NACHT

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?.

Nucleotide-binding oligomerization domain-NODEpithelial cells- bacterial muropeptides- NF-kB

inflammasomes

Pyrin dom

PRR’S-NOD-like receptors (NLR’s) and inflammasomes.

Pattern Recognitions ReceptorsPattern Recognitions Receptors

NOD aggregation and inflammasome activation

Recognition of RNA and RNA Viruses by RIG-Like-Receptors

(RLR’s).

NLRP1 MDPNLRC4 virulence factorsNLRP3- 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 intracellularythat are engulfed.

NOD-like Receptors III-Inflammasomes

Recognition of DNA Viruses [DNA Pathogens]

dMyD88

Cactus

Dif Dorsal

Host Defense Genes / Development

Pelle IRAKMyD88

TRAF6

IKKIKK

IKK

TIR

Toll 3VirusToll

TIR

Spaetzle

IBN FB

Tak1

Tab1

Tab2

P38,JNK

Tube

TBK-1

IRF3

IFN IFNR

Type I IFN (IFNa, IFNb)

Signaling

Sensor

Anti-viral genes

JAK-STAT signaling

nucleic acids

Trigger anti-viral responsesRegulate adaptive immunity

Appropriate induction of IFN

Inappropriate induction of IFN Autoimmune disease

Virus infection

Type I IFNs-dependent innate immunity

PAMPS Recognized by the TLRs and their Adaptors