key to resistance to virus/bacteria infection and perhaps cancer disease progression lies within the...
TRANSCRIPT
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
Brai
nBr
ain
Skel
etal
mus
cle
Skel
etal
mus
cle
Col
onC
olon
Thym
us
Thym
usSp
leen
Sple
enKi
dney
Kidn
eyLi
ver
Live
rSm
all I
ntes
tine
Smal
l Int
estin
e
Plac
enta
Plac
enta
Lung
Lung
Perip
hera
l leu
kocy
te
Perip
hera
l leu
kocy
te
Hea
rtH
eart
hSTINGhSTING
β-actinβ-actin
2.4 2.4 --
1.35 1.35 -- lung lung
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