Transcript viruses - Alergia e Imunopatologia

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
NFB/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
IB
N FB
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