Innate Immune Signaling and Pathogen Recognition

PATHOGEN RECOGNITION AND
INFLAMMATOY SIGNALING IN
INNATE IMMUNE RESPONSE

What is the Innate Immune Response?
 A universal and evolutionarily conserved mechanism of host defense
against infection
 First line of Defense
 Predates the adaptive immune response
 Found in all multicellular organisms

 Adaptive only in vertebrates

 Uses receptors and effectors that are ancient in their lineage

 Must provide protection against a wide variety of pathogens

 Distinguishes self from non-self perfectly
 Defects in innate immunity are very rare and almost always lethal

PAMPs: P athogen A ssociated M olecular
P atterns

PRRs: P attern R ecognition R eceptors

The Innate Immune Response:
Common Misconceptions
 The innate immune system is an evolutionary rudiment whose
only function is to contain the infection until the “real”
immune response can kick in.

 Adaptive immunity developed because of the inflexibility of
the nonclonal receptors used by the innate immune response.
The innate system cannot cope with the high mutational rate
and heterogeneity of pathogenic organisms.

The Innate immune system instructs the adaptive
immune response to respond to microbial infection

The major decision to respond or not respond to a
particular ligand is decided by the genome-encoded
receptors of the innate immune system

Innate immune recognition of
bacterial cell wall components

Gram-negative bacteria Gram-positive bacteria

PRRs common characteristics
1.Recognize microbial components, known as
pathogen associated molecular patterns
(PAMPs), that are essential for the survival of
the microorganism and are therefore difficult
for the microorganism to alter.
2.Expressed constitutively in the host and
detect the pathogens regardless of their life-
cycle stage.
3.Germline encoded, nonclonal, expressed on all
cells of a given type, and independent of
immunologic memory.

Adapted from Medzhitov and Janeway
PAMP Cur. Opin. Immunol. 1997 9:4-9

Phagocytosis
Y PRR

APC
Complement Endosome

Direct Bactericidal Activity
MHC B7 Phagocytosis
Pathogen-specific
Antibody Y Oxygen burst
Anti-microbial peptides
Naive Inflammatory
T Cell
and effector
cytokines

Activated
CD40L, FasL, CD30L, CD27L
T Cell

B Cell

 Machineries underlying innate immune
recognition are highly conserved among
species, from plants and fruit flies to
mammals.

Recognition mechanisms of innate
immunity (concepts)
 What mediates the recognition of PAMPs?
 Diverse recognition elements; 4 key families of “Pattern Recognition
Receptors”:
 Toll-like receptors (TLRs; transmembrane receptors)
 RigI-like receptors (RLRs; cytoplasmic RNA helicases)
 NOD-like receptors (NLRs; cytoplasmic sensors)
 C-type lectin receptors (CLRs; transmembrane receptors)
 Also, recognition of molecules released from necrotic cells, tissue
damage (“damage associated molecular patterns” DAMPs or
“danger”; recognized by some NLRs, TLRs, CLRs)
 New hypothesis: recognition of perturbations induced by
pathogens (“patterns of pathogenicity”) such as bacterial pore-
forming toxins, perturbations of the cytoskeleton, various types
of cell stress etc.)--recognition mechanisms less well understood
(inflammasome, etc.)

(Lectin: a protein that binds to carbohydrates)

TLRS-DISCOVERY
 Toll, the founding member of the TLR
family, was initially identified as a gene
product essential for the development
of embryonic dorsoventral polarity in
Drosophila.
 Later, it was also shown to play a
critical role in the antifungal response
of flies (Lemaitre et al., 1996).
 To date, 12 members of the TLR family
have been identified in mammals.

Discovery of the mammalian Toll-like
receptors (TLR):

1997: Janeway and Medzhitov discovered a human protein
with structural similarity to drosophila Toll that could
activate immune response genes human cells (TLR4).

1998: Beutler discovered that a mouse strain with an
altered response to bacterial lipopolysaccharide (called LPS
or endotoxin) was due to a mutation in the TLR4 gene.

There are 11 TLR family members in human and 12 in
mice. Each responds to a distinct set of microbial
products.

STRUCTURE
 TLRs are type I integral membrane
glycoproteins characterized
-extracellular domains containing
varying numbers of leucine-rich-repeat
(LRR) motifs
 a cytoplasmic signaling domain
homologous to that of the interleukin 1
receptor (IL-1R), termed the Toll/IL-1R
homology (TIR) domain (Bowie and
O’Neill, 2000).

 The LRR domains are composed of 19–25
tandem LRR motifs, each of which is 24–
29 amino acids in length, containing the
motif XLXXLXLXX as well as other
conserved amino acid residues.
 Each LRR consists of a β strand and an α
helix connected by loops.

SUBFAMILIES
 Based on their primary sequences,
 subfamily of TLR1, TLR2, and TLR6-
recognizes lipids,
 TLR7,TLR8, and TLR9 -recognize nucleic acids
.
 However, the TLRs are unusual in that some
can recognize several structurally unrelated
ligands.

Expressed on
 Various immune cells- macrophages,
dendritic cells (DCs), B cells, specific
types of T cells
 Nonimmune cells - fibroblasts and
epithelial cells.
 Expression is not static but rather is
modulated rapidly in response to
pathogens, a variety of cytokines, and
environmental stresses.

 TLRs may be expressed -
 Extracellularly on the cell surface
-TLRs 1, 2, 4, 5, and 6 are
 Intracellularly-TLRs 3, 7, 8, and 9
almost exclusively in intracellular
compartments such as endosomes, and
their ligands, mainly nucleic acids,
require internalization to the endosome
before signaling is possible.

Different mammalian Toll-like receptors (TLRs) are
specific for different classes of microbial products

Insert Fig 3-11

Toll-like receptors and
recognition of pathogensssRNA
Viral

LRR extracellular domain
TIR domain inside K. Takeda & S. Akira, Cell. Microbiol. 5: 143-53, 2003

TLR1, TLR2 and TLR6
 TLR2 recognizes- lipoproteins/lipopeptides
from various pathogens, peptidoglycan and
lipoteichoic acid from Gram-positive
bacteria, lipoarabinomannan from
mycobacteria, glycosylphosphatidylinositol
anchors from Trypanosoma cruzi, a phenol-
soluble modulin from Staphylococcus
epidermis, zymosan from fungi and
glycolipids from Treponema maltophilum .

 Also recognises- LPS preparations from
non-enterobacteria such as Leptospira
interrogans, Porphyromonas gingivalis
and Helicobacter pylori .
 TLR2 recognizes a wide range of
microbial products through functional
cooperation with several proteins that
are either structurally related or
unrelated.

TLR3
 TLR3 is implicated in the recognition of
dsRNA and viruses.
 However, TLR3-independent
mechanisms of dsRNA recognition exist.

TLR4
 TLR4 is an essential receptor for LPS
recognition , recognition of taxol, a
diterpene purified from the bark of the
western yew (Taxus brevifolia)
,endogenous ligands, such as heat shock
proteins (HSP60 and HSP70), the extra
domain A of fibronectins,
oligosaccharides of hyaluronic acid,
heparan sulfate and fibrinogen.

TLR5
 TLR5 recognize an evolutionarily conserved
domain of flagellin.
 TLR5 expression is also observed in the
intestinal endothelial cells of the subepithelial
compartment.
 In addition, flagellin activates lung epithelial
cells to induce inflammatory cytokine
production.
 These findings indicate the important role of
TLR5 in microbial recognition at the mucosal
surface.

TLR9
 Its stimulatory effect is due to the presence
of unmethylated CpG dinucleotides in a
particular base context designated CpG-DNA.
 Although the CpG motif is abundant in
bacterial genomes, its frequency is
suppressed and it is highly methylated in
mammalian genomes.
 The methylated CpG motif does not activate
mammalian immune cells.

Toll-like
receptors
(TLRs)
link microbial
products
(PAMPs) to
transcription
factor
activation
in a signaling
pathway that is
conserved
between
mammals and
insects

Adaptor molecules
 MyD88
 TIR-associated protein (TIRAP)/MyD88-adaptor-like
(MAL)
 TIR-domain-containing adaptor protein- inducing
IFN-β (TRIF)/TIR-domain-containing molecule 1
(TICAM1) (Oshiumi et al., 2003; Yamamoto et al.,
2002b)
 TRIF-related adaptor molecule (TRAM).
 The differential responses mediated by distinct TLR
ligands can be explained in part by the selective usage
of these adaptor molecules.

Toll-like receptor
signaling
pathways

•Ligand induced
dimerization of TLR-->
induced assembly with TIR-
domain containing adaptors
•MyD88 pathway and
TRIF pathway;
•Activate Transcription
factors and MAP kinases

A more detailed look at the signaling pathway down-stream
of Toll-like Receptors (TLRs)

Type I IFN Production via
TLRs
 TRIF-Dependent Pathway- Stimulation
with TLR3, TLR4, TLR7, and TLR9
ligands, but not the TLR2 ligand, induces
type I IFN production in addition to
proinflammatory signals.

Triggering of PRRs on macrophage or dendritic cells can
induce a LARGE variety of events including:

Increased phagocytosis

Production of cytokines and inflammatory mediators:
Interferons to induce anti-viral state
Chemokines to attract migrating cells
Etc, etc.

Increased cell migration

Changes in expression of molecules involved in T cell
antigen presenting cell function.

TLR signaling within phagosomes determines fate of
that phagosome (destruction vs antigen presentation).

Pathogen Dying infected cell
(non-self, TLR signaling) (self, no TLR signaling)

Material in
Material in phagosome phagosome disposed
enters antigen of inside cell-no
presentation pathway- presentation to T cells
presentation to T cells
Blander and Medzhitov 2006 Nature v440 p808

Common and Distinct Themes in TLR Signaling
TLR4/4
TLR2 TLR1/6 CD14 MD-2

Rac PI3K TRIF
PI3K
TIRAP MyD88 IRF3
MyD88 TIRAP

IFN-β
IRAK IRAK
TRAF6

TAK1/
AKT
NIK
Death domain
MAP kinases IKK
Complex TIR domain
IkB
p65 p50

 MyD88 is used by all TLRs except TLR3;
TIRAP is used by TLR2 and TLR4; TRIF
is used by TLR3 and TLR4; and TRAM is
used only by TLR4 .

TLR2- Common TLR4-
Specific Specific
Responses

Pathways of NF-κ B activation
NF-κB is a family of transcription factors: p50, p52,
p65 (Rel-A), c-Rel, Rel-B; plus inhibitors (I-κB)

Canonical Non-
pathway canonical
Pathway
(activated by
some TNF
receptor
family
members)

The NF-kB Family of Transcription Factors

 Eukaryotic transcription factor found in essentially all cell types
 First described in 1986 as a nuclear factor required for the
transcription of the immunoglobulin kappa light chain in B cells.
 Binds to a 10-bp sequence GGGGYNNCCY
 Important component in the inducible expression of many
proteins: cytokines, acute phase proteins, adhesion molecules
 The NF-kB signaling system is evolutionarily conserved
 Three NF-kB molecules in Drosophila
 dorsal
 controls dorsal/ventral polarity during development
 Regulates antifungal gene expression
 dif and relish: regulate expression of antifungal and antibacterial genes

NF-κB exists in the cytoplasm as an inactive
heterotrimer composed of 2 Rel family proteins and an
inhibitory IkB molecule

Stress, infection, or cytokine

IKK
P P (Ub)n
IkB
p65 p50
26S proteosome

Nuclear
Translocation

Activation of NF-KB
Responsive genes

Luke A.J. O’Neil www.stke.org/cgi/content/full/sigtrans;2003/171/re3

Cytoplasmic Pathogen
Recognition System
 A large family of cytoplasmic PRRs has
been cloned to date. Currently, they are
roughly subclassified into the
 NOD-LRR proteins and the CARD
helicase proteins.
 These protein families are implicated in
the recognition of bacterial and viral
components, respectively.

NOD-LRR Proteins and Their
Functions
 Proteins in this family possess LRRs
that mediate ligand sensing; a
nucleotide binding oligomerization
domain (NOD); and a domain for the
initiation of signaling, such as CARDs,
PYRIN, or baculovirus inhibitor of
apoptosis repeat (BIR) domains
(Inohara et al., 2005; Martinon and
Tschopp, 2005).

NOD-Like Receptors - NLRs

 The cytosolic NOD-Like Receptors
(NLRs, also known as
CATERPILLERs, NODs or
NALP/PAN/PYPAFs) are nucleotide-
binding oligomerization domain
containing receptors.
 22 NLRs have been identified in
humans and constitute a major class of
intracellular pattern recognition
receptors (PRRs).

 The designated
subfamilies are
(based on the
initial of the
domain name):
NLRC (formely
known as NODs),
NLRP (formerly
known as NALPs),
NLRB (formely
known as NAIP or
Birc) and NLRA.

 These proteins include NOD1 and
NOD2, which both contain N-terminal
CARD domains.
 NOD1 and NOD2 detect g-D-glutamyl-
meso-diaminopimelic acid (iE-DAP) and
muramyl dipeptide (MDP), found in
bacterial PG, respectively (Chamaillard
et al., 2003; Girardin et al., 2003).

Common alleles of NOD2 are a genetic
risk factor for Crohn’s disease

•Several moderately common alleles of the NOD2 gene (7% of total
alleles) increase susceptibility to Crohn’s disease (a form of
inflammatory bowel disease)
•Two copies of these alleles increase susceptibility by 40X
•Pretty strong evidence that these alleles of are “loss of function”
alleles
•NOD1/2 have been shown to have 4 immune functions: activation of
inflammatory cytokine gene expression; induction of anti-microbial
peptide synthesis by Paneth cells in intestines; activation of
inflammasome; autophagy of bacteria in cytoplasm

Processing of IL-1 and related
cytokines: an important regulatory
step
•Some “NLRs” assemble to form the “inflammasome” which
proteolytically processes IL-1 and related cytokines to their active,
secreted forms.
•Inflammasome is activated by cellular stress or recognition of
microbial components in the cytoplasm
•Genetic periodic fever syndromes are due to activating mutations
in inflammasome
•Activated by small crystals, important role in Gout
•Suggestive evidence that inflammasome may be activated by
cholesterol crystals (atherosclerotic lesions?); possible role in type
2 diabetes? Possible role in alzheimer’s disease?

CYTOPLASMIC BACTERIAL DETECTERS
AND SIGNALLING

RNA Helicases and Double-
Stranded RNA
 Fibroblasts and cDCs lacking MyD88 and TRIF
are still capable of inducing type I IFNs after
viral infection, indicating that the TLR system
is not required for viral detection in at least
several cell types (Kato et al., 2005).
 Retinoic-acid-inducible protein I (RIG-I) is an
IFN inducible protein containing CARDs and a
DExD/H box helicase domain and has been
identified as a cytoplasmic dsRNA detector
(Yoneyama et al., 2004).

RIG-I and MDA-5

 RIG-I (retinoic-acid-inducible protein 1, also
known as Ddx58) and MDA-5 (melanoma-
differentiation-associated gene 5, also known
as Ifih1 or Helicard) sense double-stranded
RNA (dsRNA), a replication intermediate for
RNA viruses, leading to production of type I
interferons (IFNs) in infected cells.
 In cDCs, macrophages and fibroblasts, RLRs
are the major sensors for viral infection,
while in pDCs, TLRs play a more important
role.

 RIG-I participates in the recognition of
Paramyxoviruses (Newcastle disease
virus (NDV), Sendai virus (SeV)),
Rhabdoviruses (vesicular stomatitis
virus (VSV)), Flaviviruses (hepatitis C
(HCV)) and Orthomyxoviruses
(Influenza), whereas MDA-5 is essential
for the recognition of Picornaviruses
(encephalo-myocarditis virus (EMCV))
and poly(I:C), a synthetic analog of viral

 Downstream of RIG-I-IPS-1,
 TBK1 and IKK-i are activated to
phosphorylate IRF-3 and IRF-7,
indicating that the signaling pathways
triggered by TLR stimulation and RIG-I
converge at the level of TBK1/IKK-i.

 These suggest that RNA viruses
actively replicating in the cytoplasm are
recognized by RIG-I, but not TLR3,
irrespective of their route of entry.
 On the other hand, TLR3 has been
suggested to be responsible for the
recognition of dsRNA contained in the
apoptotic bodies of virus-infected cells
taken up by DCs.

C-Type Lectin Receptors
 large family of receptors that bind to
carbohydrates in a calcium-dependent
manner.
The lectin activity of these receptors is
mediated by conserved carbohydrate-
recognition domains (CRDs).
On the basis of their molecular
structure, two groups of membrane-
bound CLRs can be distinguished and a
group of soluble CLRs.

Type I transmembrane
proteins
 Containing several CRDs or CRD-like
domains
 DEC-205 and the macrophage mannose
receptor (MMR)

Type II transmembrane CLRs
 Carry a single CRD domain Dectin-1,
Dectin-2, macrophage-inducible C-
type lectin (Mincle), the dendritic cell-
specific ICAM3-grabbing nonintegrin
(DC-SIGN), and DC NK lectin group
receptor-1 (DNGR-1).

Soluble CLRs
 MBL, an oligermeric protein that binds
an array of carbohydrate patterns on
pathogen surfaces.
 CLRs expressed by most cell types
including macrophages and dendritic
cells (DCs), which phagocytoze various
glycoproteins and microbes for the
purposes of clearance and antigen
presentation to T lymphocytes

MBL
 MBL (Mannose-binding lectin) is a soluble C-
type lectin.
MBL plays a crucial role in innate immunity
against yeast by enhanced complement
activation and enhanced uptake of
polymorphonuclear cells.
 MBL binds to repetitive mannose and/or N-
acetylglucosamine residues on microorganisms,
leading to opsonization and activation of the
lectin complement pathway.

 MBL also interacts with carbohydrates
on the glycoprotein (gp)120 of HIV-1.
MBL may inhibit DC-SIGN-mediated
uptake and spread of HIV.

Dectin-1

 Antifungal innate immunity. Dectin-1 is a
specific receptor for β-glucans .
 Dectin-1 signaling has been shown to
collaborate with TLR2 signaling to
enhance the responses triggered by
each receptor

Mincle
 Is a member of the Dectin-2 family .
 Variety of exogenous and endogenous
stimuli, such as mycobacteria, certain
fungi and necrotic cells

DC-SIGN

 The recognition of several viruses (HIV-1,
HCV, dengue virus, CMV, ebola virus) and
other microbes of the Leishmania and Candida
species.
 This type II transmembrane protein has a
single C-type lectin domain and is expressed
on immature monocyte-derived DCs. DC-SIGN
modulates TLR signaling at the level of
the transcription factor NF-κB, however,
prior TLR activation of NF-κB is required.

DNGR-1

 DNGR-1 binds damaged or dead cells
via exposed actin filaments .
 DNGR-1 is therefore considered to be
DAMPs receptor since no microbial
ligand has yet been identified.

Plasmacytoid dendritic cells
 Many cell types produce small amounts of
type 1 interferons upon infection
 There is a dendritic cell subtype
(“plasmacytoid dendritic cell”; “natural
interferon-producing cell”) that produces
100-1000x more interferon upon contact with
viruses, does not need a productive infection.
 Also produces a large amount of TNF
 Recognition mechanism: probably TLR7, TLR9

Inflammasome
 Inflammasomes are large intracellular
multiprotein complexes that play a central
role in innate immunity.
 Inflammasomes comprise a member of the
NOD-like receptor (NLR) family, such as
NLRP3 and IPAF, and are defined by the NLR
protein that they contain.
 The NLR protein recruits the inflammasome-
adaptor protein ASC, which in turn interacts with
caspase-1 leading to its activation.

 Once activated, caspase-1 promotes the
maturation of the proinflammatory
cytokines interleukin (IL)-1β and IL-18.

Innate Immune Signaling and Pathogen Recognition

Innate Immune Signaling and Pathogen Recognition

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