Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single-spanning receptors usually expressed on sentinel cells such as macrophages and that recognize structurally conserved molecules derived from microbes. TLRs are pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are molecular structures associated with pathogens, such as bacteria, viruses, and fungi, that are recognized by the innate immune system. DAMPs are molecules that are released into the extracellular space when cells are injured or damaged. TLRs play a crucial role in the recognition of PAMPs and DAMPs and the initiation of immune responses, such as the production of pro-inflammatory cytokines, type I interferons, and other molecules that enhance the immune response. TLRs are a bridge between the innate and adaptive immune systems by regulating the activation of antigen-presenting cells and key cytokines. Upon recognition of their specific ligands, TLRs initiate downstream signaling cascades, leading to the production of pro-inflammatory and antiviral factors and the upregulation of co-stimulatory molecules, promoting the maturation of antigen-presenting cells and linking innate immunity to adaptive immunity. TLRs are widely distributed in both immune and other body cells and are a critical target for the development of immunotherapies and vaccines. Further research is needed to fully understand the .mechanisms underlying TLR signaling and its potential applications in the field of immunology.Toll-like receptors (TLRs) are a bridge between the innate and adaptive immune systems. TLRs are expressed on all innate immune cells and a large majority of non-hematopoietic cells, such as macrophages, neutrophils, dendritic cells, natural killer cells, mast cells, basophils, eosinophils, and epithelial cells. Importantly, TLRs can also be detected on adaptive immune cells, including T and B cells. Adaptive immunity consists of humoral immunity and cell-mediated immunity, which are mainly mediated by B lymphocytes and T lymphocytes, respectively. TLRs critically link innate immunity to adaptive immunity by regulating the activation of antigen-presenting cells and key cytokines. Upon recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by TLRs, downstream signaling cascades are initiated, leading to the production of pro-inflammatory cytokines, such as IL-6 and INF-α, and the upregulation of co-stimulatory molecules, promoting the maturation of antigen-presenting cells and linking innate immunity to adaptive immunity. TLR signaling is also being studied for its direct regulatory roles in effector T cells and regulatory T cells, as well as its involvement in various diseases, including infectious diseases, autoimmune conditions, and cancer.

1. Presentation Title:
Toll-like Receptors &
Signaling
Presented By:
Saleem Ahmad,
BS-BC-09-F23
Presented To:
Dr. Shehbaz
Date:
Jan 8th, 2023

2. • IMMUNITY
• Our immune host defenses can
be divided into two major
categories:
• innate (natural) and
• adaptive (acquired).
• Innate Immunity
• Properties of Innate Immunity
• Innate immunity is resistance
that exists prior to exposure to
the microbe (antigen).
• It is nonspecific and includes
host defenses such as barriers
to infectious agents (e.g., skin
and mucous membranes),
certain cells (e.g., natural killer
cells), and certain proteins (e.g.,
the complement cascade and
interferons) and involves
processes such as phagocytosis
and inflammation .

3. Innate immunity does not improve after
exposure to the organism, in contrast to
acquired immunity, which does.
In addition, innate immune processes have
no memory, whereas acquired
immunity is characterized by long-term
memory.
Note that the innate arm of our host defenses
performs two major functions: killing invading
microbes and activating adaptive immune
processes.
Some components of the innate arm, such as
neutrophils, only kill microbes, whereas
others, such as macrophages and dendritic
cells, perform both functions (i.e., they kill
microbes and present antigen to helper T
cells, which activates adaptive immune
processes).
Although innate immunity is often successful
in eliminating microbes and preventing
infectious diseases, it is not sufficient for
human survival. This conclusion is based on
the observation that children with severe
combined immunodeficiency disease (SCID),
who have intact innate immunity but no
adaptive immunity, suffer from repeated, life-
threatening infections.

4. Our
understanding
of Innate
Immune
System

5. How does Immune system(Cells)
Communicate/Recognise?
• Communication through chemical signal
• In and out transfer by Pagocytosis,Transporst systems,diffusions,etc.
• Doors/check points which recognize and communicate with
environment
• These check points are Called Receptors
• Signals also sensed by Receptors
• Pagocytosis is also mediated by receptors

6. Pattern Recognition Receptors Activate Responses to Microbes and Cell Damage
Several families of cellular PRRs contribute to the activation of innate immune responses
that combat infections.
Some of these PRRs are expressed on the plasma membrane, while others are actually
found inside our cells, either in endosomes/lysosomes or in the cytosol.
Th is array of PRRs ensures that the cell can recognize PAMPs on both extracellular and
intracellular pathogens.
DAMPs released by cell and tissue damage also can be recognized by both cell surface and
intracellular PRRs.
Many cell types in the body express these PRRs,
I. including all types of myeloid white blood cells (monocytes, macrophages, neutrophils,
eosinophils, mast cells, basophils, dendritic cells) and
II. subsets of the three types of lymphocytes (B cells, T cells, and NK cells).
III. PRRs are also expressed by some other cell types, especially those commonly exposed to
infectious agents; examples include the skin, mucosal and glandular epithelial cells,
vascular endothelial cells that line the blood vessels, and fi broblasts and stromal support
cells in various tissues.

7. Pathogen-associated molecular patterns (PAMPs)
 are molecular structures associated with pathogens, such as
bacteria, viruses, and fungi, that are recognized by the innate
immune system.
 PAMPs are typically conserved structures or products that are
not self-present in the host organism and can be detected by
the host's pattern recognition receptors (PRRs).
 These molecules help the immune system distinguish between
foreign pathogens and self-components, allowing for an
appropriate immune response.
 Examples of PAMPs include
i. lipopolysaccharides (LPS) in gram-negative bacteria,
ii. flagellin in motile bacteria, and
iii. double-stranded RNA in viruses.

8. Damage-associated molecular patterns (DAMPs)
 are molecules that are released into the extracellular space when cells
are injured or damaged.
 They are recognized by the innate immune system, similar to pathogen-
associated molecular patterns (PAMPs), and can be detected by the
host's Toll-like receptors (TLRs) and other pattern recognition receptors
(PRRs).
 DAMPs help the immune system distinguish between foreign pathogens
and self-components, allowing for an appropriate immune response.
 Examples of DAMPs include
i. high-mobility group box 1 (HMGB1),
ii. interferon-alpha inducible factor 16 (IAF16), and
iii. extracellular polymeric matrix proteins (e.g., hyaline keratin and
fibrinogen).
The recognition of DAMPs and PAMPs by PRRs is crucial for the initiation

9. How does Immune system recognize Pathogens?
The immune system relies on a complex network of
receptors to recognize and respond to pathogens
and other foreign entities.
PRRs as key Element in recognition
Pattern recognition receptors (PRRs) are a key
element of the immune system, including Toll-like
receptors (TLRs), RIG-I-like receptors, Nod-like
receptors (NLRs), AIM2-like receptors, C-type lectin
receptors, and intracellular DNA and RNA sensors.
These receptors play a critical role in the recognition
of pathogen-associated molecular patterns (PAMPs)
as well as endogenous ligands released from
damaged cells (damage-associated molecular
patterns or DAMPs).
• Mutations in the genes encoding these pattern
receptors result in a failure to recognize the
pathogen and predispose to severe bacterial,
viral, and fungal infections.
• The most important of these pattern-recognition
receptors are the Toll-like receptors (TLR).

10. • Types of Receptors in Innate
Immunity:
• Toll-like Receptors (TLRs):
• Function: TLRs recognize
specific molecular patterns
associated with pathogens,
known as pathogen-associated
molecular patterns (PAMPs).
These patterns are common to
many microorganisms but not
found in the host.
• Location: TLRs are present on
the surface of cells and within
endosomes.
• Activation: Binding of TLRs to
PAMPs triggers signaling
cascades that lead to the
production of cytokines and
other immune response
mediators.

11. • C-type Lectin Receptors
(CLRs):
• Function: CLRs
recognize carbohydrate
structures on the
surface of pathogens.
• Location: Found on the
surface of dendritic
cells, macrophages,
and other immune
cells.
• Activation: Binding of
CLRs to pathogens
triggers phagocytosis
and activates immune
responses.

12. • Scavenger Receptors:
• Function: Scavenger receptors recognize and bind to a variety of ligands, including
modified or damaged host cells and microbial pathogens.
• Location: Present on the surface of macrophages and other phagocytic cells.
• Activation: Engagement of scavenger receptors leads to the clearance of cellular
debris and pathogens.

13. • Complement Receptors:
• Function: Complement receptors
recognize complement proteins
that are activated during the
complement cascade.
• Location: Found on the surface of
phagocytic cells.
• Activation: Binding to complement
proteins enhances phagocytosis
and the clearance of pathogens.

14. • RIG-I-like Receptors (RLRs):
• Function: RLRs detect viral
RNA in the cytoplasm of
infected cells.
• Location: Present in the
cytoplasm.
• Activation: Binding to viral
RNA activates RLRs, leading to
the production of interferons
and other antiviral molecules.

15. • Nucleotide-binding
Oligomerization Domain (NOD)-like
Receptors (NLRs):
• Function: NLRs are
intracellular receptors that
recognize PAMPs and danger-
associated molecular patterns
(DAMPs) released by stressed
or damaged cells.
• Location: Found in the
cytoplasm of cells.
• Activation: Activation of NLRs
leads to the formation of
inflammasomes, which initiate
inflammatory responses and
the processing of pro-
inflammatory cytokines like IL-
1β.

16. Table: Types of Receptors in Innate Immunity
Receptor Type Function Location Activation
Toll-like Receptors
(TLRs)
Recognize PAMPs;
initiate immune
responses
Surface of cells; within
endosomes
Binding to PAMPs triggers signaling
cascades, leading to the production of
cytokines and immune responses.
NOD-like Receptors
(NLRs)
Recognize PAMPs and
DAMPs; form
inflammasomes
Cytoplasm
Activation leads to the formation of
inflammasomes, initiating
inflammatory responses.
RIG-I-like Receptors
(RLRs)
Detect viral RNA in
infected cells
Cytoplasm
Binding to viral RNA activates RLRs,
leading to the production of interferons
and antiviral molecules.
C-type Lectin
Receptors (CLRs)
Recognize carbohydrate
structures on pathogens
Surface of dendritic
cells, macrophages
Binding to pathogens triggers
phagocytosis and immune responses.
Scavenger Receptors
Recognize a variety of
ligands
Surface of macrophages
and phagocytic cells
Engagement leads to the clearance of
cellular debris and pathogens.
Complement
Receptors
Recognize activated
complement proteins
Surface of phagocytic
cells
Binding enhances phagocytosis and the
clearance of pathogens.

17. Toll-like receptors (TLR)
• This is a family of 10 to 13 receptors found
mainly on the surface of three types of cells:
• macrophages,
• dendritic cells, and
• mast cells.
• Monocytes
• Etc.

18. • Intensive work over the last decade and a half has identifi ed
multiple TLR family members in mice and humans—as of 2011,
13 TLRs that function as PRRs have been identifi ed in these
species.
• TLRs 1-10 are conserved between mice and humans, although
TLR10 is not functional in mice, while TLRs 11-13 are expressed
in mice but not in humans.
• While TLRs have not been shown to be involved in vertebrate
development, unlike in fruit fl ies, the set of TLRs present in a
human or mouse can detect a wide variety of PAMPs from
bacteria, viruses, fungi, and parasites, as well as DAMPs from
damaged cells and tissues.
• Each TLR has a distinct repertoire of specifi cities for conserved
PAMPs;

19. • TLRs exist both on the
• plasma membrane and
• in the membranes of endosomes and lysosomes;
• Their cellular location is tailored to enable them to
respond optimally to the particular microbial ligands
they recognize.
• TLRs that recognize PAMPs on the outer surfaces of
extracellular microbes (see Table 5-4) are found on
the plasma membrane, where they can bind these
PAMPs and induce responses.
• In contrast, TLRs that recognize internal microbial
components that have to be exposed by the
dismantling or degradation of endocytosed
pathogens— nucleic acids in particular—are found
in endosomes and lysosomes.
• Interestingly:
• Unique among the TLRs, TLR4 has been shown to
move from the plasma membrane to endosomes/
lysosomes after binding LPS or other PAMPs. As we
will see below, it activates different signaling
pathways from the two locations

20. • TLRs recognize various microbial components
and then activate transcription factors that
enhance the synthesis of several
proinflammatory cytokines. This initiates an
immune response appropriate to defend against
that type of microbe.
• Note that the type of host defense mounted
by the body differs depending on the type of
organism.
• For example, a humoral (antibody-mediated)
response is produced against one type of
bacteria, but a cell-mediated response occurs in
response to a different type of bacteria.
• The process that determines the type of
response depends on the cytokines produced by
the macrophages, and this in turn depends on
which pattern-recognition receptor is activated
by the organism, as described in the next
paragraph. Some important examples of this
pattern recognition are as follows

21. TLR1:
Recognition:
Recognizes
bacterial
lipopeptides.
Association: Often
forms
heterodimers with
TLR2.
TLR2:
Recognition:
Recognizes a
variety of PAMPs,
including bacterial
lipoproteins,
lipoteichoic acid,
and
peptidoglycan.
Association:
Forms
heterodimers with
TLR1 or TLR6.
TLR3:
Recognition:
Recognizes
double-stranded
RNA, a common
viral product.
Location: Found in
endosomes.
TLR4:
Recognition:
Recognizes
lipopolysaccharide
(LPS), a
component of the
outer membrane
of Gram-negative
bacteria.
Association:
Interacts with
accessory proteins
MD-2 and CD14.
TLR5:
Recognition:
Recognizes
bacterial flagellin,
a protein in
bacterial flagella.

22. TLR6:
• Recognition:
Recognizes
diacylated
lipopeptides,
often in
conjunction
with TLR2.
• Association:
Forms
heterodimers
with TLR2.
TLR7:
• Recognition:
Recognizes
single-
stranded RNA,
typically
found in
viruses.
• Location:
Found in
endosomes.
TLR8:
• Recognition:
Recognizes
single-
stranded RNA,
similar to
TLR7.
• Location:
Found in
endosomes.
TLR9:
• Recognition:
Recognizes
unmethylated
CpG motifs
found in
bacterial and
viral DNA.
• Location:
Found in
endosomes.
TLR10:
• Function: Its
exact function
is still under
investigation,
and its ligands
are not well
defined.

24. Structure of TLRs
• Biochemical studies have
revealed the structure of
several TLRs and how they bind
their specific ligands.
• TLRs are membrane spanning
proteins that share a common
structural element in their
extracellular region called
leucine-rich repeats (LRRs);
multiple LRRs make up the
horseshoe-shaped extracellular
ligand-binding domain of the
TLR polypeptide chain

25. Cont…..
• When TLRs bind their PAMP or DAMP ligands via
their extracellular LRR domains, they are induced to
dimerize.
• In most cases each TLR dimerizes with itself,
forming a homodimer, but TLR2 forms heterodimers
by pairing with either TLR1 or TLR6.
• How TLRs bind their ligands was not known until
complexes of the extracellular LRR domain dimers
with bound ligands were analyzed by x-ray
crystallography.
• Structures of TLR2/1 with a bound lipopeptide and
TLR3 with dsRNA are shown in Figure ; the
characteristic “m”-shaped conformation of TLR
dimers is apparent

26. • MODE OF ACTION of Toll-like Receptors:
• Upon recognition of PAMPs, TLRs initiate
intracellular signaling cascades that lead to
the activation of transcription factors, such as
NF-κB and IRFs (interferon regulatory factors).
• Activation of these transcription factors
results in the production of pro-inflammatory
cytokines, type I interferons, and other
molecules that enhance the immune
response.
• TLR activation also upregulates the
expression of co-stimulatory molecules,
promoting the maturation of antigen-
presenting cells.

27. Signaling Through TLRs:
•The innate immune system recognizes a diverse range of pathogens.
•TLRs play a key role in pathogen recognition and initiating appropriate immune
responses.
•Signaling through TLRs involves shared and unique components in downstream
pathways
•Detailed studies of TLR signaling pathways reveal shared components and gene
activation.
•NF-κB is a shared transcription factor inducing innate and inflammatory genes.
•TLRs activate expression of genes encoding defensins, enzymes, chemokines, and
cytokines.
•Combinations of transcription factors contribute to gene expression.
•TLR activation is determined by the TLR and the initial adaptor binding to the
TLR's cytoplasmic domain.

28. MyD88-
Dependent
Signaling
Pathways
• MyD88 initiates signaling activating NF-κB and MAPK
pathways.
• MyD88 recruits and activates IRAK protein kinases, leading
to TRAF6 activation.
• TRAF6 ubiquitinates proteins leading to TAK1 activation and
downstream signaling.
• Endosomal TLRs 7, 8, and 9 trigger pathways activating IRFs
(especially IRF7).
• MyD88/IRAK4/TRAF6 complex activates TRAF3, IRAK1, and
IKKε, leading to IRF7 activation.
• IRF7 induces transcription of Type I interferons with potent
antiviral activity.

30. TRIF-Dependent
Signaling Pathways
• TLR3 and endosomal TLR4 recruit TRIF
instead of MyD88, leading to distinct
pathways.
• TRIF recruits RIP1 kinase, activating TRAF6
and initiating similar steps as MyD88-
dependent pathway.
• TRIF also activates PI3K, enhancing MAPK
pathway activation.
• TRIF activates TRAF3, TBK-1, and IKKε,
phosphorylating and activating IRF7 and
IRF3.
• IRF3 and IRF7 dimerize, move into the
nucleus, and induce transcription of IFN-α
and -β genes.
• Intracellular TLRs induce synthesis and
secretion of Type I interferons, inhibiting
virus replication.

33. TLRs as a Bridge Between Innate and
Adaptive Immunity
•Toll-like receptors (TLRs) serve as
a crucial link between the innate
and adaptive immune systems.
•TLRs are expressed on diverse
immune cells, including
macrophages, dendritic cells,
natural killer cells, T and B cells, as
well as non-hematopoietic cells.
•Recognition of pathogen-
associated molecular patterns
(PAMPs) or damage-associated
molecular patterns (DAMPs) by
TLRs initiates signaling cascades.
•TLR signaling regulates antigen-
presenting cells and cytokine
production, crucial for linking innate and
adaptive immunity.
•Activation of TLRs leads to the production
of pro-inflammatory cytokines (e.g., IL-6,
INF-α) and upregulation of co-stimulatory
molecules.
•TLRs play direct regulatory roles in
effector T cells and regulatory T cells.
•Ongoing research explores TLR signaling
in various diseases, including infections,
autoimmune conditions, and cancer,

35. DO you think you’re more interested in knowing more about “How TLRs
contribute to Adaptive immunity and it’s application?
Encouraged to read following:
Flagellin A Toll-Like Receptor 5 Agonist as an Adjuvant in Chicken Vaccines
•March 2021
•Clinical and vaccine Immunology: CVI 21(3):261-270
DOI:10.1128/CVI.00669-13
.
Toll-Like Receptor-Based Strategies for Cancer Immunotherapy
J Immunol Res. 2021; 2021: 9912188.
Published online 2021 May 22. doi: 10.1155/2021/9912188
PMCID: PMC8166496
PMID: 34124272
Toll-Like Receptors in Adaptive Immunity Vijay Kumar PMID: 34510306 DOI: 10.1007/164_2021_543
Toll-like receptor control of the adaptive immune responses
•Akiko Iwasaki &
•Ruslan Medzhitov
Nature Immunology volume 5, pages987–995 (2004)

36. • That would be all.......................
• Thank you for your time.
&
• Thanks for Listening..........