Endogenous ligands[edit]

The stereotypic inflammatory response provoked by toll-like receptor activation has prompted
speculation that endogenous activators of toll-like receptors might participate in autoimmune
diseases. TLRs have been suspected of binding to host molecules including fibrinogen (involved
in blood clotting), heat shock proteins (HSPs), HMGB1, extracellular matrix components and self
DNA (it is normally degraded by nucleases, but under inflammatory and autoimmune conditions it
can form a complex with endogenous proteins, become resistant to these nucleases and gain
access to endosomal TLRs as TLR7 or TLR9). These endogenous ligands are usually produced as
a result of non-physiological cell death.[49]

Signaling[edit]

Signaling pathway
of toll-like receptors. Dashed grey lines represent unknown associations.
TLRs are believed to function as dimers. Though most TLRs appear to function as homodimers,
TLR2 forms heterodimers with TLR1 or TLR6, each dimer having a different ligand specificity. TLRs
may also depend on other co-receptors for full ligand sensitivity, such as in the case of TLR4's
recognition of LPS, which requires MD-2. CD14 and LPS-Binding Protein (LBP) are known to
facilitate the presentation of LPS to MD-2.
A set of endosomal TLRs comprising TLR3, TLR7, TLR8 and TLR9 recognize nucleic acid derived
from viruses as well as endogenous nucleic acids in context of pathogenic events. Activation of
these receptor leads to production of inflammatory cytokines as well as type I interferons (interferon
type I) to help fight viral infection.
The adapter proteins and kinases that mediate TLR signaling have also been targeted. In addition,
random germline mutagenesis with ENU has been used to decipher the TLR signaling pathways.
When activated, TLRs recruit adapter molecules within the cytoplasm of cells to propagate a signal.
Four adapter molecules are known to be involved in signaling. These proteins are known
as MyD88, TIRAP (also called Mal), TRIF, and TRAM (TRIF-related adaptor molecule).[50][51][52]
TLR signaling is divided into two distinct signaling pathways, the MyD88-dependent and TRIF-
dependent pathway.
MyD88-dependent pathway[edit]
The MyD88-dependent response occurs on dimerization of TLRs, and is used by every TLR except
TLR3. Its primary effect is activation of NFκB and Mitogen-activated protein kinase. Ligand binding
and conformational change that occurs in the receptor recruits the adaptor protein MyD88, a
member of the TIR family. MyD88 then recruits IRAK4, IRAK1 and IRAK2. IRAK kinases then
phosphorylate and activate the protein TRAF6, which in turn polyubiquinates the protein TAK1, as
well as itself to facilitate binding to IKK-β. On binding, TAK1 phosphorylates IKK-β, which then
phosphorylates IκB causing its degradation and allowing NFκB to diffuse into the cell nucleus and
activate transcription and consequent induction of inflammatory cytokines.[49]
TRIF-dependent pathway[edit]
Both TLR3 and TLR4 use the TRIF-dependent pathway, which is triggered by dsRNA and LPS,
respectively. For TLR3, dsRNA leads to activation of the receptor, recruiting the adaptor TRIF. TRIF
activates the kinases TBK1 and RIPK1, which creates a branch in the signaling pathway. The
TRIF/TBK1 signaling complex phosphorylates IRF3 allowing its translocation into the nucleus and
production of Interferon type I. Meanwhile, activation of RIPK1 causes the polyubiquitination and
activation of TAK1 and NFκB transcription in the same manner as the MyD88-dependent pathway. [49]
TLR signaling ultimately leads to the induction or suppression of genes that orchestrate the
inflammatory response. In all, thousands of genes are activated by TLR signaling, and collectively,
the TLRs constitute one of the most pleiotropic yet tightly regulated gateways for gene modulation.
TLR4 is the only TLR that uses all four adaptors. Complex consisting of TLR4, MD2 and LPS
recruits TIR domain-containing adaptors TIRAP and MyD88 and thus initiates activation of NFκB
(early phase) and MAPK. TLR4-MD2-LPS complex then undergoes endocytosis and in endosome it
forms a signalling complex with TRAM and TRIF adaptors. This TRIF-dependent pathway again
leads to IRF3 activation and production of type I interferons, but it also activates late-phase NFκB
activation. Both late and early phase activation of NFκB is required for production of inflammatory
cytokines.[49]

Medical relevance[edit]
Imiquimod (cardinally used in dermatology) is a TLR7 agonist, and its successor resiquimod, is a
TLR7 and TLR8 agonist.[53] Recently, resiquimod has been explored as an agent for cancer
immunotherapy,[54] acting through stimulation of tumor-associated macrophages.
Several TLR ligands are in clinical development or being tested in animal models as vaccine
adjuvants,[55] with the first clinical use in humans in a recombinant herpes zoster vaccine in 2017,
which contains a monophosphoryl lipid A component.
TLR7 messenger RNA expression levels in dairy animals in a natural outbreak of foot-and-mouth
disease have been reported.[56]
TLR4 has been shown to be important for the long-term side-effects of opioids. Its activation leads to
downstream release of inflammatory modulators including TNF-α and IL-1β, and constant low-level
release of these modulators is thought to reduce the efficacy of opioid drug treatment with time, and
is involved in opioid tolerance,[57][58] hyperalgesia and allodynia.[59][60] Morphine induced TLR4 activation
attenuates pain suppression by opioids and enhances the development of
opioid tolerance and addiction, drug abuse, and other negative side effects such as respiratory
depression and hyperalgesia.[61] Drugs that block the action of TNF-α or IL-1β have been shown to
increase the analgesic effects of opioids and reduce the development of tolerance and other side-
effects,[62][63] and this has also been demonstrated with drugs that block TLR4 itself.
The "unnatural" enantiomers of opioid drugs such as (+)-morphine and (+)-naloxone lack affinity for
opioid receptors, still produce the same activity at TLR4 as their "normal" enantiomers. [64][65] So,
"unnatural" entianomers of opioids such as (+)-naloxone, can be used to block the TLR4 activity of
opioid analgesic drugs without having any affinity for μ-opioid receptor[66][65][67]