So far in this series, we’ve talked about various aspects of innate immunity,
but we still have a lot to discuss regarding the actual pathways by which threats are detected by
immune cells. So let’s do a deep dive into pattern recognition receptors. These are protein sensors
in and on cells that detect evidence of infection or tissue damage, which then launch a signaling
cascade designed to deal with the threat. We’ve introduced the general concept of these pathogen
and danger-sensing molecules before, so in this tutorial, we are going to learn precisely what
these molecules sense, and how they translate detection into immunological action. As we
already know, the signals that pattern recognition receptors sense are called pathogen-associated
molecular patterns or PAMPs, and damage-associated molecular patterns, or DAMPs. PAMPs are specific
molecular sequences or patterns that are only found in pathogens; for example,
components of bacterial cell walls or unique forms of nucleic acids that are found in viral genomes,
like single-stranded DNA or double-stranded RNA. PAMPs also tend to be highly conserved,
meaning that many different pathogens have them, and as we have already discussed,
they tend to be molecules that a pathogen needs in order to survive. This prevents a pathogen
from evolving away from innate immune recognition. DAMPs, on the other hand, are host molecules that
appear in the wrong place at the wrong time. Think about ATP. ATP is all over the place inside cells,
but it is rarely found outside of cells. If a cell senses extracellular ATP, it suggests that
there is a damaged cell nearby, and in this case, the extracellular ATP would be acting as a DAMP.
Not only is the location of PAMPs and DAMPs important for immune signaling,
but the location of pattern recognition receptors is also extremely important.
Some are on the surface of cells, and can sense extracellular PAMPs and DAMPs. Others are
cytosolic for intracellular sensing, and still others can be found in endosomes,
an organelle formed during endocytosis. Endosomal pattern recognition receptors
sense PAMPs or DAMPs that have been endocytosed from the extracellular space. The distribution of
membrane-bound pattern recognition receptors can be extremely polarized. For example,
intestinal epithelial cells are constantly exposed to the microbes in the intestinal microbiome. If
they were constantly sensing the extracellular bacteria in the gut, the intestine would always be
inflamed. Instead, many of these bacterial-sensing pattern recognition receptors are only located on
the basolateral side of the cell, which is the side not facing the inside of the gut.
In this way, these cells will only sense bacteria that have crossed the epithelial barrier and
that need to be contained by the immune system. One of the most well-studied families of pattern
recognition receptors are the Toll-like receptors or TLRs. These receptors are homologs of a fruit
fly protein called Toll, which is involved in defense against bacterial and fungal pathogens.
TLR homologs can be found in many mammals, other invertebrates, and even plants,
which tells us that this family of sensors is very ancient in an evolutionary context.
Let’s take a look at the structure of a TLR. TLRs are single-pass transmembrane proteins,
meaning that part of the protein passes through a lipid membrane one time. The extracellular
domain of a TLR has leucine-rich repeats that form a horseshoe or C-shape, and this is the
part of the protein that binds to ligands. The intracellular side has a distinct region called
a TIR domain. This region is special, because it can interact with TIR domains on other proteins
that are essential for signal transduction. When a TLR binds its ligand, it either homo-dimerizes,
or forms a heterodimer with other TLRs. Dimerization brings together cytoplasmic
tails of the receptors, which allows for docking of TIR domain-containing adaptor proteins MyD88,
TRIF, Mal, and TRAM, with each adaptor having different endpoints to their signaling cascades.
Let’s discuss the differences between the signaling cascades
of the two major adaptor proteins, MyD88 and TRIF. The MyD88 pathway begins when the cytoplasmic
tails of dimerized TLRs are brought together, allowing MyD88 to bind. Here,
MyD88 recruits the kinases IRAK4, IRAK1, and IRAK2. The IRAKs phosphorylate and activate TRAF6.
TRAF6 is an E3 ubiquitin ligase, which is a type of enzyme that tags other proteins with ubiquitin
groups. TRAF6 polyubiquinates itself and the protein NEMO, which recruits and activates TAK1.
TAK1 then phosphorylates and activates the IKK complex. Activated IKK phosphorylates IκB,
which leads to the degradation of IκB. Normally, IκB is bound to a protein called NF-κB
in the cytosol. However, when IκB gets degraded, NF-κB translocates to the nucleus,
where it acts as a transcription factor for inflammatory cytokines such as TNF-a and IL-6.
TRIF is the other main adaptor protein for TLRs. TRIF recruits TRAF6 and TRAF3.
TRAF6 recruits RIP-1, which activates TAK1. From here,
the pathway looks similar to the MyD88 pathway, and results in NF-kB activation.
TRAF3, on the other hand, recruits TBK1 and IKKi. Together, these phosphorylate and activate IRF3.
Activated IRF3 forms a dimer and then moves into the nucleus, where it drives expression of type I
interferons, which are cytokines that are critical for antiviral responses.
Different TLRs use different adaptors or combinations of adaptors, which allows the cell to
tailor its response to the type of threat at hand. Now that we’ve talked generally about how TLRs
work, let’s go through some of the most well-studied TLRs, to get an idea of what
kinds of PAMPs they recognize, and to help build our familiarity with these receptors.
First, TLR-2 heterodimerizes with TLR-1 or TLR-6 to bind bacterial lipoproteins and lipoteichoic
acid, a common component of Gram-positive bacterial cell walls. TLR-3 is located on
the inner surface of the endosome. It recognizes double-stranded RNA, which is a feature of many
viral genomes. Because TLR-3 is located in the endosome and not the cytosol, it can’t directly
sense intracellular viral infection; however, it can sense extracellular viruses that have been
endocytosed, or viruses that have infected a neighboring cell that gets phagocytosed.
TLR-4 is one of the most well-studied of the mammalian TLRs, and it senses lipopolysaccharide,
or LPS, which is found in the outer cell membrane of Gram-negative bacteria. It is unique in that
it is the only known TLR to use all four adaptor proteins, and it uses an accessory protein called
MD-2 to sense LPS. TLR-5 is expressed on the surface of myeloid cells and intestinal epithelial
cells, and binds to bacterial flagellin. Flagellin is a protein that is the main subunit of flagella,
the whip-like structures many bacteria use to move around. Like TLR-3, TLR-7 is also an endosomal
PRR, and it binds to single-stranded RNA. Now of course, you might be thinking: “Mammalian cells
have single-stranded RNA too!” This is absolutely true, and this is where it’s important to remember
that TLR location is key. Mammalian cells do have single-stranded RNA in the nucleus
and the cytoplasm, but not in the endosome. If there is single-stranded RNA in the endosome,
it’s usually the result of phagocytosing single-stranded RNA viral particles. Finally,
TLR-9 is another endosomal sensor, and it recognizes what is called unmethylated CpG
DNA. CpG DNA is a sequence involving cytosine and guanine being adjacent to each other,
with the cytosine being at the 5’ end. In mammals, this sequence is heavily covered in methyl groups,
but not in bacterial or some viral DNA, hence the term “unmethylated.”
There are a few other TLRs that are common in mammals, but we’ll stop here for now.
TLRs are not the only pattern recognition receptors used by mammalian cells. While TLRs
are confined to lipid membranes, there is another family of pattern recognition receptors that can
sense microbial products in host cell cytoplasm. These receptors are called NOD-like receptors,
or NLRs. NLRs are particularly good at sensing intracellular bacteria, and are often expressed
in epithelial cells and myeloid cells. Like TLRs, NLRs are also ancient pathogen sensors with
leucine rich repeats. However, instead of having a TIR domain, they have an amino-terminal CARD
domain, which is a docking site for other proteins with CARD domains. NLRs recognize components of
peptidoglycan from both Gram-positive and Gram-negative bacteria. When the NLR senses
its target, a protein called RIPK2 binds to the NLR’s CARD domain. This leads to downstream
activation of TAK1, IKK, and then NF-κB, similar to the TRIF-dependent pathway of TLR signaling.
So if NLRs can sense intracellular bacteria and TLRs 3, 7, and 9 can sense endocytosed viruses,
how do cells sense intracellular viruses? For this, they use the RIG-I-like helicases.
RIG-I-like helicases sense viral nucleic acids.
They have a helicase-like domain that can bind to viral RNA and not one,
but two amino terminal CARD domains that are used for signal transduction. Some examples
of RIG-I-like helicases include RIG-I, which can specifically sense viral single-stranded RNA,
and MDA-5, which senses cytosolic double-stranded DNA. Activation of the RIG-I like helicases
leads to production of type I interferons, which are important antiviral cytokines.
So we’ve discussed the TLRs, NLRs, and RIG-I-like helicases, which all sense PAMPs, but what about
sensing of DAMPs? There are also some dedicated DAMP receptors. The receptor P2X7 senses
extracellular ATP. RAGE is a receptor that senses HMGB1, which is a DNA-binding protein that should
only be outside the nucleus but within the cell. Once it is extracellular, it becomes a DAMP. RAGE
also senses a family of cytosolic calcium-binding proteins called the S100s. DAMP sensing generally
leads to pro-inflammatory signaling cascades similar to the ones we’ve already discussed.
Interestingly, many of the PRRs we’ve already discussed can also recognize DAMPs. For example,
TLR 4 can also sense HMGB1, the S100 proteins, and many other DAMPs. The broad range of PAMP and DAMP
recognition is one of the features that allows the innate immune system to respond quickly to
threats. Later on, when we talk about the adaptive immune system, we’ll learn about the benefits of
antigen specific responses. But for now, let’s continue learning about the innate immune system.