There Are Several Classes Of Pattern Recognition Receptors
PRRs on phagocytic cells recognize and are activated by PAMPs
Because the ability to discriminate friend from foe is of paramount importance for any self‐respecting phagocyte, macrophages are fairly bristling with receptors capable of recognizing diverse PAMPs. Many of the PRRs are also expressed on DCs, NK cells, neutrophils and mast cells, as well as cells of the adaptive immune system. Several of these PRRs are lectin‐like and bind multivalently with considerable specificity to exposed microbial surface sugars with their characteristic rigid three‐dimensional geometric configurations. They do not bind appreciably to the array of galactose or sialic acid groups that are commonly the penultimate and ultimate sugars that decorate mammalian surface polysaccharides, so providing the molecular basis for discriminating between self and nonself microbial cells. Other PRRs detect nucleic acids derived from bacterial and viral genomes by virtue of modifications not commonly found within vertebrate nucleic acids or conformations not normally found in the cytoplasm (e.g., double‐stranded RNA).
PRRs are a diverse group of receptors that can be subdivided into at least five distinct families (TLRs, CTLRs, NLRs, RLRs, and scavenger receptors) based upon structural features. Another class of sensors has also emerged in recent years, the cytosolic DNA sensors (CDSs), which contains a structurally diverse set of cytosolic DNA‐sensing receptors that are pre- dominantly involved in detecting intracellular bacteria and viruses. Multiple receptors also exist in each class, with the result that in excess of 50 distinct PRRs may be expressed by a phagocyte at any given time.
Cell‐associated PRRs decode the nature of infection
As noted earlier, there are several classes of cell‐associated PRRs, some of which are plasma membrane‐associated (e.g., many of the Toll‐like receptors as well as the C‐type lectin receptors and scavenger receptors), some of which face the luminal space of endosomes (TLR3, 7, 8, 9) and some of which are cytoplasmic (NOD‐like receptors, RIG‐I‐like receptors, cytoplasmic DNA sensors). In general terms, each PRR is specific for a distinct PAMP and, combined with the different cellular compartments that PRRs reside in, this conveys considerable information concerning the nature of the pathogen and whether it is extracellular, has been captured through phagocytosis (i.e., is within endosomes) or has invaded the cytoplasm. This information helps to tailor the response towards what will be most effective for the particular class of pathogen by influencing the nature of the cytokines that are produced by the responding cell.
Engagement of several categories of PRR simultaneously may be required for effective immune responses
Although this is an area of ongoing research, combinatorial PRR signaling is probably very important for the initiation of effective immune responses. Thus, the triggering of a single type of PRR, in a DC for example, may not be fully effective for the initiation of a robust adaptive immune response, as this could indicate either a low level of infection, or that the DC is at a considerable distance from the site of infection (and has simply encountered a few stray PAMPs that have been released owing to lysis of the infectious agent). However, phagocytosis of a single bacterium by a DC is likely to stimulate multiple categories of PRR simultaneously, leading to synergistic activation of several signal transduction pathways, thereby signifying that a robust response is warranted. Furthermore, it is likely that engagement of different combinations of PRRs underpins the different types of immune response that are required to successfully contain different types of infection: intracellular, extracellular, large parasite, yeast, bacterial, viral, etc.
As we shall see throughout this book, delivery of two (or more) different signals in tandem is a common theme in immune reactions and can lead to very different outcomes compared with delivery of either signal on its own. We will now look at the various PRR families in more detail.
Toll‐like receptors (TLRs)
A major subset of the PRRs belong to the Toll‐like receptor (TLR) family, named on the basis of their similarity to the Toll receptor in the fruit fly, Drosophila. The history of the discovery of the TLR family is interesting, as it perfectly illustrates the serendipitous nature of scientific discovery and illustrates how very important findings can originate in the most unlikely places. Lipopolysaccharide (LPS, also called endotoxin), a major component of the cell walls of Gram‐ negative bacteria, was long known to provoke strong immune responses in animals and is a good example of a classical PAMP. Indeed, LPS is one of the major contributors to septic shock, the severe immune reaction that results when a bacterial infection reaches the bloodstream, and which is often fatal. For these reasons, immunologists tried to identify the LPS receptor in human and mouse for many years, largely without success. However, a major breakthrough came when the Toll receptor was found to be involved in sensing microbial infection in adult fruit flies. This in itself was quite a surprise because the Toll receptor had already been identified, many years before, as a major regulator of dorsal– ventral patterning (i.e., specifying which surface of the fly is the back and which is the underside) during early embryonic development of Drosophila. A curious fact that emerged was that the intracellular domain of Drosophila Toll contained a motif, now known as the Toll/IL‐1 receptor (TIR) signaling motif, that was very similar to the cytoplasmic signaling domain identified in the IL‐1 receptor, a molecule that was already well known to be involved in immune signaling in mammals. Putting two and two together, this led to the identification of the whole TLR family in mammals, as these receptors all possess a TIR domain within their cytoplasmic regions.
A series of TLRs have now been identified (there are 10 distinct TLRs in humans), all of which act as sensors for PAMPs (Figure 1.16). TLR ligands include peptidoglycan, lipoproteins, mycobacterial lipoarabinomannan, yeast zymosan, flagellin, microbial DNA, microbial RNAs, as well as other pathogen‐derived ligands (Table 1.1). Although many TLRs are displayed on the cell surface, some, such as TLR3 and TLR7/8/9 that are responsive to intracellular viral RNA and unmethylated bacterial DNA, are located in endosomes and become engaged upon encounter with phagocytosed material (Figure 1.16). Engagement of TLRs with their respective ligands drives activation of nuclear factor kB (NFkB) and several members of the interferon‐regulated factor (IRF) family of transcription factors, depending on the specific TLR. Combinatorial activation of TLRs is also possible, for example TLR2 is capable of responding to a wide diversity of PAMPs and typically functions within heterodimeric TLR2/TLR1 or TLR2/TLR6 complexes (Table 1.1).
All TLRs have the same basic structural features, with multiple N‐terminal leucine‐rich repeats (LRRs) arranged in a horseshoe or crescent‐shaped solenoid structure that acts as the PAMP‐binding domain (Figure 1.17). Upon binding of a PAMP, TLRs transduce signals into the cell via their TIR domains, which recruit adaptor proteins within the cytoplasm (such as MyD88) that possess similar TIR motifs. These adaptors propagate the signal downstream, culminating in activation of NFκB and interferon regulatory family (IRF) transcription factors, which regulate the transcription of a whole battery of inflammatory cytokines and chemokines (Figure 1.16 and Figure 1.18). As we will discuss later in this chapter, the IRF transcription factors control the expression of, among other things, type I interferons. The latter cytokines are especially important in defense against viral infections as they can induce the expression of a series of proteins that can interfere with viral mRNA translation and viral replication, as well as induce the degradation of viral RNA genomes.
C‐type lectin receptors (CTLRs)
Phagocytes also display another set of PRRs, the cell‐bound C‐type (calcium‐dependent) lectins, of which the macrophage mannose receptor is an example. Other members of this diverse and large family include Dectin‐1, Dectin‐2, Mincle, DC‐ SIGN, Clec9a, and numerous others. These transmembrane proteins possess multiple carbohydrate recognition domains whose engagement with their cognate microbial PAMPs generates intracellular activation signals through a variety of signaling pathways. However, some C‐type lectin receptors (CTLRs) do not trigger robust transcriptional responses and function primarily as phagocytic receptors. The CTLR family is highly diverse and the ligands for many receptors in this category are the subject of ongoing research. But it can be said that members of the CTLR family broadly serve as sensors for extracellular fungal species. Some examples of ligands for CTLRs include β‐glucans (which binds Dectin‐1), mannose (which binds Dectin‐2), and α NOD‐like receptors (NLRs)
Turning now to the sensing of infectious agents that have succeeded in gaining access to the interior of a cell, microbial products can be recognized by the so‐called NOD‐like receptors (NLRs). Unlike TLRs and CTLRs that reside within the plasma membrane or intracellular membrane compartments, NLRs are soluble proteins that reside in the cytoplasm, where they also act as receptors for PAMPs. Although a diverse family of receptors (Figure 1.19), NLRs typically contain an N‐terminal protein–protein interaction motif that enables these proteins to recruit proteases or kinases upon activation, followed by a central oligomerization domain and multiple C‐terminal leucine‐rich repeats (LRRs) that act as the sensor for pathogen products (Figure 1.19). The NLRs can be subdivided into four subfamilies on the basis of the motifs present at their N‐termini. NLRs are thought to exist in an autoinhibited state with their N‐terminal domains folded back upon their C‐terminal LRRs, a conformation that prevents the N‐terminal region from interacting with its binding partners in the cytoplasm. Activation of these receptors is most likely triggered through direct binding of a PAMP to the C‐terminal LRRs which has the effect of disrupting the interaction between the N‐ and C‐ termini of the NLR and permits oligomerization into a complex that is now capable of recruiting either an NFkB‐activating kinase (such as RIP‐2) or members of the caspase family of proteases that can proteolytically process and activate the IL‐1β precursor into the mature, biologically active cytokine.
A very well‐studied NLR complex, called the inflammasome, is assembled from NLRP3 in response to LPS in combination with bacterial virulence factors, and is important for the production of IL‐1β as well as IL‐18. However, for full activation of the inflammasome and liberation of IL‐1β, a second signal in the form of a membrane‐damaging bacterial toxin (which can also be mimicked by a variety of noxious agents) is required. This second signal appears to permit the efflux of K+ ions from the cytosol, which permits full assembly of the inflammasome, caspase‐1 activation, and processing of IL‐1β and IL‐18 downstream (Figure 1.20).
RIG‐I‐like helicase receptors (RLRs)
The RIG‐I‐like helicases are a relatively recently discovered family that act as intracellular sensors for viral‐derived RNA (Figure 1.21). Similar to the NLRs, RIG‐I‐like helicase receptors (RLRs) are found in the cytoplasm and are activated in response to double‐stranded RNA and are capable of directing the activation of NFkB and IRF3/4 that cooperatively induce antiviral type I interferons (IFNα and β). RIG‐I (retinoic acid‐ inducible gene I) and the related MDA‐5 (also called Helicard) protein can directly bind to different forms of viral RNA (either unmodified 5′‐triphosphate ssRNA or dsRNA, respectively) in the cytoplasm, followed by propagation of their signals via MAVS (mitochondrial‐associated viral sensor), again leading to activation of IRFs and NFkB (Figure 1.22).
Cytosolic DNA sensors
A number of proteins belonging to different families are capable of sensing cytosolic DNA or cyclic dinucleotides. Host cell DNA is normally sequestered safely in the nuclear or mitochondrial compartments and cannot trigger these sensors, except under pathological conditions that involve release of mitochondrial DNA into the cytosol, for example. However, bacterial or viral DNA can trigger the activation of the AIM2 or IFI16 DNA sensors and this can lead to assembly of a complex involving the Pyrin‐domain‐containing adaptor (ASC), leading to activation of caspase‐1 and IL‐1β Activation of the AIM2 inflammasome can also lead to death of the cell. IFI16 can also recognize cytosolic DNA and can either propagate signaling by forming a complex with ASC and caspase‐1, similar to the AIM2 inflammasone, or via STING, which is discussed below. Two additional DNA‐sensing path- ways have also been discovered very recently and both make use of STING (stimulator of interferon genes) a molecule that can either directly bind to cytoplasmic DNA or can respond to cyclic GAMP, a molecule that is generated by an upstream enzyme called cGAS, which detects cytoplasmic DNA and synthesizes cGAMP in response (Figure 1.23). In response to STING activation, type I IFNs are generated which have potent antiviral properties.
Scavenger receptors represent yet a further class of phagocytic receptors that recognize a variety of anionic polymers and acetylated low‐density proteins. The role of the CD14 scavenger molecule in the handling of Gram‐negative LPS (lipopolysaccharide endotoxin) merits some attention, as failure to do so can result in septic shock. The biologically reactive lipid A moiety of LPS is recognized by a plasma LPS‐binding protein, and the complex that is captured by the CD14 scavenger molecule on the phagocytic cell then activates TLR4. However, unlike the PRRs discussed above, engagement of scavenger receptors are typically insufficient on their own to initiate cytokine activation cascades.
PRR engagement results in cell activation and proinflammatory cytokine production
Upon encountering ligands of any of the aforementioned PRRs, the end result is a switch in cell behavior from a quiescent state to an activated one. Activated macrophages and neutrophils are capable of phagocytosing particles that engage their PRRs and, as we have seen from our discussion of the various classes of PRRs, upon engagement of the latter they also release a range of cytokines and chemokines that amplify the immune response further (see Figure 1.12). As the reader will no doubt have noticed, engagement of many of the above PPRs results in a signal transduction cascade culminating in activation of NFkB, a transcription factor that controls the expression of numerous immunologically important molecules such as cytokines and chemokines. In resting cells, NFκB is sequestered in the cytoplasm by its inhibitor IkB, which masks a nuclear localization signal on the former. Upon binding of a PAMP to its cognate PRR, NFκB is liberated from IκB because of the actions of a kinase that phosphorylates IκB and promotes its destruction. NFκB is now free to translocate to the nucleus, seek out its target genes, and initiate transcription (see Figure 1.18).
Some of the most important inflammatory mediators synthesized and released in response to PRR engagement include the antiviral interferons (also called type I interferons), the small protein cytokines IL‐1β, IL‐6, IL‐12, and tumor necrosis factor α (TNFα), which activate other cells through binding to specific receptors, and chemokines, such as IL‐8, which represent a subset of chemoattractant cytokines. Collectively, these molecules amplify the immune response further and have effects on the local blood capillaries that permit extravasation of neutrophils, which come rushing into the tissue to assist the macrophage in dealing with the situation (see Figure 1.15).
Dying cells also release molecules capable of engaging PRRs
As we have mentioned earlier, cells undergoing necrosis (but not apoptosis) are also capable of releasing molecules (i.e., DAMPs) that are capable of engaging PRRs (see Figure 1.3). The identity of these molecules is only slowly emerging, but includes HMGB1, members of the S100 calcium‐binding protein family, HSP60 and the classical cytokines IL‐1α and IL‐33. Certain DAMPs appear to be able to bind to members of the TLR family (i.e., HMGB1 has been suggested to signal via TLR4), while others such as IL‐1α and IL‐33 bind to specific cell surface receptors that possess similar intracellular signaling motifs to the TLR receptors.
DAMPs are involved in amplifying immune responses to infectious agents that provoke cell death and also play a role in the phenomenon of sterile injury, where an immune response occurs in the absence of any discernable infectious agent (e.g., the bruising that occurs in response to a compression injury that does not breach the skin barrier represents an innate immune response). Indeed, Polly Matzinger has proposed that robust immune responses are only seen when nonself is detected in combination with tissue damage (i.e., a source of DAMPs). The thinking here is that the immune system does not need to respond if an infectious agent is not causing any harm. Thus, PAMPs and DAMPs may act synergistically to provoke more robust and effective immune responses than would occur in response to either alone.