Transcript of Lecture 2b: Innate Immune Receptors and Signaling

hi everyone this is lecture 2b for applied immunology today we'll be learning about innate immune receptors and signaling modules of the innate immune system these are broadly referred to as pattern recognition receptors or prrs we'll discuss different categories of prrs how their localization correlates with pathogen niches within a cell and which signaling modules are engaged downstream of prr activation we'll get into a fair amount of immunology definitions in jargon today so i want to briefly revisit this conceptual schematic from week one as we learn about different types and functions of pattern recognition receptors please try to focus on thinking about how each of these contribute to the central goals of a successful immune response now first given a massive diversity of potential pathogens a given innate immune cell needs to be able to recognize or detect different types of microbes which is accomplished through pattern recognition receptors next the innate immune cell needs to be able to engage transcriptional programs or other effector programs in response to infection that allow it to assume a cellular activation state where effector molecules are expressed lastly the expression of these effector molecules constitute any of a number of mechanisms that can be deployed in order to destroy pathogens these can consist of direct cytotoxic mechanisms such as reactive oxygen species and antimicrobial peptide production that we learned about in the last lecture as well as indirect mechanisms that function by communicating information about an infection to another cell an example of this is the cytokine and chemokine production that are used to generate an inflammatory state in infected tissue so if any of the immunology vocabulary that we covered today gets a little bit overwhelming i suggest that you really try to center your focus on how each pattern recognition receptor and downstream signaling pathway relate to these relatively simple goals of the immune response first let's learn about some key principles of pattern recognition receptors historically the theory of pattern recognition was first proposed by charlie janeway back in 1989 and this name should sound familiar if you've purchased the textbook for this course now janeway observed that antigen alone was insufficient to stimulate lymphocytes of the adaptive immune system and hypothesized that animals must have a specialized set of receptors that are capable of triggering an innate immune response following their activation upon binding to conserved classes of microbial molecules now we refer to these conserved molecules as pathogen-associated molecular patterns or pamps which again are molecules that are only found in pathogens and not in host eukaryotic cells common pamps include cell wall components of bacteria fungi and parasites as well as certain types of nucleic acids found in viruses and bacteria as well as a motility and secretion machinery that are used by bacteria to establish infection since prrs recognize pamps that are shared by a category of pathogen these receptors must be able to respond to an enormous diversity of microbes using a rather limited set of host genes and importantly these receptors need to remain ignorant to self-molecules in order to preserve the concept of immunological tolerance that we've discussed already considering the genetics and evolution of prrs we know that these receptors are germline encoded and are not subject to any type of rearrangement so the repertoire remains fixed over the course of an individual's lifetime prrs are also an evolutionarily ancient form of host protection and their importance is demonstrated through the fact that these proteins are relatively conserved across many plant and animal species lastly i want to emphasize that host pathogen interactions are a major driver of metazoan evolution as the evolutionary pressure of infection and its associated risks with mortality have really helped shape the specificity and repertoire of pr ors now pattern recognition receptors can be divided into a few different categories that sample distinct cellular compartments toll-like receptors or tlrs are found both at the plasma membrane and within organelles called endosomes that originate from the golgi complex tlrs are primarily responsible for sensing microbes that are present in the extracellular space so this is pretty obvious for plasma membrane tlrs where their curve detection domain kind of sticks out into the extracellular space but for endosoma endosomal tlrs endosomes will actually fuse with fatal lysosomes and therefore these tlrs can sample extracellular contents that have been phagocytosed we also have a handful of prrs that sample the cytosol of a host cell these include nod-like receptors or analars which upon activation assemble into these large multi-protein complexes called inflammasomes there are also rig i like receptors or rlrs as well as the sensor sea gas which all of which patrol the site is all for signs of infection considering the cellular compartments where these sensors are located we can categorize the types of pathogens that are capable of activating each type of pattern recognition receptor again tlrs detect extracellular molecules with plasma membrane tlr's directly sampling sampling the extracellular space while endosomal tlr's sample phagocytose material nlrs rlrs and sea gas all bind to intracellular molecules that are floating around in the cytoplasm so given how we discussed different classes of pathogens in the previous lecture and categorized them based on their host niche such as intracellular versus extracellular bacteria i want you to start thinking about which different types of microbes might be sensed by extracellular pattern recognition receptors versus those that are present in the cytosol once these prrs bind to their cognate ligand they can then undergo signal transduction pathways that upregulate transcriptional programs that are specific to that pamp prr interaction one exception to this are the nlrs and they're associated inflammasomes which activate proteolytic cleavage cascades rather than transcriptional programs for the rest of these however although one pamp prr interaction typically only activates one signal transduction pathway there's a considerable amount of overlap between some pattern recognition receptors and therefore some fairly conserved transcriptional outputs not surprisingly these transcriptional programs relate to the production of molecules required for the um primary effector functions of the innate immune system which includes the production of inflammatory cytokines and chemokines the synthesis of antimicrobial molecules and antigen processing and presentations help stimulate adaptive immunity so let's learn more about the specifics behind each of these types of pattern recognition receptors first we'll start with toll-like receptors or tlr's these are some of the most well characterized pattern recognition receptors as they were the first prs to be experimentally discovered by janeway and his lab members the plasma membrane tlrs consists of consist of tlr1 2 and 6 which form heterodimers that recognize both lipopeptides from gram-positive bacterial cell walls as well as beta-glucan which is found in fungal cell walls tlr-5 forms a homodimer and detects and detects monomeric flagellin which is the protein subunit for flagella which are these little whips that some mito motile bacteria use to propel themselves through liquids such as mucus and there's also tlr4 which recognizes a cell wall component of gram-negative bacteria called lipopolysaccharide or lps as a side note tlr4 requires a co-receptor called md2 in order to form its activated homodimer now for these tlrs their ability to to distinguish self versus non-self is fairly straightforward as the bacterial and fungal pamps that they bind consist of molecules that simply aren't made by host cells and they're only found extracellularly next let's learn about the endosomal tlrs these include tlr3 which recognizes double-stranded rna generated by some viruses tlr 7 and 8 which detect single stranded rna used by other classes of rna viruses and something called cpg dna which is a nucleic acid motif found in some dna viruses again these tlr's fuse um or these tlrs are present on the endosome which then fuses with the phagolysosome in order to sample the extracellular space but the reasoning behind their sequestration and endosomes makes a lot more sense when you think about the types of pamps that they detect now again these tlr's need to remain ignorant to self-nucleic acids which are obviously abundant in other host compartments such as the cytosol in the nucleus and so this distinction is accomplished through a few different strategies one is the fact that they are sequestered in endosomes where their sensing domains aren't exposed to the cytosol and so this restricts their access to self-rna and self-dna the second is through the recognition of certain nucleic acid motifs that are only found in pathogens or primarily found in pathogens and a good example of this is that unmethylated cpg dna is relatively rare in eukaryotic genomes but this motif is enriched in large dna viruses lastly the endosomal tlrs exist as inactive proreceptors in their endosomes which means that when they're first produced at the golgi complex they lack the ability to undergo activation and signaling within endosomes however once they fuse with the acidic lysosome they undergo proteolytic cleavage events that process them into active forms that are signaling competent so in summary the fact that endosomal tlrs are potentially cross-reactive with respect to detecting both host and pathogen nucleic acids has really necessitated the evolution and development of additional layers of regulation compared to the plasma membrane tlrs which only detect bonafide foreign pamps in the extracellular space now once tlrs bind to their cognate pamp they trigger signal transduction cascades to induce infect effector mechanisms that are appropriate for the upstream pathogen most tlrs recruit the adapter protein mighty 88 which leads to the phosphorylation of an ikk complex that then phosphorylates into creates a protein called i kappa b which is a negative regulator of this key transcription factor called nf kappaby tlr signaling therefore leads to the liberation of nf kappab in the cytoplasm which allows it to then enter the nucleus and bind to a large number of genes that are extremely relevant for immune responses so transcriptional targets of nf kappa b include general pro-survival programs as well as inflammatory chemokines and cytokines machinery that's related to antigen processing and presentation as well as co-stimulatory marker expression which is also related to antigen presentation and if kappa b is therefore a major pro-inflammatory transcription factor that can be activated upon tlr signaling in addition to nf kappa b the endosomal tlrs can also activate transcription factors which upregulate antiviral responses this makes sense considering that the endosomal tlrs here on the left we have tlr3 and tlr7 as examples sense nucleic acids that are associated with viral infection tlr-3 signals independently of mighty 88 and instead uses an adapter called trif and downstream signaling cascades to phosphorylate and activate a transcription factor called irf3 which can then translocate into the nucleus for target gene expression tlr-7 also signals through mighty 88 but uses different downstream signaling mechanisms to phosphorylate and activate a transcription factor called irf7 instead of targeting nf kappa b both irf3 and irf7 promote the transcription of type 1 interferon which encompasses two different cytokines either interferon alpha or interferon beta that are critical master regulators of antiviral immune responses once interferon signals in paracrine fashion on neighboring cells it can induce antiviral programs that confer resistance to intracellular viral replication that can activate antigen presenting cells or apcs to stimulate adaptive immunity they can also activate a type of lymphocyte called an nk cell which is important for killing virally infected cells and they can also increase the expression of a molecule called mhc class 1 which renders cells susceptible to killing by cytotoxic t cells these last two points are ones that we'll discuss uh later on in summary tlrs can activate either nf kappa b or irf transcription factors which have some slight differences in the effector mechanisms that are engaged in that these correspond with the upstream pathogen that triggered this specific tlr beyond membrane-bound tlrs we also express several cytosolic pattern recognition receptors that detect microbial components floating around in the cytoplasm first let's go over the rig-i-like receptors or rlrs there are two rlrs obviously rig-i as well as another receptor called mda5 that is structurally similar to rig-eye the rlrs detect a modification on viral rna that is specific to viruses which is a triphosphate group on the five prime end of the rna molecule and this modification is only found on viral rnas and not on the host rnas resulting from genome transcription that are abundant in the cytosol as for signaling activated rig i and mda5 function similarly and that they bind to a mitochondrial protein called mavs and this leads to the aggregation of mavs complexes at the mitochondrial outer membrane mavs aggregates then trigger two key signaling outcomes the first is the phosphorylation of the kinase tbk1 which then phosphorylates and activates irf3 and similar to signaling downstream of endosomal tlrs irf3 activation leads to the production of antiviral type 1 interferon second mavs aggregates trigger the degradation of i kappa b which is again the negative regulator negative regulator of nf kappa b that we introduced on the last slide and this event allows nf kappa b to translocate into the nucleus and upregulate various pro-survival and cellular activation programs that we discussed previously so in summary here on the left rlr activation induces the activation rlr activation induces uh activation of both nf kappa b and irf3 transcription factors another key set of cytosolic pattern recognition receptors are sea gas and sting which coordinate responses to cytosolic dna introduced by either by either intracellular bacteria or dna viruses the dna here interestingly does not have any distinct molecular differences from host cell dna but this pattern recognition receptor is logically located in a part of the host cell where self-dna shouldn't really be found and this is the cytosol um because the host dna should be restricted to the nucleus so these prrs function under the premise that any cytosolic dna is likely associated with an active infection sea gas sea gas stands for cyclic gmp amp synthase which describes the activity of this pattern recognition receptor once bound to double-stranded dna so sea gas takes a molecule of atp and a molecule of gtp and binds them together to form a small dinucleotide called cyclic gmp-amp or c-gamp note that this molecule is also structurally similar to cyclic dinucleotides that are found in certain species of bacteria so c-gamp or bacterial dinucleotides can then bind to a second prr that's located at the end of endoplasmic reticulum called sting which stands for stimulator of interferon genes secant binding brings two sting molecules together which then triggers their activation and downstream signaling now like mavs activation downstream of rlr activation up here on the top sting activation also leads to the phosphorylation of the kinase tbk1 which in turn activates the transcription factor irf3 leading to type 1 interferon production now the sea gas sting pathway is interesting in that this is the first mechanism we've learned about that consists of the sequential activation of two separate pattern recognition receptors first sea gas which then generates the second messenger c gamp that is then detected by sting and this leads to the signal transduction cascade responsible for generating type 1 interferon together the rlrs and sea gas sting pathway make up two important methods for for detecting cytosolic pamps the last set of cytosolic sensors that we'll cover today are nod-like receptors or analars which when activated form large protein complexes called inflammasomes there are several types of nlrs but we'll focus on three for the purposes of this lecture nlrs are interesting because although there are some that function more like classic prs and that they bind specific pamps nlrp3 is an nlr that's activated in response to cellular stress and damage signals that could potentially be indicative of a pathogenic infection over on the left here we have a schematic of nlrp3 activation taken from the textbook although nlrp3 is in the host cell cytosol it can become it can become activated in response to signs of membrane integrity damage at the plasma membrane including pore formation that's caused by the insertion of certain bacterial toxins into the plasma membrane or the opening of plasma membrane channel proteins such as p2x7 either of these activating stimuli allow for the efflux of potassium ions from the host cell which through an undefined mechanism leads to the oligomerization of nlrp3 molecules in the cytosol this oligomerization allows for the recruitment of certain adapter proteins such as ask which generates a very large intracellular protein complex that we call the inflammasome inflammasomes function as sites of proteolytic cleavage specifically for a substrate called caspase-1 and this is a shared downstream substrate of all inflammasomes so caspase-1 cleavage and activation into its active form then allows it to conduct its own enzymatic activity by targeting the inactive forms of some inflammatory interleukins called il1 family members this family includes the cytokines il-1 il-18 and il-1 beta which exist as inactive pro-proteins in the host cell that are transcribed and translated downstream of other priming signals such as tlr activation caspase-1 functions by processing il-18 and il-1 beta into their active forms which then allows them to be released out of the cell so that they can execute their inflammatory cytokine functions on neighboring cells so the consequence of nlrp3 activation is caspase-1 activation which leads to the secretion of pro-inflammatory il-18 and il-1 beta and interestingly this same output can be triggered by a diverse array of upstream stimuli that have been shown to trigger nlrp3 activation including pore formation and subsequent potassium efflux which we just covered as well as exposure to crystals such as uric acid and even elevated levels of intracellular reactive oxygen species or ross so all of these activating stimuli are interesting because they aren't specific molecules that are produced like a by a pathogen like classical pamps and instead these are considered to be sterile insults of stress or damage that could be triggered in the absence of infection so just keep in mind that nlrp3 is kind of a unique type of pattern recognition receptor in this respect there are also inflammasomes that function more like classic prrs and that they're activated by specific pamps so there's nlrc4 which specifically recognizes intracellular bacterial flagellin remember that nlrc4 therefore shares pamp detection with tlr5 which binds to extracellular bacterial flagellin there's also aim2 which detects intracellular dna so remember that aim2 therefore shares pamp detection with the sea gas and sting pathway that we just talked about on the last slide in all cases these nlrs patrol the intracellular niche for their cognate pamps and lead to the same downstream consequences of as nlrp3 activation so you get inflammasome assembly caspase-1 activation and the secretion of il-1 beta and il-18 just as a side note i also want to say that caspase-1 activation also triggers a form of programmed cell death that further supports inflammation and we'll discuss that in more detail in our lecture for next lecture for week two it's worth noting that while other prrs we've introduced today the tlrs rlrs and seagas sting all function through specific signal transduction cascades whose main outcome is the activation of transcription factors and consequential transcriptional programs inflammasomes are distinct from these prs and that they don't actually trigger de novo transcription in translation and so rather they enable the activation of intracellular proteins that are already expressed within the cell i next want to emphasize a conceptual point that you've probably realized by now which is that infection with most pathogens can lead to the activation of more than one prr typically those that sample distinct cellular compartments or niches a good example of this is double-stranded rna that's associated with viral infection so if you remember double-stranded rna can be detected both by tlr3 which is an endosomal tlr this pamp can also be recognized by the cytosolic rlrs rig i and mda5 which recognize the virus-specific triphosphate group on the prime end of the rna molecule it's worth noting that while tlr3 and the rlrs sample distinct cellular niches the extracellular space versus the host cytosol they still detect the same type of pathogen which is a virus so it makes sense that they lead to the same downstream signaling output which is anti-viral type one interferon production please note that while this example centers upon the same pamp triggering multiple pr's based on its localization there are also examples where a single pathogen can express multiple pumps that activate multiple so the main take-home point that i want um for you to focus on here with this slide is the fact that these represent redundant detection mechanisms where a given pathogen can be detected by more than one single prr it's important to have these types of backup and eight immune signaling pathways that can be engaged in case of pathogen successfully evolves mechanisms that block one form of prr detection which is often the case we'll get into this idea in more depth when we discuss virulence factors and how these relate to host pathogen interactions next week we'll end today with this table which i think nicely summarizes the modular nature of innate immune responses that are triggered by prrs and by modular i mean that the innate immune system needs to be able to respond to the four major classes of pathogens using a fixed set of detectors and responding transcriptional programs and then we can kind of mix and match these modules depending on the type of pathogen so you end up seeing some shared responses between different classes of pathogens for viruses bacteria fungi and parasites i've summarized the prrs and downstream cytokine modules that are engaged by each major class of microbe as well as some of the major responding cell types that first respond to prr activation we've talked about some of these responding cell types already such as phagocytes and we'll learn more about some of these other leukocytes in upcoming lectures such as nk cells and cytotoxic t cells which are essential for host defense from viral infection today we've learned that the innate immune system is capable of distilling or condensing a massive repertoire of potential pathogens into a relatively small number of innate immune response modules and that this is primarily accomplished through pattern recognition receptors we've learned about different classes of prs and how they sample different subcellular compartments including tlrs which are exposed to extracellular molecules as well as rlrs and nlrs which sample the host cytosol we've also summarized some of the key downstream responses triggered by prs including master transcriptional factors such as nf kappa b and the irs 3 and 7 as well as the proteolytic enzyme caspase-1 which regulates il-1 family cytokine processing and release now that we've learned about some of the specifics of innate immune signaling i want you to think a little bit more about this question here with what might some of the consequences be of genetic mutations to components of pr signaling pathways as well as cases where you have direct inhibition of these pathways and this question is something that i want you to keep in mind over the next couple of weeks since we'll eventually be having a discussion board prompt that's related to it that's it for this lecture um please remember to watch the final lecture for week two where we'll discuss programmed cell death in the complement system which are two other key components of innate immunity