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Neutrophils are short-lived cells of the innate immune system that play numerous roles in defense against infection, regulation of immune responses, tissue damage and repair, autoimmunity, and other non-communicable diseases. Understanding neutrophil function at a mechanistic level has been hampered by the difficulty of working with primary neutrophils, which die rapidly upon isolation, and the relative paucity of neutrophil cell lines. Murine neutrophil progenitors that are immortalized with estrogen-regulated expression of Hoxb8 differentiate into neutrophils upon withdrawal of estrogen and facilitate the quantitative production of neutrophils in vitro. Here we report the creation of a Cas9+ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils for the first time. By editing progenitors via transduction with sgRNAs, and then withdrawing estrogen, Cas9 edited neutrophils are produced with high efficiency. Importantly, neutrophil differentiation of edited progenitors occurs both in vitro in cell culture and when transferred into murine recipients. To demonstrate the utility of Cas9+ER-Hoxb8 progenitors for forward genetics, we performed a pooled CRISPR screen to identify factors required for survival during neutrophil differentiation. This screen identified hundreds of genes both negatively and positively selected under differentiation conditions. One of the top hits from this screen was Cebpe, a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using the progenitor cell line, we also confirmed that Cepbe is required for neutrophil differentiation in vivo, validating the utility of this cell line both for screening and for studying in vivo phenotypes. The genome-wide screen also identified all components of the WASH complex as being required for neutrophil differentiation, a finding that extends the known role of WASH in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, we demonstrate that Cas9+ER-Hoxb8 immortalized neutrophils can be used to study neutrophil function both in vitro and in vivo. This new resource will enable the analysis of the role of neutrophils in numerous disease states using genetics for the first time.

The multipass transmembrane protein UNC93B1 is critical for the proper trafficking and function of many members of the Toll-like receptor (TLR) family of innate immune receptors. A new study reports two structures of UNC93B1 in complex with full-length TLR3 or TLR7 and sheds light on how this single chaperone may differentially interact with and regulate the function of individual TLRs.

Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate the recognition of microbial DNA and RNA while limiting the recognition of an organism's own nucleic acids1. Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory diseases2-6. Intracellular localization of these receptors is thought to be crucial for the discrimination between self and non-self7, but the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a mechanism that prevents the activation of TLR9 from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone UNC93B1, which occurs only within endosomes and is required for ligand binding and signal transduction. Preventing release of TLR9 from UNC93B1, either by mutations in UNC93B1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signalling. Whereas TLR9 and TLR3 are released from UNC93B1, TLR7 does not dissociate from UNC93B1 in endosomes and is regulated by distinct mechanisms. This work defines a checkpoint that reinforces the compartmentalized activation of TLR9, and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated.

Many of the ligands for Toll-like receptors (TLRs) are unique to microorganisms, such that receptor activation unequivocally indicates the presence of something foreign. However, a subset of TLRs recognizes nucleic acids, which are present in both the host and foreign microorganisms. This specificity enables broad recognition by virtue of the ubiquity of nucleic acids but also introduces the possibility of self-recognition and autoinflammatory or autoimmune disease. Defining the regulatory mechanisms required to ensure proper discrimination between foreign and self-nucleic acids by TLRs is an area of intense research. Progress over the past decade has revealed a complex array of regulatory mechanisms that ensure maintenance of this delicate balance. These regulatory mechanisms can be divided into a conceptual framework with four categories: compartmentalization, ligand availability, receptor expression and signal transduction. In this Review, we discuss our current understanding of each of these layers of regulation., Activation of nucleic acid-sensing Toll-like receptors is finely tuned to limit self-reactivity while maintaining recognition of foreign microorganisms. The authors describe recent progress made in defining the regulatory mechanisms that facilitate this delicate balance.

The mucophilic anaerobic bacterium Akkermansia muciniphila is a prominent member of the gastrointestinal (GI) microbiota and the only known species of the Verrucomicrobia phylum in the mammalian gut. A high prevalence of A. muciniphila in adult humans is associated with leanness and a lower risk for the development of obesity and diabetes. Four distinct A. muciniphila phylogenetic groups have been described, but little is known about their relative abundance in humans or how they impact human metabolic health. In this study, we isolated and characterized 71 new A. muciniphila strains from a cohort of children and adolescents undergoing treatment for obesity. Based on genomic and phenotypic analysis of these strains, we found several phylogroup-specific phenotypes that may impact the colonization of the GI tract or modulate host functions, such as oxygen tolerance, adherence to epithelial cells, iron and sulfur metabolism, and bacterial aggregation. In antibiotic-treated mice, phylogroups AmIV and AmII outcompeted AmI strains. In children and adolescents, AmI strains were most prominent, but we observed high variance in A. muciniphila abundance and single phylogroup dominance, with phylogroup switching occurring in a small subset of patients. Overall, these results highlight that the ecological principles determining which A. muciniphila phylogroup predominates in humans are complex and that A. muciniphila strain genetic and phenotypic diversity may represent an important variable that should be taken into account when making inferences as to this microbe's impact on its host's health.IMPORTANCE The abundance of Akkermansia muciniphila in the gastrointestinal (GI) tract is linked to multiple positive health outcomes. There are four known A. muciniphila phylogroups, yet the prevalence of these phylogroups and how they vary in their ability to influence human health is largely unknown. In this study, we performed a genomic and phenotypic analysis of 71 A. muciniphila strains and identified phylogroup-specific traits such as oxygen tolerance, adherence, and sulfur acquisition that likely influence colonization of the GI tract and differentially impact metabolic and immunological health. In humans, we observed that single Akkermansia phylogroups predominate at a given time but that the phylotype can switch in an individual. This collection of strains provides the foundation for the functional characterization of A. muciniphila phylogroup-specific effects on the multitude of host outcomes associated with Akkermansia colonization, including protection from obesity, diabetes, colitis, and neurological diseases, as well as enhanced responses to cancer immunotherapies.

Neutrophils are generally the first immune cells recruited during the development of sterile or microbial inflammation. As these cells express many innate immune receptors with the potential to directly recognize microbial or endogenous signals, we set out to assess whether their functions are locally influenced by the signals present at the onset of inflammation. Using a mouse model of peritonitis, we demonstrate that neutrophils elicited in the presence of C-type lectin receptor ligands have an increased ability to produce cytokines, chemokines, and lipid mediators in response to subsequent TLR stimulation. Importantly, we found that licensing of cytokine production was mediated by paracrine TNF-α-TNFR1 signaling rather than direct ligand sensing, suggesting a form of quorum sensing among neutrophils. Mechanistically, licensing was largely imparted by changes in the posttranscriptional regulation of inflammatory cytokines, whereas production of IL-10 was regulated at the transcriptional level. Altogether, our data suggest that neutrophils rapidly adapt their functions to the local inflammatory milieu. These phenotypic changes may promote rapid neutrophil recruitment in the presence of pathogens but limit inflammation in their absence.

Chronic itch remains a highly prevalent disorder with limited treatment options. Most chronic itch diseases are thought to be driven by both the nervous and immune systems, but the fundamental molecular and cellular interactions that trigger the development of itch and the acute-to-chronic itch transition remain unknown. Here, we show that skin-infiltrating neutrophils are key initiators of itch in atopic dermatitis, the most prevalent chronic itch disorder. Neutrophil depletion significantly attenuated itch-evoked scratching in a mouse model of atopic dermatitis. Neutrophils were also required for several key hallmarks of chronic itch, including skin hyperinnervation, enhanced expression of itch signaling molecules, and upregulation of inflammatory cytokines, activity-induced genes, and markers of neuropathic itch. Finally, we demonstrate that neutrophils are required for induction of CXCL10, a ligand of the CXCR3 receptor that promotes itch via activation of sensory neurons, and we find that that CXCR3 antagonism attenuates chronic itch.

At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA and self-DNA, respectively. Despite the structural and functional similarities between these receptors, their contributions to autoimmune diseases such as systemic lupus erythematosus can differ. For example, TLR7 and TLR9 have opposing effects in mouse models of systemic lupus erythematosus-disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice1. However, the mechanisms of negative regulation that differentiate between TLR7 and TLR9 are unknown. Here we report a function for the TLR trafficking chaperone UNC93B1 that specifically limits signalling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. Mutations in UNC93B1 that lead to enhanced TLR7 signalling also disrupt binding of UNC93B1 to syntenin-1, which has been implicated in the biogenesis of exosomes2. Both UNC93B1 and TLR7 can be detected in exosomes, suggesting that recruitment of syntenin-1 by UNC93B1 facilitates the sorting of TLR7 into intralumenal vesicles of multivesicular bodies, which terminates signalling. Binding of syntenin-1 requires phosphorylation of UNC93B1 and provides a mechanism for dynamic regulation of TLR7 activation and signalling. Thus, UNC93B1 not only enables the proper trafficking of nucleic acid-sensing TLRs, but also sets the activation threshold of potentially self-reactive TLR7.

B-1a cells play an important role in mediating tissue homeostasis and protecting against infections. They are the main producers of ‘natural’ IgM, spontaneously secreted serum antibodies predominately reactive to self antigens, like phosphatidylcholine (PtC), or antigens expressed by the intestinal microbiota. The mechanisms that regulate the B-1a immunoglobulin (Ig) repertoire and their antibody secretion remain poorly understood. Here, we use a novel reporter mouse to demonstrate that production of self- and microbiota-reactive antibodies is linked to BCR signaling in B-1a cells. Moreover, we show that Toll-like receptors (TLRs) are critical for shaping the Ig repertoire of B-1a cells as well as regulating their antibody production. Strikingly, we find that both the colonization of a microbiota as well as microbial-sensing TLRs are required for anti-microbiota B-1a responses, whereas nucleic-acid sensing TLRs are required for anti-PtC responses, demonstrating that linked activation of BCR and TLRs controls steady state B-1a responses to both self and microbiota-derived antigens.

Context shapes anticommensal immunity The gut bacterium Akkermansia muciniphila is associated with protection from obesity, enhanced wound healing, and augmented antitumor responses. Ansaldo et al. found that this microbe induces antigen-specific immunoglobulin G1 (IgG1) antibodies generated by B cells with CD4 + T cell help. This is in contrast to most anticommensal responses, which involve the T cell–independent production of IgA antibodies. In a gnotobiotic setting in which all components of the microbiome are defined, A. muciniphila –specific T cells expanded only when A. muciniphila was present. The T cells primarily displayed a phenotype associated with B cell help. However, in mice with a conventional gut microbiota, other proinflammatory A. muciniphila –specific T cell populations also expanded. Thus, anti– A. muciniphila immunity is context dependent, which may explain the variable immune responses to this microbe reported in patients. Science , this issue p. 1179

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.

Recognition of nucleic acids enables detection of diverse pathogens by a limited number of innate immune receptors but also exposes the host to potential autoimmunity. At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self RNA or DNA, respectively. Despite the structural and functional similarities between these receptors, their contribution to autoimmune diseases such as SLE can be quite different. However, mechanisms of negative regulation that differentiate between TLR7 and TLR9 have not been described. Here we report a new function for the TLR trafficking chaperone Unc93b1 that specifically limits TLR7 signaling and prevents TLR7-dependent autoimmunity. Unc93b1 is known to traffic TLRs from the endoplasmic reticulum to endosomes, but this new regulatory function does not affect TLR7 localization. Instead, Unc93b1 recruits Syntenin-1, which inhibits TLR7, but not TLR9, signaling. Syntenin-1 binding requires phosphorylation of two serine residues on Unc93b1, providing a mechanism for dynamic regulation of the activation threshold of TLR7. Disruption of the Unc93b1/Syntenin-1 interaction in mice results in TLR7-dependent autoimmunity. Thus, Unc93b1 not only enables proper trafficking of nucleic acid sensing TLRs but also sets the activation threshold of these potentially self-reactive receptors.

Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate recognition of microbial DNA and RNA while limiting recognition of self-nucleic acids1. Failure to properly regulate nucleic acid-sensing TLRs can lead to autoimmune and autoinflammatory disease2-6. Intracellular localization of these receptors is thought to be critical for self vs. non-self discrimination7, yet the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a new mechanism that prevents TLR9 activation from locations other than endosomes. This control is achieved through the regulated release of TLR9 from its trafficking chaperone Unc93b1, which only occurs within endosomes and is required for ligand binding and signal transduction. Mutations in Unc93b1 that increase affinity for TLR9 impair release and result in defective signaling. The release is specific to TLR9, as TLR7 does not dissociate from Unc93b1 in endosomes. This work defines a novel checkpoint that reinforces self vs. non-self discrimination by TLR9 and provides a mechanism by which TLR9 and TLR7 activation can be distinctly regulated.

Summary Signaling by Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) is critical for intestinal homeostasis. To visualize epithelial expression of individual TLRs in vivo, we generated five strains of reporter mice. These mice revealed that TLR expression varied dramatically along the length of the intestine. Indeed, small intestine (SI) IECs expressed low levels of multiple TLRs that were highly expressed by colonic IECs. TLR5 expression was restricted to Paneth cells in the SI epithelium. Intestinal organoid experiments revealed that TLR signaling in Paneth cells or colonic IECs induced a core set of host defense genes, but this set did not include antimicrobial peptides, which instead were induced indirectly by inflammatory cytokines. This comprehensive blueprint of TLR expression and function in IECs reveals unexpected diversity in the responsiveness of IECs to microbial stimuli, and together with the associated reporter strains, provides a resource for further study of innate immunity.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.

SUMMARYAlthough apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. To better understand this phenomenon, we identified and characterized macrophage populations that continually engulf ACs in several distinct tissues. These macrophages share characteristics compatible with immunologically silent clearance of ACs, including high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. When removed from tissues these macrophages lose many of these characteristics and generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment are required to program macrophages for silent AC clearance. We show that KLF2 and KLF4 control expression of many genes within this AC clearance program. Coordinated expression of AC receptors with genes that limit responses to nucleic acids may represent a central feature of tissue macrophages that ensures maintenance of homeostasis.

Toll-like receptors (TLRs) play an important role in innate immune responses against pathogenic microorganisms or tissue damage. Nucleic acid (NA)-sensing TLRs localize in intracellular vesicular compartments and recognize foreign-derived and host-derived nucleic acid ligands. Inappropriate activation of NA-sensing TLRs can cause pathogenic inflammation and autoimmunity. Multiple regulatory mechanisms exist to limit recognition of self-NAs. This review summarizes recent progress that has been made in understanding how NA-sensing TLRs are regulated via trafficking, proteolytic cleavage, as well as ligand processing and recognition.

The classic anti-viral cytokine interferon-β (IFN-β) can be induced during parasitic infection, but relatively little is know about the cell types and signaling pathways involved. Here we show that inflammatory monocytes (IMs), but not neutrophils, produce IFN-β in response to T. gondii infection. This difference correlated with the mode of parasite entry into host cells, with phagocytic uptake predominating in IMs and active invasion predominating in neutrophils. We also show that expression of IFN-β requires phagocytic uptake of the parasite by IMs, and signaling through Toll-like receptors (TLRs) and MyD88. Finally, we show that IMs are major producers of IFN-β in mesenteric lymph nodes following in vivo oral infection of mice, and mice lacking the receptor for type I interferon (IFN-1) show higher parasite loads and reduced survival. Our data reveal a TLR and internalization-dependent pathway in IMs for IFN-β induction to a non-viral pathogen.

When normal, healthy cells die by apoptosis, they must be cleared to ensure tissue homeostasis. Failure to clear apoptotic cells (ACs) efficiently enables the release of inflammatory stimuli, such as nucleic acids, leading to tissue injury and, ultimately, autoimmunity. Clearance of ACs is largely performed by tissue-resident macrophages. Despite expressing many innate immune receptors, these cells do not respond to the potentially stimulatory ligands within ACs. Recently, the Barton Lab has identified several populations of AC-engulfing macrophages with many common features. These features include the expression of AC-recognition receptors and reduced TLR responsiveness to nucleic acids. The transcription factors KLF2 and KLF4 appear to contribute to this expression program both in vitro and in vivo in distinct AC-clearing macrophage populations. Macrophages that are removed from their environments lose this program and the program can be adopted by transferring TLR responsive macrophages into environments where endogenous silent residents are maintained. This coordinated silent immune program allows tissue-resident macrophages to maintain homeostasis while efficiently clearing ACs. Citation Format: Kathleen Pestal, Gregory M. Barton. The regulation of silent apoptotic cell-clearance in tissue-resident macrophages [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B179.

To maintain a symbiotic relationship between the host and its resident intestinal microbiota, appropriate mucosal Tcell responses to commensal antigens must be established. Mice acquire both IgG and IgA maternally; the former has primarily been implicated in passive immunity to pathogens while the latter mediates host-commensal mutualism. Here, we report the surprising observation that mice generate Tcell-independent and largely Toll-like receptor (TLR)-dependent IgG2b and IgG3 antibody responses against their gut microbiota. We demonstrate that maternal acquisition of these antibodies dampens mucosal T follicular helper responses and subsequent germinal center B cell responses following birth. This work reveals a feedback loop whereby Tcell-independent, TLR-dependent antibodies limit mucosal adaptive immune responses to newly acquired commensal antigens and uncovers a broader function for maternal IgG.

The innate immune system detects diverse microbial species with a limited repertoire of immune receptors that recognize nucleic acids. The cost of this immune surveillance strategy is the potential for inappropriate recognition of self-derived nucleic acids and subsequent autoimmune disease. The relative expression of two closely related receptors, Toll-like receptor (TLR) 7 and TLR9, is balanced to allow recognition of microbial nucleic acids while limiting recognition of self-derived nucleic acids. Situations that tilt this balance toward TLR7 promote inappropriate responses, including autoimmunity; therefore, tight control of expression is critical for proper homeostasis. Here we report that differences in codon bias limit TLR7 expression relative to TLR9. Codon optimization of Tlr7 increases protein levels as well as responses to ligands, but, unexpectedly, these changes only modestly affect translation. Instead, we find that much of the benefit attributed to codon optimization is actually the result of enhanced transcription. Our findings, together with other recent examples, challenge the dogma that codon optimization primarily increases translation. We propose that suboptimal codon bias, which correlates with low guanine-cytosine (GC) content, limits transcription of certain genes. This mechanism may establish low levels of proteins whose overexpression leads to particularly deleterious effects, such as TLR7.

SummaryPathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.

SummaryThe transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.

Despite the paradigm that the innate immune system uses nucleic acid-specific receptors to detect viruses because of a lack of other conserved features, many viruses are recognized by Toll-like receptor 2 (TLR2) and TLR4. The relevance of this recognition for antiviral immunity remains largely unexplained. Here we report that TLR2 activation by viruses led to the production of type I interferon. TLR2-dependent induction of type I interferon occurred only in response to viral ligands, which indicates that TLR2 is able to discriminate between pathogen classes. We demonstrate that this specialized response was mediated by Ly6C(hi) inflammatory monocytes. Thus, the innate immune system can detect certain non-nucleic acid features of viruses and links this recognition to the induction of specific antiviral genes.

An emerging paradigm in innate immune signalling is that cell biological context can influence the outcome of a ligand-receptor interaction. In this Review we discuss how Toll-like receptor (TLR) activation and signal transduction are regulated by subcellular compartmentalization of receptors and downstream signalling components. In particular, we focus on the functional specialization of TLRs in the endosomal system. We discuss recent studies that illustrate how basic aspects of the cellular machinery contribute to TLR function and regulation. This emerging area of research will provide important information on how immune signal transduction networks depend on (and in some cases influence) the generic regulators that organize eukaryotic cells.

Inflammation is a rapid yet coordinated response that can lead to the destruction of microbes and host tissue. Triggers capable of inducing an inflammatory response include tissue damage and infection by pathogenic and nonpathogenic microbes. Each of these triggers represents a qualitatively distinct stress to the host immune system, yet our understanding of whether they are interpreted as such remains poor. Accumulating evidence suggests that recognition of these distinct stimuli converges on many of the same receptors of the innate immune system. Here I provide an overview of these innate receptors and suggest that the innate immune system can interpret the context of an inflammatory trigger and direct inflammation accordingly.

Toll-like receptors (TLRs) sense infection by detecting molecular structures of microbial origin. TLR3, TLR7 and TLR9 recognize nucleic acids and are localized to intracellular compartments where they normally respond to viral nucleic acids. The purpose for this intracellular localization, however, is not clear. Here we describe a chimeric TLR9 receptor that localized to the cell surface and responded normally to synthetic TLR9 ligands but not to viral nucleic acids. However, the 'relocated' chimeric TLR9 receptor was able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid.

Mammalian Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses1,2,3. Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defence responses through their intracellular signalling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88 (refs 1–3). Although members of the TLR and the interleukin-1 (IL-1) receptor families all signal through MyD88, the signalling pathways induced by individual receptors differ. TIRAP, an adaptor protein in the TLR signalling pathway, has been identified and shown to function downstream of TLR4 (refs 4, 5). Here we report the generation of mice deficient in the Tirap gene. TIRAP-deficient mice respond normally to the TLR5, TLR7 and TLR9 ligands, as well as to IL-1 and IL-18, but have defects in cytokine production and in activation of the nuclear factor NF-κB and mitogen-activated protein kinases in response to lipopolysaccharide, a ligand for TLR4. In addition, TIRAP-deficient mice are also impaired in their responses to ligands for TLR2, TLR1 and TLR6. Thus, TIRAP is differentially involved in signalling by members of the TLR family and may account for specificity in the downstream signalling of individual TLRs.

If T cells require specific interactions with MHC-bound peptides during positive selection, then the specificities of T cells selected by one peptide should be distinct from those selected by another. We have examined positive selection of CD4 T cells in four strains of mice, each overexpressing a different peptide–1-A b (A b ) complex. We show that a subset of CD4 T cells is selected by the overexpressed peptide and that the specificities of the CD4 T cells, as measured by reactivity to wild-type antigen-presenting cells, vary greatly depending on which peptide is overexpressed. These differences in specificity are mediated through positive selection not negative selection. Each of the four peptide–A b complexes appears to adopt a different conformation, and these differences correlate with the differences in reactivity. Our results suggest that individual peptide–MHC complexes positively select different subsets of self-MHC-reactive T cells and that the conformation of the peptide–MHC complex may contribute to this process.

MyD88 is the canonical adaptor for inflammatory signaling pathways downstream of members of the Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor families. MyD88 links IL-1 receptor (IL-1R) or TLR family members to IL-1R-associated kinase (IRAK) family kinases via homotypic protein-protein interaction. Activation of IRAK family kinases leads to a variety of functional outputs, including the activation of nuclear factor-kappa B (NFκB), mitogen-activated protein kinases, and activator protein 1, making MyD88 a central node of inflammatory pathways. As more details of MyD88-dependent signaling have been elucidated, it has become clear that the functions of this critical signaling component can be influenced by multiple interaction partners in distinct subcellular compartments. In this review, we will focus on recent developments in the understanding of the assembly of MyD88 signaling complexes and the mechanisms leading to the diversification of MyD88-based signaling.

This chapter focuses on how pattern recognition is used by the innate immune system to distinguish self from nonself and how this discrimination is translated into induction of adaptive immunity. The past few years have seen significant advances in our understanding of how adaptive immune responses are controlled by the initial innate recognition of microbial infection. In particular, the identification of the Toll-like receptor (TLR) family as the critical receptor family involved in the recognition of infectious nonself has enabled researchers to examine the mechanisms by which adaptive responses are controlled by the innate immune system. Before discussing the specific mechanisms by which TLRs control adaptive immunity, the chapter talks about the general mechanisms by which self/nonself discrimination is regulated within the adaptive immune system. The second half of the chapter focuses on how TLRs control some of these mechanisms and link microbial recognition to self and nonself discrimination by the adaptive immune system. Upon activation, lymphocytes undergo a period of rapid proliferation. The innate immune system keeps the infection in check long enough for lymphocytes to expand and eventually eliminate the microbial challenge. Immature B cells that have receptors capable of recognizing membrane-bound selfantigens receive signals leading to apoptosis of the self-reactive B lymphocyte. IgG2 antibodies are effective at eliminating a variety of intracellular and extracellular pathogens because they can fix complement and direct the lysis of infected cells in a process called antibody-dependent cellular cytotoxicity.

Summary Pathogens utilize features of the host response as cues to regulate virulence gene expression. Salmonella enterica serovar Typhimurium (ST) sense Toll-like receptor (TLR)-dependent signals to induce Salmonella Pathogenicity Island 2 (SPI2), a locus required for intracellular replication. To examine pathogenicity in the absence of such cues, we evaluated ST virulence in mice lacking all TLR function ( Tlr2 −/− x Tlr4 −/− x Unc93b1 3d/3d ). When delivered systemically to TLR-deficient mice, ST do not require SPI2 and maintain virulence by replicating extracellularly. In contrast, SPI2 mutant ST are highly attenuated after oral infection of the same mice, revealing a role for SPI2 in the earliest stages of infection, even when intracellular replication is not required. This early requirement for SPI2 is abolished in MyD88 −/− x TRIF −/− mice lacking both TLR- and other MyD88-dependent signaling pathways, a potential consequence of compromised intestinal permeability. These results demonstrate how pathogens use plasticity in virulence strategies to respond to different host immune environments.

Over the past decade we have learned much about nucleic acid recognition by the innate immune system and in particular by Toll-like receptors (TLRs). These receptors localize to endosomal compartments where they are poised to recognize microbial nucleic acids. Multiple regulatory mechanisms function to limit responses to self DNA or RNA, and breakdowns in these mechanisms can contribute to autoimmune or inflammatory disorders. In this review we discuss our current understanding of the cell biology of TLRs involved in nucleic acid recognition and how localization and trafficking of these receptors regulates their function.

Mammalian Toll-like receptors (TLRs) recognize conserved products of microbial metabolism and activate NF-kappa B and other signaling pathways through the adapter protein MyD88. Although some cellular responses are completely abolished in MyD88-deficient mice, TLR4, but not TLR9, can activate NF-kappa B and mitogen-activated protein kinases and induce dendritic cell maturation in the absence of MyD88. These differences suggest that another adapter must exist that can mediate MyD88-independent signaling in response to TLR4 ligation. We have identified and characterized a Toll-interleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP) and have shown that it controls activation of MyD88-independent signaling pathways downstream of TLR4. We have also shown that the double-stranded RNA-binding protein kinase PKR is a component of both the TIRAP- and MyD88-dependent signaling pathways.

Intrathymic self-peptide–major histocompatibility complex class II (MHC) molecules shape the T cell repertoire through positive and negative selection of immature CD4+CD8+ thymocytes. By analyzing the development of MHC class II–restricted T cell receptor (TCR) transgenic T cells under conditions in which the endogenous peptide repertoire is altered, we show that self-peptide–MHC complexes are also involved in setting T cell activation thresholds. This occurs through changes in the expression level of molecules on thymocytes that influence the sensitivity of TCR signaling. Our results suggest that the endogenous peptide repertoire modulates T cell responsiveness in the thymus in order to enforce tolerance to self-antigens.

A critical aspect of innate immune function is the coordinated regulation of inflammatory gene expression. Now an unexpected discovery shows that certain signaling components of the unfolded protein response are required for optimal gene induction by Toll-like receptors.

The process of antigen presentation by MHC molecules allows T cells to sample the proteins expressed within a particular cell. This sampling is in the form of short peptides bound within the grooves of MHC molecules displayed on the surface of cells. In the context of immune surveillance, this presentation allows the identification of infected cells by displaying peptides originating from foreign proteins within the cell. However, MHC-bound peptides play additional roles beyond serving as antigenic stimuli during an immune response. In fact, it has become clear that MHC-bound peptides derived from self proteins are critically involved in the development of T cells during selective events in the thymus. In this review we will discuss the nature of the population of MHC-bound peptides as it relates to thymocyte development, with particular emphasis on the recent finding that peptide-MHC complexes present at low levels can drive the positive selection of thymocytes.

We report an experimental system for abundant expression of specific peptide-class II complexes in vivo and in vitro. We have constructed a cassette which allows for the replacement of the CLIP region of invariant chain (Ii) with an antigenic peptide. In fibroblasts expressing an altered Ii protein, in which CLIP has been replaced with peptide 52-68 from the class II I-E alpha chain (pEalpha), pEalpha-I-Ab complexes are formed with high efficiency. This peptide loading occurs in the endoplasmic reticulum (ER) when the Ii:pEalpha fusion protein associates with the I-Ab alpha and beta chains. The trimeric complexes of Ii:pEalpha and I-Ab molecules are stable in SDS and can be detected by the pEalpha-I-Ab-specific mAb, YAe, indicating that pEalpha is bound in the class II groove in the context of full-length Ii. These data strongly suggest that the CLIP region of intact Ii prevents peptide loading in the ER by binding in the peptide binding groove of newly synthesized class II alphabeta dimers.

To the editor: Unc93b1 is a critical regulator of Toll-Like Receptor (TLR) signaling1. The original description of mice with an inactivating mutation in Unc93b1 (H412R, also termed 3d) reported that these mice have defective TLR3, TLR7 and TLR9 signaling as well as major histocompatibility complex (MHC) class II presentation and cross-presentation defects1. Mutations in Unc93b1 have also been identified as a genetic etiological factor in herpes simplex virus-1 encephalitis, where susceptibility is associated with defects in TLR signaling2. Interestingly, in vitro studies of human cells have not found defects in MHC class II presentation after Unc93b1 knock-down3, raising the possibility that the role of Unc93b1 in antigen presentation, at least for MHC class II, is limited to mice. Further studies have shown that Unc93b1 controls TLR signaling by acting as a trafficking chaperone, associating with TLRs (such as TLR3, 7, 8, 9, 11 and 13) and delivering them from the endoplasmic reticulum to endosomes4, 5, 6. On the other hand, despite ample studies of Unc93b13d/3d mice, a mechanistic understanding for the role of Unc93b1 in antigen presentation is still lacking. Because of the discrepancies between human and mouse studies and the lack of any mechanistic basis for an antigen presentation defect in Unc93b13d/3d mice, we sought to confirm the antigen presentation defect reported in Tabeta et al.’s original description of Unc93b13d/3d mice1. First, we measured proliferation of ovalbumin (OVA)-specific OT-II T cells transferred into either wild-type or Unc93b13d/3d recipients, after injection of soluble OVA. In contrast to the findings reported by Tabeta et al., cells transferred into an Unc93b13d/3d host proliferated to a similar extent as in a wild-type host, both in terms of frequency of proliferating cells and average number of divisions (Fig.1a,b). Of note, the activation state of cells, followed by CD44 upregulation, was also identical in both wild-type and Unc93b1-deficient hosts (not shown). Figure 1 Antigen presentation is unaffected in Unc93b13d/3d mice Next, we tested the ability of Unc93b13d/3d hosts to cross-present antigens to CD8+ T cells, using Act-OVA cells as an antigen source, as described in the initial characterization of Unc93b13d/3d mice1. To ensure that we only measured cross-presentation, we injected OVA-expressing H-2Kbm1 cells in wild-type and Unc93b13d/3d mice. As H-2Kbm1 cells cannot directly present peptides derived from OVA on MHC class I molecules7, CD8+ T cell activation must be the result of cross-presentation by host cells. In this setting, we also failed to detect any significant differences between the proliferation profiles of OVA-specific OT-I T cells transferred in wild-type or Unc93b1-deficient hosts (Fig.1c,d). Moreover, the activation status of CD8+ T cells also appeared similar in both strains. Indeed, upregulation of CD44 and production of interferon-γ (IFNγ) after restimulation with either peptide or PMA-ionomycin (not shown) were not affected by the genotype of the host (Fig.1e, f). We also studied CD4+ T cell proliferation in this setting and found similar proliferation 7 days after activation in wild-type and Unc93b13d/3d mice (not shown). Taken together, our data do not support a direct role for Unc93b1 in antigen presentation – either MHC class II presentation or cross-presentation. We do observe defective TLR3, TLR7, and TLR9 function as originally reported for Unc93b13d/3d mice5. Given the well-recognized role of TLRs in activating antigen-presenting cells to enhance T cell priming8, it is certainly likely that Unc93b1 plays an indirect role in T cell activation in some models through its control of TLR trafficking. It is possible that differences in the level of contaminating TLR ligands may account for the discrepancy between our findings and those published by Tabeta et al. Regardless of the explanation, it is important to establish that there is no evidence to support a function for Unc93b1 beyond control of TLR localization. This clarification is particularly relevant to studies that employ Unc93b13d/3d mice as a model of multiple TLR deficiencies9 because any additional functions for Unc93b1 would complicate interpretation of such studies. Thus, Unc93b13d/3d mice, especially when crossed with other TLR-deficient strains, may offer a more straightforward model of multiple TLR deficiencies than the commonly used Myd88−/−Trif−/− mice, which also lack signaling downstream of the Interleukin-1 receptor family.