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A distinct population of Foxp3 + CD4 + regulatory T (Treg) cells promotes repair of acutely or chronically injured skeletal muscle. The accumulation of these cells depends critically on interleukin (IL)-33 produced by local mesenchymal stromal cells (mSCs). An intriguing physical association among muscle nerves, IL-33 + mSCs, and Tregs has been reported, and invites a deeper exploration of this cell triumvirate. Here we evidence a striking proximity between IL-33 + muscle mSCs and both large-fiber nerve bundles and small-fiber sensory neurons; report that muscle mSCs transcribe an array of genes encoding neuropeptides, neuropeptide receptors, and other nerve-related proteins; define muscle mSC subtypes that express both IL-33 and the receptor for the calcitonin-gene–related peptide (CGRP); and demonstrate that up- or down-tuning of CGRP signals augments or diminishes, respectively, IL-33 production by muscle mSCs and later accumulation of muscle Tregs. Indeed, a single injection of CGRP induced much of the genetic program elicited in mSCs early after acute skeletal muscle injury. These findings highlight neural/stromal/immune-cell crosstalk in tissue repair, suggesting future therapeutic approaches.

Movement induces sensory stimulation of an animal's own sensory receptors, termed reafference. With a few exceptions, notably vestibular and proprioception, this reafference is unwanted sensory noise and must be selectively filtered in order to detect relevant external sensory signals. In the cerebellum-like electrosensory nucleus of elasmobranch fish, an adaptive filter preserves novel signals by generating cancellation signals that suppress predictable reafference. A parallel fiber network supplies the principal Purkinje-like neurons (called ascending efferent neurons, AENs) with behavior-associated internal reference signals, including motor corollary discharge and sensory feedback, from which predictive cancellation signals are formed. How distinct behavior-specific cancellation signals interact within AENs when multiple behaviors co-occur and produce complex, changing patterns of reafference is unknown. Here, we show that when multiple streams of internal reference signals are available, cancellation signals form that are specific to parallel fiber inputs temporally correlated with, and therefore predictive of, sensory reafference. A single AEN has the capacity to form more than one cancellation signal, and AENs form multiple cancellation signals simultaneously and modify them independently during co-occurring behaviors. Cancellation signals update incrementally during continuous behaviors, as well as episodic bouts of behavior that last minutes to hours. Finally, individual AENs, independently of their neighbors, form unique AEN-specific cancellation signals that depend on the particular sensory reafferent input it receives. Together, these results demonstrate the capacity of the adaptive filter to utilize multiple cancellation signals to suppress dynamic patterns of reafference arising from complex co-occurring and intermittently performed behaviors.

Background Lung nociceptor neurons amplify immune cell activity and mucus metaplasia in response to an inhaled allergen challenge in sensitized mice. Objective We sought to identify the cellular mechanisms by which these sensory neurons are activated subsequent to allergen exposure. Methods We used calcium microscopy and electrophysiologic recording to assess whether vagal neurons directly respond to the model allergen ovalbumin (OVA). Next, we generated the first nociceptor-specific FceR1γ knockdown (TRPV1Cre::FceR1γfl/fl) mice to assess whether this targeted invalidation would affect the severity of allergic inflammation in response to allergen challenges. Results Lung-innervating jugular nodose complex ganglion neurons express the high-affinity IgE receptor FceR1, the levels of which increase in OVA-sensitized mice. FceR1γ-expressing vagal nociceptor neurons respond directly to OVA complexed with IgE with depolarization, action potential firing, calcium influx, and neuropeptide release. Activation of vagal neurons by IgE-allergen immune complexes, through the release of substance P from their peripheral terminals, directly amplifies TH2 cell influx and polarization in the airways. Allergic airway inflammation is decreased in TRPV1Cre::FceR1γfl/fl mice and in FceR1α–/– mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FceR1 to immune complexes in both mouse jugular nodose complex ganglion neurons and human induced pluripotent stem cell–derived nociceptors. Conclusions Allergen sensitization triggers a feedforward inflammatory loop between IgE-producing plasma cells, FceR1-expressing vagal sensory neurons, and TH2 cells, which helps to both initiate and amplify allergic airway inflammation. These data highlight a novel target for reducing allergy, namely, FceR1γ expressed by nociceptors.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.

SUMMARYGut-innervating nociceptor sensory neurons respond to noxious/tissue-damaging stimuli by initiating protective responses and releasing mediators that regulate tissue inflammation, gastrointestinal secretion, and motility. The role of nociceptors in host defense against enteric pathogens is unclear. Here, we found that gut-extrinsic nociceptor neurons are critical in protecting the host against Salmonella typhimurium (STm) infection. Nociceptors responded to STm by releasing the neuropeptide calcitonin gene-related peptide (CGRP). Targeted depletion of Nav1.8 and TRPV1 neurons from gut-extrinsic dorsal root ganglia and vagal ganglia increased STm colonization, invasion, and dissemination. Nociceptors regulated the gut microbiota at homeostasis, specifically segmented filamentous bacteria (SFB) levels in the ileum, which protected against STm by colonization resistance. Nociceptors also regulated the density of microfold epithelial cells in the Peyer’s patch via CGRP to limit points of entry for STm invasion into host tissues. Understanding how host sensory neurons crosstalk with pathogenic bacteria may impact treatments for enteric infections.HIGHLIGHTSNav1.8 and TRPV1 nociceptors defend against Salmonella typhimurium (STm) infectionNociceptors shape the gut microbiota and SFB levels which resist pathogen colonizationNociceptors suppress Peyer’s patch microfold cell density to limit pathogen invasionNeurons sense STm and release CGRP to modulate microfold cells and host defense

SummaryLung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8+ sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4+ and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma.

Sensory neurons in the gastrointestinal tract have multi-faceted roles in maintaining homeostasis, detecting danger, and initiating protective responses. The gastrointestinal tract is innervated by three types of sensory neurons: dorsal root ganglia, nodose/jugular ganglia, and intrinsic primary afferent neurons. Here, we examine how these distinct sensory neurons and their signal transducers participate in regulating gastrointestinal inflammation and host defense. Sensory neurons are equipped with molecular sensors that enable neuronal detection of diverse environmental signals including thermal and mechanical stimuli, inflammatory mediators, and tissue damage. Emerging evidence shows that sensory neurons participate in host–microbe interactions. Sensory neurons are able to detect pathogenic and commensal bacteria through specific metabolites, cell-wall components, and toxins. Here we review recent work on the mechanisms of bacterial detection by distinct subtypes of gut-innervating sensory neurons. Upon activation, sensory neurons communicate to the immune system to modulate tissue inflammation through antidromic signaling and efferent neural circuits. We discuss how this neuro-immune regulation is orchestrated through transient receptor potential ion channels and sensory neuropeptides including substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating polypeptide. Recent studies also highlight a role for sensory neurons in regulating host defense against enteric bacterial pathogens including Salmonella typhimurium, Citrobacter rodentium, and enterotoxigenic Escherichia coli. Understanding how sensory neurons respond to gastrointestinal flora and communicate with immune cells to regulate host defense enhances our knowledge of host physiology and may form the basis for new approaches to treat gastrointestinal diseases.

Dendritic cells (DCs), macrophages (MPs), and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous Ags preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum translocation, trimming, and MHC-I presentation. In this study, we compared the capacity of DCs, MPs, and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848, and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs, and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs, and monocytes. Differences in Ag-processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load.

The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells requires the presentation of viral peptides intracellularly processed and displayed by major histocompatibility complex class I. Assays to measure CTL-mediated killing often use peptides exogenously added onto target cells –which does not account for epitope processing- or follow killing of infected cells at a single time point. In this study we established a real-time fluorogenic cytotoxic assay that measures the release of the Glucose-6-phosphate-dehydrogenase by dying target cells every 5 minutes after addition of CTL. It has comparable sensitivity to 51chromium-based killing assay with the additional advantage of incorporating the kinetics of epitope presentation. We showed that HIV infection of immortalized or primary CD4 T cells leads to asynchronous killing by two CTL clones specific for epitopes located in different proteins. Real-time monitoring of killing of virus-infected cells will enable identification of immune responses efficiently preventing virus dissemination.

Induction of virus-specific CD8⁺ T cell responses is critical for the success of vaccines against chronic viral infections. Despite the large number of potential MHC-I-restricted epitopes located in viral proteins, MHC-I-restricted epitope generation is inefficient, and factors defining the production and presentation of MHC-I-restricted viral epitopes are poorly understood. Here, we have demonstrated that the half-lives of HIV-derived peptides in cytosol from primary human cells were highly variable and sequence dependent, and significantly affected the efficiency of cell recognition by CD8⁺ T cells. Furthermore, multiple clinical isolates of HLA-associated HIV epitope variants displayed reduced half-lives relative to consensus sequence. This decreased cytosolic peptide stability diminished epitope presentation and CTL recognition, illustrating a mechanism of immune escape. Chaperone complexes including Hsp90 and histone deacetylase HDAC6 enhanced peptide stability by transient protection from peptidase degradation. Based on empirical results with 166 peptides, we developed a computational approach utilizing a sequence-based algorithm to estimate the cytosolic stability of antigenic peptides. Our results identify sequence motifs able to alter the amount of peptide available for loading onto MHC-I, suggesting potential new strategies to modulate epitope production from vaccine immunogens.

Investigation of host-environment interactions in the gut would benefit from a culture system that maintained tissue architecture yet allowed tight experimental control. We devised a microfabricated organ culture system that viably preserves the normal multicellular composition of the mouse intestine, with luminal flow to control perturbations (e.g., microbes, drugs). It enables studying short-term responses of diverse gut components (immune, neuronal, etc.). We focused on the early response to bacteria that induce either Th17 or RORg+ T-regulatory (Treg) cells in vivo. Transcriptional responses partially reproduced in vivo signatures, but these microbes elicited diametrically opposite changes in expression of a neuronal-specific gene set, notably nociceptive neuropeptides. We demonstrated activation of sensory neurons by microbes, correlating with RORg+ Treg induction. Colonic RORg+ Treg frequencies increased in mice lacking TAC1 neuropeptide precursor and decreased in capsaicin-diet fed mice. Thus, differential engagement of the enteric nervous system may partake in bifurcating pro- or anti-inflammatory responses to microbes.

Background Whether HIV-infectable subsets, such as CD4 T cells, monocytes, macrophages and dendritic cells (DCs), have equivalent capacity to produce and present MHC-I restricted epitopes to HIV-specific CD8 T cells is unknown. MHC-I epitopes are processed by an intracellular degradation pathway involving multiple proteases. In this study we analyzed the effect of toll-like receptor (TLR) agonist-mediated maturation on the processing and presentation of HIV antigens in monocyte-derived DCs and macrophages.

Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected individuals have identified numerous examples of HLA-associated mutations within or adjacent to T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC class I molecules. Definition of sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. In this study, we identified cytosolic aminopeptidases cleavage preferences in primary cells and its impact on HIV Ag degradation into epitopes in primary human cell extracts by mass spectrometry and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically derived viral sequences from 1134 antiretroviral-naive HIV-infected individuals: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of Ag processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.

Background Epitopes displayed by MHC-I come from the multistep degradation of proteins by intracellular peptidases such as proteasome and aminopeptidases or cathepsins in the exogenous pathway. We hypothesize that due to structural homologies HIV protease inhibitors (PIs) used in antiretroviral therapies may affect activities of cellular peptidases involved in epitope processing and may affect epitope presentation to immune cells. Methods Using a fluorogenic assay the effect of 5 HIV-1 PIs (Ritonavir, Saquinavir, Nelfinavir, Indinavir, Atazanavir) on proteasome, aminopeptidase and cathepsin activities was tested in PBMCs from at least 6 healthy donors. Using PBMC cytosol as a source of peptidases and HPLC and mass spectrometry to define and quantify the degradation products, the effect of HIV PIs on HIV peptide processing kinetics and HIV epitope half-life was assessed. Finally we assessed the impact of PIs on the endogenous processing and presentation of epitopes by infected cells to CD8 T cells using a fluorescence-based cytotoxicity assay. Results HIV PIs variably altered proteasome, post-proteasomal aminopeptidases and cathepsin activities. Depending on the PI, some activities were inhibited (from 1.1 to 5 folds, p

Background Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. Results In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. Conclusion By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.