Intestinal stem cells (ISCs) reside in regionally variable niches that provide diverse microenvironmental cues such as tissue oxygen status, and morphogen signaling. Integration of these cues with ISC metabolism and fate remains poorly understood. Here, we show that cellular redox balance orchestrates niche factors with metabolic state to govern cell fate decisions. We demonstrate that hypoxia and Wnt signaling synergistically restrict the reactive oxygen species generating enzyme NADPH oxidase 1 (NOX1) regionally to the crypt base in the distal colon. NOX1 enables maintenance of an oxidative cell state that licenses cell cycle entry, altering the balance of asymmetric ISC self-renewal and lineage commitment. Mechanistically, cell redox state directs a self-reinforcing circuit that connects hypoxia inducible factor 1α-dependent signaling with post-translational regulation of the metabolic enzyme isocitrate dehydrogenase 1. Our studies show redox balance acts as a cellular rheostat that is central and causative for metabolic control of the ISC cell-cycle.

Infections after psychological stress are a major health care problem. Single-cell transcriptomics and lipidomic profiling in a mouse model of stress show that dermal fibroblasts undergoing adipogenesis have defective responses to Staphylococcus aureus skin infection. Adrenalectomy or adrenergic inhibition restores the fibroblast adipogenic response to S. aureus and enables mice to effectively resist infection during stress. Increased susceptibility to S. aureus from stress is attributed to suppression of the antimicrobial peptide cathelicidin (Camp) because adrenaline directly inhibits Camp production by fibroblasts, and mice lacking Camp in fibroblasts do not increase infection after stress. Transforming growth factor β (TGFβ) is induced by stress and adrenergic signaling, and inhibition of TGFβ or deletion of the TGFβ receptor on fibroblasts increases Camp expression and restores protection against infection. Together, these data show that stress initiates a brain-skin axis mediated by TGFβ that impairs the immune defense function of dermal fibroblasts to produce the Camp antimicrobial peptide.

Deciphering multicellular interactions is essential to understanding immune-mediated diseases. Myeloid cells can coordinate inflammatory responses and are central to immune crosstalk with neuronal, epithelial, and stromal cells. Here, we show that myeloid-specific loss of ten-eleven-translocation methylcytosine dioxygenase 2 (TET2) protected against colitis by limiting enterochromaffin (EC) cell differentiation and subsequent serotonin release. This protective effect was mediated by elevated interleukin (IL)-1β production by myeloid cells, which signals to tyrosine hydroxylase (TH)-positive neurons under inflammatory conditions. Neuronal IL-1R signaling dampened neuronal-epithelial interactions and consequent α₁-adrenergic signaling, thereby reducing EC differentiation. Conversely, physiological stress exacerbated colitis by enhancing catecholaminergic signals, which increased EC differentiation and serotonin production following mucosal injury. Thus, myeloid-derived IL-1β and stress exert opposing control over colitis severity through the α₁-adrenergic-EC axis, uncovering a neuro-immune-epithelial circuit that shapes intestinal inflammation.

OBJECTIVES:Bacterial musculoskeletal infections (MSKIs) can be challenging to diagnose. We compared the accuracy of calcitonin gene-related peptide (CGRP), a neuropeptide which is transcribed from the same gene as procalcitonin, to procalcitonin for the diagnosis of a MSKI in children. METHODS:We conducted a prospective cohort study of patients 21 years old or younger who underwent evaluation for MSKIs at one of 3 emergency departments. Our primary outcome was a MSKI, defined as septic arthritis, osteomyelitis, or pyomyositis. We used a Spearman correlation coefficient to measure the association between serum CGRP and procalcitonin and compared the diagnostic accuracy using area under the receiver operating characteristic curve (AUC) analysis. RESULTS:Of the 200 enrolled patients, 33 (17%) had a MSKI. Overall, median serum CGRP level did not differ between patients with and without a MSKI (13.5 pg/mL MSKI vs 10.9 pg/mL no MSKI; difference: 2.6, 95% CI: -0.6, 5.8), while PCT was higher in patients with a MSKI (0.12 ng/mL MSKIs vs 0.04 ng/mL no MSKI; difference: 0.08, 95% CI: 0.03 to 0.13). CGRP and PCT levels were not correlated (Spearman rank coefficient: -0.01, 95% CI: -0.15 to 0.13). CGRP had a lower AUC than procalcitonin [0.57, 95% CI: 0.47 to 0.66 CGRP vs 0.78, 95% CI: 0.69 to 0.87 PCT, P < 0.01]. CONCLUSIONS:Although biochemically related, CGRP was not correlated with procalcitonin in children undergoing evaluation for a MSKI. Our exploratory pilot highlights the ongoing need for novel biomarkers for the accurate and timely identification of children with a MSKI.

Influenza viruses are a major global cause of morbidity and mortality. Although vagal TRPV1+ nociceptive sensory neurons are known to mediate defenses against harmful agents, including pathogens, their function in lung antiviral defenses remains unclear. Our study demonstrates that both systemic and vagal-specific ablation of TRPV1+ nociceptors reduce survival in mice infected with influenza A virus (IAV). Despite no difference in viral load, mice lacking TRPV1+ neurons exhibited increased viral spread, exacerbated lung pathology, and elevated levels of proinflammatory cytokines. Loss of TRPV1+ neurons altered the lung immune landscape, including an expansion of neutrophils and monocyte-derived macrophages. Transcriptional analysis revealed impaired interferon signaling in myeloid cells and an imbalance in distinct neutrophil subpopulations in the absence of nociceptors. Furthermore, antibody-mediated depletion of myeloid cells during IAV infection substantially improved survival after nociceptor ablation, underscoring the role of TRPV1+ neurons in preventing pathogenic myeloid cell states that contribute to IAV-induced mortality.

The intestinal epithelium is a rapidly regenerating tissue dependent on resident stem cell self-renewal and differentiation. The reactive oxygen species (ROS) generating enzyme NADPH oxidase 1 (NOX1) is spatially restricted to the crypt base epithelium in the distal colon. Using NOX1-deletion, along with cell-specific redox reporter mice, lineage tracing, single-cell transcriptomics, cysteine proteomics, and cell metabolic studies, we investigated the role of redox balance in colonic stem cell function. We show that distal colonic stem cells uniquely require NOX1-generated ROS to maintain a relatively oxidized state that promotes stem cell self-renewal. Mechanistically, this occurs through a cellular circuit that connects hypoxia inducible factor 1 (HIF1α)-dependent signaling to ROS regulation of isocitrate dehydrogenase 1 (IDH1), to maintain efficient cell cycle entry and cell fate determination in the relatively hypoxic distal colonic niche environment. Our studies therefore provide a basis for understanding regeneration dynamics and disease propensity in the distal large intestine. ### Competing Interest Statement J.O.M. reports compensation for consulting services with Tessel Biosciences and Radera Biotherapeutics.

Bacterial musculoskeletal infections (MSKIs) can be challenging to diagnose. We compared the accuracy of calcitonin gene–related peptide (CGRP), a neuropeptide which is transcribed from the same gene as procalcitonin, to procalcitonin for the diagnosis of a MSKI in children. We conducted a prospective cohort study of patients 21 years old or younger who underwent evaluation for MSKIs at one of 3 emergency departments. Our primary outcome was a MSKI, defined as septic arthritis, osteomyelitis, or pyomyositis. We used a Spearman correlation coefficient to measure the association between serum CGRP and procalcitonin and compared the diagnostic accuracy using area under the receiver operating characteristic curve (AUC) analysis. Of the 200 enrolled patients, 33 (17%) had a MSKI. Overall, median serum CGRP level did not differ between patients with and without a MSKI (13.5 pg/mL MSKI vs 10.9 pg/mL no MSKI; difference: 2.6, 95% CI: −0.6, 5.8), while PCT was higher in patients with a MSKI (0.12 ng/mL MSKIs vs 0.04 ng/mL no MSKI; difference: 0.08, 95% CI: 0.03 to 0.13). CGRP and PCT levels were not correlated (Spearman rank coefficient: −0.01, 95% CI: −0.15 to 0.13). CGRP had a lower AUC than procalcitonin [0.57, 95% CI: 0.47 to 0.66 CGRP vs 0.78, 95% CI: 0.69 to 0.87 PCT, P < 0.01]. Although biochemically related, CGRP was not correlated with procalcitonin in children undergoing evaluation for a MSKI. Our exploratory pilot highlights the ongoing need for novel biomarkers for the accurate and timely identification of children with a MSKI.

Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.

Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (T H 17)–like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ + regulatory T (T reg ) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1 + neurons in dorsal root ganglia decreased T reg cell numbers via the neuropeptide calcitonin gene–related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut T reg cell function.

Influenza viruses are a major global cause of morbidity and mortality. Vagal TRPV1 + nociceptive sensory neurons, which innervate the airways, are known to mediate defenses against harmful agents. However, their function in lung antiviral defenses remains unclear. Our study reveals that both systemic and vagal-specific ablation of TRPV1 + nociceptors reduced survival in mice infected with influenza A virus (IAV), despite no significant changes in viral burden or weight loss. Mice lacking nociceptors showed exacerbated lung pathology and elevated levels of pro-inflammatory cytokines. The increased mortality was not attributable to the loss of the TRPV1 ion channel or neuropeptides CGRP or substance P. Immune profiling through flow cytometry and single-cell RNA sequencing identified significant nociceptor deficiency-mediated changes in the lung immune landscape, including an expansion of neutrophils and monocyte-derived macrophages. Transcriptional analysis revealed impaired interferon signaling in these myeloid cells and an imbalance in distinct neutrophil sub-populations in the absence of nociceptors. Furthermore, anti-GR1-mediated depletion of myeloid cells during IAV infection significantly improved survival, underscoring a role of nociceptors in preventing pathogenic myeloid cell states that contribute to IAV-induced mortality. One Sentence Summary : TRPV1 + neurons facilitate host survival from influenza A virus infection by controlling myeloid cell responses and immunopathology.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3–5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP–RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis. Two Streptococcus spp. can utilize a neuropeptide (CGRP) and its receptor (RAMP1) on macrophages to promote brain invasion, a finding that may help the development of therapies for bacterial meningitis.

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection with Citrobacter rodentium or enterohemorrhagic Escherichia coli, luminal C3 levels increase significantly and are required for protection. C. rodentium is remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis of C. rodentium functions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals’ intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection. Highlights Gut complement component 3 (C3) is induced by the microbiome in healthy humans and mice at a microbiota-specific level. Gut stromal cells located in intestinal lymphoid follicles are a major source of luminal C3 During enteric infections with Citrobacter rodentium or enterohemorrhagic Escherichia coli, gut luminal C3 levels increase and are required for protection. C. rodentium is significantly more invasive of the gut epithelium in C3-deficient mice when compared to WT mice. In the gut, C3-mediated opsonophagocytosis of C. rodentium functions to clear pathogens.

Epithelial response to injury is coordinated through an intricate interaction with neuronal and myeloid cells, however the signaling modules involved are not well understood. In humans, somatic mutations in Tet methylcytosine dioxygenase 2 (TET2), a DNA demethylase, are commonly observed during ageing in myeloid cells and known to modulate inflammatory responses. Using a mouse model that lacks TET2 in myeloid cells (Tet2 ΔLysM), we show that myeloid cells and sympathetic neurons form a signaling nexus that controls differentiation of enterochromaffin cells and serotonin production during colonic inflammation. Under physiological conditions, TET2 restricts IL-1β production by myeloid cells which in turn controls the intestinal sympathetic architecture. During inflammation, IL1R signaling limits sympathetic cues that drive differentiation of enterochromaffin cells through α1-adrenergic signaling. As a result, enterochromaffin differentiation and colitis progression in response to mucosal injury is attenuated in Tet2 ΔLysMmice. Further, protection from colitis in Tet2 ΔLysMmice is mediated by its catalytic activity, and dependent on sympathetic neurons and IL1R signaling. Adrenergic control of epithelial response and pro-colitic serotonin production is also evident under conditions of physiological stress that leads to increased colitis susceptibility. Overall, our study reveals a sympathetic-epithelial axis that controls the severity of colitis and is modulated by myeloid-derived IL-1β and physiological stress. Our findings may also explain why inflammatory bowel disease in the elderly, where TET2 mutations in myeloid cells are common, is less severe and suggests TET2 activity as an attractive target for IBD. Supported by the US National Institutes of Health (DK067180) to B.J, the University of Chicago's Center for Interdisciplinary Study of Inflammatory Intestinal Disorders (C-IID) Pilot & Feasibility Award (NIDDK P30 DK042086) to A.M. and D.S, Crohn’s and Colitis Foundation Career Development Award #964209 to A.M. and G.I. Research Foundation Associates Board Award to A.M. and D.S.

The gastrointestinal tract is densely innervated by the peripheral nervous system and populated by the immune system. These two systems critically coordinate the sensations of and adaptations to dietary, microbial, and damaging stimuli from the external and internal microenvironment during tissue homeostasis and inflammation. The brain receives and integrates ascending sensory signals from the gut and transduces descending signals back to the gut via autonomic neurons. Neurons regulate intestinal immune responses through the action of local axon reflexes or through neuronal circuits via the gut-brain axis. This neuroimmune crosstalk is critical for gut homeostatic maintenance and disease resolution. In this review, we discuss the roles of distinct types of gut-innervating neurons in the modulation of intestinal mucosal immunity. We will focus on the molecular mechanisms governing how different immune cells respond to neural signals in host defense and inflammation. We also discuss the therapeutic potential of strategies targeting neuroimmune crosstalk for intestinal diseases.

Neuroepithelial crosstalk is critical for gut physiology. However, the mechanisms by which sensory neurons communicate with epithelial cells to mediate gut barrier protection at homeostasis and during inflammation are not well understood. Here, we find that Nav1.8+CGRP+ nociceptor neurons are juxtaposed with and signal to intestinal goblet cells to drive mucus secretion and gut protection. Nociceptor ablation led to decreased mucus thickness and dysbiosis, while chemogenetic nociceptor activation or capsaicin treatment induced mucus growth. Mouse and human goblet cells expressed Ramp1, receptor for the neuropeptide CGRP. Nociceptors signal via the CGRP-Ramp1 pathway to induce rapid goblet cell emptying and mucus secretion. Notably, commensal microbes activated nociceptors to control homeostatic CGRP release. In the absence of nociceptors or epithelial Ramp1, mice showed increased epithelial stress and susceptibility to colitis. Conversely, CGRP administration protected nociceptor-ablated mice against colitis. Our findings demonstrate a neuron-goblet cell axis that orchestrates gut mucosal barrier protection.

In the gut, coordinated cell interactions regulate tissue repair and immunity. How enteric glial cells (EGCs) mediate these processes remained elusive. In a recent paper, Progatzky et al. demonstrate that EGCs interact with immune and mesothelial cells under homeostasis and helminth infection, revealing an indispensable role of an interferon-γ (IFNγ)-EGC-CXCL10 axis in tissue repair.

Gene therapy offers promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS, PNS). However, genetic access remains difficult, reflecting the need for development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated viral (AAV) capsid, serotype-9, in mouse models, and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents, and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver: (1) the neuronal sensor GCaMP8s to record calcium signal dynamics in nodose ganglia and evaluate vagal function in a gastric assay, and (2) the neuronal actuator DREADD to dorsal root ganglia and mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species, and their functional utility.

Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.

The gastrointestinal tract is densely innervated by a complex network of neurons that coordinate critical physiological functions. Here, we summarize recent studies investigating the crosstalk between gut-innervating neurons, resident immune cells, and epithelial cells at homeostasis and during infection, food allergy, and inflammatory bowel disease. We introduce the neuroanatomy of the gastrointestinal tract, detailing gut-extrinsic neuron populations from the spinal cord and brain stem, and neurons of the intrinsic enteric nervous system. We highlight the roles these neurons play in regulating the functions of innate immune cells, adaptive immune cells, and intestinal epithelial cells. We discuss the consequences of such signaling for mucosal immunity. Finally, we discuss how the intestinal microbiota is integrated into the neuro-immune axis by tuning neuronal and immune interactions. Understanding the molecular events governing the intestinal neuro-immune signaling axes will enhance our knowledge of physiology and may provide novel therapeutic targets to treat inflammatory diseases.

BACKGROUND:Exposure to early-life undernutrition is closely related to higher risks of adverse immunologic outcomes in adulthood. Although it has been suggested that asthma has its origins in early life, its underlying mechanisms remain largely unknown. OBJECTIVE:We characterized the effects of early-life undernutrition on T lymphocytes, which play a pivotal role in immune diseases, and we investigated whether this contributes to susceptibility to asthma in adulthood. METHODS:Pregnant mice were fed a protein restriction diet (PRD) to establish an early-life undernutrition model. Naive CD4+ T cells (CD4+CD62LhiCD44-) from offspring were used throughout the study. TH2 differentiation was examined by using fluorescence-activated cell sorting and ELISA under TH2-polarized conditions in vitro and through ovalbumin-induced experimental asthma in vivo. T-cell metabolism was measured with a Seahorse XF96 Analyzer. DNA methylation levels were measured by using bisulfite sequencing. RESULTS:PRD CD4+ T cells displayed increased activation and proliferation and were prone to differentiate into TH2 cells both in vitro and in vivo, leading to susceptibility to experimental asthma. Mechanistically, early-life undernutrition upregulated mechanistic target of rapamycin 1-dependent glycolysis and induced conserved noncoding DNA sequence 1 DNA hypomethylation in the TH2 cytokine locus of CD4+ T cells. Glycolysis blockades undermined increased TH2 skewing and alleviated experimental asthma in PRD mice. CONCLUSION:Early-life undernutrition induced mechanistic target of rapamycin 1-dependent glycolysis upregulation and TH2 cytokine locus hypomethylation in CD4+ T cells, resulting in increased T-cell activation, proliferation, and TH2 skewing and further susceptibility to experimental asthma.