Gut-Innervating Nociceptor Neurons Regulate Peyer's Patch Microfold Cells and SFB Levels to Mediate Salmonella Host Defense.

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.

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Maternal gut bacteria drive intestinal inflammation in offspring with neurodevelopmental disorders by altering the chromatin landscape of CD4+ T cells.

Children with autism spectrum disorders often display dysregulated immune responses and related gastrointestinal symptoms. However, the underlying mechanisms leading to the development of both phenotypes have not been elucidated. Here, we show that mouse offspring exhibiting autism-like phenotypes due to prenatal exposure to maternal inflammation were more susceptible to developing intestinal inflammation following challenges later in life. In contrast to its prenatal role in neurodevelopmental phenotypes, interleukin-17A (IL-17A) generated immune-primed phenotypes in offspring through changes in the maternal gut microbiota that led to postnatal alterations in the chromatin landscape of naive CD4+ T cells. The transfer of stool samples from pregnant mice with enhanced IL-17A responses into germ-free dams produced immune-primed phenotypes in offspring. Our study provides mechanistic insights into why children exposed to heightened inflammation in the womb might have an increased risk of developing inflammatory diseases in addition to neurodevelopmental disorders.

Human gut bacteria produce ΤΗ17-modulating bile acid metabolites.

The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed TH17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key TH17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of TH17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit TH17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease.

The Kinase IKKß Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKß and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKß and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKß to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.

Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

SLC46 Family Transporters Facilitate Cytosolic Innate Immune Recognition of Monomeric Peptidoglycans.

Tracheal cytotoxin (TCT), a monomer of DAP-type peptidoglycan from Bordetella pertussis, causes cytopathology in the respiratory epithelia of mammals and robustly triggers the Drosophila Imd pathway. PGRP-LE, a cytosolic innate immune sensor in Drosophila, directly recognizes TCT and triggers the Imd pathway, yet the mechanisms by which TCT accesses the cytosol are poorly understood. In this study, we report that CG8046, a Drosophila SLC46 family transporter, is a novel transporter facilitating cytosolic recognition of TCT, and plays a crucial role in protecting flies against systemic Escherichia coli infection. In addition, mammalian SLC46A2s promote TCT-triggered NOD1 activation in human epithelial cell lines, indicating that SLC46As is a conserved group of peptidoglycan transporter contributing to cytosolic immune recognition.

The caspase-8 homolog Dredd cleaves Imd and Relish but is not inhibited by p35.

In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo.

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan.

Overexpression of malic enzyme in the larval stage extends Drosophila lifespan.

Metabolic modifications during the developmental period can extend longevity. We found that malic enzyme (Men) overexpression during the larval period lengthened the lifespan of Drosophila. Men overexpression by S106-GeneSwitch-Gal4 driver increased pyruvate content and NADPH/NADP(+) ratio but reduced triglyceride, glycogen, and ATP levels in the larvae. ROS levels increased unexpectedly in Men-overexpressing larvae. Interestingly, adults exposed to larval Men-overexpression maintained ROS tolerance with enhanced expression levels of glutathione-S-transferase D2 and thioredoxin-2. Our results suggest that metabolic changes mediated by Men during development might be related to the control of ROS tolerance and the longevity of Drosophila.

cAMP signalling in mushroom bodies modulates temperature preference behaviour in Drosophila.

Homoiotherms, for example mammals, regulate their body temperature with physiological responses such as a change of metabolic rate and sweating. In contrast, the body temperature of poikilotherms, for example Drosophila, is the result of heat exchange with the surrounding environment as a result of the large ratio of surface area to volume of their bodies. Accordingly, these animals must instinctively move to places with an environmental temperature as close as possible to their genetically determined desired temperature. The temperature that Drosophila instinctively prefers has a function equivalent to the 'set point' temperature in mammals. Although various temperature-gated TRP channels have been discovered, molecular and cellular components in Drosophila brain responsible for determining the desired temperature remain unknown. We identified these components by performing a large-scale genetic screen of temperature preference behaviour (TPB) in Drosophila. In parallel, we mapped areas of the Drosophila brain controlling TPB by targeted inactivation of neurons with tetanus toxin and a potassium channel (Kir2.1) driven with various brain-specific GAL4s. Here we show that mushroom bodies (MBs) and the cyclic AMP-cAMP-dependent protein kinase A (cAMP-PKA) pathway are essential for controlling TPB. Furthermore, targeted expression of cAMP-PKA pathway components in only the MB was sufficient to rescue abnormal TPB of the corresponding mutants. Preferred temperatures were affected by the level of cAMP and PKA activity in the MBs in various PKA pathway mutants.

A bacterial bile acid metabolite modulates Treg activity through the nuclear hormone receptor NR4A1.

Bile acids act as signaling molecules that regulate immune homeostasis, including the differentiation of CD4+ T cells into distinct T cell subsets. The bile acid metabolite isoallolithocholic acid (isoalloLCA) enhances the differentiation of anti-inflammatory regulatory T cells (Treg cells) by facilitating the formation of a permissive chromatin structure in the promoter region of the transcription factor forkhead box P3 (Foxp3). Here, we identify gut bacteria that synthesize isoalloLCA from 3-oxolithocholic acid and uncover a gene cluster responsible for the conversion in members of the abundant human gut bacterial phylum Bacteroidetes. We also show that the nuclear hormone receptor NR4A1 is required for the effect of isoalloLCA on Treg cells. Moreover, the levels of isoalloLCA and its biosynthetic genes are significantly reduced in patients with inflammatory bowel diseases, suggesting that isoalloLCA and its bacterial producers may play a critical role in maintaining immune homeostasis in humans.

A microbial metabolite remodels the gut-liver axis following bariatric surgery.

Bariatric surgery is the most effective treatment for type 2 diabetes and is associated with changes in gut metabolites. Previous work uncovered a gut-restricted TGR5 agonist with anti-diabetic properties-cholic acid-7-sulfate (CA7S)-that is elevated following sleeve gastrectomy (SG). Here, we elucidate a microbiome-dependent pathway by which SG increases CA7S production. We show that a microbial metabolite, lithocholic acid (LCA), is increased in murine portal veins post-SG and by activating the vitamin D receptor, induces hepatic mSult2A1/hSULT2A expression to drive CA7S production. An SG-induced shift in the microbiome increases gut expression of the bile acid transporters Asbt and Ostα, which in turn facilitate selective transport of LCA across the gut epithelium. Cecal microbiota transplant from SG animals is sufficient to recreate the pathway in germ-free (GF) animals. Activation of this gut-liver pathway leads to CA7S synthesis and GLP-1 secretion, causally connecting a microbial metabolite with the improvement of diabetic phenotypes.

Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF.

The pigment-dispersing factor (PDF) is a neuropeptide controlling circadian behavioral rhythms in Drosophila, but its receptor is not yet known. From a large-scale temperature preference behavior screen in Drosophila, we isolated a P insertion mutant that preferred different temperatures during the day and night. This mutation, which we named han, reduced the transcript level of CG13758. We found that Han was expressed specifically in 13 pairs of circadian clock neurons in the adult brain. han null flies showed arrhythmic circadian behavior in constant darkness. The behavioral characteristics of han null mutants were similar to those of pdf null mutants. We also found that PDF binds specifically to S2 cells expressing Han, which results in the elevation of cAMP synthesis. Therefore, we herein propose that Han is a PDF receptor regulating circadian behavioral rhythm through coordination of activities of clock neurons.

Fibroblast activation protein alpha identifies mesenchymal stromal cells from human bone marrow.

Mesenchymal stromal cells (MSCs) have gained widespread popularity in cell therapy, but their development into clinical products has been impeded by the scarcity of cell-specific markers. We previously explored transcriptome and membrane proteome of MSCs, from which fibroblast activation protein alpha (FAP) was recognized as a prime surface marker candidate. The present study showed that FAP was constitutively expressed on MSCs, but not on other cells. FAP immunoselection yielded homogeneous MSCs from cryopreserved bone marrow (BM). These results suggest that FAP serves as a surface protein marker that can singly define MSCs from BM and possibly from other sources.

Histamine and its receptors modulate temperature-preference behaviors in Drosophila.

Temperature profoundly influences various life phenomena, and most animals have developed mechanisms to respond properly to environmental temperature fluctuations. To identify genes involved in sensing ambient temperature and in responding to its change, >27,000 independent P-element insertion mutants of Drosophila were screened. As a result, we found that defects in the genes encoding for proteins involved in histamine signaling [histidine decarboxylase (hdc), histamine-gated chloride channel subunit 1 (hisCl1), ora transientless (ort)] cause abnormal temperature preferences. The abnormal preferences shown in these mutants were restored by genetic and pharmacological rescue and could be reproduced in wild type using the histamine receptor inhibitors cimetidine and hydroxyzine. Spatial expression of these genes was observed in various brain regions including pars intercerebralis, fan-shaped body, and circadian clock neurons but not in dTRPA1-expressing neurons, an essential element for thermotaxis. We also found that the histaminergic mutants showed reduced tolerance for high temperature and enhanced tolerance for cold temperature. Together, these results suggest that histamine signaling may have important roles in modulating temperature preference and in controlling tolerance of low and high temperature.

Development of a Reporter System for In Vivo Monitoring of γ-Secretase Activity in Drosophila.

The γ-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the ß-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the γ-secretase catalytic component, presenilin, which lead to increased amyloid ßpeptide production, are responsible for early-onset familial Alzheimer's disease. ß-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. Here, we created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of γ-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, we find that apoptosis partly contributes to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate γ-secretase activity and/or APPL metabolism.