C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Sox10-cre BAC transgenes reveal temporal restriction of mesenchymal cranial neural crest and identify glandular Sox10 expression.

Diversity of neural crest derivatives has been studied with a variety of approaches during embryonic development. In mammals Cre-LoxP lineage tracing is a robust means to fate map neural crest relying on cre driven from regulatory elements of early neural crest genes. Sox10 is an essential transcription factor for normal neural crest development. A variety of efforts have been made to label neural crest derivatives using partial Sox10 regulatory elements to drive cre expression. To date published Sox10-cre lines have focused primarily on lineage tracing in specific tissues or during early fetal development. We describe two new Sox10-cre BAC transgenes, constitutive (cre) and inducible (cre/ERT2), that contain the complete repertoire of Sox10 regulatory elements. We present a thorough expression profile of each, identifying a few novel sites of Sox10 expression not captured by other neural crest cre drivers. Comparative mapping of expression patterns between the Sox10-cre and Sox10-cre/ERT2 transgenes identified a narrow temporal window in which Sox10 expression is present in mesenchymal derivatives prior to becoming restricted to neural elements during embryogenesis. In more caudal structures, such as the intestine and lower urinary tract, our Sox10-cre BAC transgene appears to be more efficient in labeling neural crest-derived cell types than Wnt1-cre. The analysis reveals consistent expression of Sox10 in non-neural crest derived glandular epithelium, including salivary, mammary, and urethral glands of adult mice. These Sox10-cre and Sox10-cre/ERT2 transgenic lines are verified tools that will enable refined temporal and cell-type specific lineage analysis of neural crest derivatives as well as glandular tissues that rely on Sox10 for proper development and function.

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.

Mechanical Skin Injury Promotes Food Anaphylaxis by Driving Intestinal Mast Cell Expansion.

Mast cell (MC) mediator release after crosslinking of surface-bound IgE antibody by ingested antigen underlies food allergy. However, IgE antibodies are not uniformly associated with food allergy, and intestinal MC load is an important determinant. Atopic dermatitis (AD), characterized by pruritis and cutaneous sensitization to allergens, including foods, is strongly associated with food allergy. Tape stripping mouse skin, a surrogate for scratching, caused expansion and activation of small intestinal MCs, increased intestinal permeability, and promoted food anaphylaxis in sensitized mice. Tape stripping caused keratinocytes to systemically release interleukin-33 (IL-33), which synergized with intestinal tuft-cell-derived IL-25 to drive the expansion and activation of intestinal type-2 innate lymphoid cells (ILC2s). These provided IL-4, which targeted MCs to expand in the intestine. Duodenal MCs were expanded in AD. In addition to promoting cutaneous sensitization to foods, scratching may promote food anaphylaxis in AD by expanding and activating intestinal MCs.

A workshop on leadership for senior MD-PhD students.

Leadership skills are essential for a successful career as a physician-scientist, yet many MD-PhD training programs do not offer formal training in leadership. The Vanderbilt Medical Scientist Training Program (MSTP) previously established a 2-day leadership workshop that has been held biennially since 2006 for students in the first and second years of the graduate school portion of combined MD and PhD training (G1/G2 students). Workshop attendees have consistently rated this workshop as a highly effective experience. However, opportunities for structured training in leadership competencies during the subsequent 3-5 years of MD-PhD training are limited. Given the success of the G1/G2 leadership workshop and the need for continuity in this model of leadership training, we developed a half-day workshop for MSTP students in the clinical years of medical school (M3/M4 students) to foster continued training in leadership. Our workshop curriculum, based in part on original cases drafted by Vanderbilt MSTP students, provides concrete strategies to manage conflict and navigate leadership transitions in the physician-scientist career path. The curriculum emphasizes both short-term competencies, such as effective participation as a member of a clinical team, and long-term competencies, such as leadership of a research team, division, or department. Our inaugural senior leadership workshop, held in August, 2015, was judged by student participants to be well organized and highly relevant to leadership concepts and skills. It will be offered biennially in our training curriculum for M3 and M4 MSTP students.

Enteric neuron imbalance and proximal dysmotility in ganglionated intestine of the Sox10Dom/+ Hirschsprung mouse model.

BACKGROUND & AIMS: In Hirschsprung disease (HSCR), neural crest-derived progenitors (NCPs) fail to completely colonize the intestine so that the enteric nervous system (ENS) is absent from distal bowel. Despite removal of the aganglionic region, many HSCR patients suffer from residual intestinal dysmotility. To test the hypothesis that inappropriate lineage segregation of NCPs in proximal ganglionated regions of the bowel could contribute to such postoperative disease, we investigated neural crest (NC)-derived lineages and motility in ganglionated, postnatal intestine of the Sox10Dom/+ HSCR mouse model. METHODS: Cre-mediated fate-mapping was applied to evaluate relative proportions of NC-derived cell types. Motility assays were performed to assess gastric emptying and small intestine motility while colonic inflammation was assessed by histopathology for Sox10Dom/+ mutants relative to wildtype controls. RESULTS: Sox10Dom/+ mice showed regional alterations in neuron and glia proportions as well as Calretinin+ and nNOS+ neuronal subtypes. In the colon, imbalance of enteric NC derivatives correlated with the extent of aganglionosis. All Sox10Dom/+ mice exhibited reduced small intestinal transit at 4-weeks of age, and at 6-weeks, Sox10Dom/+ males had increased gastric emptying rates. Sox10Dom/+ mice surviving to 6-weeks of age had little or no colonic inflammation when compared to wildtype littermates, suggesting that these changes in GI motility are neurally mediated. CONCLUSIONS: The Sox10Dom mutation disrupts the balance of NC-derived lineages and affects GI motility in the proximal, ganglionated intestine of adult animals. This is the first report identifying alterations in enteric neuronal classes in Sox10Dom/+ mutants, which suggests a previously unrecognized role for Sox10 in neuronal subtype specification.

LRIG1 Regulates Ontogeny of Smooth Muscle-Derived Subsets of Interstitial Cells of Cajal in Mice.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) control intestinal smooth muscle contraction to regulate gut motility. ICC within the plane of the myenteric plexus (ICC-MY) arise from KIT-positive progenitor cells during mouse embryogenesis. However, little is known about the ontogeny of ICC associated with the deep muscular plexus (ICC-DMP) in the small intestine and ICC associated with the submucosal plexus (ICC-SMP) in the colon. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) marks intestinal epithelial stem cells, but the role of LRIG1 in nonepithelial intestinal cells has not been identified. We sought to determine the ontogeny of ICC-DMP and ICC-SMP, and whether LRIG1 has a role in their development. METHODS: Lrig1-null mice (homozygous Lrig1-CreERT2) and wild-type mice were analyzed by immunofluorescence and transit assays. Transit was evaluated by passage of orally administered rhodamine B-conjugated dextran. Lrig1-CreERT2 mice or mice with CreERT2 under control of an inducible smooth muscle promoter (Myh11-CreERT2) were crossed with Rosa26-LSL-YFP mice for lineage tracing analysis. RESULTS: In immunofluorescence assays, ICC-DMP and ICC-SMP were found to express LRIG1. Based on lineage tracing, ICC-DMP and ICC-SMP each arose from LRIG1-positive smooth muscle progenitors. In Lrig1-null mice, there was loss of staining for KIT in DMP and SMP regions, as well as for 2 additional ICC markers (anoctamin-1 and neurokinin 1 receptor). Lrig1-null mice had significant delays in small intestinal transit compared with control mice. CONCLUSIONS: LRIG1 regulates the postnatal development of ICC-DMP and ICC-SMP from smooth muscle progenitors in mice. Slowed small intestinal transit observed in Lrig1-null mice may be due, at least in part, to loss of the ICC-DMP population.

Balancing on the crest - Evidence for disruption of the enteric ganglia via inappropriate lineage segregation and consequences for gastrointestinal function.

Normal enteric nervous system (ENS) development relies on numerous factors, including appropriate migration, proliferation, differentiation, and maturation of neural crest (NC) derivatives. Incomplete rostral to caudal migration of enteric neural crest-derived progenitors (ENPs) down the gut is at least partially responsible for the absence of enteric ganglia that is a hallmark feature of Hirschsprung disease (HSCR). The thought that ganglia proximal to aganglionosis are normal has guided surgical procedures for HSCR patients. However, chronic gastrointestinal dysfunction suffered by a subset of patients after surgery as well as studies in HSCR mouse models suggest that aberrant NC segregation and differentiation may be occurring in ganglionated regions of the intestine. Studies in mouse models that possess enteric ganglia throughout the length of the intestine (non-HSCR) have also found that certain genetic alterations affect neural crest lineage balance and interestingly many of these mutants also have functional gastrointestinal (GI) defects. It is possible that many GI disorders can be explained in part by imbalances in NC-derived lineages. Here we review studies evaluating ENS defects in HSCR and non-HSCR mouse models, concluding with clinical implications while highlighting areas requiring further study.

Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation.