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, mediated by receptors including Frizzled 1/2/7 (FZD1/2/7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB action stimulates 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 (DT), into peptidergic sensory neurons that express exogeneous DT receptor (an approach we termed toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP, or the SP receptor (NK1R) show reduced pathology in both models of cecal TcdB injection and CDI. Blocking SP or CGRP signaling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.
Publications
2023
Manion, John, Melissa A Musser, Gavin A Kuziel, Min Liu, Amy Shepherd, Siyu Wang, Pyung-Gang Lee, et al. (2023) 2023. “C. Difficile Intoxicates Neurons and Pericytes to Drive Neurogenic Inflammation.”. Nature. https://doi.org/10.1038/s41586-023-06607-2.
Prochera, Aleksandra, and Meenakshi Rao. (2023) 2023. “Mini-Review: Enteric Glial Regulation of the Gastrointestinal Epithelium.”. Neuroscience Letters 805: 137215. https://doi.org/10.1016/j.neulet.2023.137215.
Many enteric glia are located along nerve fibers in the gut mucosa where they form close associations with the epithelium lining the gastrointestinal tract. The gut epithelium is essential for absorbing nutrients, regulating fluid flux, forming a physical barrier to prevent the entry of pathogens and toxins into the host, and participating in immune responses. Disruptions to this epithelium are linked to numerous diseases, highlighting its central importance in maintaining health. Accumulating evidence indicates that glia regulate gut epithelial homeostasis. Observations from glial-epithelial co-cultures in vitro and mouse genetic models in vivo suggest that enteric glia influence several important features of the gut epithelium including barrier integrity, ion transport, and capacity for self-renewal. Here we review the evidence for enteric glial regulation of the intestinal epithelium, with a focus on these three features of its biology.
2022
Rastelli, Daniella, Ariel Robinson, Valentina Lagomarsino, Lynley Matthews, Rafla Hassan, Kristina Perez, William Dan, et al. 2022. “Diminished Androgen Levels Are Linked to Irritable Bowel Syndrome and Cause Bowel Dysfunction in Mice”. J Clin Invest 132 (2). https://doi.org/10.1172/JCI150789.
Functional gastrointestinal disorders (FGIDs) have prominent sex differences in incidence, symptoms, and treatment response that are not well understood. Androgens are steroid hormones present at much higher levels in males than females and could be involved in these differences. In adults with irritable bowel syndrome (IBS), a FGID that affects 5% to 10% of the population worldwide, we found that free testosterone levels were lower than those in healthy controls and inversely correlated with symptom severity. To determine how this diminished androgen signaling could contribute to bowel dysfunction, we depleted gonadal androgens in adult mice and found that this caused a profound deficit in gastrointestinal transit. Restoring a single androgen hormone was sufficient to rescue this deficit, suggesting that circulating androgens are essential for normal bowel motility in vivo. To determine the site of action, we probed androgen receptor expression in the intestine and discovered, unexpectedly, that a large subset of enteric neurons became androgen-responsive upon puberty. Androgen signaling to these neurons was required for normal colonic motility in adult mice. Taken together, these observations establish a role for gonadal androgens in the neural regulation of bowel function and link altered androgen levels with a common digestive disorder.
2021
Rao, Meenakshi, and Milena Bogunovic. 2021. “Enteric Glia Worm Their Way into Gut Immunity”. Immunity 54 (12): 2698-2700. https://doi.org/10.1016/j.immuni.2021.11.014.
The gut houses one of the largest populations of glia in the nervous system, yet their essential functions remain unclear. New work by Progatzky et al. (2021) in Nature reveals that these enteric glia orchestrate an IFNγ-dependent immune response to helminth infection that promotes tissue repair.
Dan, William, Ga Hyun Park, Shruti Vemaraju, Amy Wu, Kristina Perez, Meenakshi Rao, Dan Berkowitz, Richard Lang, and Peter Yim. (2021) 2021. “Light-Mediated Inhibition of Colonic Smooth Muscle Constriction and Colonic MotilityOpsin 3”. Front Physiol 12: 744294. https://doi.org/10.3389/fphys.2021.744294.
Opsin photoreceptors outside of the central nervous system have been shown to mediate smooth muscle photorelaxation in several organs. We hypothesized that opsin receptor activation in the colon would have a similar effect and influence colonic motility. We detected Opsin 3 (OPN3) protein expression in the colonic wall and demonstrated that OPN3 was present in enteric neurons in the muscularis propria of the murine colon. Precontracted murine colon segments demonstrated blue light (BL) -mediated relaxation ex vivo. This photorelaxation was wavelength specific and was increased with the administration of the chromophore 9-cis retinal and a G protein receptor kinase 2 (GRK2) inhibitor. Light-mediated relaxation of the colon was not inhibited by L-NAME or tetrodotoxin (TTX). Furthermore, BL exposure in the presence of 9-cis retinal decreased the frequency of colonic migrating motor complexes (CMMC) in spontaneously contracting mouse colons ex vivo. These results demonstrate for the first time a receptor-mediated photorelaxation of colonic smooth muscle and implicate opsins as possible new targets in the treatment of spasmodic gastrointestinal dysmotility.
Rosenberg, Harry, and Meenakshi Rao. 2021. “Enteric Glia in Homeostasis and Disease: From Fundamental Biology to Human Pathology”. IScience 24 (8): 102863. https://doi.org/10.1016/j.isci.2021.102863.
Glia, the non-neuronal cells of the nervous system, were long considered secondary cells only necessary for supporting the functions of their more important neuronal neighbors. Work by many groups over the past two decades has completely overturned this notion, revealing the myriad and vital functions of glia in nervous system development, plasticity, and health. The largest population of glia outside the brain is in the enteric nervous system, a division of the autonomic nervous system that constitutes a key node of the gut-brain axis. Here, we review the latest in the understanding of these enteric glia in mammals with a focus on their putative roles in human health and disease.
Yan, Yiqing, Deepshika Ramanan, Milena Rozenberg, Kelly McGovern, Daniella Rastelli, Brinda Vijaykumar, Omar Yaghi, et al. 2021. “Interleukin-6 Produced by Enteric Neurons Regulates the Number and Phenotype of Microbe-Responsive Regulatory T cells in the Gut”. Immunity 54 (3): 499-513.e5. https://doi.org/10.1016/j.immuni.2021.02.002.
The immune and enteric nervous (ENS) systems monitor the frontier with commensal and pathogenic microbes in the colon. We investigated whether FoxP3 regulatory T (Treg) cells functionally interact with the ENS. Indeed, microbe-responsive RORγ and Helios subsets localized in close apposition to nitrergic and peptidergic nerve fibers in the colon lamina propria (LP). Enteric neurons inhibited in vitro Treg (iTreg) differentiation in a cell-contact-independent manner. A screen of neuron-secreted factors revealed a role for interleukin-6 (IL-6) in modulating iTreg formation and their RORγ proportion. Colonization of germfree mice with commensals, especially RORγ Treg inducers, broadly diminished colon neuronal density. Closing the triangle, conditional ablation of IL-6 in neurons increased total Treg cells but decreased the RORγ subset, as did depletion of two ENS neurotransmitters. Our findings suggest a regulatory circuit wherein microbial signals condition neuronal density and activation, thus tuning Treg cell generation and immunological tolerance in the gut.
2020
Jazayeri, Amir, Dieudonne Nonga, and Meenakshi Rao. 2020. “The Key to a Boy’s Heart Is Through His Intestine”. Gastroenterology 158 (5): 1226-28. https://doi.org/10.1053/j.gastro.2019.08.056.
Lindahl, Maria, Alcmène Chalazonitis, Erik Palm, Emmi Pakarinen, Tatiana Danilova, Tuan Pham, Wanda Setlik, et al. 2020. “Cerebral Dopamine Neurotrophic Factor-Deficiency Leads to Degeneration of Enteric Neurons and Altered Brain Dopamine Neuronal Function in Mice”. Neurobiol Dis 134: 104696. https://doi.org/10.1016/j.nbd.2019.104696.
Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf mouse are much greater than in those of Cdnf mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf mouse brain is altered. The deficiencies of Cdnf mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.
Chalazonitis, Alcmène, ZhiShan Li, Tuan Pham, Jason Chen, Meenakshi Rao, Päivi Lindholm, Mart Saarma, Maria Lindahl, and Michael Gershon. 2020. “Cerebral Dopamine Neurotrophic Factor Is Essential for Enteric Neuronal Development, Maintenance, and Regulation of Gastrointestinal Transit”. J Comp Neurol. https://doi.org/10.1002/cne.24901.
Cerebral dopamine neurotrophic factor (CDNF) is expressed in the brain and is neuroprotective. We have previously shown that CDNF is also expressed in the bowel and that its absence leads to degeneration and autophagy in the enteric nervous system (ENS), particularly in the submucosal plexus. We now demonstrate that enteric CDNF immunoreactivity is restricted to neurons (submucosal > myenteric) and is not seen in glia, interstitial cells of Cajal, or smooth muscle. Expression of CDNF, moreover, is essential for the normal development and survival of enteric dopaminergic neurons; thus, expression of the dopaminergic neuronal markers, dopamine, tyrosine hydroxylase, and dopamine transporter are deficient in the ileum of Cdnf mice. The normal age-related decline in proportions of submucosal dopaminergic neurons is exacerbated in Cdnf animals. The defect in Cdnf animals is not dopamine-restricted; proportions of other submucosal neurons (NOS-, GABA-, and CGRP-expressing), are also deficient. The deficits in submucosal neurons are reflected functionally in delayed gastric emptying, slowed colonic motility, and prolonged total gastrointestinal transit. CDNF is expressed selectively in isolated enteric neural crest-derived cells (ENCDC), which also express the dopamine-related transcription factor Foxa2. Addition of CDNF to ENCDC promotes development of dopaminergic neurons; moreover, survival of these neurons becomes CDNF-dependent after exposure to bone morphogenetic protein 4. The effects of neither glial cell-derived neurotrophic factor (GDNF) nor serotonin are additive with CDNF. We suggest that CDNF plays a critical role in development and long-term maintenance of dopaminergic and other sets of submucosal neurons.