Introduction Commensal gut microbiota play an important role in regulating metabolic and inflammatory conditions. Reshaping intestinal microbiota through pharmacologic means may be a viable treatment option. We sought to delineate the functional characteristics of glucocorticoid-mediated alterations on gut microbiota and their subsequent repercussions on host mucin regulation and colonic inflammation. Methods Adult male C57Bl/6 mice, germ-free (GF), Muc2-heterozygote (+/−), or Muc2-knockout (−/−) were injected with dexamethasone, a synthetic glucocorticoid, for four weeks. Fecal samples were collected for gut microbiota analysis via 16S rRNA T-RFLP and amplicon sequencing. Intestinal mucosa was collected for mucin gene expression studies. GF mice were conventionalized with gut microbes from treated- and non-treated groups to determine their functional capacities in recipient hosts. Results Exposure to DEX in WT mice led to substantial shifts in gut microbiota over a four-week period. Furthermore, a significant down-regulation of colonic Muc2 gene expression was observed after treatment. Muc2-knockout mice harbored a pro-inflammatory environment of gut microbes, characterized by the increase or decrease in prevalence of specific microbiota populations such as Clostridiales and Lactobacillaceae, respectively. This colitogenic phenotype was transmissible to IL10-knockout (IL10-KO) mice, a genetically susceptible model of colonic inflammatory disorders. Microbiota from donors pre-treated with DEX, however, ameliorated symptoms of inflammation. Conclusions Commensal gut bacteria may be a key mediator of the anti-inflammatory effects observed in the large intestine after GC exposure. These findings underscore the notion that intestinal microbes comprise a “microbial organ” essential for host physiology that can be targeted by therapeutic approaches to restore intestinal homeostasis.

Chronic diseases arise when there is mutual reinforcement of pathophysiological processes that cause an aberrant steady state. Such a sequence of events may underlie chronic constipation, which has been associated with dysbiosis of the gut. In this study we hypothesized that assemblage of microbial communities, directed by slow gastrointestinal transit, affects host function in a way that reinforces constipation and further maintains selection on microbial communities. In our study, we used two models – an opioid‐induced constipation model in mice, and a humanized mouse model where germ‐free mice were colonized with stool from a patient with constipation‐predominant irritable bowel syndrome (IBS‐C) in humans. We examined the impact of pharmacologically (loperamide)‐induced constipation (PIC) and IBS‐C on the structural and functional profile of the gut microbiota. Germ‐free (GF) mice were colonized with microbiota from PIC donor mice and IBS‐C patients to determine how the microbiota affects the host. PIC and IBS‐C promoted changes in the gut microbiota, characterized by increased relative abundance of Bacteroides ovatus and Parabacteroides distasonis in both models. PIC mice exhibited decreased luminal concentrations of butyrate in the cecum and altered metabolic profiles of the gut microbiota. Colonization of GF mice with PIC‐associated mice cecal or human IBS‐C fecal microbiota significantly increased GI transit time when compared to control microbiota recipients. IBS‐C‐associated gut microbiota also impacted colonic contractile properties. Our findings support the concept that constipation is characterized by disease‐associated steady states caused by reinforcement of pathophysiological factors in host‐microbe interactions.

Gunst SJ, Herring BP. Altered calcium signaling in colonic smooth muscle of type 1 diabetic mice. Am J Physiol Gastrointest Liver Physiol 302: G66 -G76, 2012. First published October 6, 2011 doi:10.1152/ajpgi.00183.2011.-Seventy-six percent of diabetic patients develop gastrointestinal symptoms, such as constipation. However, the direct effects of diabetes on intestinal smooth muscle are poorly described. This study aimed to identify the role played by smooth muscle in mediating diabetes-induced colonic dysmotility. To induce type 1 diabetes, mice were injected intraperitoneally with low-dose streptozotocin once a day for 5 days. Animals developed hyperglycemia (Ͼ200 mg/dl) 1 wk after the last injection and were euthanized 7-8 wk after the last treatment. Computed tomography demonstrated decreased overall gastrointestinal motility in the diabetic mice. In vitro contractility of colonic smooth muscle rings from diabetic mice was also decreased. Fura-2 ratiometric Ca 2ϩ imaging showed attenuated Ca 2ϩ increases in response to KCl stimulation that were associated with decreased light chain phosphorylation in diabetic mice. The diabetic mice also exhibited elevated basal Ca 2ϩ levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca 2ϩ handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for Ͻ1 wk also showed decreased colonic contractile responses that were associated with decreased Ca 2ϩ increases in response to KCl stimulation, although without an elevation in basal Ca 2ϩ levels or a significant change in the expression of Ca 2ϩ signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca 2ϩ influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility. streptozotocin; colon; voltage-gated calcium channel AS MANY AS 76% OF DIABETIC patients develop gastrointestinal (GI) symptoms, such as dysphagia, vomiting, constipation, diarrhea, or fecal incontinence, that have been linked to poor glycemic control, rather than duration of the disease (3,8). Of these, constipation resulting from impaired colonic motility is the most common symptom and affects ϳ60% of patients (8,24). Animal studies have shown that diabetes can lead to accelerated or delayed GI motility, depending on the animal model used and the specific parts of the GI tract tested. Several studies have used streptozotocin (STZ) to cause specific loss of pancreatic ␤-cells creating type 1 diabetes-like animal models. STZ-induced diabetic rats have been reported to exhibit increased small intestine smooth muscle mass and increased colon contractility (10, 26). The spontaneous contractile activity in STZ-induced diabetic rat colon smooth muscle was increased (12) without a change in intracellular Ca 2ϩ handling (11), while, in the ileum, intracellular Ca 2ϩ handling was decreased (11). Conversely, in STZ-induced diabetic mice, the ...

Acetylcholine (ACh)-synthesizing neurons are major components of the enteric nervous system (ENS). They release ACh and peptidergic neurotransmitters onto enteric neurons and muscle. However, pharmacological interrogation has proven inadequate to demonstrate an essential role for ACh. Our objective was to determine whether elimination of ACh synthesis during embryogenesis alters prenatal viability, intestinal function, the neurotransmitter complement, and the microbiome. Conditional deletion of choline acetyltransferase ( ChAT), the ACh synthetic enzyme, in neural crest-derived neurons ( ChAT-Null) was performed. Survival, ChAT activity, gut motility, and the microbiome were studied. ChAT was conditionally deleted in ENS neural crest-derived cells. Despite ChAT absence, mice were born live and survived the first 2 wk. They failed to gain significant weight in the third postnatal week, dying between postnatal d 18 and 30. Small intestinal transit of carmine red was 50% slower in ChAT-Nulls vs. WT and ChAT- Het. The colons of many neonatal ChAT-Null mice contained compacted feces, suggesting dysmotility. Microbiome analysis revealed dysbiosis in ChAT-Null mice. Developmental deletion of ChAT activity in enteric neurons results in proximal gastrointestinal tract dysmotility, critically diminished colonic transit, failure to thrive, intestinal dysbiosis, and death. ACh is necessary for sustained gut motility and survival of neonatal mice after weaning.-Johnson, C. D., Barlow-Anacker, A. J., Pierre, J. F., Touw, K., Erickson, C. S., Furness, J. B., Epstein, M. L., Gosain, A. Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Rodenberg JM, Hoggatt AM, Chen M, Touw K, Jones R, Herring BP. Regulation of serum response factor activity and smooth muscle cell apoptosis by chromodomain helicase DNA-binding protein 8. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation, migration, and apoptosis. It is generally accepted that one mechanism by which SRF regulates these diverse functions is through pathwayspecific cofactor interactions. A novel SRF cofactor, chromodomain helicase DNA binding protein 8 (CHD8), was isolated from a yeast two-hybrid screen using SRF as bait. CHD8 is highly expressed in adult smooth muscle tissues. Coimmunoprecipitation assays from A10 smooth muscle cells demonstrated binding of endogenous SRF and CHD8. Data from GST-pulldown assays indicate that the NH2terminus of CHD8 can interact directly with the MADS domain of SRF. Adenoviral-mediated knockdown of CHD8 in smooth muscle cells resulted in attenuated expression of SRF-dependent, smooth muscle-specific genes. Knockdown of CHD8, SRF, or CTCF, a previously described binding partner of CHD8, in A10 VSMCs also resulted in a marked induction of apoptosis. Mechanistically, apoptosis induced by CHD8 knockdown was accompanied by attenuated expression of the anti-apoptotic proteins, Birc5, and CARD10, whereas SRF knockdown attenuated expression of CARD10 and Mcl-1, but not Birc5, and CTCF knockdown attenuated expression of Birc5. These data suggest that CHD8 plays a dual role in smooth muscle cells modulating SRF activity toward differentiation genes and promoting cell survival through interactions with both SRF and CTCF to regulate expression of Birc5 and CARD10.

Touw K, Hoggatt AM, Simon G, Herring BP. Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity.

Gut microbes play a significant role in development and maintenance of intestinal functions, but can contribute to pathogenesis of gastrointestinal (GI) disorders when host‐microbiome relationship is perturbed. In this study we sought to examine how constipation affects microbiome, and how these alterations in turn affect the host. Adult C57Bl/6 mice were treated with 0.1% Loperamide in drinking water for 7 days to induce constipation. Loperamide‐treated mice had increased GI transit time, indicating constipation. Bacterial DNA analysis by HiSeq sequencing showed decreased Firmicutes and increased Bacteroidetes in constipated mice. Microbiome metabolic potential was measured with tetrazolium dye assay and showed shift toward increased sugar metabolism in Loperamide‐treated mice. Microbial metabolites were measured by GC‐MS, and showed lower butyrate concentration in constipated mice. To determine constipation effects on the host, we introduced microbiota from constipated donors to germ‐free recipients. These mice had delayed GI transit time. To determine mechanism for delayed GI transit we analyzed colon tissues by qRT‐PCR and western blotting analysis, and determined lower expression of nNos mRNA and protein levels in these mice. Overall, these results suggest that delayed GI transit shifts microbiome taxonomic and metabolic profile, and these changes further contribute to development of constipation. Grant Funding Source: NIH NIDDK T32 DK07074

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