Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

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.

Altered calcium signaling in colonic smooth muscle of type 1 diabetic mice.

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 (>200 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(2+) imaging showed attenuated Ca(2+) 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(2+) levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca(2+) handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for <1 wk also showed decreased colonic contractile responses that were associated with decreased Ca(2+) increases in response to KCl stimulation, although without an elevation in basal Ca(2+) levels or a significant change in the expression of Ca(2+) signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca(2+) influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility.

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 pathway-specific 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 NH(2)-terminus 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.

Mutual reinforcement of pathophysiological host-microbe interactions in intestinal stasis models.

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.

Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases.

BACKGROUND: 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, Muc2-heterozygote (±), or Muc2-knockout (-/-) were injected with dexamethasone, a synthetic glucocorticoid, for 4 weeks. Fecal samples were collected for gut microbiota analysis through 16S rRNA terminal restriction fragment length polymorphism and amplicon sequencing. Intestinal mucosa was collected for mucin gene expression studies. Germ-free mice were conventionalized with gut microbes from treated and nontreated groups to determine their functional capacities in recipient hosts. RESULTS: Exposure to dexamethasone in wild-type mice led to substantial shifts in gut microbiota over a 4-week period. Furthermore, a significant downregulation of colonic Muc2 gene expression was observed after treatment. Muc2-knockout mice harbored a proinflammatory 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 mice, a genetically susceptible model of colonic inflammatory disorders. Microbiota from donors pretreated with dexamethasone, 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 glucocorticoid 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.

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity.

Mouse telokin and SM22alpha promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22alpha promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22alpha transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22alpha transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells.