Dynamic regulation of actin‐binding protein synaptopodin by butyrate promotes intestinal epithelial barrier function

Intestinal epithelial cells (IECs) form a dynamic barrier that maintains homeostasis by separating the host immune system from an external environment of pathogenic and commensal microorganisms. Disruption of epithelial barrier in diseases such as inflammatory bowel diseases (IBD) can increase bacterial translocation and result in inappropriate immune responses. Under such conditions, rapid wound healing to restore this barrier is central to inflammation resolution. Mucosal barrier disruption is often associated with dysbiosis, particularly decreases in species producing short chain fatty acids (SCFAs). While SCFAs are an established energy source for IECs, it was hypothesized that microbial‐derived SCFAs could promote IEC barrier function through specific gene regulation. The SCFA butyrate was shown to selectively augment barrier formation, monitored by increased electrical resistance, and enhance wound healing, measured by increased scratch‐wound closure, in T84 model IEC monolayers. An unbiased single cell RNA sequencing analysis (scRNAseq) was performed to define potential mechanisms of butyrate regulation and identified a number of gene targets that could coordinate epithelial barrier function. Of particular interest was the prominent butyrate‐induced expression of synaptopodin (SYNPO), an actin‐associated protein involved in cell shape and motility previously shown to be expressed in neuronal dendrites and kidney podocytes. Validation of the scRNAseq revealed that butyrate induces IEC SYNPO mRNA and protein expression by nearly 10‐fold in IECs, and that lentiviral knockdown of SYNPO results in diminished barrier formation and wound healing not rescued with butyrate treatment. Immunofluorescence studies revealed that SYNPO distinctly localizes to the IEC tight junction (e.g. co‐localization with ZO‐1), the linkage between cells responsible for regulating paracellular flux. In vivo studies using antibiotic‐depletion of microbiota followed by butyrate add‐back in mice confirmed SYNPO induction by butyrate, and mice subjected to dextran sulfate sodium (DSS)‐induced colitis showed decreased SYNPO expression. Studies in Synpo ‐deficient mice demonstrated exacerbated disease susceptibility and increased intestinal permeability in the DSS colitis model. These findings establish a critical role for the microbiota and their products, specifically butyrate, in the regulated expression of SYNPO for intestinal homeostasis and reveal a direct mechanistic link between microbiota‐derived butyrate and barrier restoration. Understanding butyrate regulation of barrier function in the intestinal mucosa may offer insight into new IBD therapeutics to promote wound healing. Support or Funding Information This work was supported by NIH grants DK1047893, DK50189, DK095491, DK103712, and by the VA Merit Award BX002182. R.X.W. was supported by an NIH NRSA fellowship F30DK120072 and NIH MSTP training grant T32GM008497. J.S.L. was supported by an NIH NRSA fellowship F32DK122741.