BACKGROUND: Around 20-25% of ulcerative colitis patients undergo restorative proctocolectomy with ileal pouch-anal anastomosis. Pouchitis is an idiopathic inflammatory disease that may occur in ileal pouches, and it can lead to ileal pouch failure. We aim to evaluate the effect of Lactobacillus casei (L. casei) DG, a probiotic strain, on the intestinal microbiota in the ileal pouch mucosa to determine the crosstalk between microbiota and mucosal immune system.
METHODS: Fifty-two patients who had restorative proctocolectomy were randomized to receive a daily oral supplementation of L. casei DG or placebo for 8 weeks from the ileostomy closure (T0) to a pouch endoscopy after 8 weeks (T1, 26 L. casei DG and 26 placebo). Pouchitis symptoms were quantified with the modified pouchitis activity Index (mPDAI). Biopsies were collected from the pouch mucosa and activation (CD40 and CD80 expression) of dendritic cells (CD1a+), macrophage (CD163+) and T cells (CD69 expression on CD4+ and CD8+ lymphocytes) was assessed with dual staining flow cytometry. The V3-V4 region of the 16S rRNA gene was sequenced utilizing a MiSeq Platform (Illumina, USA). Sequences were processed using QIIME2 pipeline, the taxonomy was assigned using Greengenes database, and statistical analysis was performed in R.
RESULTS: No difference in pouchitis episodes was observed between the two groups. The L. casei DG supplemented group had a significantly higher alpha-diversity evaluated by using species richness index (P= 0.043) compared to the placebo group 8 weeks after ileostomy closure. Bray-Curtis β-diversity showed a significant difference between ileostomy closure (T0) and 8 weeks after ileostomy (T1) mucosal samples, independently from the treatment. Enterobacteriaceae and Clostridium were significantly more abundant at T1 compared to T0, while the Fusobacterium genus was less frequently present at T1 compared to T0. At the species level, Prevotella was significantly more frequent at T0 while Ruminococcus was significantly more abundant at T1. The L. casei DG supplemented group had a higher frequency of patients with a decrease of CD1a+CD80+ activated dendritic cells rate in the ileal pouch mucosa in the T0-T1 time frame than the placebo group (p=0.049). Moreover, at T1, the mean fluorescence intensity of CD1a+CD80+ dendritic cells tended to correlate with mPDAI (rho=0.31, p=0.07).
CONCLUSIONS: Manipulation of mucosal microbiota by L. casei DG after stoma closure in patients who underwent restorative proctocolectomy significantly increased alpha diversity compared to placebo and decreased the infiltration of activated dendritic cells. Moreover, this increase in species diversity included the presence of Bifidus species. Despite the lack of long-term clinical effects these results suggest the efficacy of L. casei DG supplementation to maintain a favorable ileal pouch microenvironment.

Francesco Celotto

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