Probiotic Escherichia coli Nissle 1917 Expressing Elafin Protects Against Inflammation and Restores the Gut Microbiota

Guigen Teng, Zilin Liu, Yun Liu, Ting Wu, Yun Dai, Huahong Wang, Weihong Wang, Guigen Teng, Zilin Liu, Yun Liu, Ting Wu, Yun Dai, Huahong Wang, Weihong Wang

Abstract

Intestinal mucosal inflammation and epithelial barrier dysfunction have been implicated as pathological factors in inflammatory bowel disease (IBD). An emerging area of IBD research focuses on probiotics. The probiotic Escherichia coli Nissle 1917 (EcN) is an excellent choice for engineering therapeutic microbes. Elafin is an endogenous specific inhibitor of neutrophil elastase (NE) and proteinase 3, and we previously found Elafin can effectively suppress the development of colitis. Here, we genetically engineered EcN to deliver Elafin (EcN-Elafin) directly to the colonic mucosa and explored the protective effects of EcN-Elafin against colitis in mice. EcN-Elafin significantly alleviated dextran sodium sulfate (DSS) induced colitis. Compared with wild-type EcN, oral administration of EcN-Elafin displayed better effects on loss of weight, colon length shortening, elevated expression of myeloperoxidase (MPO), and proinflammatory cytokines and chemokine in colonic tissues. In addition, EcN-Elafin restored the expression and distribution of tight junction protein ZO-1 in colonic tissues back to normal. In a damaged colonic epithelial model utilizing Caco-2 cells stimulated with TNF-α, EcN-Elafin efficiently downregulated the activation level of NF-κB signaling. EcN-Elafin was also found to have restored the dysbiosis in gut caused by DSS administration. Moreover, EcN-Elafin significantly enhanced the concentrations of butyrate and valerate in the gut lumen. Thus, our findings demonstrated that EcN-Elafin enhanced the colonic epithelial barrier, promoted the resolution of inflammation, modulated the gut microbiota, and elevated concentrations of short-chain fatty acids (SCFAs) in the gut. EcN-Elafin may be a potential therapeutic method for IBD.

Keywords: EcN; Elafin; gut microbiota; inflammatory bowel disease; intestinal epithelial barrier.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Teng, Liu, Liu, Wu, Dai, Wang and Wang.

Figures

Figure 1
Figure 1
EcN-Elafin ameliorated DSS-induced chronic colitis in mice. (A) Detection of Elafin in supernatants and bacterial homogenates of EcN-WT and EcN-Elafin using Western blotting. (B) Schematic of administration schedule. EcN-WT (109 CFU) and EcN-Elafin (109 CFU) were administered orally once daily. (C) Body weight changes of the mice in each group after DSS administration. ap < 0.05 and bp < 0.01 vs. DSS group; cp < 0.05 vs. EcN-WT group. (D) Representative photo of colons and lengths of the colons in each group. (E) Histopathological scores of colonic sections in each group. (F) Representative photos of H&E-stained sections of colonic tissues from each group. Image markers indicate loss of crypt and goblet cell depletion (X), immune cell infiltration (I). n = 6–8 in each group. The data are presented as the mean ± SEM. *p < 0.05; **p < 0.01.
Figure 2
Figure 2
EcN-Elafin downregulated the expression of proinflammatory mediators in the colonic tissues from DSS-treated mice. (A) The expression of MPO in the colons from each group. Arrows indicate MPO positive cells in each group. (B) Relative mRNA expression and (C) protein levels of TNF-α, IL-6, and CXCL-1 in conic tissues from each group. 2ΔCT method was used to calculate relative expression of mRNA. Protein levels were presented as pg/mg of proteins. n = 6–8 in each group. The data are presented as the mean ± SEM. *p < 0.05; **p < 0.01.
Figure 3
Figure 3
EcN-Elafin enhanced the intestinal epithelial barrier in mice with colitis. (A) Representative photo of immunofluorescence staining for the epithelial tight junction protein ZO-1 in mouse colonic tissues. Scale bar = 20 μm. (B) Caco-2 cells were cultured and then stimulated with recombinant TNF-α (7.5 ng/ml) in the presence or absence of either EcN-WT or EcN-Elafin bacterial extracts for 30 min. Then, the cell lysates were analyzed for p-IκBα, IκBα, p-IKKβ, IKKβ, p-P65, P65, and GAPDH expression by Western blotting.
Figure 4
Figure 4
EcN-Elafin restored the disturbed gut microbiota in DSS-induced chronic colitis mice. (A) The Venn diagram exhibiting the overlaps of OTUs from four groups; (B) α-diversity was calculated by the observed-species diversity, PD-whole-tree diversity, and Shannon and Simpson indices. ns: not significant. *p < 0.05 and **p < 0.01 by Mann–Whitney test; (C) Heatmap with clusters displaying the results of unweighted Unifrac analysis among four groups (D) β-diversity analysis of the gut microbiota of each group. Ordination plots based on the PCoA and NMDS analysis using unweighted UniFrac. (E,F) Relative abundances of bacterial taxa at the family and genus level. n = 6 per group.
Figure 5
Figure 5
Effect of EcN-Elafin on the bacterial composition in the gut of DSS-induced chronic colitis mice. (A) Relative abundances of a common set of microbial families; (B) Relative abundances of a common set of microbial genera; (C) Differences in microbiota in each group were calculated by LDA effect size (LEfSe). n = 6 per group. Kruskal-Wallis test was used with a statistical significance cutoff of p < 0.05 and the threshold of LDA score is 2. p, phylum; c, class; o, order; f, family; and g: genus.
Figure 6
Figure 6
EcN-Elafin increased the levels of SCFAs in the gut of DSS challenged mice. Levels of SCFAs in fecal samples, including (A) acetic acid, (B) propionic acid, (C) isobutyric acid, (D) butyric acid, (E) valeric acid, and (F) caproic acid in each mouse were determined. n = 6 in each group. *p < 0.05; **p < 0.01.

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