Society: AGA

The microbiome plays a central role in intestinal inflammation in IBD. It is influenced by environmental factors included diet. In turn, the microbiome can incite intestinal inflammation

Background Mounting evidence suggests that food additives, especially emulsifier polysorbate-80 (P80), promote colitis via gut microbiota. Here, we explored the underlying mechanism of P80 and specific species Proteus mirabilis (P. mirabilis), a pathobiont in gut, in pathogenesis and aggravation of intestinal inflammation.

Methods This study includes two independent cohorts comprised of Crohn’s disease (CD) and healthy controls, respectively Hong Kong (92 CD, 21 controls) and Australian (98 CD, 21 controls). Previous and current consumption of P80 was quantified using a validated 26-item food additive questionnaire. Fecal microbiota composition was assessed by shotgun metagenomic sequencing. The interaction between P80 and P. mirabilis was investigated in both in vitro and in vivo models. The effect of P80 on P. mirabilis was determined by bacterial transcriptome sequencing. The interested protein was identified by silver straining followed by mass spectrometry.

Results P. mirabilis was identified as the enriched microbiota in the CD patients with high food additive consumption when compared to controls in both Hong Kong and Australia cohorts. Co-cultivation of P. mirabilis and P80 induced more cell death on human epithelial cells lines, INT 407 and NCM 460, compared to P. mirabilis alone or Escherichia coli control groups (Figure 1A). P80 exposure increased expression of virulence genes in P. mirabilis by RNA-seq. The co-cultured conditional medium of P. mirabilis and P80 (Plus.CM) triggered cell death, which was attenuated by Proteinase K, implying that the secreted protein is the functional participant in this process (Figure 1B). Compared to other fractions, the proteins with molecular weight over 100kDa exhibited the most notable function. By proteomics identification, P. mirabilis-derived enzyme X was uncovered as the potential deleterious protein in arising and aggravating inflammation (Figure 1C). In mouse models, the mice orally challenged with P. mirabilis and fed with P80 diet had shortened colon length (Figure 2A) and higher Lipocalin-2 (LCN2) levels (Figure 2B) compared to P. mirabilis solely and control groups.

Conclusion We have explored that with the addition of P80, P. mirabilis exacerbated the inflammation both in vitro and in vivo. The enzyme X, derived from P. mirabilis, was identified being the possible deleterious participant in this process and may be targeted for future precision therapy.

This work is supported by the Croucher Senior Research Fellowship and The Leona M. and Harry B. Helmsley Charitable Trust.

$<b>Figure 1. (A)</b> Exposure to both <i>P. mirabilis </i>and P80 induced more cell death, in comparison with <i>P. mirabilis</i> alone or other control groups. <b>(B)</b> The Proteinase K treatment attenuated the function of co-cultured conditional medium in triggering cell death. <b>(C) </b>Silver staining of protein fractions above 100kDa indicated the targeted enzyme. Statistical significance was determined by 1-way or 2-way analysis of variance appropriately. ****<i>P </i>< .0001.$

Figure 1. (A) Exposure to both P. mirabilis and P80 induced more cell death, in comparison with P. mirabilis alone or other control groups. (B) The Proteinase K treatment attenuated the function of co-cultured conditional medium in triggering cell death. (C) Silver staining of protein fractions above 100kDa indicated the targeted enzyme. Statistical significance was determined by 1-way or 2-way analysis of variance appropriately. ****P < .0001.

<b>Figure 2. </b>Mice challenged with <i>P. mirabilis</i> and P80 diet <b>(A)</b> shortened colon length and <b>(B)</b> induced higher level of Lcn-2 expression. Statistical significance was determined by 1-way or 2-way analysis of variance appropriately. *<i>P</i> < .05.

Figure 2. Mice challenged with P. mirabilis and P80 diet (A) shortened colon length and (B) induced higher level of Lcn-2 expression. Statistical significance was determined by 1-way or 2-way analysis of variance appropriately. *P < .05.

Increasing evidence suggest that environmental factors, including the gut microbiome, are important in the pathogenesis of inflammatory bowel disease in genetically predisposed individuals. The Winnie mouse is a model of spontaneous chronic colitis due to a point mutation in the Muc2 gene that disrupts the colonic mucus layer, hence, increasing the permeability to microbes in conjunction with dysbiosis. The aim of this study was to test the hypothesis that the environment in which these mice are reared might be a significant modifier of colitis severity when Muc2 function is abnormal. To test this hypothesis, we aseptically rederived Winnie mice from a conventional (Conv) animal facility in Italy into our specific pathogen free (SPF) vivarium at CWRU in Cleveland and performed a time-course of colitis severity in both colonies. In addition, we carried out microbiome and metabolomic analyses to investigate potential mechanistic differences in the two colonies. Surprisingly, Winnie mice housed in our SPF animal facility developed more severe colitis compared to the Conv facility. In addition, SPF Winnie mice developed colon tumors starting at 8 weeks of age in the proximal and medial part of the colon, while Conv housed Winnie mice developed only mild inflammation. Winnie mice raised under germ-free conditions showed no signs of colitis up to 40 weeks of age. We then analyzed the stool microbiome and metabolome of these mice. We found many differentially represented bacterial genera between SPF and conventional Winnie mice. Anaerosporobacter, Dubosiella,Faecalibaculum, Mucispirillum and Prevotellaceae_NK3B31 were more frequent in SPF Winnie mice while Rikenellaand Prevotellaceae_UCG-001 were more represented in Conv mice. Metabolomic analyses showed marked differences between the two colonies. Metabolites related to purine and nitrogen recycling were significantly increased in SPF Winnie mice compared to Conv Winnie mice. Of great interest, analysis of lipidomes revealed an increased presence of sphingomyelins and decreased amounts of ceramides in the SPF group compared to Conv Winnie mice. This class of lipids shares important roles in the onset and development of intestinal inflammation and interact with bacterial species to cause severe dysbiosis, inflammation and tumorigenesis. Further characterization through fecal material transplantation (FMT) experiments and culturomic studies are in progress to better clarify the role of specific bacterial strains and their metabolites in the development of chronic colitis and colitis-associated cancer in this model.

Luca Di Martino

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