Karima Relizani, Katy Le Corf, Camille Kropp, Rebeca Martin-Rosique, Déborah Kissi, Guillaume Déjean, Lisa Bruno, Ccori Martinez, Georges Rawadi, Frédéric Elustondo, Wilfrid Mazier & Sandrine P. Claus
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Scientific Reports volume 12, Article number: 6017 (2022)
Microbiome-based therapies for inflammatory bowel diseases offer a novel and promising therapeutic approach. The human commensal bacteria of the species Christensenella minuta(C. minuta) have been reported consistently missing in patients affected by Crohn’s disease (CD) and have been documented to induce anti-inflammatory effects in human epithelial cells, supporting their potential as a novel biotherapy. This work aimed at selecting the most promising strain of C. minuta for future development as a clinical candidate for CD therapy. Here, we describe a complete screening process combining in vitro and in vivo assays to conduct a rational selection of a live strain of C. minuta with strong immunomodulatory properties. Starting from a collection of 32 strains, a panel of in vitro screening assays was used to narrow it down to five preclinical candidates that were further screened in vivo in an acute TNBS-induced rat colitis model. The most promising candidate was validated in vivo in two mouse models of colitis. The validated clinical candidate strain, C. minuta DSM 33715, was then fully characterized. Hence, applying a rationally designed screening algorithm, a novel strain of C. minuta was successfully identified as the most promising clinical candidate for CD.
This work was intended to select a C. minuta strain candidate as a future live biotherapeutic product using a method based on non-biased screening steps followed by in vivo validation. For this purpose, a collection of 32 C. minuta strains were screened based on rigorous selection criteria using in vitro and in vivo assays that allowed to identify a promising bacterial strain with high anti-inflammatory potential.
Our screening program included an evaluation of the protective action of the strains on gut epithelium upon challenge with the pro-inflammatory cytokine TNF-α. For this, we used a monolayer of human Caco-2 cells that constitute the gold standard in mimicking the human intestinal barrier (STEP 1—TEER assay). Interestingly, we observed that all tested C. minutastrains had a significant protective action on intestinal barrier integrity. This is consistent with other observations in similar screening assays19. As a consequence, it was not possible to use this assay to discriminate between strains. In addition, to favor the inclusion of genetically distinct C. minuta strains, we prioritized strains donor diversity to select between strains with similar scoring. We also evaluated the anti-inflammatory potential of the C. minuta strains using the HT-29 human intestinal cell line and human-derived PBMCs by quantifying the production of relevant cytokines. The HT-29 cells, deriving from a human colorectal adenocarcinoma, are good representatives of the intestinal membrane at both structural and functional levels20 and were selected here for their ability to secrete high amounts of IL-8 when challenged with TNF-α. IL-8 is a strong neutrophil chemoattractant produced by epithelial intestinal cells (EIC) that has been found overexpressed in the mucosa of all intestinal segments from the ileum to the rectum in active CD patients21 as well as in the serum of both CD and UC patients22. We therefore considered that strains with a strong ability to prevent IL-8 release would be of high therapeutic potential. Similarly, IL-10 has long been known for its anti-inflammatory action as it down-regulates the secretion of the pro-inflammatory cytokines TNF-α and IL-1β23. In addition, the discovery that mice lacking IL-10 and IL-10 receptor expression develop spontaneous enterocolitis established the crucial role of IL-10 in intestinal inflammation and in the etiology of IBD24. IL-10 being mostly produced by leukocytes, we chose to screen our collection of C. minuta strains on human-derived PBMC as an indicator of a systemic anti-inflammatory potential in humans.
Based on this rationale, we were able to select 5 pre-clinical candidates with high potential to protect the intestinal mucosa in IBD. We thus progressed with the selection of the clinical candidate in vivo in a chemically induced colitis model. Out of the 5 pre-selected strains, two significantly reduced the macroscopic score in the TNBS-induced colitis rat model, and only C. min 22 (DSM 33715) managed to successfully reduce the microscopic score (Fig. 4). On one hand, this result indicates that our screening strategy was appropriate to identify a strong clinical candidate for future drug development. On another hand, the panel of chosen in vitro assays may be complemented to consider other markers of active enterocolitis, such as reduction of TNF-α, of the interferon gamma-induced protein (IP)-10 expression or of IL-1β, which have all been reported as being massively increased in active CD biopsies21.
C. min 22 (DSM 33715), our selected candidate strain, limited clinical degradation in the DSS-induced colitis mouse model, a gold-standard for IBD modeling25. Interestingly, it also strongly reduced colonic IL-1β in two in vivo models suggesting that screening on the ability to reduce IL-1β secretion by macrophages in vitro might improve predictability. From a therapeutic perspective, low serum baseline concentrations of IL-1β have been associated with a higher response rate to the anti-TNF-α drug infliximab in CD patients, but not in UC26. This observation opens perspectives of potential use of C. minuta DSM 33715 (C. min 22) as an add-on to anti-TNF-α therapy for CD patients.
Observations made in animal models should always be interpreted with caution due to their limited ability to predict human response. Even if we have a strong rationale based on epidemiological data to target specifically CD where Christensenellaceae have been consistently reported as being reduced11, there is currently no established accurate preclinical model of CD that would discriminate from UC. For this reason, we diversified the models used and paid particular attention to use different animal species (i.e., rats and mice). We chose these chemically induced acute models of colitis primarily because they are well characterized, reproducible, simple to set up and deliver quick readouts, which are all essential criteria for a screening process. In addition, combining these models provides some interesting insights into potential mechanisms of action. Indeed, TNBS- and DNBS-induced colitis models (so called hapten-induced colitis) provoke a cell-mediated acute inflammation mostly affecting the distal colon that resembles human IBD27. Both TNBS and DNBS induce a similar immune response in rodents as demonstrated by Wallace et al.28, involving activation of the Th1- and Th17-mediated immune system29. To the contrary, the DSS-mouse model induces erosion of the mucosal layer in the lower part of the intestine, provoking an acute colitis through epithelial damage27. Although the immune response to DSS is first characterized by a strong Th1-mediated response with high TNFα secretion, it rapidly converts into a Th2-dominant response30. The fact that we were able to prevent gut inflammation in both models indicates that the bacteria mediate their effects through a common immune pathway that will need to be explored in the future. Nevertheless, these models mostly involve the innate immune response and therefore limit our ability to anticipate responses of the adaptive immune system31. Thus, follow-up studies focusing on detailed mechanisms of action will also need to consider chronic models of colitis, ideally combined with genetic models of IBD and humanized animal models to improve prediction of outcomes in patients.
From a mechanistic perspective, we observed a reduction of caecal SCFAs after DNBS-induced colitis in vehicle-treated animals, which is aligned with clinical observations where fecal SCFAs and SCFA-producing bacteria have been shown to be reduced in IBD32. Interestingly, both DSM 33715 (C. min 22) and 5-ASA prevented these colitis-induced reductions of SCFAs, which may potentially mediate the beneficial effects observed on gut inflammation in this study since SCFAs have strong immunomodulatory properties. These are mediated through stimulation of GPCR41 receptors, which induce production of protective IL22 by CD4+ T cells33 and GPCR43 receptors that promote Treg cells, the immune sentinels of healthy gut epithelia34. Accordingly, the pharmacological stimulation of SCFA receptors has also been shown to reduce susceptibility to develop colitis in a DSS mouse model through GPCR43 stimulation35. Even if the modulatory effect of 5-ASA on IBD patients’ gut microbiota has been documented36, its impact on SCFA levels has never been described to date. Considering the previously described keystone role of C. minuta species18,37, we assume that the beneficial effect of DSM 33715 (C. min 22) on SCFA levels is mediated through a modulation of gut microbial function leading to an increased production of SCFA. Yet, further metagenomics studies on fecal material collected during colitis studies are necessary to elucidate this point.
Finally, the selected C. minuta clinical candidate, strain DSM 33715 (C. min 22), was characterized following current regulatory guidelines appropriate for further drug development38. The characterization panel revealed high similarity with previously described C. minuta strains5,18 but DSM 33715 (C. min 22) slightly differed from the others at the biochemical level (negative for salicin and positive for mannose metabolism) but these modest differences are not sufficient to explain differences in anti-inflammatory effects. Hence, a complete comparative genome analysis will be needed to identify specific regions that may convey unique functions to this particular strain.
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