Gut bacteria potential key to IBD and colon cancer prevention
Authors: Rabina Giri, Emily C. Hoedt, Shamsunnahar Khushi, Angela A. Salim, Anne-Sophie Bergot, Veronika Schreiber, Ranjeny Thomas, Michael A. McGuckin, Timothy H. Florin, Mark Morrison, Robert J. Capon, Páraic Ó Cuív, Jakob Begun
Link to research article: Secreted NF-κB suppressive microbial metabolites modulate gut inflammation: Cell Reports
Emerging evidence suggests that microbiome-host crosstalk regulates intestinal immune activity and predisposition to inflammatory bowel disease (IBD). NF-κB is a master regulator of immune function and a validated target for the treatment of IBD. Here, we identify five Clostridium strains that suppress immune-mediated NF-κB activation in epithelial cell lines, PBMCs, and gut epithelial organoids from healthy human subjects and patients with IBD. Cell-free culture supernatant from Clostridium bolteae AHG0001 strain, but not the reference C. bolteae BAA-613 strain, suppresses inflammatory responses and endoplasmic reticulum stress in gut epithelial organoids derived from Winnie mice. The in vivo responses to Clostridium bolteae AHG0001 and BAA-613 mirror the in vitro activity. Thus, using our in vitro screening of bacteria capable of suppressing NF-κB in the context of IBD and using an ex vivo organoid-based approach, we identify a strain capable of alleviating colitis in a relevant pre-clinical animal model of IBD.
The human gut is the largest immune organ of the body, and gut epithelial cells play a key role in the establishment and maintenance of gut homeostasis, as well as rapid responses to infection (Peterson and Artis, 2014). The gut is colonized by a diverse microbiota that has co-evolved with its host and forms a symbiotic relationship through its modulation of innate and adaptive immune responses (Geva-Zatorsky et al., 2017; Kabat et al., 2014). However, with a few notable exceptions (Mazmanian et al., 2008; Wlodarska et al., 2017), the microbes and microbial determinants of immune homeostasis remain cryptic.Inflammatory bowel diseases (IBDs) are composed of two predominant subtypes, Crohn disease (CD) and ulcerative colitis (UC), that are characterized by relapsing and remitting gut inflammation.
The nuclear factor-κB (NF-κB) family of transcription factors are master regulators of gut epithelial integrity and inflammation and activation of antigen-presenting cells and effector leukocytes. Upon activation, NF-κB dimers translocate to the nucleus, where they regulate transcription of a wide range of genes, including those involved in immune and inflammatory responses (Wullaert et al., 2011). In the healthy gut, NF-κB activation is tightly regulated (Renner and Schmitz, 2009), but several IBD genetic risk alleles, including NOD2, TOLLIP, and A20, exert their pathogenic effects at least in part through dysregulated NF-κB signaling (Zaidi and Wine, 2018). Additionally, macrophages and epithelial cells isolated from the inflamed intestine of patients with IBD show increased activation, and nuclear localization of NF-κB-p65 (Rogler et al., 1998).
NF-κB signaling contributes significantly to multiple host responses underlying the pathogenesis of IBD.The gut microbiota is increasingly recognized as an important contributory risk factor for IBD development and activity, as healthy and IBD gut microbiota differ and are characterized by structure and function alterations (Costello et al., 2017; Wilson et al., 2019). Several bacterial taxa are abundant in the healthy gut that can suppress inflammatory responses and alleviate inflammation in animal models of disease (Sokol et al., 2008; Eeckhaut et al., 2012; Takeshita et al., 2016). These “anti-inflammatory” properties are best characterized for the gut bacterium Faecalibacterium prausnitzii A2-165, which produces secreted peptides derived from the Mam protein that suppress NF-κB in vitro and in vivo via stabilization of the IKK complex (Quevrain et al., 2016).
However, although Firmicutes-affiliated Clostridia are among the most abundant and functionally diverse gut bacteria, Mam expression is largely restricted to members of Faecalibacterium spp., and much remains to be discovered about the immunomodulatory capacities inherent to other Firmicutes.Here, we identified five new Firmicutes isolates that are comparable or superior to F. prausnitzii A2-165 in their NF-κB suppressive activity and whose activities are characterized by strain-specific differences.
Notably, these bacteria suppressed cytokine-mediated IL-8 secretion in CD and UC gut-derived organoid cultures and peripheral blood mononuclear cells (PBMCs). On the basis of these observations, we demonstrated using two Clostridium bolteae strains and ex vivo assays that ex vivo activity in organoids predicts immunomodulatory bioactivity in vivo using the Winnie murine model of spontaneous colitis. Furthermore, using an activity-guided high-performance liquid chromatography (HPLC) purification method, we have shown that non-polar and hydrophobic bacterial supernatant derived fractions from Clostridium bolteae can suppress both cytokine and lipopolysaccharide (LPS)-driven chemokine/cytokine expression on human organoids and PBMCs, as well as non-canonical NF-κB in vitro and in vivo. These data demonstrate the potential of bioprospecting the human microbiome for novel therapeutic leads targeting immunologically relevant pathways for treating gut inflammation.
Firmicutes-affiliated bacteria are among the most abundant gut microbes, and these taxa are widely recognized to possess immunomodulatory capacities (Atarashi et al., 2011, Atarashi et al., 2013; Sokol et al., 2008). However, they are poorly represented in culture collections, and their ability to modulate immune responses remains largely undefined. In this study, we identified five gut bacterial strains affiliated with Clostridium clusters IV, XIVa, and XV that have NF-κB suppressive activity comparable or superior to the well-characterized F. prausnitzii A2-165 strain. The NF-κB suppressive bioactivities were characterized by significant biochemical and intraspecies variations suggesting that NF-κB suppressive capacity may be more prevalent than previously appreciated and not adequately captured in existing genetic-based studies of the gut microbiome.
This is consistent with Geva-Zatorsky et al. (Geva-Zatorsky et al., 2017), who determined that as few as 53 isolates were associated with more than 24,000 immune phenotypes that include functionalities relevant to IBD such as Treg induction. Modulating host immune responses may support the ability of gut bacteria to colonize and persist in the gut environment. Furthermore, the ability of the microbiota to act as an extrinsic regulator of host immunity may underpin immune homeostasis, and therefore alterations in the gut microbial composition may contribute to disease risk in genetically susceptible individuals.IBD is characterized by a dysregulated immune response with select genetic susceptibilities affecting therapeutic responsiveness (Niess et al., 2012; Barber et al., 2016).
Given this variability in IBD, we used patient-derived gut epithelial organoids and immune cells to confirm bacterial capability to suppress cytokine-mediated inflammatory responses. The heat- and Proteinase K-resilient bioactives showed strong suppression of IL-8 secretion in organoids and immune cells from non-IBD, CD, and UC subjects. Interestingly, the proteinase sensitive bioactives produced by F. prausnitzii A2-165 and C. aldenense AHG0011 appeared less suppressive in UC-derived organoids and PBMCs, and in CD organoids, compared with organoids derived from non-IBD controls, which may be reflective of the increased endogenous protease activity observed in IBD (Vergnolle, 2016). Our in vitro and ex vivo data also suggested that functional capacity rather than phylogeny may be the key determinant of biologic effects. To explore this hypothesis, we capitalized on the C. bolteae intraspecies differences in activity and demonstrated that ex vivo effects observed in Winnie organoids, where CS from C. bolteae AHG0001 but not ATCC BAA-613 suppressed immune responses, predicted in vivo efficacy in ameliorating established colitis in Winnie mice.
Notably, treatment with C. bolteae AHG0001 CS was associated with a rapid onset of action with improvement in diarrhea, alleviation of inflammation and ER stress, as well as restoration of mucin production. Mucosal and histologic healing are among the best predictors of long-term outcomes in IBD (Turner et al., 2021) and taken together, our data suggest a precision medicine approach using ex vivo patient-derived organoids could be applied to select effective microbiome derived IBD treatments.The NF-κB suppressive strains we have identified carry multiple BGCs, many of whose products remain cryptic, underlining the inherent challenges in applying genomic-based approaches to map genotype with phenotype.
In addition, the biosynthesis of bioactives by gut bacteria may be driven principally through modest modifications of common primary metabolites that are underpinned by small BGCs (Cohen et al., 2015, Cohen et al., 2017). As the medium dependent effects on NF-κB suppression may affect the therapeutic efficacy of live biotherapeutics for IBD, we used a bioassay-guided ethyl acetate extraction coupled with reversed-phase analytical high-performance liquid chromatography fractionation to further characterize low-molecular weight non-polar bioactive associated with the NF-κB suppressive activity of C. bolteae AHG0001. Consistent with other microbial bioactives, the C. bolteae AHG0001 bioactive acts independently of the bacterial cell and suppresses the inflammatory response in animals. In summary, our functional approach provides new opportunities to rationally bioprospect the gut microbiota for precision live biotherapeutic strains and/or bioactives that could be used to expedite the development of safer and more efficacious therapeutics in IBD.
Limitations of the study
In this study, we showed that Clostridium species isolated from a single individual have NF-κB suppressive ability, but the immunomodulatory properties are likely not limited to Clostridium. Although we identified multiple strains with secreted immunomodulatory activity, it remains undetermined if the immunomodulatory properties of Clostridium can be generalized to other species of bacteria. Similarly, we used one mouse model of colitis to validate the in vitro activity of culture supernatants, although the activity was validated in multiple cell lines as well as patient-derived organoids. Future studies should use other models of murine colitis.