Background: This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. Results: MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0–6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. Conclusions: Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation.

In milk-fed calves, the effects of sodium-butyrate (Na-butyrate) to replace flavomycin on growth performance and some mechanisms involved were studied. Pancreatic and intestinal morphology, digestive enzyme activities, plasma gut regulatory peptide concentrations, and expression of their receptors in the gastrointestinal tract were measured. Gastrointestinal tract defense systems were examined by measuring protein levels of 2 heat-shock proteins (HSP27 and HSP70). The calves were randomly allocated into 2 groups fed the same basic diet with flavomycin as an antimicrobial growth promoter or with Na-butyrate (3 g/kg of dry matter). Sodium-butyrate disappeared quickly in the upper gut and was not found in circulating blood. Supplementation with Na-butyrate enhanced growth rate and improved feed conversion into body weight gain compared with the flavomycin group. Supplementation with Na-butyrate was likely associated with an improvement in efficacy of the gastrointestinal tract digestive capacities expressed by enhanced production of digestive enzymes and increased absorptive capacities in the upper small intestine. The effects could have been controlled by insulin-like growth factor-1 but probably not by any of the cholecystokinin/gastrin peptide family. Concentrations of HSP27 and HSP70 were increased in stomach and colon of calves receiving Na-butyrate, thereby assuring protection of cells with intensive metabolism (chaperone function). In conclusion, beneficial effects of Na-butyrate on maturation of gastrointestinal functions were shown in milk-fed calves and may be applied to young mammals of other species.

Background & Aims Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. Methods We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10−/−), and colitis induced by adoptive transfer of CD4⁺CD45RB^high T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-γ on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). Results Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-γ. The combination of TNF and IFN-γ increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-γ was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-γ, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. Conclusions The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

Na⁺/H⁺exchanger 3 (NHE3) provides a major route for intestinal Na⁺absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3^−/−mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2′-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer²⁷⁶-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-α (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3^−/−mice. NHE3^−/−mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.

Chronic inflammation and enteric infections are frequently associated with epithelial Na⁺/H⁺exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3^−/−mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3^−/−mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3^−/−mice compared with WT. Rederivation of NHE3^−/−mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3^−/−mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3^−/−mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.