Peris M. Munyaka

External influence of early childhood establishment of gut microbiota and subsequent health implications.

Postnatal maturation of immune regulation is largely driven by exposure to microbes. The gastrointestinal tract is the largest source of microbial exposure, as the human gut microbiome contains up to 1014 bacteria, which is 10 times the number of cells in the human body. Several studies in recent years have shown differences in the composition of the gut microbiota in children who are exposed to different conditions before, during, and early after birth. A number of maternal factors are responsible for the establishment and colonization of gut microbiota in infants, such as the conditions surrounding the prenatal period, time and mode of delivery, diet, mother’s age, BMI, smoking status, household milieu, socioeconomic status, breastfeeding and antibiotic use, as well as other environmental factors that have profound effects on the microbiota and on immunoregulation during early life. Early exposures impacting the intestinal microbiota are associated with the development of childhood diseases that may persist to adulthood such as asthma, allergic disorders (atopic dermatitis, rhinitis), chronic immune-mediated inflammatory diseases, type 1 diabetes, obesity, and eczema. This overview highlights some of the exposures during the pre- and postnatal time periods that are key in the colonization and development of the gastrointestinal microbiota of infants as well as some of the diseases or disorders that occur due to the pattern of initial gut colonization.

Acute dextran sulfate sodium (DSS)-induced colitis promotes gut microbial dysbiosis in mice.

The most widely used and characterized experimental model of ulcerative colitis (UC) is the epithelial erosion, dextran sulfate sodium (DSS)‐induced colitis, which is developed by administration of DSS in drinking water. We investigated fecal and colonic mucosa microbial composition and functional changes in mice treated with DSS. C57Bl/6 mice received 5% DSS in drinking water for 5 days. Inflammation was evaluated clinically and by analysis of colonic tissue cytokine levels and C‐reactive protein (CRP) in the serum. Colonic mucosa and fecal samples were used for DNA extraction and the V4 region of bacterial 16S rRNA gene was subjected to MiSeq Illumina sequencing. Alpha‐ and beta‐diversities, and compositional differences at phylum and genus levels were determined, and bacterial functional pathways were predicted. DSS increased disease severity, serum CRP and cytokines IL‐1β and IL‐6, but decreased bacterial species richness, and shifted bacterial community composition. Bacteroides, Turicibacter, Escherichia, Clostridium, Enterobacteriaceae, Clostridiaceae, Bacteroidaceae, Bacteroidales, among other taxa were associated with DSS treatment in fecal and colonic samples. Also, DSS altered microbial functional pathways in both colonic mucosa and fecal samples. Conclusions: The development of colitis in DSS model was accompanied with reduced microbial diversity and dysbiosis of gut microbiota at lower taxonomical levels.

Central muscarinic cholinergic activation alters interaction between splenic dendritic cell and CD4+CD25- T cells in experimental colitis.

Background The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4+/CD25−T cell activation in the context of experimental colitis. Methods The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25−T cell co-culture were determined. Results McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25−T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy. Conclusions Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.

Antepartum Antibiotic Treatment Increases Offspring Susceptibility to Experimental Colitis: A Role of the Gut Microbiota

Background and aims Postnatal maturation of the immune system is largely driven by exposure to microbes, and thus the nature of intestinal colonization may be associated with development of childhood diseases that may persist into adulthood. We investigated whether antepartum antibiotic (ATB) therapy can increase offspring susceptibility to experimental colitis through alteration of the gut microbiota. Methods Pregnant C57Bl/6 mice were treated with cefazolin at 160 mg/kg body weight or with saline starting six days before due date. At 7 weeks, fecal samples were collected from male offspring after which they received 4% dextran sulfate sodium (DSS) in drinking water for 5 days. Disease activity index, histology, colonic IL-6, IL-1β and serum C-reactive protein (CRP) were determined. The V3-V4 region of colonic and fecal bacterial 16S rRNA was sequenced. Alpha-, beta-diversity and differences at the phylum and genus levels were determined, while functional pathways of classified bacteria were predicted. Results ATB influenced fecal bacterial composition and hence bacterial functional pathways before induction of colitis. After induction of colitis, ATB increased onset of clinical disease, histologic score, and colonic IL-6. In addition, ATB decreased fecal microbial richness, changed fecal and colon microbial composition, which was accompanied by a modification of microbial functional pathways. Also, several taxa were associated with ATB at lower taxonomical levels. Conclusions The results support the hypothesis that antepartum antibiotics modulate offspring intestinal bacterial colonization and increase susceptibility to develop colonic inflammation in a murine model of colitis, and may guide future interventions to restore physiologic intestinal colonization in offspring born by antibiotic-exposed mothers.

Carrageenan Gum and Adherent Invasive Escherichia coli in a Piglet Model of Inflammatory Bowel Disease: Impact on Intestinal Mucosa-associated Microbiota

Inflammatory bowel diseases (IBD) including Crohns disease (CD), and ulcerative colitis (UC), are chronic conditions characterized by chronic intestinal inflammation. Adherent invasive Escherichia coli (AIEC) pathotype has been increasingly implicated in the etiopathogenesis of IBD. In a 21-day study, we investigated the effects of AIEC strain UM146 inoculation on microbiota profile of the ileal, cecal, ascending and descending colon in a pig model of experimental colitis. Carrageenan gum (CG) was used to induce colitis in weaner piglets whereas AIEC strain UM146 previously isolated from a CD patient was included to investigate a cause or consequence effect in IBD. Treatments were: (1) control; (2) CG; (3) AIEC strain UM146; and (4) CG+UM146. Pigs in groups 2 and 4 received 1% CG in drinking water from day 1 of the study while pigs in groups 3 and 4 were inoculated with UM146 on day 8. Following euthanization on day 21, tissue mucosal scrapings were collected and used for DNA extraction. The V4 region of bacterial 16S rRNA gene was then subjected to Illumina sequencing. Microbial diversity, composition, and the predicted functional metagenome were determined in addition to short chain fatty acids profiles in the digesta and inflammatory cytokines in the intestinal tissue. CG-induced colitis decreased bacterial species richness and shifted community composition. At the phylum level, an increase in Proteobacteria and Deferribacteres and a decrease in Firmicutes, Actinobacteria, and Bacteroidetes were observed in CG and CGUM146 compared to control and UM146. The metabolic capacity of the microbiome was also altered in CG and CGUM146 compared to UM146 and control in the colon. We demonstrated that CG resulted in bacterial dysbiosis and shifted community composition similar to what has been previously observed in IBD patients. However, AIEC strain UM146 alone did not cause any clear changes compared to CG or control in our experimental IBD pig model.

Effect of crowding stress and Escherichia coli K88+ challenge in nursery pigs supplemented with anti-Escherichia coli K88+ probiotics.

Under commercial conditions and during production periods, pigs are generally exposed to a number of stressors that may have direct or indirect influence on their performance and general health. As a result, environmental stressors can influence the productivity as well as the onset and severity of infectious diseases. The aim of this study was to investigate the effects of crowding stress on performance and some immunological responses in piglets challenged with enterotoxigenic Escherichia coli (ETEC) K88(+) and fed diets containing anti-ETEC probiotic strains derived from E. coli in a 3-wk trial. Ninety-six piglets at 20 ± 1 d of age were randomly assigned to 6 experimental treatments with 3 pigs/pen and 5 replicates/treatment and fed a basal mash diet. To model stress, piglets were housed in 2 kinds of pens: standard floor allowance (SFA; no stress) and half standard floor allowance (HSFA; stressed). The treatments were 1) control pigs with SFA, 2) control pigs with HSFA, 3) pigs with SFA and challenged with ETEC (ESFA), 4) pigs with HSFA and challenged with ETEC (EHSFA), 5) pigs with ESFA and supplemented with a cocktail of E. coli probiotics (PSFA), and 6) pigs with EHSFA and supplemented with a cocktail of E. coli probiotics (PHSFA). After 4 d acclimatization, crowding stress was started on d 5, E. coli probiotics were given daily from d 5, and ETEC challenge was administered on d 8 and 10. Body weight and feed disappearance were determined weekly and severity of diarrhea was characterized daily. Blood samples were collected for biochemistry and inflammatory analysis and pigs were euthanized to obtain digesta for bacterial enumeration. The ADG and ADFI decreased (P < 0.05) in stressed and ETEC-challenged pigs 5 d after ETEC challenge. The ETEC population was higher in both ileal and colon digesta of stressed pigs whereas ETEC-challenged and probiotic-fed pigs had higher fecal consistency scores 96 h and 7 d after ETEC challenge. Stressed pigs had higher (P < 0.001) levels of neutrophils, neutrophil:lymphocyte ratio, albumin:globulin ratio, fibrinogen, total protein:fibrinogen ratio, and cortisol. Generally, the levels of cortisol, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) differed over time among some treatment groups. In conclusion, reduced space allowance stress and ETEC challenge depressed certain aspects of performance and immunological responses and increased E. coli counts; however, probiotics feeding had little positive effects on these measures suggesting further research.

Reactivation of Intestinal Inflammation Is Suppressed by Catestatin in a Murine Model of Colitis via M1 Macrophages and Not the Gut Microbiota

While there is growing awareness of a relationship between chromogranin-A (CHGA) and susceptibility to inflammatory conditions, the role of human catestatin [(hCTS); CHGA352–67] in the natural history of established inflammatory bowel disease is not known. Recently, using two different experimental models, we demonstrated that hCTS-treated mice develop less severe acute colitis. We have also shown the implication of the macrophages in this effect. The aims of this study were to determine (1) whether hCTS treatment could attenuate the reactivation of inflammation in adult mice with previously established chronic colitis; (2) whether this effect is mediated through macrophages or the gut microbiota. Quiescent colitis was induced in 7–8-week-old C57BL6 mice using four cycles (2–4%) of dextran sulfate sodium. hCTS (1.5 mg/kg/day) treatment or vehicle started 2 days before the last induction of colitis and continuing for 7 days. At sacrifice, macro- and microscopic scores were determined. Colonic pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, and TNF- α], anti-inflammatory cytokines (IL-10, TGF- β), classically activated (M1) (iNOS, Mcp1), and alternatively activated (M2) (Ym1, Arg1) macrophages markers were studied using ELISA and/or RT-qPCR. In vitro, peritoneal macrophages isolated from naïve mice and treated with hCTS (10−5 M, 12 h) were exposed to either lipopolysaccharide (100 ng/ml, 12 h) to polarize M1 macrophages or to IL-4/IL-13 (20 ng/ml) to polarize M2 macrophages. M1/M2 macrophage markers along with cytokine gene expression were determined using RT-qPCR. Feces and mucosa-associated microbiota (MAM) samples were collected, and the V4 region of 16 s rRNA was sequenced. Micro- and macroscopic scores, colonic IL-6, IL-1β, TNF- α, and M1 macrophages markers were significantly decreased in the hCTS-treated group. Treatment did not have any effect on colonic IL-10, TGF-β, and M2 markers nor modified the bacterial richness, diversity, or the major phyla in colitic fecal and MAM samples. In vitro, pro-inflammatory cytokines levels, as well as their gene expression, were significantly reduced in hCTS-treated M1 macrophages. hCTS treatment did not affect M2 macrophage markers. These findings suggest that hCTS treatment attenuates the severity of inflammatory relapse through the modulation of the M1 macrophages and the release of pro-inflammatory cytokines.

Human Catestatin Alters Gut Microbiota Composition in Mice

The mammalian intestinal tract is heavily colonized with a dense, complex, and diversified microbial populations. In healthy individuals, an array of epithelial antimicrobial agents is secreted in the gut to aid intestinal homeostasis. Enterochromaffin cells (EC) in the intestinal epithelium are a major source of chromogranin A (CgA), which is a pro-hormone and can be cleaved into many bioactive peptides that include catestatin (CST). This study was carried out to evaluate the possible impact of CST on gut microbiota in vivo using a mouse model. The CST (Human CgA352−372) or normal saline was intrarectally administered in C57BL/6 male mice for 6 days and then sacrificed. Feces and colonic mucosa tissue samples were collected, DNA was extracted, the V4 region of bacterial 16S rRNA gene was amplified and subjected to MiSeq Illumina sequencing. The α-diversity was calculated using Chao 1 and β-diversity was determined using QIIME. Differences at the genus level were determined using partial least square discriminant analysis (PLS-DA). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict functional capacity of bacterial community. CST treatment did not modify bacterial richness in fecal and colonic mucosa-associated microbiota; however, treatment significantly modified bacterial community composition between the groups. Also, CST-treated mice had a significantly lower relative abundance of Firmicutes and higher abundance of Bacteroidetes, observed only in fecal samples. However, at lower phylogenetic levels, PLS-DA analysis revealed that some bacterial taxa were significantly associated with the CST-treated mice in both fecal and colonic mucosa samples. In addition, differences in predicted microbial functional pathways in both fecal and colonic mucosa samples were detected. The results support the hypothesis that CST treatment modulates gut microbiota composition under non-pathophysiological conditions, however, the result of this study needs to be further validated in a larger experiment. The data may open new avenues for the development of a potential new line of antimicrobial peptides and their use as therapeutic agents to treat several inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or other health conditions.

Mo1699 Denosumab: A Monoclonal Antibody to Receptor Activator of Nuclear Factor-Kappa B Ligand (RANKL) Decreases Experimental Colitis

The gastrointestinal microbial communities of C57BL/6 mice between Taconic Farms (Tac) and Jackson Laboratory (Jax) are highly variable; most notably, only the Tac mice are colonized with segmented filamentous bacteria (SFB), a potent inducer of proinflammatory TH17 cells. To examine whether Jax and Tac mice have a different intestinal microbiome and respond differently to H. pylori (Hp) infection, C57BL/6 mice from Tac and Jax were dosed with Hp PMSS1. As measured by Illumina sequencing, OTUs in fecal microbiomes were richer in Tac than Jax mice (p<0.01); the ratio of Bacteriodetes to Firmicutes, two predominant phyla, were 1.86 for Jax but 0.33 for Tac mice. In addition, the results obtained by qPCR demonstrated that colonization status of 5 mucosa-associated species ASF356, ASF361, ASF457, ASF500, ASF519 (members of altered schaedler flora) in the stomachs of the control mice from the 2 vendors were comparable, while there were significantly lower levels of ASF457 and ASF519 but higher levels of ASF361 and ASF500 in the ceca of control Jax mice compared to the Tac counterparts (P<0.01). By 16 weeks-post-inoculation, there were significantly lower levels of gastric and cecal ASF361, cecal ASF457, ASF500 and ASF519, but higher levels of cecal ASF356 in the infected Jax mice compared to their controls (P<0.01). Hp infection in Tac mice also increased SFB levels in the large intestine but did not influence ileal SFB levels compared to the controls. In contrast, Hp infection was only associated with higher levels of cecal ASF356 in the infected Tac mice compared to the sham controls (P<0.0001). Interestingly, Hp infection significantly altered fecal microbial compositions of Tac mice with an increase of the class Bacilli and a decrease in the classes Clostridia and Bacteroidia but this was not noted in Jax mice. Hp colonization levels and mRNA expression of gastric Il-1β, Il-17A, and RegIIIγwere significantly lower in Tac compared to Jax mice (P<0.05). In contrast, mRNA levels of gastric Inf-γ, Tnf-α, and Foxp-3 were comparable between the infected Jax and Tac groups. There were no significant differences in gastric pathology including inflammation, epithelial defects, oxyntic atrophy, hyperplasia, and dysplasia between the infected Jax and Tac groups, except for mucus metaplasia that was more severe in the infected Tac mice vs. the infected Jax mice (P<0.05). Our data indicate that the overall gastrointestinal microbiomes in Tac mice are more diverse and prone to Hp perturbation compared to Jax mice, while there is more profound Hp-associated change in colonization levels of the 5 mucosa-associated ASF species in Jax mice than Tac mice. Long-term clinical effects of the different responses in the microbiome and inflammation-associated mediators to Hp infection between Jax and Tac mice needs further study.