G. De Palma

Microbiota and host determinants of behavioural phenotype in maternally separated mice

Early-life stress is a determinant of vulnerability to a variety of disorders that include dysfunction of the brain and gut. Here we exploit a model of early-life stress, maternal separation (MS) in mice, to investigate the role of the intestinal microbiota in the development of impaired gut function and altered behaviour later in life. Using germ-free and specific pathogen-free mice, we demonstrate that MS alters the hypothalamic-pituitary-adrenal axis and colonic cholinergic neural regulation in a microbiota-independent fashion. However, microbiota is required for the induction of anxiety-like behaviour and behavioural despair. Colonization of adult germ-free MS and control mice with the same microbiota produces distinct microbial profiles, which are associated with altered behaviour in MS, but not in control mice. These results indicate that MS-induced changes in host physiology lead to intestinal dysbiosis, which is a critical determinant of the abnormal behaviour that characterizes this model of early-life stress.

The HLA-DQ2 genotype selects for early intestinal microbiota composition in infants at high risk of developing coeliac disease

Objective Intestinal dysbiosis has been associated with coeliac disease (CD), but whether the alterations are cause or consequence of the disease is unknown. This study investigated whether the human leukocyte antigen (HLA)-DQ2 genotype is an independent factor influencing the early gut microbiota composition of healthy infants at family risk of CD. Design As part of a larger prospective study, a subset (n=22) of exclusively breastfed and vaginally delivered infants with either high genetic risk (HLA-DQ2 carriers) or low genetic risk (non-HLA-DQ2/8 carriers) of developing CD were selected from a cohort of healthy infants with at least one first-degree relative with CD. Infant faecal microbiota was analysed by 16S rRNA gene pyrosequencing and real time quantitative PCR. Results Infants with a high genetic risk had significantly higher proportions of Firmicutes and Proteobacteria and lower proportions of Actinobacteria compared with low-risk infants. At genus level, high-risk infants had significantly less Bifidobacterium and unclassified Bifidobacteriaceae proportions and more Corynebacterium, Gemella, Clostridium sensu stricto, unclassified Clostridiaceae, unclassified Enterobacteriaceae and Raoultella proportions. Quantitative real time PCR also revealed lower numbers of Bifidobacterium species in infants with high genetic risk than in those with low genetic risk. In high-risk infants negative correlations were identified between Bifidobacterium species and several genera of Proteobacteria (Escherichia/Shigella) and Firmicutes (Clostridium). Conclusions The genotype of infants at family risk of developing CD, carrying the HLA-DQ2 haplotypes, influences the early gut microbiota composition. This finding suggests that a specific disease-biased host genotype may also select for the first gut colonisers and could contribute to determining disease risk.

Pivotal Advance: Bifidobacteria and Gram-negative bacteria differentially influence immune responses in the proinflammatory milieu of celiac disease.

CD is a chronic inflammatory disorder of the small intestine that presents in genetically predisposed individuals following gluten consumption. In this study, the effects of Bifidobacterium (Bifidobacterium bifidum IATA‐ES2 and Bifidobacterium longum ATCC15707) and Gram‐negative bacteria (Bacteroides fragilis DSM2451, Escherichia coli CBL2, and Shigella CBD8 isolated from CD patients), alone and in the presence of CD triggers (gliadins and/or IFN‐γ) on surface marker expression and cytokine production by PBMCs, were determined. These effects were also evaluated in cocultures of PBMCs and Caco‐2 cells. The Gram‐negative bacteria induced higher secretion of Th1‐type proinflammatory cytokines (IL‐12 and/or IFN‐γ) than the Bifidobacterium strains. Shigella CBD8 and E. coli CBL2 up‐regulated mainly HLA‐DR and CD40 expression involved in Th1 activation, and Bifidobacterium strains up‐regulated CD83 expression. Specific interactions among the studied bacteria, gliadins, and IFN‐γ, which favored the CD immune features, were also detected. Therefore, intestinal bacteria could be additional factors that regulate the ability of monocytes recruited to the mucosa to respond to gliadins and IFN‐γ in CD patients, influencing the course of the disease.

Modulation of phenotypic and functional maturation of dendritic cells by intestinal bacteria and gliadin: relevance for celiac disease.

DC maturation and functions are influenced by microbial and environmental stimuli, which could contribute to immune dysfunction. Here, we have investigated the role of enterobacteria (Escherichia coli CBL2 and Shigella CBD8) isolated from CD patients, bifidobacteria (Bifidobacterium longum CECT 7347 and Bifidobacterium bifidum CECT 7365), and gliadins on phenotypic and functional features of MDDCs and in coculture with Caco‐2 cells. The ultimate goal of our study is to understand the roles played by specific components of the gut microbiota in CD. Enterobacteria induced marked alterations in MDDC morphology, inducing podosome dissolution and dendrites, and activated MDDC adhesion and spreading. Enterobacteria also induced inflammatory cytokine production (IFN‐γ, TNF‐α, and IL‐12), partially resembling the gliadin‐induced Th1‐type cytokine profile. B. longum CECT 7347 and B. bifidum CECT 7365 induced minor MDDC morphological changes and activated adhesion and spreading and inflammatory cytokine production to a lesser extent compared with enterobacteria. B. longum CECT 7347 also induced lower CD86 and CD40 expression on MDDCs than the two enterobacteria. The aforementioned bifidobacterial strain also reduced gliadin‐induced IFN‐γ production and increased IL‐10 secretion when both stimuli were combined. Similar trends were detected for MDDCs cocultured with Caco‐2 cells. B. longum CECT 7347 reversed the gliadin‐reduced ZO‐1 expression in Caco‐2 cells. Thus, our results suggest that specific components of the gut microbiota may influence phenotypic and functional maturation of DCs differently and their interactions with epithelial cells. This could ultimately define the role of DCs in CD progression.

Functional impact of IBS-D microbiota on the gut-brain axis

Irritable Bowel Syndrome (IBS) is a common disorder characterized by altered gut function and frequent psychiatric co-morbidity. Although altered intestinal microbiome profiles have been documented, their relevance to the clinical expression of IBS is unknown. To assess the functional role of the microbiota, we colonized germ-free mice with fecal microbiota from healthy controls or from IBS patients with diarrhea (IBS-D) with or without accompanying anxiety, and monitored gut function (gastrointestinal transit, expression of several genes related to inflammation, and barrier function) and behavior. Specific microbial profiles were transferred from the human donors into mouse recipients. Despite having taxonomically similar composition as controls, mice with IBS-D microbiota had distinct serum metabolomic profiles related to neuro- and immunomodulation. Mice with IBS-D, but not control microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation and anxiety-like behavior. Our study thus supports the notion that gut microbiota contributes to both intestinal and behavioral manifestations of IBS-D and supports the use of microbiota-directed therapies in ameliorating this condition.

Impaired responses to gliadin and gut microbes of immune cells from mice with altered stress-related behavior and premature immune senescence

Stress is associated with impaired communication between the nervous and immune systems leading to immunosenescence and increased disease risk. We investigated whether leukocytes from mice with altered stress-related behavior and premature immunosenescence, as well as from chronologically aged mice differently responded ex vivo to celiac disease (CD) triggers (gliadin) and intestinal bacteria by ELISA and flow cytometry and differed in microbiota composition. We found that altered stress-related behavior and premature immunosenescence led to alterations in T lymphocytes and cytokine release of immune cells basally and in response to peptic fragments of gliadin and commensal and pathogenic bacteria, possibly increasing susceptibility to CD in adulthood.

Abstract # 1850 Early life stress predisposes to increased sensitivity to inflammatory stimuli through intestinal dysbiosis

Stress, anxiety and depression are considered risk factors for Inflammatory Bowel Disease. We have previously shown in mice that early life stress induced by maternal separation (MS) changes host physiology leading to intestinal dysbiosis, which in turns causes anxiety and depression-like behavior. Using this murine model, we investigated the role of stress and microbiota in susceptibility to intestinal inflammation. Adult MS and control C57Bl/6 mice were used. Experimental colitis was induced by 1.5% or 3.5% dextran sulfate sodium (DSS) in drinking water in germ-free or colonized (ex-germ-free) mice, respectively. After their behavior and clinical scores were assessed, mice were sacrificed and samples collected. Colonized but not germ-free MS mice displayed anxiety and depression-like behavior, higher secretory IgA levels, decreased Paneth cell counts and dysbiosis compared to controls. In colonized mice, DSS colitis clinical scores were higher in MS mice accompanied by marked dysbiosis, lower secretory IgA and increased serum MCP-1 levels compared to controls, although the colonic microscopic scores and inflammatory cytokine levels were similar. In germ-free conditions, no difference in clinical colitis scores, histology or cytokine levels were found. In summary, MS leads to intestinal dysbiosis that sustains and exacerbates an impaired cellular and humoral immunity, leading to increased sensitivity to intestinal injury. Our results further support the role of gut-microbiota-brain axis in the genesis of chronic gastrointestinal inflammatory disorders.

Abstract # 1849 The role of immune system maturation in development of normal behavior in mice

Previous studies demonstrated differences in behavior and brain biochemistry between germ-free and conventional mice. We showed that mono-colonization with E. coli induces similar changes in behavior as with complex microbiota. BDNF and cFos expression in the amygdala and hippocampus regions were also changed after mono-colonization. Furthermore, using permanent colonizer E. coli JM83 and transient colonizer E. coli HA107 (mutant form of E. coli JM83) we demonstrated that changes in behavior persist despite mice reverting to germ-free status. Using mono-colonized SCID and MyD88-/- Ticam-/- mice we now show that signaling via the innate, and not adaptive immune pathway is crucial for this change in behavior. Analysis of colonic and brain tissues using NanoString technique has revealed that multiple innate immunity and neural system related genes are significantly altered after colonization, including networks previously linked to neuronal plasticity. To further investigate the role of Toll-like receptors, we gavaged germ-free mice with LPS and poly I:C for two weeks. Only mice given LPS displayed altered behavior suggesting that TLR-4 related pathways are crucial for the development of normal mouse behavior. In conclusion, bacterial colonization induces long lasting changes in behavior and brain chemistry, which are associated with alterations in multiple innate immunity and neural system related gene networks. Signalling via TLR-4 pathway appears to be crucial for gut-microbiota-brain axis communication and for the development of normal behavioral.

Adrenergic innervation regulates intestinal microbiota diversity via generation of cholinergic Th17 lymphocytes

Background: Although, it is usually difficult to differentiate multiple system atrophy (MSA) from Parkinson’s disease (PD), we have previously reported that post-void residuals and sphincter electromyography were useful for differentiation 1), . However, we did not know which is more appropriate in differentiating MSA from PD. Aims: We aimed to perform receiver operating characteristic analysis to determine the ability of sphincter electromyography (mean duration) and post-void residuals (free flow study and pressure-flow study) for distinguishing multiple system atrophy from Parkinson’s disease. Methods: We retrospectively reviewed 241 case records where both urodynamic study and sphincter electromyography were performed in patients with multiple system atrophy (n = 147) and Parkinsons disease (n = 94). We performed receiver operating characteristic analysis of the data sets. Results: The area under the curve (AUC) used to differentiate multiple system atrophy from Parkinson’s disease was 0.79 in post-void residuals during pressure flow study, 0.73 in post-void residuals during freeflow study and 0.69 in mean duration of sphincter electromyography, respectively; these values were statistically significant. Conclusion: The present results suggested that post-void residuals were more appropriate than mean duration of sphincter electromyography in differentiating MSA from PD. In particular, the AUC in postvoid residuals under pressure flow study was larger than that of under free-flow study. 1) Yamamoto T, Sakakibara R, Uchiyama T, Yamaguchi C, Ohno S, Nomura F, Yanagisawa M, Hattori T, Kuwabara S. Time-dependent changes and gender differences in urinary dysfunction in patients with multiple system atrophy. Neurourol Urodyn. 2014 Jun;33(5):516-23. 2) Yamamoto T, Sakakibara R, Uchiyama T, Yamaguchi C, Nomura F, Ito T, YanagisawaM, YanoM, Awa Y, Yamanishi T, Hattori T, Kuwabara S. Receiver operating characteristic analysis of sphincter electromyography for parkinsonian syndrome. Neurourol Urodyn. 2012 Sep;31(7):1128-34.

Influence of early environmental factors on peripheral lymphocyte subsets and gut microbiota in infants at risk for celiac disease

Genetic risk linked to the HLA (Human Leucocyte Antigen) system is not enough to explain the incidence of celiac disease (CD) and other genetic and environmental factors have been suggested to play a role . The immune system and microbiota are not fully developed at birth and single antigen encounter in the intestine is a key process to achieve full development . Environmental factors through this or other route are believed to impact on the immune system and microbiota as well. We sought to assess the effect of several early environmental factors on lymphocyte subsets and microbiota composition in infants at familial risk for CD. For this purpose, 55 infants with a first degree relative suffering CD were recruited before birth. Information on early environmental factors was prospectively collected including type of delivery, mother’s antibiotic intake during pregnancy, mother’s antibiotic administration during labour, milkfeeding practices, infections and antibiotic intake in the first 4 months of life and rotavirus vaccine administration. Lymphocyte subsets in peripheral blood and gut microbiota composition in faeces samples were studied at the age of 4 months by flow cytometry analysis and qPCR respectively. Linear regression analysis showed that, the absolute counts of total lymphocytes, CD3 + , CD4 + , CD4 +CD38+ , CD4+CD28+ , were positively associated with formula-fed infants and infant’s infections in the first 4 months of age. The percentage of CD4 +CD25 + was positively associated with vaginal delivery and antibiotic use by mothers during pregnancy, and negatively with rotavirus vaccine, and their absolute counts were associated positively with infant’s infections. The absolute counts of CD3+CD4+CD45RO + were positively associated with formula-feeding and infant’s infections, and negatively with infant’s antibiotic intake. CD4 +HLA-DR + cell counts were positively associated with infant’s infections. The percentages and absolute counts of NK cells were positively associated with infant’s infections and antibiotic administration in mothers during labor. Regarding microbiota, infant’s antibiotic intake is associated with higher counts of Bacteroides spp. and lower counts of B. longum. Cesarean delivery is associated with higher counts of B. angulatum and lower counts of B. catenulatum. B. angulatum counts were also positively associated with infant’s antibiotic administration in the first 4 months of life and negatively with formula feeding and antibiotic administration during pregnancy. In conclusion, the balance between the effects of the preand post-natal factors on lymphocyte subsets and microbiota composition might modify the risk for future development of immune-related diseases such as celiac disease.