Human milk oligosaccharides alleviate stress-induced visceral hypersensitivity and associated microbiota dysbiosis.

Pain-related functional gastrointestinal disorders (FGIDs) are characterized by visceral hypersensitivity (VHS) associated with alterations in the microbiota-gut-brain axis. Since human milk oligosaccharides (HMOs) modulate microbiota, gut and brain, we investigated whether HMOs impact VHS, and explored the role of gut microbiota. To induce VHS, C57BL/6JRj mice received hourly water avoidance stress (WAS) sessions for 10 d, or antibiotics (ATB) for 12 d. Challenged and unchallenged (Sham) animals were fed AIN93M diet (Cont) or AIN93M containing 1% of a 6-HMO mix (HMO6). VHS was assessed by monitoring the visceromotor response to colorectal distension. Fecal microbiome was analyzed by shotgun metagenomics. The effect of HMO6 sub-blends on VHS and nociceptive pathways was further tested using the WAS model. In mice fed Cont, WAS and ATB increased the visceromotor response to distension. HMO6 decreased WAS-mediated electromyographic rise at most distension volumes and overall Area Under Curve (AUC=6.12±0.50 in WAS/HMO6 vs. 9.46±0.50 in WAS/Cont; P<.0001). In contrast, VHS in ATB animals was not improved by HMO6. In WAS, HMO6 promoted most microbiota taxa and several functional pathways associated with low VHS and decreased those associated with high VHS. Among the sub-blends, 2'FL+DFL and LNT+6'SL reduced visceromotor response close to Sham/Cont values and modulated serotoninergic and CGRPα-related pathways. This research further substantiates the capacity of HMOs to modulate the microbiota-gut-brain communication and identifies mitigation of abdominal pain as a new HMO benefit. Ultimately, our findings suggest the value of specific HMO blends to alleviate pain associated FGIDs such as infantile colic or Irritable Bowel Syndrome.

Secretory IgA in complex with Lactobacillus rhamnosus potentiates mucosal dendritic cell-mediated Treg cell differentiation via TLR regulatory proteins, RALDH2 and secretion of IL-10 and TGF-ß.

The importance of secretory IgA in controlling the microbiota is well known, yet how the antibody affects the perception of the commensals by the local immune system is still poorly defined. We have previously shown that the transport of secretory IgA in complex with bacteria across intestinal microfold cells results in an association with dendritic cells in Peyer's patches. However, the consequences of such an interaction on dendritic cell conditioning have not been elucidated. In this study, we analyzed the impact of the commensal Lactobacillus rhamnosus, alone or associated with secretory IgA, on the responsiveness of dendritic cells freshly recovered from mouse Peyer's patches, mesenteric lymph nodes, and spleen. Lactobacillus rhamnosus-conditioned mucosal dendritic cells are characterized by increased expression of Toll-like receptor regulatory proteins [including single immunoglobulin interleukin-1 receptor-related molecule, suppressor of cytokine signaling 1, and Toll-interacting molecule] and retinaldehyde dehydrogenase 2, low surface expression of co-stimulatory markers, high anti- versus pro-inflammatory cytokine production ratios, and induction of T regulatory cells with suppressive function. Association with secretory IgA enhanced the anti-inflammatory/regulatory Lactobacillus rhamnosus-induced conditioning of mucosal dendritic cells, particularly in Peyer's patches. At the systemic level, activation of splenic dendritic cells exposed to Lactobacillus rhamnosus was partially dampened upon association with secretory IgA. These data suggest that secretory IgA, through coating of commensal bacteria, contributes to the conditioning of mucosal dendritic cells toward tolerogenic profiles essential for the maintenance of intestinal homeostasis.

Temporal Changes of Protein Composition in Breast Milk of Chinese Urban Mothers and Impact of Caesarean Section Delivery.

Human breast milk (BM) protein composition may be impacted by lactation stage or factors related to geographical location. The present study aimed at assessing the temporal changes of BM major proteins over lactation stages and the impact of mode of delivery on immune factors, in a large cohort of urban mothers in China. 450 BM samples, collected in three Chinese cities, covering 8 months of lactation were analyzed for α-lactalbumin, lactoferrin, serum albumin, total caseins, immunoglobulins (IgA, IgM and IgG) and transforming growth factor (TGF) ß1 and ß2 content by microfluidic chip- or ELISA-based quantitative methods. Concentrations and changes over lactation were aligned with previous reports. α-lactalbumin, lactoferrin, IgA, IgM and TGF-ß1 contents followed similar variations characterized by highest concentrations in early lactation that rapidly decreased before remaining stable up to end of lactation. TGF-ß2 content displayed same early dynamics before increasing again. Total caseins followed a different pattern, showing initial increase before decreasing back to starting values. Serum albumin and IgG levels appeared stable throughout lactation. In conclusion, BM content in major proteins of urban mothers in China was comparable with previous studies carried out in other parts of the world and C-section delivery had only very limited impact on BM immune factors.

Role of secretory IgA in the mucosal sensing of commensal bacteria.

While the gut epithelium represents the largest mucosal tissue, the mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple outcomes that remain poorly understood at the molecular level. Deciphering such events may provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the intestinal immune system include maturation processes prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. As commensal bacteria are naturally coated by natural and antigen-specific SIgA in the gut lumen, understanding the consequences of such an interaction may provide new clues on how the antibody contributes to homeostasis at mucosal surfaces. This review discusses several aspects of the role of SIgA in the essential communication existing between the host epithelium and members of its microbiota.

The role of secretory immunoglobulin A in the natural sensing of commensal bacteria by mouse Peyer's patch dendritic cells.

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer's patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c(+)CD11b(+)CD8(-) dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.

Influence of gut microbiota on mouse B2 B cell ontogeny and function.

A complex interplay between the microbiota and the host immune system is evidenced to shape the immune system throughout life, but little is known about the microbial effect on key players of the adaptive immune system, the B2 B cells. In the presented study, we have evaluated the effect of commensal bacteria on B cell ontogeny and function, with the focus on B2 B cells of spleen and Peyer's patches. We have compared germ-free mice to mice that are exposed to a normal complex bacterial community from the day of birth and combined classical immunological assessment with advanced genome-wide expression profiling. Despite a preservation of all B cell subsets and phenotype, our results show that microbiota strongly impact mucosal B cell physiology and lead to higher serum Ig concentrations. We show that this microbial influence comprises downregulation of transcription factors involved in early B cell activation steps and upregulation of genes and proteins involved in later stages of B cell response. In summary, we show an influence of the gut microbiota on function of mucosal B2 B cells, involving mechanisms downstream of B cell activation and proliferation.

Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis.

AIM: To assess the anti-inflammatory effect of the probiotic Bifidobacterium lactis (B. lactis) in an adoptive transfer model of colitis. METHODS: Donor and recipient mice received either B. lactis or bacterial culture medium as control (deMan Rogosa Sharpe) in drinking water for one week prior to transfer of a mix of naive and regulatory T cells until sacrifice. RESULTS: All recipient mice developed signs of colonic inflammation, but a significant reduction of weight loss was observed in B. lactis-fed recipient mice compared to control mice. Moreover, a trend toward a diminution of mucosal thickness and attenuated epithelial damage was revealed. Colonic expression of pro-inflammatory and T cell markers was significantly reduced in B. lactis-fed recipient mice compared to controls. Concomitantly, forkhead box protein 3, a marker of regulatory T cells, was significantly up-regulated by B. lactis. CONCLUSION: Daily oral administration of B. lactis was able to reduce inflammatory and T cells mediators and to promote regulatory T cells specific markers in a mouse model of colitis.

Time-resolved quantitative proteome analysis of in vivo intestinal development.

Postnatal intestinal development is a very dynamic process characterized by substantial morphological changes that coincide with functional adaption to the nutritional change from a diet rich in fat (milk) to a diet rich in carbohydrates on from weaning. Time-resolved studies of intestinal development have so far been limited to investigation at the transcription level or to single or few proteins at a time. In the present study, we elucidate proteomic changes of primary intestinal epithelial cells from jejunum during early suckling (1-7 days of age), middle suckling (7-14 days), and weaning period (14-35 days) in mice, using a label-free proteomics approach. We show differential expression of 520 proteins during intestinal development and a pronounced change of the proteome during the middle suckling period and weaning. Proteins involved in several metabolic processes were found differentially expressed along the development. The temporal expression profiles of enzymes of the glycolysis were found to correlate with the increase in carbohydrate uptake at weaning, whereas the abundance changes of proteins involved in fatty acid metabolism as well as lactose metabolism indicated a nondiet driven preparation for the nutritional change at weaning. Further, we report the developmental abundance changes of proteins playing a vital role in the neonatal acquisition of passive immunity. In addition, different isoforms of several proteins were quantified, which may contribute to a better understanding of the roles of the specific isoforms in the small intestine. In summary, we provide a first, time-resolved proteome profile of intestinal epithelial cells along postnatal intestinal development.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy is effective in treating some Crohn's disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. METHODS: DSS colitic mice were treated with daily pegylated GM-CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. RESULTS: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts, and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b(+) monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF-induced CD11b(+) myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wildtype bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF-induced CD11b(+) myeloid cells were shown to promote in vitro wound repair. CONCLUSIONS: Our study shows that GM-CSF-dependent stimulation of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM-CSF therapy in Crohn's disease.

Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model.

BACKGROUND & AIMS: Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS: In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS: Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS: IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

Ascites and highly elevated CA-125 levels in a case of peritoneal tuberculosis.

Peritoneal tuberculosis (PT) is uncommon in industrialized countries. We report the case of a 35-y-old female with a 1-y history of abdominal discomfort, ascites, systemic symptoms, and highly elevated CA-125 suggesting malignancy, in whom the diagnosis of PT was considered. Both clinical symptoms and CA-125 levels regressed under tuberculostatic treatment.

[Chronic cough: practical aspects]. / Toux chronique: de la théorie à to pratique.

A cough is defined as chronic, if it exceeds 8 weeks in length. Post-nasal drip (PND), bronchial asthma and gastro-esophageal reflux (GERD) must be systematically investigated, as these account for 90 percent of chronic cough cases. In addition to medical history and examination which should exclude either a postinfectious cough or coughing related to ACE inhibitor medication, a new evaluation model suggests chest X ray and spirometry as the initial step. A chronic cough is rarely due to one cause as in at least 25% of cases 2 etiologies are present. An effective treatment of chronic cough often relies on several medication trials until its disappearance. After a multidisciplinary approach to chronic cough using this investigative model, the diagnosis of idiopathic or psychogenic cough should remain exceptional.

Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses.

Secretory IgA (SIgA) is essential in protecting mucosal surfaces by ensuring immune exclusion. In addition, SIgA binds selectively to M cells in Peyer's patches (PP), resulting in transport across the epithelium and targeting of dendritic cells (DC) in the dome region. The immunological consequences of such an interaction are unknown. In this study, we find that oral delivery of SIgA comprising human secretory component and mouse IgA induces human secretory component-specific Ab and cellular responses in mucosal and peripheral tissues in mice. This takes place in the absence of co-addition of cholera toxin, identifying so far unraveled properties in SIgA. Specific immune responses are accompanied by sustained IL-10 and TGF-beta expression in draining mesenteric lymph nodes and spleen. SIgA also triggers migration of DC to the T cell-rich regions of PP, and regulates expression of CD80 and CD86 on DC in PP, mesenteric lymph nodes, and spleen. These results provide evidence that mucosal SIgA re-entering the body exerts a function of Ag delivery that contributes to effector and/or regulatory pathways characteristic of the intestinal mucosal compartment.

Simplified procedure to recover recombinant antigenized secretory IgA to be used as a vaccine vector.

Induced protection mechanisms at mucosal surfaces involve secretory IgA (SIgA), a complex structure made of polymeric-dimeric IgA (IgA(p/d)) antibody associated with secretory component (SC). SIgA can adhere to M cells of the intestinal and nasal epithelia, are transported across these latter, and are thus available to the immune cells underlying the epithelia. This property makes SIgA suitable as potential mucosal vaccine delivery vector. It remains that production and purification of SIgA is a complex task since IgA(p/d) and SC are naturally synthesized by two different cell types. Furthermore, only IgA(p/d) are capable to associate with SC. Thus, we sought to separate IgA(p/d) and monomeric IgA (IgA(m)) antibodies secreted by hybridoma cells in CELLine bioreactors. To this aim, we connected together two 1-m long columns filled with Sephacryl S-300 beads and placed them under the control of a automatized chromatographic system. In parallel, we produced recombinant antigenized human SC (ra-hSC) in Chinese hamster ovary (CHO) cells adapted to suspension culture in CELLine bioreactors. To avoid intermediate purification of ra-hSC, culture supernatants (SN) containing this latter were combined with purified IgA(p/d), and the recombinant antigenized SIgA (raSIgA) complex was resolved on a 1-m long column filled with Superdex 200 beads. Biochemical characterization based on SDS-PAGE, silver staining, immunodetection and enzyme-linked immunosorbent assay (ELISA) indicates that highly purified raSIgA can be recovered using this simple two-step procedure. Such preparations are currently used to immunize mice to induce mucosal and systemic responses.