Exercise improves intestinal IgA production by T-dependent cell pathway in adults but not in aged mice.

Introduction: Hypermutated high-affinity immunoglobulin A (IgA), neutralizes toxins and drives the diversification of bacteria communities to maintain intestinal homeostasis although the mechanism underlies the impact of moderate aerobic exercise (MAE) on the IgA-generation via T-dependent (TD) is not fully know. Therefore, the aim of this study was to determine the effect of long-time MAE on the production of IgA through the TD pathway in Peyer´s patches of the small intestine from aged mice. Methods: MAE protocol consisted of twenty 3-month-old (young) BALB/c mice running in an endless band at 0° inclination and a speed of 10 m/h for 5 days a week and resting 2 days on the weekend until reaching 6-month-old (adulthood, n=10) or 24-month-old (aging, n=10). Groups of young, adult, or elderly mice were included as sedentary controls (n=10/per group). At 6 or 24 months old, all were sacrificed, and small intestine samples were dissected to prepare intestinal lavages for IgA quantitation by ELISA and to obtain suspensions from Peyer´s patches (PP) and lamina propria (LP) cells for analysis of T, B, and plasma cell subpopulations by flow cytometry and mRNA analysis expression by RT-qPCR of molecular factors related to differentiation of B cells to IgA+ plasma cells, class switch recombination, and IgA-synthesis. Statistical analysis was computed with two-way ANOVA (factor A=age, factor B=group) and p<0.05 was considered for statistically significant differences. Results: Compared to age-matched sedentary control, in exercised elderly mice, parameters were either increased (IgA concentration, IL-21, IL-10 and RDH mRNA expression), decreased (α-chain mRNA, B cells, mIgA+ B cells, mIgM+ B cells and IL-4 mRNA) or unchanged (PP mIgA+ plasmablasts and LP cyt-IgA+ plasma cells). Regarding the exercised adult mice, they showed an up-modulation of IgA-concentration, mRNA expression IL-21, IL-10, and RDH and cells (PP B and T cells, mIgM+ plasmablasts and LP cyt-IgA+plasma cells). Conclusion: Our findings suggest that MAE restored the IgA production in adult mice via the TD cell pathway but does not in aged mice. Other studies are necessary to know in more detail the impact of long-time MAE on the TD pathway to produce IgA in aging.

Outcomes of nicotinic modulation on markers of intestinal IgA antibody response.

Acetylcholine (ACh), as a ligand of nicotinic acetylcholine receptors (nAChRs), plays a key role in the cholinergic anti-inflammatory pathway; however, its role in the immunoglobulin A (IgA) response remains unknown. Therefore, the present study aimed to investigate the role of ACh in the intestinal biomarkers involved in IgA synthesis and the polymeric immunoglobulin receptor (pIgR) involved in IgA transcytosis. Groups of mice were administered GTS-21 (an α7nAChR agonist) or mecamylamine (a non-selective nAChR antagonist) intraperitoneally for 7 days. Intestinal fluids were used for antibody concentration assessment by ELISA, cell suspensions from Peyer's patches and the lamina propria were obtained for flow cytometric analysis of plasma cells, and CD4+ T-cells expressing intracellular transforming growth factor (TGF)-ß and IgA-producing interleukin (IL)-4, -5, -6 and -10, and isolated epithelial cells to determine the levels of pIgR mRNA using reverse transcription-quantitative PCR. Regarding to the untreated control group, the concentration of IgA was reduced in the mecamylamine group and unaltered in the GTS-21 group while IgM levels exhibited no differences; the percentage of IgA+ plasma cells from Peyer's patches and the lamina propria, and the percentage of TGF-ß+/CD4+ T-cells from Peyer's patches were greater in the GTS-21-group. In both treatment groups, the percentages of IgM+ plasma cells and IL-6+/IL-10+ CD4+ T cells were greater in both compartments; pIgR mRNA expression levels decreased in epithelial cells. The percentage of IL-4 CD4+ T-cells were greater in Peyer's patches and lower in the lamina propria in the mecamylamine group, and the percentage of IL-5 CD4+ T-cells in the lamina propria were decreased in both treatment groups. These findings require further examination to address the impact of cholinergic modulation on IgA-transcytosis via pIgR. The present study may be an experimental reference for clinical trials that address the role of nicotinic system in intestinal dysfunctions as postoperative ileus.

Temporal Dynamics of T Helper Populations in the Proximal Small Intestine after Oral Bovine Lactoferrin Administration in BALB/c Mice.

Bovine lactoferrin (bLf), a component of milk and a dietary supplement, modulates intestinal immunity at effector and inductor sites. Considering the regional difference in intestinal compartments and the dynamics of local cytokine-producing cells in the gut across time, the aim of this work was to characterize the effects of bLf on the proximal small intestine in a BALB/c murine model of oral administration. Male BALB/c mice were treated with oral bLf vs. saline control as mock by buccal deposition for 28 days. Intestinal secretions were obtained at different time points and cells were isolated from Peyer's patches (PP) and lamina propria (LP) of the proximal small intestine as representative inductor and effector sites, respectively. Total and specific anti-bLF IgA and IgM were determined by enzyme-immuno assay; the percentages of IgA+ and IgM+ plasma cells (PC) and cytokine-producing CD4+ T cells of PP and LP were analyzed by flow cytometry. We found that total and bLf-specific IgA and IgM levels were increased in the intestinal secretions of the bLf group in comparison to mock group and day 0. LP IgA+ PC and IgM+ PC presented an initial elevation on day 7 and day 21, respectively, followed by a decrease on day 28 in comparison to mock. Higher percentages of CD4+ T cells in LP were found in the bLf group. Cytokines-producing CD4+ T cells populations presented a pattern of increases and decreases in the bLf group in both LP and PP. Transforming growth factor beta (TGF-ß)+ CD4+ T cells showed higher percentages after bLf administration with a marked peak at day 21 in both LP and PP in comparison to mock-treated mice. Oral bLf exhibits complex immune properties in the proximal small intestine, where temporal monitoring of the inductor and effector compartments reveals patterns of rises and falls of different cell populations. Exceptionally, TGF-ß+ CD4+ T cells show consistent higher numbers after bLf intervention across time. Our work suggests that isolated measurements do not show the complete picture of the modulatory effects of oral bLf in immunological sites as dynamic as the proximal small intestine.

Anterior subdiaphragmatic vagotomy decreases the IgA antibody response in the small intestines of BALB/c mice.

To assess the impact of vagotomy on the IgA-response, male BALB/c mice underwent anterior vagotomy or a sham procedure were sacrificed on day 14 post-operation and the proximal and distal small-gut segments were dissected. In intestinal lavages IgA/IgM antibodies were analysed by ELISA; in Peyer's-patches and lamina-propria cell suspensions the intracellular IgA-associated interleukins (ILs) and pro-inflammatory cytokines in CD4+ T cells were analysed by cytofluorometry. Vagotomy reduced the IgA or increased the IgM antibody concentration in both segments and reduced or increased the lamina- propria CD4+ T cell pro-inflammatory cytokine responses in the distal or proximal segments, respectively. Data show the role of the vagus nerve on the IgA response.

Modulatory Effects of Oral Bovine Lactoferrin on the IgA Response at Inductor and Effector Sites of Distal Small Intestine from BALB/c Mice.

Bovine lactoferrin (bLf) up-modulates intestinal IgA that is essential for homeostasis and which might confer protection to the distal small intestine that is vulnerable to inflammation. This study analyzed the effects of bLf administered orally on the IgA response at inductive (Peyer's patches) and effector (lamina propria) sites of the distal small intestine in mice. Groups of five healthy male BALB/c mice were orally treated with 5 mg of bLf for 7, 14, 21, or 28 days. Then, mice were killed and the distal small intestine was dissected. Intestinal fluid samples were analyzed to determine IgA and IgM levels by enzyme-immuno assay. Peyer's patches and lamina propria were analyzed for IgA(+) or IgM(+) plasma cells, B, CD4(+) T and CD8(+) T cells as well as CD4(+) T cells positive for either pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon-γ and interleukin (IL)-12] or for IgA-producing ILs (IL-4, -5, -10 and -6) by cytofluorometry. Antibodies, antibody-secreting cells, and B and T responses in both Peyer's patches and lamina propria were higher in bLf-treated than bLf-untreated mice. The generation of IL-10 and IL-6 CD4(+) T cells in Peyer's patches or TNF-α and IL-12 CD4(+) T cells in lamina propria showed similar response patterns. On days 14 and 28, cytokine/IL CD4(+) T cell responses were increased in Peyer's patches or decreased in lamina propria. The effect of bLf on the elicitation of IgA indicates a potential application of bLf as a nutraceutical to control inflammation in the distal small intestine.