Patrick Serrant

Bacterial imprinting of the neonatal immune system: lessons from maternal cells?

OBJECTIVE. We examined the presence of a natural bacterial inoculum in breast milk and its intracellular transport from the maternal intestine to the breast through the circulation. METHODS. Breast milk and peripheral blood were collected aseptically from healthy donors at various times after delivery, and the presence of viable bacteria was determined through plating. Temporal temperature gradient gel electrophoresis was used to examine the bacterial ribosomal DNA content in milk cells, maternal peripheral blood mononuclear cells, and feces and in corresponding infant feces. Blood from nongravid nonlactating women served as control samples. Bacterial translocation to extraintestinal tissues was also evaluated in virgin, pregnant, and lactating mice. RESULTS. Breast milk contained a low total concentration of microbes of <103 colony-forming units per mL. Temporal temperature gradient gel electrophoresis revealed that maternal blood and milk cells contained the genetic material of a greater biodiversity of enteric bacteria. Some bacterial signatures were common to infant feces and to samples of maternal origin. Bacterial translocation from the gut to mesenteric lymph nodes and mammary gland occurred during late pregnancy and lactation in mice. CONCLUSIONS. Bacterial translocation is a unique physiologic event, which is increased during pregnancy and lactation in rodents. Human breast milk cells contain a limited number of viable bacteria but a range of bacterial DNA signatures, as also found in maternal peripheral blood mononuclear cells. Those peripheral blood mononuclear cells showed greater biodiversity than did peripheral blood mononuclear cells from control women. Taken together, our results suggest that intestinally derived bacterial components are transported to the lactating breast within mononuclear cells. We speculate that this programs the neonatal immune system to recognize specific bacterial molecular patterns and to respond appropriately to pathogens and commensal organisms.

Potential role of the intestinal microbiota of the mother in neonatal immune education.

Mucosal dendritic cells are at the heart of decision-making processes that dictate immune reactivity to intestinal microbes. They ensure tolerance to commensal bacteria and a vigorous immune response to pathogens. It has recently been demonstrated that the former involves a limited migration of bacterially loaded dendritic cells from the Peyers patches to the mesenteric lymph nodes. During lactation, cells from gut-associated lymphoid tissue travel to the breast via the lymphatics and peripheral blood. Here, we show that human peripheral blood mononuclear cells and breast milk cells contain bacteria and their genetic material during lactation. Furthermore, we show an increased bacterial translocation from the mouse gut during pregnancy and lactation and the presence of bacterially loaded dendritic cells in lactating breast tissue. Our observations show bacterial translocation as a unique physiological event, which is increased during pregnancy and lactation. They suggest endogenous transport of intestinally derived bacterial components within dendritic cells destined for the lactating mammary gland. They also suggest neonatal immune imprinting by milk cells containing commensal-associated molecular patterns.

Hazard assessment through hybrid in vitro/in silico approach: the case of zearalenone

Within the framework of reduction, refinement and replacement of animal experiments, new approaches for identification and characterization of chemical hazards have been developed. Grouping and read across has been promoted as a most promising alternative approach. It uses existing toxicological information on a group of chemicals to make predictions on the toxicity of uncharacterized ones. In the present work, the feasibility of applying in vitro and in silico techniques to group chemicals for read across was studied using the food mycotoxin zearalenone (ZEN) and metabolites as a case study. ZEN and its reduced metabolites are known to act through activation of the estrogen receptor α (ERα). The ranking of their estrogenic potencies appeared highly conserved across test systems including binding, in vitro and in vivo assays. This data suggests that activation of ERα may play a role in the molecular initiating event (MIE) and be predictive of adverse effects and provides the rationale to model receptor-binding for hazard identification. The investigation of receptor-ligand interactions through docking simulation proved to accurately rank estrogenic potencies of ZEN and reduced metabolites, showing the suitability of the model to address estrogenic potency for this group of compounds. Therefore, the model was further applied to biologically uncharacterized, commercially unavailable, oxidized ZEN metabolites (6α-, 6β-, 8α-, 8β-, 13- and 15-OH-ZEN). Except for 15-OH-ZEN, the data indicate that in general, the oxidized metabolites would be considered a lower estrogenic concern than ZEN and reduced metabolites.

Effect of Lactobacillus paracasei NCC2461 on Antigen-Specific T-Cell Mediated Immune Responses in Aged Mice

Aging is associated with a reduced capacity to mount proper immune responses, in particular to vaccines. Probiotic lactic acid bacteria may improve the immune status of the elderly; however, there is little evidence showing an effect of these bacteria on humoral and cellular immune responses. In the present study, the immunomodulatory capacity of the probiotic Lactobacillus paracasei NCC2461 combined or not with a prebiotic composition, FOS/inulin, was examined in aged mice. Male C57BL/6J mice (21-months-old) were allocated to one of three groups fed ad libitum for 44 days with different diets: a normal diet (control), a normal diet plus NCC2461 given in the drinking water, or a diet containing FOS/inulin plus NCC2461 in the drinking water. All mice were immunized on day 15 and challenged on day 22 with keyhole limpet hemocyanin (KLH). T helper (Th)1 cell-dependent immune responses (anti-KLH immunoglobulin G(2a) [IgG(2a)] levels and delayed type hypersensitivity response) were increased significantly in NCC2461-supplemented mice when compared to controls. Supplementation with FOS/inulin did not further improve the immune-enhancing effect mediated by the probiotic. Splenocyte proliferation, T cell subsets, systemic total IgG levels, and mucosal total IgA responses were not affected. Interestingly, supplementation with NCC2461 modulated the intestinal microbiota composition by increasing the numbers of bifidobacteria and lactobacilli. In conclusion, oral intake of L. paracasei NCC2461 by aged mice enhanced the specific adaptive immune response to in vivo antigenic challenge without altering other cellular and humoral immune responses. The poor responsiveness to antigenic challenge, frequently observed in elderly people, may be improved by supplementation with L. paracasei NCC2461.

Intake of a milk-based wolfberry formulation enhances the immune response of young-adult and aged mice.

Aging is associated with alterations of immune responses. Wolfberry, a popular Chinese functional ingredient, is prized for its anti-aging properties; however, little is known about the immunological effect of wolfberry intake. The purpose of this study was to examine the effect of dietary intake of a milk-based formulation of wolfberry, named Lacto-Wolfberry, on in vivo and ex vivo parameters of adaptive immunity in young-adult and aged mice. Over 44 days, young-adult (2 months) and aged (21 months) C57BL/6J mice were fed ad libitum with a controlled diet and received drinking water supplemented or not with 0.5% (wt/vol) Lacto-Wolfberry. All mice were immunized on day 15 and challenged on day 22 with a T cell- dependent antigen, keyhole limpet hemocyanin (KLH). Lacto-Wolfberry supplementation significantly increased in vivo systemic immune markers that are known to decline with aging. Indeed, both antigen-(KLH) specific humoral response and cell-mediated immune responses in young-adult and aged mice were enhanced when compared to their respective controls. No significant effect of Lacto-Wolfberry supplementation was observed on ex vivo spleen cells proliferative response to mitogens and on splenocyte T cell subsets. In conclusion, dietary intake of Lacto-Wolfberry may favorably modulate the poor responsiveness to antigenic challenge observed with aging.

Anti-inflammatory effects of Lacto-Wolfberry in a mouse model of experimental colitis

AIM To investigate the anti-inflammatory properties of Lacto-Wolfberry (LWB), both in vitro and using a mouse model of experimental colitis. METHODS The effects of LWB on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) and interleukin (IL)-6 secretion were assessed in a murine macrophage cell line. in vitro assessment also included characterizing the effects of LWB on the activation of NF-E2 related 2 pathway and inhibition of tumor necrosis factor-α (TNF-α)-induced nuclear factor-κB (NF-κB) activation, utilizing reporter cell lines. Following the in vitro assessment, the anti-inflammatory efficacy of an oral intervention with LWB was tested in vivo using a preclinical model of intestinal inflammation. Multiple outcomes including body weight, intestinal histology, colonic cytokine levels and anti-oxidative measures were investigated. RESULTS LWB reduced the LPS-mediated induction of ROS production [+LPS vs 1% LWB + LPS, 1590 ± 188.5 relative luminescence units (RLU) vs 389 ± 5.9 RLU, P < 0.001]. LWB was more effective than wolfberry alone in reducing LPS-induced IL-6 secretion in vitro (wolfberry vs 0.5% LWB, 15% ± 7.8% vs 64% ± 5%, P < 0.001). In addition, LWB increased reporter gene expression via the anti-oxidant response element activation (wolfberry vs LWB, 73% ± 6.9% vs 148% ± 28.3%, P < 0.001) and inhibited the TNF-α-induced activation of the NF-κB pathway (milk vs LWB, 10% ± 6.7% vs 35% ± 3.3%, P < 0.05). Furthermore, oral supplementation with LWB resulted in a reduction of macroscopic (-LWB vs +LWB, 5.39 ± 0.61 vs 3.66 ± 0.59, P = 0.0445) and histological scores (-LWB vs +LWB, 5.44 ± 0.32 vs 3.66 ± 0.59, P = 0.0087) in colitic mice. These effects were associated with a significant decrease in levels of inflammatory cytokines such as IL-1β (-LWB vs +LWB, 570 ± 245 μg/L vs 89 ± 38 μg/L, P = 0.0106), keratinocyte-derived chemokine/growth regulated protein-α (-LWB vs +LWB, 184 ± 49 μg/L vs 75 ± 20 μg/L, P = 0.0244), IL-6 (-LWB vs +LWB, 318 ± 99 μg/L vs 117 ± 18 μg/L, P = 0.0315) and other pro-inflammatory proteins such as cyclooxygenase-2 (-LWB vs +LWB, 0.95 ± 0.12 AU vs 0.36 ± 0.11 AU, P = 0.0036) and phosphorylated signal transducer and activator of transcription-3 (-LWB vs +LWB, 0.51 ± 0.15 AU vs 0.1 ± 0.04 AU, P = 0.057). Moreover, antioxidant biomarkers, including expression of gene encoding for the glutathione peroxidase, in the colon and the plasma anti-oxidant capacity were significantly increased by supplementation with LWB (-LWB vs +LWB, 1.2 ± 0.21 mmol/L vs 2.1 ± 0.19 mmol/L, P = 0.0095). CONCLUSION These results demonstrate the anti-inflammatory properties of LWB and suggest that the underlying mechanism is at least in part due to NF-κB inhibition and improved anti-oxidative capacity.

Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro

In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. Its necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect. To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance.

Integrating bioassays and analytical chemistry as an improved approach to support safety assessment of food contact materials

ABSTRACT Food contact materials (FCM) contain chemicals which can migrate into food and result in human exposure. Although it is mandatory to ensure that migration does not endanger human health, there is still no consensus on how to pragmatically assess the safety of FCM since traditional approaches would require extensive toxicological and analytical testing which are expensive and time consuming. Recently, the combination of bioassays, analytical chemistry and risk assessment has been promoted as a new paradigm to identify toxicologically relevant molecules and address safety issues. However, there has been debate on the actual value of bioassays in that framework. In the present work, a FCM anticipated to release the endocrine active chemical 4-nonyphenol (4NP) was used as a model. In a migration study, the leaching of 4NP was confirmed by LC-MS/MS and GC-MS. This was correlated with an increase in both estrogenic and anti-androgenic activities as measured with bioassays. A standard risk assessment indicated that according to the food intake scenario applied, the level of 4NP measured was lower, close or slightly above the acceptable daily intake. Altogether these results show that bioassays could reveal the presence of an endocrine active chemical in a real-case FCM migration study. The levels reported were relevant for safety assessment. In addition, this work also highlighted that bioactivity measured in migrate does not necessarily represent a safety issue. In conclusion, together with analytics, bioassays contribute to identify toxicologically relevant molecules leaching from FCM and enable improved safety assessment.