Anne Donnet-Hughes

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.

Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

ABSTRACT Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.

The inflammatory status of the elderly: the intestinal contribution.

A common finding in the elderly population is a chronic subclinical inflammatory status that coexists with immune dysfunction. These interconnected processes are of sufficient magnitude to impact health and survival time. In this review we discuss the different signals that may stimulate the inflammatory process in the aging population as well as the molecular and cellular components that can participate in the initiation, the modulation or termination of the said process. A special interest has been devoted to the intestine as a source of signals that can amplify local and systemic inflammation. Sentinel cells in the splanchnic area are normally exposed to more than one stimulus at a given time. In the intestine of the elderly, endogenous molecules produced by the cellular aging process and stress as well as exogenous evolutionarily conserved molecules from bacteria, are integrated into a network of receptors and molecular signalling pathways that result in chronic inflammatory activation. It is thus possible that nutritional interventions which modify the intestinal ecology can diminish the pro-inflammatory effects of the microbiota and thereby reinforce the mucosal barrier or modulate the cellular activation pathways.

Osteoprotegerin in Human Milk: A Potential Role in the Regulation of Bone Metabolism and Immune Development

Osteoprotegerin (OPG) is a member of the tumor necrosis factor superfamily. It is a soluble “decoy” receptor for tumor necrosis factor–related apoptosis-inducing ligand and ligand of the receptor activator of NF-κB. As such, OPG inhibits osteoclast activity and regulates the immune system. Human milk is a complex biologic fluid that supplies nutritional and protective factors to the breast-fed infant. In the present study, human milk samples at various times postpartum were assessed for the presence of OPG. Using biochemical as well as immunologic and biologic techniques we showed that human milk contains OPG at a level that is 1000-fold higher than that found in normal human serum. We observed that human breast milk cells and the human mammary epithelial cell line MCF-7 express OPG, indicating that both cell types are possible sources of milk OPG in vivo. In vitro studies demonstrated that milk OPG is biologically active and suggested that it may contribute to the antiresorptive activity of milk on bone, as well as tumor necrosis factor–related apoptosis-inducing ligand–induced inhibition of T cell proliferation. OPG-like activity was also observed in bovine colostrum and milk. Furthermore, we were able to detect human OPG in the sera of rats gavaged with human milk. We discuss the relevance of our findings for the breast-fed infant and for the prevention of immune and bone disorders.

The intestinal mucosa as a target for dietary polyunsaturated fatty acids

Several studies have reported beneficial effects of dietary polyunsaturated fatty acids (PUFA) on various aspects of both human and animal health, and particular reference has been made to their effects on systemic immune responses. Both immune stimulation and immune suppression have been reported, with the outcome dependent on the type of PUFA, the target cell, as well as the immune competence of the cells before exposure. The systemic and the mucosal immune systems are discrete entities, which have evolved specific approaches in the defense of the host. The latter comprises several interconnected tissues, which communicate with one another through the action of soluble mediators and the trafficking of cellular components. After the oral mucosa, the intestinal epithelium and its associated gutassociated lymphoid tissue are the primary targets of dietary components. Absorption of dietary PUFA and its incorporation into intestinal tissues has been well studied, but the consequences of these events in relation to local immune responses have received little attention. This article describes some of the immune mechanisms operating at this barrier and, where possible, pinpoints areas for which a modulatory role for PUFA has already been demonstrated. Although not an exhaustive treatise of the subject, it is hoped that this review will foster research into the specific interaction between dietary PUFA and cell populations comprising the intestinal barrier.

Generation of Bioactive Peptides Derived from Caseins Using a Lactobacillus helveticus Strain

Bovine milk is known to contain a number of peptide fractions that can affect physiological functions. Bioactive peptides have to be hydrolytically derived from native proteins. Various biological effects of peptides generated upon in vitro enzymatic or in vivo gastrointestinal digests have been demonstrated [1].

Modulation of the MHC Class I and II Molecules by Bacterial Products on Intestinal Epithelial Cells

Communicating signals between different cells of the immune system involve both direct cell-cell interactions and soluble factors. In the intraepithelial compartment, enterocytes and lymphocytes are closely associated and can interact through both mechanisms. An example of such interactions is the ability of epithelial cells to present antigen to specific T lymphocytes in vitro.1,2 Class I and Class II major histocompatibility complex molecules displaying antigenic peptides are recognized by specific T cell subsets.3 MHC class II molecules on antigen presenting cells interact with CD4+ helper T cells while MHC class I molecules interact with CD8+ suppressor cytotoxic lymphocytes. Enterocytes constitutively express Class I and II molecules whose levels are modulated by environmental factors. Since epithelial cells of the gut are in permanent contact with food and bacterial products we examined the modulation of MHC molecules expression on these cells by bacterial products.

Microbiota, Probiotics and Natural Immunity of the Gut

The mammalian intestine is host to a rich microbiota whose composition is determined by the host’s diet and by the various physiological and immunological forces at play in the local microenvironment. In turn, the complex bacterial communities which form the microbiota bestow important nutritional, metabolic and protective functions. The precise mechanisms underlying this intimate relationship are slowly being uncovered and the knowledge gained is being used in the selection of probiotic microorganisms.

The Modulation of Class I and Class II MHC Molecules on Intestinal Epithelial Cells at Different Stages Of Differentiation

Two classes of effector T cells exist. The first is normally CD4+, has a TCR associated with CD3 and interacts with foreign peptides bound to MHC Class II antigens on antigen presenting cells. The second, is CD8+, has TCR-CD3 and interacts with foreign peptides associated with Class I MHC molecules on target cells. However, little is known of the interaction between these differenteffector cells and intestinal epithelial cells. For this reason, the regulation of MHC molecules by two T cell products, IFN-γ and IL-4, was examined. Cytokines such as the IFN-γ produced by Th1 cells, induce MHC Class II expression on intestinl epithelial cells (IEC) both “in vitro”1 and “in vivo”2 but the effect on MHC Class I, and on cells at different stages of differentiation has received essattention. IL-4, a product of Th2 cells, is known to up-regulate Class II expression on B cells and monocytes3 but its effect on that of IEC has not been studied. We, therefore, used FACScan analysis to compare the expression of MHC antigens on human intestinal epithelial cell lines and the subsequent effect of a 48hr exposure to IFN-γ or IL-4. Furthermore, since IEC “in vivo” are present at different stages of differentiation depending on their location within the intestinal villus, two epithelial cell lines, representing different maturation states were compared.