Oral combined therapy with probiotics and alloantigen induces B cell-dependent long-lasting specific tolerance.

Allogeneic hematopietic stem cell transplantation (aHSCT) is widely used for the treatment of hematologic malignancies. Although aHSCT provides a good response against the malignant cells (graft-versus-leukemia [GVL]), it also leads to the development of graft-versus-host disease (GVHD), a severe disease with high mortality and morbidity rates. Therapy for GVHD is commonly based on nonspecific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. In this study, we propose an alternative approach to specifically suppress GVHD while sparing the GVL, based on oral treatment of transplant donors with recipient Ags, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined therapy). We show that treatment of C57BL/6 donor mice with combined therapy before the transplant protects the recipients F1 (C57BL/6 × BAL/c) mice from clinical and pathological manifestations of disease, resulting in 100% survival rate. Importantly, the animals keep the immunological competence maintaining the GVL response as well as the response to third-party Ags. The protection is specific, long lasting and dependent on donor IL-10-sufficient B cells activity, which induces regulatory T cells in the host. These data suggest that combined therapy is a promising strategy for prevention of GVHD with preservation of GVL, opening new possibilities to treat human patients subjected to transplantation.

Previous Ingestion of Lactococcus lactis by Ethanol-Treated Mice Preserves Antigen Presentation Hierarchy in the Gut and Oral Tolerance Susceptibility.

BACKGROUND: Ethanol (EtOH) consumption is able to disturb the ovalbumin (OVA)-oral tolerance induction by interfering on the function of antigen presenting cells (APC), down-regulating dendritic cells (DCs) and macrophages and up-regulating B-lymphocytes and their function, which results in an overall allergic-type immune status. In this study, the potential of a priori administration of Lactococcus lactis (LL) in avoiding loss of oral tolerance in EtOH-treated mice was investigated. METHODS: Female C57BL/6 mice received, by oral route, ad libitum wild-type (WT) LL or heat-shock protein producer (Hsp65) LL for 4 consecutive days. Seven days later, mice were submitted to short-term high-dose EtOH treatment. After 24 hours, stomach, intestine, spleen, mesenteric lymph nodes (mLN) specimens were collected for biomarkers analysis. Following EtOH-treatment protocol, a group of animals underwent single-gavage OVA-tolerance protocol and sera samples collected for antibody analysis. RESULTS: The ingestion of WT LL or Hsp65 LL is able to restore oral tolerance to OVA in EtOH-treated mice, by reducing local and systemic allergic outcomes such as gastric mast cells and gut-interleukin-4, as well as serum IgE. WT LL treatment prevents the decrease of mLN regulatory T cells induced by the EtOH treatment. Moreover, LL treatment preserves APC hierarchy and antigen presentation commitment in EtOH-treated mice, with conserved DC and macrophage activity over B lymphocytes in mLN and preserved macrophage activity over DC and B-cell subsets in the spleen. CONCLUSIONS: The present findings suggest that a priori ingestion of LL preserves essential mechanisms associated with oral tolerance induction that are disturbed by EtOH ingestion. Maintenance of mucosal homeostasis by preserving APC hierarchy and antigen presentation commitment could be associated with T-regulatory subset activities in the gastrointestinal tract.

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-ß - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.

Antigenic dietary protein guides maturation of the host immune system promoting resistance to Leishmania major infection in C57BL/6 mice.

The immature immune system requires constant stimulation by foreign antigens during the early stages of life to develop properly and to create efficient immune responses against later infections. We have previously shown that intake of antigenic dietary protein is critical for inducing maturation of the immune system as well as for the development of T helper type 1 (Th1) immunity. In this study, we show that administration of an amino acid (aa)-based diet during the development of the immune system subsequently resulted in inefficient control of Leishmania major infection in adult C57BL/6 mice. Compared with mice fed a control protein-containing diet, adult aa-fed mice showed a decreased interferon (IFN)-gamma response to parasite antigens and insufficient production of nitric oxide (NO), which is crucial to parasite death. However, no deviation towards Th2-specific immunity to L. major was observed. Phenotypic analysis of antigen-presenting cells (APCs) from aa-fed mice revealed deficient levels of the costimulatory molecules CD40 and CD80, and low levels of interleukin (IL)-12 produced by peritoneal macrophages, revealing an early stage of maturation of these cells. APCs isolated from aa-fed mice were unable to stimulate a Th1 response in vitro. Both phenotypic features of T cells from aa-fed mice and their ability to produce a Th1 response in the presence of mature APCs were unaffected when compared with T cells from control mice. The results presented here support the notion that regulation of Th1 immunity to infection includes environmental factors such as dietary proteins, which provide a natural source of stimulation that contributes to the process of maturation of APCs.

Milk fermented with a 15-lipoxygenase-1-producing Lactococcus lactis alleviates symptoms of colitis in a murine model.

Inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the administration of milks fermented by a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a trinitrobenzenesulfonic acid-induced IBD mouse model. The results obtained demonstrated that 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model. The work also confirmed previous studies showing that fermented milk is an effective form of administration of recombinant lactic acid bacteria expressing beneficial molecules.

Oral administration of sodium butyrate attenuates inflammation and mucosal lesion in experimental acute ulcerative colitis.

Butyrate is a four-carbon short-chain fatty acid that improves colonic trophism. Although several studies have shown the benefits of butyrate enemas in ulcerative colitis (UC), studies using the oral route are rare in the literature. In the present study, we evaluated the effect of butyrate intake in the immune response associated to UC. For that, mice were fed control or butyrate (0.5% sodium butyrate) diets for 14 days. Acute UC was induced by dextran sulphate sodium (DSS, 2.5%), replacing drinking water. The results showed that, in UC animals, oral butyrate significantly improved trophism and reduced leukocyte (eosinophil and neutrophil) infiltration in the colon mucosa and improved the inflammatory profile (activated macrophage, B and T lymphocytes) in cecal lymph nodes. In the small intestine, although mucosa histology was similar among groups, DSS treatment reduced duodenal transforming growth factor-ß, increased interleukin-10 concentrations and increased memory T lymphocytes and dendritic cells in Peyer's patches. Butyrate supplementation was able to revert these alterations. When cecal butyrate concentration was analyzed in cecal content, it was still higher in the healthy animals receiving butyrate than in the UC+butyrate and control groups. In conclusion, our results show that oral administration of sodium butyrate improves mucosa lesion and attenuates the inflammatory profile of intestinal mucosa, local draining lymph nodes and Peyer's patches of DSS-induced UC. Our results also highlight the potential use of butyrate supplements as adjuvant in UC treatment.