Rafael P. Oliveira

Aging correlates with reduction in regulatory-type cytokines and T cells in the gut mucosa

Aging is reported to be associated with decline in oral tolerance induction, which is initiated at the intestinal mucosal surface. Herein, we examined the effect of aging in T cells and cytokines at the intestinal mucosa that might be involved in oral tolerance induction. Frequencies of regulatory-type IEL subsets such as TCRγδ(+) and TCRαβ(+)CD8αα(+) were lower in aged mice. Mucosal CD4(+)CD25(+)Foxp3(+) and CD4(+)LAP(+) T cells increased with aging but activated CD44(+)CD4(+) mucosal T cells also augmented. Production of TGF-β and IL-10 in the small intestine of old mice was reduced. Moreover, the ability of mucosal dendritic cells of aged mice to stimulate TGF-β secretion and differentiation of CD4(+)LAP(+) T cells in co-culture studies also declined with aging. Reduction in these regulatory-type cytokines and T cells may help to explain the decline in susceptibility to oral induction during aging. However, not all mucosal regulatory elements were altered by aging and CD4(+)CD25(+)Foxp3(+) T cells were especially resistant to changes. Persistence of some mechanisms of regulation may play a critical role in maintaining mucosal homeostasis during aging.

Identification and characterization of latency-associated peptide-expressing γδ T cells

γδ T cells are a subset of lymphocytes specialized in protecting the host against pathogens and tumours. Here we describe a subset of regulatory γδ T cells that express the latency-associated peptide (LAP), a membrane-bound TGF-β1. Thymic CD27+IFN-γ+CCR9+α4β7+TCRγδ+ cells migrate to the periphery, particularly to Peyers patches and small intestine lamina propria, where they upregulate LAP, downregulate IFN-γ via ATF-3 expression and acquire a regulatory phenotype. TCRγδ+LAP+ cells express antigen presentation molecules and function as antigen presenting cells that induce CD4+Foxp3+ regulatory T cells, although TCRγδ+LAP+ cells do not themselves express Foxp3. Identification of TCRγδ+LAP+ regulatory cells provides an avenue for understanding immune regulation and biologic processes linked to intestinal function and disease.

Antigen administration by continuous feeding enhances oral tolerance and leads to long-lasting effects.

The ability to avoid inflammatory responses to dietary components and microbiota antigens in the gut mucosa is achieved by a mechanism termed oral tolerance. This phenomenon is crucial to maintain the physiological immune activity in the gut and to prevent inflammatory disorders such as food allergy and inflammatory bowel diseases. Moreover, orally administered antigens induce regulatory cells that control systemic inflammatory responses as well. Given its specific, systemic and long-lasting effects, oral tolerance represents a promising approach for immunotherapies that aim to modulate inflammatory and autoimmune diseases. However, there are different protocols of feeding for induction of oral tolerance, and they have an impact in tolerance efficiency and length. Herein, we present and discuss different experimental feeding protocols and how they influence the outcome of oral administration of antigens.

Role of mesenteric lymph nodes and aging in secretory IgA production in mice.

Although it is known that Peyers patches are the major inductive site for S-IgA production and B1 cells contribute to half of the IgA plasma cells detected in the gut lamina propria, the type of contribution of mesenteric lymph nodes to the process is still unclear. Cytokines such as TGF-beta, IL-10, IL-4, IL-5, and IL-6, are required to promote IgA class switching and IgA synthesis. Aging-related alterations in T and B cells and in cytokine production are already known. Some reports have also proposed that S-IgA production might be altered in aged animals. Herein, we investigated the role of MLN and aging in S-IgA production. Two- to 18-month-old BALB/c mice were used to evaluate aging-related alterations and MLN were removed to study its role in S-IgA production. We found that MLN are important, although not essential for S-IgA production. In addition, we showed that production of IgA-related cytokines are well preserved in MLN but not in PP of aged mice and that S-IgA levels are not affected by aging. Our results suggest that MLN may play a complementary role in S-IgA production mostly in aged animals.

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells

BackgroundInflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn’s disease.ResultsThe murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid—pValac::dts::IL-4—was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA+ in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80+MHCII+Ly6C−IL-4+, F4/80+MHCII+Ly6C−IL-10+, F4/80+MHCII+Ly6C−CD206+CD124+IL-10+ and CD4+Foxp3+IL10+ cells compared to the other groups.ConclusionsThis study shows that L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels of IL-10-secreting regulatory cells and, thus, demonstrating the effectiveness of this novel DNA delivery-based strategy.

Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways

Heat shock proteins (Hsps) are highly expressed at all sites of inflammation. As they are ubiquitous and immunodominant antigens, these molecules represent good candidates for the therapeutic use of oral tolerance in autoimmune and chronic inflammatory diseases. Evidences from human and animal studies indicate that inflammatory bowel disease (IBD) results from uncontrolled inflammatory responses to intestinal microbiota. Hsps are immunodominant proteins expressed by several immune cells and by commensal bacteria. Using an IBD mouse model, we showed that oral pretreatment with genetically modified Lactococcus lactis that produces and releases Mycobacterium Hsp65, completely prevented DSS-induced colitis in C57BL/6 mice. Protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; increased IL-10 production in colonic tissue; and expansion of CD4+Foxp3+ and CD4+LAP+ regulatory T cells in spleen and mesenteric lymph nodes. This effect was dependent on IL-10 and toll-like receptor 2. Thus, this approach may open alternative options for long-term management of IBD.

Effect of a protein-free diet in the development of food allergy and oral tolerance in BALB/c mice

The aim of the present study was to investigate the effect of a protein-free diet in the induction of food allergy and oral tolerance in BALB/c mice. The experimental model used was mice that were fed, since weaning up to adulthood, a balanced diet in which all dietary proteins were replaced by amino acid diet (Aa). The absence of dietary proteins did not prevent the development of food allergy to ovalbumin (OVA) in these mice. However, Aa-fed mice produced lower levels of IgE, secretory IgA and cytokines. In addition, when compared with mice from control group, Aa-fed mice had a milder aversive reaction to the allergen measured by consumption of OVA-containing solution and weight loss during food allergy development. In addition, mice that did not have dietary proteins in their diets were less susceptible to induction of oral tolerance. One single oral administration was not enough to suppress specific serum Ig and IgG1 levels in the Aa-fed group, although it was efficient to induce suppression in the control group. The present results indicate that the stimulation by dietary proteins alters both inflammatory reactivity and regulatory immune reactivity in mice probably due to their effect in the maturation of the immune system.

Trichoderma asperelloides Spores Downregulate dectin1/2 and TLR2 Receptors of Mice Macrophages and Decrease Candida parapsilosis Phagocytosis Independent of the M1/M2 Polarization

The intensive use of pesticides to control pests in agriculture has promoted several issues relating to environment. As chemical pesticides remain controversial, biocontrol agents originating from fungi could be an alternative. Among them, we highlight biocontrol agents derived from the fungi genus Trichoderma, which have been documented in limiting the growth of other phytopathogenic fungus in the roots and leaves of several plant species. An important member of this genus is Trichoderma asperelloides, whose biocontrol agents have been used to promote plant growth while also treating soil diseases caused by microorganisms in both greenhouses and outdoor crops. To evaluate the safety of fungal biological agents for human health, tests to detect potentially adverse effects, such as allergenicity, toxicity, infectivity and pathogenicity, are crucial. In addition, identifying possible immunomodulating properties of fungal biocontrol agents merits further investigation. Thus, the aim of this study was to evaluate the effects of T. asperelloides spores in the internalization of Candida parapsilosis yeast by mice phagocytes, in order to elucidate the cellular and molecular mechanism of this interaction, as a model to understand possible in vivo effects of this fungus. For this, mice were exposed to a fungal spore suspension through-intraperitoneal injection, euthanized and cells from the peripheral blood and peritoneal cavity were collected for functional, quantitative and phenotypic analysis, throughout analysis of membrane receptors gene expression, phagocytosis ability and cells immunophenotyping M1 (CCR7 and CD86) and M2 (CCR2 and CD206). Our analyses showed that phagocytes exposed to fungal spores had reduced phagocytic capacity, as well as a decrease in the quantity of neutrophils and monocytes in the peripheral blood and peritoneal cavity. Moreover, macrophages exposed to T. asperelloides spores did not display the phenotypic profile M1/M2, and had reduced expression of pattern recognition receptors, such as TLR2, dectin-1 and dectin-2, all involved in the first line of defense against clinically important yeasts. Our data could infer that T. asperelloides spores may confer susceptibility to infection by C. parapsilosis.

CLA-supplemented diet accelerates experimental colorectal cancer by inducing TGF-β-producing macrophages and T cells

Conjugated linoleic acid (CLA) has been shown to activate the nuclear receptor PPAR-γ and modulate metabolic and immune functions. Despite the worldwide use of CLA dietary supplementation, strong scientific evidence for its proposed beneficial actions are missing. We found that CLA-supplemented diet reduced mucosal damage and inflammatory infiltrate in the dextran sodium sulfate (DSS)-induced colitis model. Conditional deletion of PPAR-γ in macrophages from mice supplemented with CLA diet resulted in loss of this protective effect of CLA, suggesting a PPAR-γ-dependent mechanism mediated by macrophages. However, CLA supplementation significantly worsened colorectal tumor formation induced by azoxymethane and DSS by inducing macrophage and T-cell-producing TGF-β via PPAR-γ activation. Accordingly, either macrophage-specific deletion of PPAR-γ or in vivo neutralization of latency-associated peptide (LAP, a membrane-bound TGF-β)-expressing cells abrogated the protumorigenic effect of CLA. Thus, the anti-inflammatory properties of CLA are associated with prevention of colitis but also with development of colorectal cancer.