RESUMEN
Oral tolerance is the physiological process that enables the immune system to differentiate between harmless dietary and microbiota antigens from pathogen derived antigens. It develops at the mucosal surfaces and can result in local and systemic regulatory and anti-inflammatory effects. Translation of these benefits to the clinical practice faces limitations involving specificity and doses of antigen as well as regimens of feeding. To circumvent these problems, we developed a recombinant Hsp65 delivered by the acid lactic bacteria Lactococcus lactis NCDO 2118 directy in the intestinal mucosa. Hsp65 is a ubiquitous protein overexpressed in inflamed tissues and capable of inducing immunoregulatory mechanisms. L. lactis has probiotic properties and is commonly and safely used in dairy products. In this study, we showed that continuous delivery of HSP65 in the gut mucosa by L. lactis is a potent tolerogenic stimulus inducing regulatory CD4+LAP+ T cells that prevented collagen-induced and methylated bovine serum albumin-induced arthritis in mice. Clinical and histological signs of arthritis were inhibited as well as levels of inflammatory cytokines such as IL-17 and IFN-γ, serum titers of anti-collagen antibodies and rheumatoid factor. Oral administration of L. lactis induced alterations in microbiota composition toward an increased abundance of anaerobic bacteria such as Bifidobacterium and Lactobacillus. Tolerance to HSP65 and arthritis prevention induced by the recombinant L. lactis was associated with increase in IL-10 production by B cells and it was dependent on LAP+ T cells, IL-10 and TLR2 signaling. Therefore, HSP65-producing treatment induced effective tolerance and prevented arthritis development suggesting it can be used as a therapeutic tool for autoimmune diseases.
Asunto(s)
Artritis/inducido químicamente , Artritis/prevención & control , Proteínas Bacterianas/metabolismo , Colágeno/efectos adversos , Proteínas de Choque Térmico/metabolismo , Lactococcus lactis/metabolismo , Albúmina Sérica Bovina/efectos adversos , Administración Oral , Animales , Artritis/inmunología , Enfermedades Autoinmunes/prevención & control , Proteínas Bacterianas/genética , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Microbioma Gastrointestinal , Proteínas de Choque Térmico/genética , Tolerancia Inmunológica , Mucosa Intestinal/inmunología , Lactococcus lactis/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Probióticos/administración & dosificación , Proteínas Recombinantes/metabolismoRESUMEN
IFN-γ is a pleotropic cytokine produced in the bone microenvironment. Although IFN-γ is known to play a critical role on bone remodeling, its function is not fully elucidated. Consistently, outcomes on the effects of IFN-γ recombinant protein on bone loss are contradictory among reports. In our work we explored, for the first time, the role of IFN-γ encoding plasmid (pIFN-γ) in a mouse model of osteopenia induced by ovariectomy and in the sham-operated counterpart to estimate its effects in skeletal homeostasis. Ovariectomy produced a dramatic decrease of bone mineral density (BMD). pINF-γ injected mice showed a pathologic bone and bone marrow phenotype; the disrupted cortical and trabecular bone microarchitecture was accompanied by an increased release of pro-inflammatory cytokine by bone marrow cells. Moreover, mesenchymal stem cells' (MSCs) commitment to osteoblast was found impaired, as evidenced by the decline of osterix-positive (Osx+) cells within the mid-diaphyseal area of femurs. For instance, a reduction and redistribution of CXCL12 cells have been found, in accordance with bone marrow morphological alterations. As similar effects were observed both in sham-operated and in ovariectomized mice, our studies proved that an increased IFN-γ synthesis in bone marrow might be sufficient to induce inflammatory and catabolic responses even in the absence of pathologic predisposing substrates. In addition, the obtained data might raise questions about pIFN-γ's safety when it is used as vaccine adjuvant.