Lactococcus lactis expressing HSP65 protects against the development of type 1 diabetes through the induction of tolerogenic dendritic cells via TLR2

Author(s)

Jefferson Elias Oliveira

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-04-30

Examining board members

Daniela Carlos Sartori; Maria Cristina Foss de Freitas; Luiz Osório Silveira Leiria

• Advisor: Daniela Carlos Sartori

Abstract

Type 1 Diabetes mellitus is characterized by the destruction of insulin-producing beta cells, resulting in hyperglycemia. New therapeutic strategies are important to control the disease and prevent/mitigate associated complications. In recent years, the use of natural or recombinant probiotics has shown beneficial effects in autoimmune diseases, mainly by modulating the intestinal microbiota, inducing tolerance or immunoregulatory mechanisms. It has been shown that heat shock protein 65 (HSP65), a molecular chaperone that maintains the structure and function of proteins, has anti-inflammatory activity in autoimmune and chronic inflammatory diseases. In this sense, the objective of the work was to evaluate the prophylactic-therapeutic effect of Lactococcus lactis expressing HSP65 during the development of DM1 in an experimental model. First, we observed that recombinant HSP65 induces the differentiation of two subtypes of conventional dendritic cells in vitro, cDC1 and cDC2, with distinct activation profiles. Bone marrow-derived dendritic cells (BMDCs) stimulated with rHSP65 produced more IL-10 compared to those stimulated with LPS. Furthermore, rHSP65-stimulated CD103+ BMDCs co-cultured with naïve CD4+ T cells induced differentiation of regulatory T cells (Tregs). Subsequently, we used a prophylactic-therapeutic model involving the administration of L. lactis expressing HSP65 in mice with streptozotocin (STZ)-induced DM1 and in non-obese diabetic mice (NOD), which develop spontaneous DM1. In both models, we observed that administration of L. lactis-HSP65 reduced hyperglycemia and disease incidence. In the STZ induction model, we observed an increase in insulin expression in pancreatic islets and a decrease in serum levels of IgG anti-HSP65 compared to diabetic mice that did not receive probiotics. Furthermore, L. lactis increased the expression of genes associated with intestinal barrier function in the colon, such as Reg3g and Tjp1. We observed an increase in the populations of cDC1 TLR2+ and PD-1 + Tregs in the cecal lymph node (LC) and LAP+ Tregs in the pancreatic lymph nodes (LP), associated with increased IL-10 and TGF-β in the pancreas of mice that received L. lactis-HSP65. On the other hand, the administration of L. lactis-HSP65 in TLR2-deficient mice was not able to reduce the incidence of the disease and control hyperglycemia as observed in wild-type mice. These data were associated with lower percentages of cDC1 and Tregs and reduced IL-10 production in the pancreas. In BMDCs, we observed that TLR2 is essential for the gene expression of Irf8 and Il-10, and also for the generation of cDC1 and IL-10 production. Finally, we observed that TLR2- or IL-10-deficient CD103+ BMDCs stimulated with rHSP65 and co-cultured with naïve CD4+ T cells induced a lower percentage of Tregs when compared to BMDCs from wild-type mice. In summary, the results demonstrate that L. lactis-HSP65 induced protection against T1D through immunoregulation mediated by regulatory T lymphocytes and tolerogenic dendritic cells through a TLR2/IL-10-dependent mechanism. (AU)

• FAPESP's process: 22/02762-4 - Effect of the administration of Lactococcus lactis expressing HSP65 on the induction of tolerance mediated by regulatory T cells and on the immunoregulation of DM1 in an experimental model.
• Grantee: Jefferson Elias Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate