Lactococcus lactis-HSP65 requires TLR2 to induce tolerogenic dendritic cells and confer protection against type 1 diabetes in a murine model

Type 1 Diabetes (T1D) is characterized by the destruction of insulin-producing β cells, leading to hyperglycemia. New therapeutic strategies are crucial for preventing or managing the disease and its complications. Natural or recombinant probiotics, by modulating the gut microbiota and dictating the immunological tolerance, have been recently employed as tolerogenic immunotherapy for autoimmune diseases. Heat shock protein 65 (HSP65) exhibits regulatory and anti-inflammatory activity in autoimmune and chronic inflammatory conditions. Here, we show that dendritic cells from T1D patients exhibit reduced expression of immunological tolerance-associated genes and diminished TLR2 expression. Recombinant HSP65 (rHSP65) upregulated Tlr2 expression and promoted the differentiation of classical dendritic cell subsets-cDC1 and cDC2-capable of inducing regulatory T cells (Tregs) in vitro. Mice administered L. lactis-HSP65 became resistant to T1D, proved by reduced hyperglycemia and lower disease incidence compared to diabetic group that did not receive probiotic. These mice exhibited increased TLR2⁺ cDC1 and PD-1⁺ Treg cells in the cecum-associated mesenteric lymph node. Furthermore, L. lactis-rHSP65 increased Treg population, IL-10, and TGF-β production in the pancreas. In TLR2-deficient mice, the tolerogenic effects of L. lactis-HSP65 were not observed. TLR2 and IL-10-deficient DCs cannot drive the Treg differentiation in vitro. Overall, the results indicate that L. lactis-HSP65 protects against T1D onset by immunoregulatory effects mediated by tolerogenic cDC1 through a mechanism involving TLR2 and IL-10.