Lactococcus lactis subsp. cremoris C60 creates a type I regulatory T cell-dominant anti-inflammatory intestinal environment through functional modification of dendritic cells

Lactic acid bacteria (LAB)-mediated probiotics have the capability to modulate intestinal immunity, creating an anti-inflammatory environment. Among the immune-modulatory effects, interleukin (IL)-10-producing CD4 ⁺ T cells are often discussed for their anti-inflammatory roles. While regulatory T cells (Tregs) are a well-known subset, type I regulatory T (Tr1) cells, which also produce abundant IL-10, receive less attention in LAB-induced anti-inflammatory effects. Here, we report that Lactococcus lactis subsp. cremoris C60, a probiotic LAB strain, increases IL-10-producing Tr1 cells in the intestinal environment, contributing to resistance against inflammatory conditions such as dextran sodium sulfate (DSS)-induced colitis. Intragastric administration of heat-killed (HK)-C60 significantly increased IL-10 ⁺ CD4 ⁺ T cells in intestinal tissues compared to control treatment. These populations predominantly consisted of Forkhead box protein P3 (Foxp3)-negative cells, indicating that Tr1 cells were primarily expanded by HK-C60 stimulation. Further analysis revealed that IL-10 ⁺ Tr1 cells could be classified into two major subpopulations: IL-10 single-positive (SP) and IFN-γ/IL-10 double-positive (DP) cells. An in vitro co-culture system using CD4 ⁺ T cells and bone marrow-derived dendritic cells (BMDCs) demonstrated that HK-C60 stimulation modified dendritic cell function, promoting Tr1 cell differentiation. Moreover, pro-inflammatory IFN-γ ⁺ CD4 ⁺ T (Th1) cells differentiated into IL-10 ⁺ Tr1 cells in the presence of HK-C60-stimulated BMDCs, with the effect being particularly pronounced in DP Tr1 cells. HK-C60-administration attenuated inflammation and reduced pro-inflammatory leukocyte infiltration in the colon of DSS-induced colitis mice, accompanied by the expansion of DP Tr1 cells. The suppressive effect of intestinal CD4 ⁺ T cells in HK-C60-treated mice was IL-10-dependent, as demonstrated by an in vitro IL-10 neutralizing assay. Thus, C60-based probiotics promote the creation of an anti-inflammatory intestinal environment by increasing IL-10 ⁺ Tr1 cells rather than Tregs.