B10 cells promote pro-resolving macrophage function through direct cell-cell contact and IL-10 secretion in Raw 264.7 cells

It is well known that regulatory B cells (Breg), especially IL-10-producing regulatory cells (B10), play an important role in immune regulation during inflammatory and infectious diseases. Although it has been revealed that the immune regulatory function of B10 can be exerted through cognate cell-cell contact with T cells, more research is needed to delineate its impact on other key cellular immune components within the immune microenvironment. In this study, we evaluated the effect of B10 on the phenotypic change of macrophages and their pro-resolving functional activities using various co-culture systems. The roles of cell-cell contact and the IL-10 secretion by B10 on macrophage differentiation and function were determined. Splenocyte-derived B10 cells from wild-type or IL-10 knockout (KO) mice were co-cultured with RAW 264.7 cells in the presence or absence of trans-well inserts. Macrophage polarization, programmed cell death 1 (PD-1) expression, production of specialized pro-resolving mediators (SPMs), and phagocytic activity were evaluated. The results showed that direct B10-macrophage co-culture enhanced the macrophage polarization towards a pro-resolving phenotype and their PD-1 expression, which was diminished when the cultured B10 and macrophages were separated by trans-well inserts, or when B cells from IL-10 KO mice were used for the co-culture. In addition, B10 was found to promote the release of specific SPM [resolvin D series 5 (RvD5)] and phagocytic activity by macrophages after co-culture. These effects were compromised in trans-well co-culture or co-cultures with IL-10-deficient B cells. Our results suggest that B10 promotes pro-resolving macrophage differentiation and function through direct cell-cell contact and IL-10 secretion.