Extracellular Inosine Induces Anergy in B Cells to Alleviate Autoimmune Hepatitis

Dysregulation of naïve B cell receptor (BCR) signaling and the generation of antibody-secreting B cells (ASCs) have been implicated in the development of autoimmune diseases. Anergic B cells (B_NDs) are naïve B cells with a low-density of surface IgM-BCR, thus demonstrating attenuated autoantigen responsiveness. However, potential regulatory mechanisms of B cell anergy and their roles in autoimmune hepatitis (AIH) remain unestablished.

The frequency of circulating B cell subsets and comparative phenotypic analyses were conducted using flow cytometry. Primary human CD19⁺ B cells were differentiated in vitro with inosine or specific inhibitors, followed by quantitative polymerase chain reaction (qPCR), Western blotting, and flow cytometry analyses. The effects of inosine were evaluated in a concanavalin A-induced AIH mouse model, and a specific equilibrative nucleoside transporter 1 (ENT1) inhibitor was utilized both in vitro and in vivo.

An elevated frequency of ASCs but a diminish of B_NDs were observed in AIH. B_NDs showed attenuated activated status compared with C_NBs. B_NDs uniquely exhibited high-level expression of CD73, the rate-limiting enzyme in purinergic metabolism. Inosine, as the end-product of the extracellular purinergic pathway, significantly enhanced B_NDs expansion and inhibited ASCs differentiation in vitro. Mechanically, extracellular inosine was taken up via ENT1, promoting surface IgM internalization by inhibiting the PARP14-STAT6 signaling pathway. Pharmacological inhibition of ENT1 with dipyridamole reversed therapeutic effects of inosine both in vitro and in vivo.

Our findings revealed that inosine was a crucial metabolite that induced immune tolerance of B cells, thus proposing a potential intervention strategy for AIH.