B CELL-INTRINSIC TOLL-LIKE RECEPTOR 7 SIGNALING PROMOTES TERTIARY LYMPHOID STRUCTURES FORMATION IN INJURED KIDNEY

Elderly patients are prone to progress from acute kidney injury to chronic kidney disease. We previously demonstrated that tertiary lymphoid structures (TLSs), organized ectopic lymphoid aggregates, form in aged, but not young, kidneys, with tissue maladaptive repair. In addition to aging, TLSs are observed in various kidney diseases and is associated with disease severity and poor renal prognosis. We also found that the interactions between CD153+PD-1+CD4+ senescence-associated T (SA-T) cells and CD30+T-bet+ age-associated B cells (ABCs) drive TLS expansion and exacerbate kidney dysfunction. Both lymphocytes are induced not only by aging but also by toll-like receptor 7 (TLR7) stimulation. However, how TLR7 signaling contributes to TLS formation in injured kidneys remains unclear. Therefore, we investigated the role of TLR7 in TLS formation, focusing on the underlying cellular and molecular mechanisms.

To assess TLR7-expressing cell types, we conducted in situ hybridization and flow cytometry (FCM) of immune cells in aged injured kidney with TLSs and spleens after renal ischemia-reperfusion injury (IRI). TLS size, maturation stage, gene expressions of TLS-related cytokines/chemokines, and frequencies of ABCs and SA-T cells were evaluated by PAS staining, immunostaining, qPCR, and FCM in kidney injury models of the following mice: (1) aged TLR7 knockout (KO) mice, (2) aged C57BL/6J (B6J) mice treated with TLR7 inhibitors, (3) young B6J mice treated with TLR7 agonists, and (4) B cell-specific TLR7 KO mice generated by bone marrow transplantation. Furthermore, TLR7 expression in B cells and their capacity to proliferate and differentiate into ABCs were compared between young and aged mice.

TLR7 was expressed in B cells, macrophages, and dendritic cells in aged injured kidneys with TLSs and in the spleens. In the kidney of aged TLR7 KO mice and TLR7 inhibitor-treated B6J mice post-IRI, TLS expansion and maturation were suppressed, and frequencies of ABCs and SA-T cells were reduced, suggesting the pivotal role of TLR7 signaling in TLS formation. In young B6J mice post-IRI, the administration of TLR7 agonists induced the formation of advanced-stage TLSs, with upregulated Tnfrsf8 (encoding CD30), Tnfsf8 (encoding CD153), Ifng, Cxcl13, and Ccl19 expressions and increased frequencies of ABCs and SA-T cells in the kidney, recapitulating the changes observed in aged injured kidneys with TLSs. On the other hand, in young mice with B cell-specific TLR7 deficiency, TLR7 agonists induced only small immature TLSs, indicating that TLR7 in B cells was essential for TLS expansion.

We demonstrated that TLR7 in B cells is essential for TLS formation and maturation. Genetic deletion or pharmacological inhibition of TLR7 suppressed TLS formation in aged mice, whereas the administration of TLR7 agonists to young mice recapitulated renal TLS formation in aged mice. Furthermore, B cell-specific deletion of TLR7 suppressed TLS formation in young mice treated with agonists, suggesting that increased TLR7 signaling plays a pivotal role in renal TLS formation. Our findings will facilitate further research to elucidate mechanisms underlying TLS formation in various kidney diseases.