PMN-MDSCs modulated B cells via BAFF
We found that PMN-MDSCs from CIA mice had a stronger ability to support B cells than those from DBA/1J mice. To verify the differences in PMN-MDSCs from DBA/1J and CIA mice, we isolated PMN-MDSCs from the spleens of the mice, and PMN-MDSCs were assayed by RNA-Seq. The heatmap of differentially-expressed genes associated with rheumatoid arthritis were shown, revealing thatTnfsf13b (Baff ) was highly expressed in PMN-MDSC from CIA mice (Fig. 5A). BAFF belongs to the TNF family and plays a vital role in the survival of B-lymphocytes (15). We confirmed that PMN-MDSCs from CIA mice expressed higher levels of BAFF than those from DBA/1J mice (Fig. 5B). To confirm that PMN-MDSC-derived BAFF supports B cells, we added an anti-BAFF antibody to block the function of BAFF. After neutralizing the BAFF in the coculture system, the concentration of TNF-𝛂 not IgG in the supernatants decreased significantly (Fig. 5C), which indicated that BAFF mainly promoted the secretion of TNF-𝛂, the IgG secretion was probably compensated by other signaling pathway. Furthermore, the PMN-MDSCs mediated increased TNF-𝛂 expression and proliferation in B cells weakened (Fig. 5D, 5E), and the decreased apoptosis was also counteracted by anti-BAFF antibody in vitro (Fig. 5F, 5G).
BAFF stimulatedTNF-𝛂 expression of B cell throughBTK/N F-𝛋B signaling pathway.
Bruton’s tyrosine kinase (BTK) belongs to the TEC tyrosine kinase family and plays a major role in B-cell activation, proliferation, maturation, and differentiation(16). NF-𝛋B signaling is known as a downstream target of BTK(17). Activation of canonical NF-𝛋B leads to the expression of proinflammatory cytokines(18). BAFF plays essential role in activation and survival of B cells(19-23), mainly through activating NF-𝛋B pathway(24), however, less is known about its role in the induction of cytokine production of B cells. Therefore, we were here to explore whether BAFF stimulate TNF-𝛂 through BTK/NF-𝛋B pathway. We found that TNF-𝛂+ B cells increased in the coculture assay after adding BAFF 3 days later (Fig. 6A). And BAFF enhanced the phosphorylation level of BTK, I𝛋B𝛂 and p65 after stimulating with BAFF for 5min, 30min and 60min (Fig. 6B). BTK inhibitor Ibrutinib dampened TNF-𝛂 increase in a dose dependent manner (Fig. 6C). What’s more, the phosphorylation of BTK, I𝛋B𝛂 and p65 was also inhibited by Ibrutinib (Fig. 6D). Thus, these results demonstrated that BAFF enhanced TNF-𝛂 expression of B cells through BTK/NF-𝛋B signaling pathway.