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-π not IgG in the supernatants
decreased significantly (Fig. 5C), which indicated that BAFF mainly
promoted the secretion of TNF-π, the IgG secretion was probably
compensated by other signaling pathway. Furthermore, the PMN-MDSCs
mediated increased
TNF-π
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-π
expression of B cell throughBTK/N F-πB
signaling pathway.
Brutonβs 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-πB
signaling is known as a downstream target of BTK(17). Activation of
canonical NF-πB 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-πB 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-π
through BTK/NF-πB pathway. We found that TNF-π+ B
cells increased in the coculture assay after adding BAFF 3 days later
(Fig. 6A). And BAFF enhanced the phosphorylation level of
BTK,
IπBπ and p65 after stimulating with BAFF for 5min, 30min and 60min (Fig.
6B). BTK inhibitor Ibrutinib dampened TNF-π increase in a dose dependent
manner (Fig. 6C). Whatβs more, the phosphorylation of BTK, IπBπ and p65
was also inhibited by Ibrutinib (Fig. 6D). Thus, these results
demonstrated that BAFF enhanced
TNF-π
expression of B cells through
BTK/NF-πB
signaling pathway.