CCR2 depletion triggers the activation of STAT1 to enhance BCR signaling
CCR2 is associated with the activity of the JAK/STAT pathway and its downstream transcriptional factors.21 Furthermore, the inhibition on the CCL2-CCR2 axis was shown to affect the activity of NF-κB.30 To ascertain the effect of CCR2 KO on the transcriptional factors downstream of JAK/STAT pathway, we determined the activation of STAT1, NF-κB, and STAT5 in B cells using CFm and western blotting. Upon sAg stimulation, the colocalization between pSTAT1 and BCR in CCR2 deficient B cells significantly increased within 10 min after stimulation (Fig. 6A-C ), which was in contrast to the observations for WT B cells. For pNF-κB, its colocalization with BCR was notably higher in CCR2 deficient B cells at 5 and 30 min post-stimulation (Fig. 6D-F ). Lastly, we observed increased colocalization of pSTAT5 and BCR at 5 min after stimulation in CCR2 depleted B cells (Fig. 6G-I ). While the pSTAT1, pNF-κB, and pIKKB protein expression levels increased in CCR2 KO B cells upon sAg stimulation, there was no difference in pSTAT5 protein expression (Fig. 6J ).
Previous studies showed that Mst1 plays a role in cell proliferation by influencing the AKT/mTOR signaling pathway,31 and the JAK/STAT downstream transcriptional factor activation.32 To identify the possible sequential effect of Mst1-mTORC1-STAT1 on BCR signaling, we pretreated CCR2 KO B cells with the STAT1 inhibitor, fludarabine, prior to sAg stimulation. Following the fludarabine treatment, the levels of pCD19, pSHIP, pAkt, pS6, Dock8, pNF-kB, pIKKB, and pMst1 were corrected towards the expression levels observed in WT B cells (Fig. 6K ). Similarly, following pretreatment with XMU-MP-1 (Fig. 6L) or rapamycin (Fig. 6M) , the levels of pSTAT1 and pNF-kB in CCR2 KO B cells were rescued to levels comparable to WT B cells, with the exception of pSTAT5 levels, which displayed little to no changes before and after the rapamycin treatment. This exception may reflect that STAT5 is not particularly affected by Mst1 and mTORC1 suppression. Altogether, by connecting the upstream and downstream signaling pathways, CCR2 might regulate BCR signaling via the Mst1-mTORC1-STAT1 pathway.