CCR2 deficiency boosts F-actin accumulation and BCR internalization
Actin remodeling has been shown to play a crucial role during the process of BCR clustering and formation of B cell morphological features.6 To examine the involvement of CCR2 in actin remodeling and polymerization, we assessed F-actin accumulation and the activation of actin nucleation promoting factor-WASP in CCR2 KO B cells after sAg stimulation using confocal microscopy (CFm). The F-actin levels observed in activated CCR2 deficient B cells were consistently higher than those of the control B cells (Fig. 4A-B ). The colocalization between F-actin and BCR was significantly increased after 5 min of stimulation (Fig. 4C ). Similarly, the pWASP levels in activated CCR2 deficient B cells remained higher than those in WT B cells (Fig. 4D ). Furthermore, after 5 min of stimulation, the colocalization of pWASP and BCR in CCR2 KO B cells was enhanced (Fig. 4E ). Additionally, phosflow was performed to measure the pWASP and F-actin levels which coincided with the CFm results. Examining the dynamics of actin polymerization, we found that the mean fluorescence intensity (MFI) of F-actin in CCR2 KO B cells increased slower during the first 5 min of stimulation, after which it accelerated rapidly and peaked at 10 min (Fig. 4F ). The MFI of pWASP raised rapidly during the first 5 min of stimulation and declined thereafter (Fig. 4G ). Further, the Mst1-Dock8-WASP axis protein expression levels were examined using western blotting. In the absence of CCR2, the levels of Dock8, pMst1, and pWASP were increased (Fig. 4H ). To get clearer image of the B cell membrane morphology, we utilized scanning electron microscopy (SEM) to observe B cell morphology and quantify their filopodia (Fig. 4I ). Compared to the WT B cells, the filopodia number and length were remarkably increased in CCR2 KO B cells (Fig. 4J-K ). Furthermore, in order to illustrate the latent mechanism through which CCR2 regulates the BCR signaling via the Mst1/Dock8/WASP axis, we pretreated CCR2 KO B cells with the Mst1 inhibitor, XMU-MP-1, and reexamined the BCR signaling molecules upon sAg stimulation. Remarkably, the levels of Dock8, pMst1, pSHIP, pBtk, pPI3K, pAKT, pFoxo-1, pmTOR, and pWASP in CCR2 KO B cells decreased following XMU-MP-1 treatment (Fig. 4L ). Subsequently, to verify whether the inhibition of mTORC1 in CCR2 KO B cells could also adjust the Dock8/WASP axis, we pretreated CCR2 KO B cells with rapamycin. After the inhibition, the levels of Dock8, pMst1, and pWASP in CCR2 KO B cells were rescued to a comparable degree to WT B cells (Fig. 4M ). Taken together, CCR2 deletion augments actin accumulation, which is achieved through the modulation of the Dock8-WASP activity, and the latter, at least in part, implemented via the Mst1-mTORC1 pathway.