Discussion
In this study, we dissected the influence of CCR2 deficiency on BCR signaling pathways by using germinal CCR2-deletion mice. This is the first study focusing on the correlation between CCR2 and BCR signaling, and the first to systematically illustrate the sequential involvement of three typical signaling pathways regulated by CCR2 which ultimately influence BCR signaling. We discovered that CCR2 participates in a series of biological signal changes following B cell activation and its absence guides the up-regulation of key BCR signaling molecules, which were associated with disturbed FO B cell differentiation, enhanced early event BCR activation, and compromised B cell function. Furthermore, we showed that these effects were mediated by CCR2 through the synergy of the Mst1/mTORC1/STAT1 signaling pathways.
Mst1, mTOR, STAT1, and their associated pathways have been extensively studied due to their general involvement in various physiological processes. Separately, they are likely responsible for the occurrence and development of autoimmunity. Defective Mst1/Foxo-1 signaling results in the collapse of immune tolerance,17 and activation of mTORC1 represent one of the major molecules responsible for the SLE pathogenesis and other autoimmune diseases caused by oxidative stress.33Additionally, in neuro-autoimmunity, STAT1 phosphorylation and CCR2 expression co-clustered with CD8+ T cells.34 In the present study, during B cells differentiation, PI3K/Akt pathway enhanced the activity of mTORC1 which promoted B cell energy metabolism; which also bring about attenuation of FO B cells in CCR2 KO mice. Alternatively, CCR2 deficiency induced Mst1 up-regulation, which promoted F-actin accumulation and the interaction of BCR with other signaling molecules via the mTORC1/Dock8/WASP axis. Moreover, by taking advantage of multiple downstream transcription factors of the JAK/STAT pathway, Mst1 and mTORC1 modulated STAT1 to control BCR signaling. Mechanistically, the pSTAT1 and pNF-kB expression levels were corrected following treatment with Mst1 and mTOR inhibitors, which indicates a feedback loop in the CCR2-regulated BCR signaling pathway. Taken together, these findings supported the notion that CCR2 regulates B cell signaling via the Mst1-mTORC1-STAT1 axis.
Based on a different study investigating the interaction between CCL2 and B cell signaling (unpublished data), there are some worth noting differences between CCL2 and CCR2 deficient mice. Amongst the similarities between CCL2 and CCR2 deficient mice, are the up-regulated BCR signaling, increased F-actin accumulation, decreased ASCs, and attenuated antibody production. However, there are significant differences in immune system characteristics. First of all, considering the peripheral B cell differentiation, CCL2 deficiency leads to a reduction in MZ B cells and increase in GC B cells as well as formation of spontaneous germinal centers (Spt-GCs), while CCR2 deficiency leads to fewer FO B cells and has no obvious impact on MZ or GC B cells. An explanation for this discrepancy may be that CCR2 is expressed on non-GC B cells while absent on GC B cells.35 Furthermore, the distinctions between CCL2 and CCR2 deficiencies might also be explained by the different stages during B cell development at which each marker is expressed. Another difference lies within the downstream activities mediated by the Mst1/mTORC1/STAT1 axis, among which the activity of STAT5 is noteworthy. The absence of CCL2 positively regulated the STAT1 and STAT5 expression levels, while CCR2 depletion only enhanced STAT1 expression levels. This might indicate that CCR2 exerts its autoimmune-inducing effects by targeting STAT1 rather than STAT5. Nevertheless, CCL2 and CCR2 might have complementary roles in the BCR downstream Mst1/mTORC1/STAT1 signaling pathway. Last but not least, their impact on B cell function also differs. Upon T cell dependent immunization, splenic lymphatic follicles of immunized CCR2 KO mice were augmented and enlarged, while that of CCL2 KO mice were atrophied and diminished, which further supports the differences in B subsets seen increased during B cell peripheral differentiation. As was the case for CCL2 deficiency, the reduction of ASCs and antibody production might be attributed to the underlying physiological thresholds required for triggering the appropriate immune responses.
Since Mst1, mTOR, and STAT1 have been linked to the pathogenesis of SLE autoimmune disorders, targeting these pathways may allow simultaneous suppression of multiple cytokines. CCR2 can directly participate in the pathogenesis of SLE by means of cooperation with its ligands. In this study, the increased level of anti-dsDNA and T-bet observed in CCR2 KO mice would definitely aggravate the autoimmune progression of SLE. Thus, the existence of a signalling feedback loop in the CCR2-regulated BCR signaling pathway is highly probable and it is worth investigating it since it might prove to be a candidate therapeutic target for autoimmune diseases. Moreover, the correction of signaling expression levels following the targeted treatment with specific inhibitors in CCR2 deficient mice further supports this hypothesis. In conclusion, our findings highlight the dual role of the CCR2-regulated BCR signaling pathway. On the one hand, as proposed elsewhere, BCR signaling pathways are strongly controlled by the synergistic effects of various signaling axes, in which our study complements the bridging role of CCR2. On the other hand, as previously suggested, biological signals remain within an immune homeostasis maintenance range and either CCR2 or CCL2 abnormalities can trigger to the development of autoimmune disorders, albeit with varied phenotypes or target molecules.