Introduction
B cells are derived from bone marrow (BM) and travel to peripheral lymphoid organs to fight pathogens.1 Following B cell receptor (BCR) activation, sequential signaling pathways involving adaptors, kinases, second messengers, and phosphatases produce a variety of B cell responses.2-4Actin cytoskeleton is a major target of BCR signaling that can regulate B cell morphological features, immune synapse formation, and BCR internalization.5, 6
As a member of the chemokine family, the contribution of chemokine (C-C motif) receptor 2 (CCR2) in autoimmunity has been previously demonstrated. A previous study suggested that CCR2 expressed on regulatory T cells (Tregs) can restrain the proliferation of T cellin vitro. 7Furthermore, CCR2 was shown to mediate arthritis progression by enhancing Tregs migration.8 Although not as well studied as T cells, a growing body of evidence indicate a potential connection between CCR2 and B cell regulation. While CXCR4 as well as functional and constitutive CCR2 are expressed in B cells, the transcription of CCR2 occurs in immature B cells and dampens in mature B cells.9 Furthermore, mice lacking CCR2 are a specifically well suited animal model to investigate B cell expansion under infection and non-infection condition.10 Following infections, the lymphatic follicles are notably enlarged, which represents an increased B cell proliferation and outgrowth.11Additionally, in CCR2 deficient immature B cells, actin polymerization is increased, as well as the migration and homing to the lymph nodes, all of which being mediated by the interaction between CCR2 and its ligand CCL2.12. Taken together, these studies suggest that CCR2 might play an important role in the B cell downstream signaling pathways.
Several downstream pathways dominated by BCR activation are particularly prominent in autoimmunity. First, the mammalian target of rapamycin (mTOR) was shown to be involved in the autoimmune pathogenesis, while rapamycin has been demonstrated to represent an effective treatment of rheumatism.13, 14 MTOR aids the expanded T follicular helper (TFH) cells to facilitate B cell activation and autoantibody production.15Furthermore, CCR2 can mediate HIF-1α expression via the PI3K-Akt-mTOR pathway; thus, controlling the cell metabolic process.16 Another pathway mainly regulated by Mst1 plays an essential role in lymphocyte migration and adhesion during immunosurveillance. During T cell-regulated B cell activation, Mst1 functions as a molecular brake to balance immune tolerance, and its depletion leads to hypergammaglobulinemia in mice.17 Mst1 deficient mice exhibited peripheral lymphoid tissue hypertrophy, decreased marginal zone (MZ) B cells in the spleen, and the emigration of single-positive thymocytes was also influenced.18 Moreover, Mst1 depleted B cells show an attenuated response to mitogens and a down-regulated BCR signaling as well as clustering.19Additionally, the Janus kinase/signal transduction and activator of transcription (JAK/STAT) signaling pathway is involved in the development of systemic lupus erythematosus (SLE), which is supported by the effective use of JAK inhibitor as a therapeutic method.20 CCL2-CCR2 has been shown to associate with the JAK/STAT signaling pathway by activating STAT1, STAT3, and STAT5.21 Furthermore, CCL2 and CCR2 dimerization induce tyrosine phosphorylation of STAT5.22 However, the interaction between these three main pathways during the course of autoimmunity and the manner in which they are regulated by CCR2 and B cell signaling activity, remains unknown.
Therefore, we hypothesized that CCR2 interacts with BCR signaling potentially through sequential Mst1-mTOR-STAT1 activation. Herein, we utilized CCR2 deficient mice and revealed that the loss of CCR2 leads to up-regulation of BCR proximal signaling molecules, enhancement of the F-actin remodeling related BCR clustering, and increased expression levels of various transcriptional factors. Changes being underlined by the effect of CCR2 on PI3K-Akt-mTORC1, Mst1-mTORC1-Dock8-WASP, and Mst1-mTORC1-STAT1 axes, respectively.