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and B cell signaling (unpublished data), there are some worth noting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.