mTORC1-mediated energy metabolism during B cell differentiation
was enhanced in mice lacking CCR2
Cell differentiation-associated molecules, mTOR complex 1 (mTORC1) have
been reported to function via the PI3K/Akt signaling pathway to mediate
hypoxia-inducible factor 1 (HIF-1) expression and consequently influence
the cell energy metabolism
process.16 Thus, we
examined PI3K-Akt-mTORC1 activity in B cells lacking CCR2.
Following
sAg stimulation, the B cell lysate was incubated with Abs specific for
phosphorylated PI3K (pPI3K), mTORC1 (pmTORC1), Akt (pAkt), S6 (pS6), and
Akt distal glucose metabolism-related transcriptional factor forkhead
box protein O1 (pFoxo-1). The protein expression levels of pPI3K, pAkt,
pS6, pmTORC1, and pFoxo-1 were increased in CCR2 KO B cells following
stimulation (Fig. 3A ). To clarify whether the effects of CCR2
on mTORC1 and its downstream pathways affected the B cell metabolic
molecular signaling, we used mTORC1 inhibitors in CCR2 KO B cells to
obstruct the upstream effectors of the mTOR pathway. Additionally, CCR2
KO B cells were pretreated with rapamycin to examine the activation of B
cell signaling molecules under the same conditions as for the
uninhibited groups. As expected, following rapamycin treatment, the
activation of pSHIP, pBtk, pCD19, pAkt, pFoxo-1, and pS6 in CCR2 KO B
cells was brought to similar levels as the ones observed in WT B cells
(Fig. 3B ). This further indicates that mTORC1 pathway activity
is necessary for CCR2-regulated B cell metabolism. In addition, Seahorse
assay was performed to analyze the real-time oxidative phosphorylation
based on the oxygen consumption rate (OCR). Following stimulation with
F(ab’)2 anti-mouse Ig (M + G), CCR2 KO B cells exhibited
higher ATP production levels, maximal respiratory capacity, and maximal
respiratory potential than those of WT B cells (Fig. 3C ). This
change in energy metabolism can facilitate either proliferation or
apoptotic processes in CCR2 KO B cells. In support of this, uponin vitro lipopolysaccharide (LPS) stimulation, CCR2 KO B cells
proliferated faster than LPS stimulated WT B cells (Fig. 3D-E ).
Furthermore, upon in vitro CPG stimulation, CCR2
KO B cells proliferated and proceeded through apoptosis at higher rates
than those of CPG stimulated WT B cells (Fig.
3F-G ).
Engagement of BCR induces heightened aerobic glycolysis by up-regulating
glucose and oxygen utilization; thus, B cell activation drives
MYC-dependent up-regulation of glucose transporter 1 (Glut1) and
HIF-1α-mediated up-regulation of oxygen
transport.27,
28 HIF-1 and MYC have been reported to
coordinately regulate immune cell metabolic
reprogramming.29 Thus
following our immunoblotting analysis of these two molecules we
discovered that CCR2 KO B cells exhibited increased levels of HIF-1α and
c-MYC (Fig. 3H ).
Taken together, loss of CCR2 disrupts the differentiation of FO B cells
through the up-regulation of the PI3K/Akt/mTORC1 metabolic pathway.