The Role Of CKIP-1in The Regulation Of Macrophage Proliferation And Polarization

Macrophages are fully differentiated cells. M-CSF induced proliferation anddifferentiation play a key role in the development of macrophages. However, maturedmacrophages in tissues hardly proliferate in response to M-CSF in homeostasis. In certainconditions, such as inflammation, fully differentiated macrophages will enter into cellcycle again. This process is regulated by IL-4and M-CSF as well. How macrophagesinitiate proliferation and when they go to rest? How macrophages keep such balance isstill not clear. M-CSF is the key regulator of macrophage proliferation, differentiation,survival and so on. Understanding the regulation of M-CSF signaling will contribute tounveil the underlying mechanisms of macrophage population control. In contrast to itsactivating mechanism, which has been intensively investigated, it is less known about thenegative regulation.Macrophages are key to the development, progression, and resolution of inflammation,with different macrophage subtypes involved at each step. Macrophage polarization israpid in response to environmental cues, with the polarized extremes (classical [M1] andalternative [M2]) present during acute and resolution phases of inflammation, respectively.Increasing evidences indicate that macrophage polarization is involved in thedevelopment and progression of tumors, atherosclerosis, non-alcoholic fatty liver disease(NAFLD), self-immune diseases, obesity and so on. Macrophage polarization is of greatimportance when they act their roles, but the molecular mechanisms underlyingmacrophage polarization remain largely unknown. Identification of new regulatingmoleculars of macrophage polarization under certain conditions will contribute to betterunderstand how macrophages play their roles.CKIP-1is a cytoskeleton-associated protein, involved in the regulation of celldifferentiation, proliferation, apoptosis and so on. Our previous studies and a chip data suggest CKIP-1abundantly expressed in immune cells, especially in macrophages. So weattempted to identify the role of CKIP-1in macrophage development and functions. Weplanned to investigate the relationship between CKIP-1and the homeostasis ofmacrophage population control and function regulation, as well as the underlyingmechanisms.Our major results and conclusions are listed as following:We identify CKIP-1as a negative regulator of macrophage proliferation in vitro andin vivo.1) CKIP-1deficiency leads to enhanced proliferation and survival ofmacrophages. We cultured BMCs from CKIP-1-deficient and wild-type (WT) mice withM-CSF and observed an excessive yield of CKIP-1–/–macrophages compared with that ofWT cells. The percentages of CKIP-1-deficient BMDMs in S and G2/M phases werehigher relative to WT BMDMs. BrdU incorporation assay confirmed thehyper-proliferative response of CKIP-1-deficient cells to M-CSF. CKIP-1-deficient cellsdisplayed a decreased percentage of apoptosis after M-CSF deprivation. In32D-CSF1Rcells, overexpression of CKIP-1decreased proliferation of cells in the presence of M-CSF,compared with empty vector control.2) CKIP-1-/-mice spontaneously developsplenomegaly and have more microglias in the CNS system. We compared microglias inthe brains and spinal cords of WT and CKIP-1-deficient mice by staining with themicroglia marker Iba-1. More microglias were observed in the brain and spinal cord ofCKIP-1–/–mice, compared with those of WT mice. We noted that most CKIP-1–/–micespontaneously developed splenomegaly when they are getting elder. Flow cytometricanalyses revealed that the numbers of splenic macrophages and monocytes wassignificantly increased in CKIP-1–/–mice compared to their WT littermates.3) CKIP-1suppresses myelopoiesis in vivo and in vitro. The percentages of monocytic cells andCD11b+myeloid progenitors in the bone morrow of CKIP-1–/–mice were increased.CKIP-1–/–splenic cells generated more G/M colonies than WT cells.4) TRAF6isinvolved in M-CSF signaling transduction. Knockdown of TRAF6by shRNAdramatically decreased the proliferation of32D-CSF1R cells when cultured with M-CSFconditioned medium. Data of BrdU incorporation assay also supported this notion. Knockdown of TRAF6aslo attenuated Akt phosphorylation upon M-CSF stimulation in32D-CSF1R.5) CKIP-1regulates macrophage proliferation by inhibiting M-CSF-inducedAkt activation. CKIP-1-deficient BMDMs exhibit prolonged Akt activation upon M-CSFstimulation. Treatment with PI3K inhibitor LY294002dramatically decreased BrdU+cellsin CKIP-1-/-BMDMs, the same with that in WT cells.6) CKIP-1is a bona-fide target ofGSK3β. CKIP-1and p-CKIP-1can be separated by phostag-containing SDS-PAGE.Either λ-phosphatase treatment or adding phos-tag to the SDS-PAGE abolished the bandof phosphorylated CKIP-1recognized by a p-CKIP-1antibody.We also showed that CKIP-1play a pivotal role in regulation of M1/M2macrophagepolarization.1) CKIP-1-deficient mice were resistant to LPS-induced sepsis. At a lethaldoes of LPS injection,80%of CKIP-1-deficient mice survived in contrast to less than20%of WT mice survived. The levels of pro-inflammatory cytokines such as TNF-α, IL-6,were lower in serums of CKIP-1-deficient mice than that of WT control.2) CKIP-1deficiency resulted in a weakened pro-inflammatory phenotype of macrophages.CKIP-1-deficient BMDMs secreted less TNF-α and IL-6upon PGN，LPS，Poly(I:C)stimulation. Experiments on PECs obtained similar results.3) CKIP-1suppresses M2polarization. CKIP-1-deficient BMDMs expressed higher levels of Ym1and Fizz1inresponse to IL-4/IL-13. Experiments on PECs obtained similar results. In the condition ofChitin-elicited Th2inflammation response, BMCs from CKIP-1-deficient mice expressedM2marker genes at a higher level.4) CKIP-1balanced M1/M2polarization in LPSinduced Th1inflammation response. BMCs from CKIP-1-deficient mice after LPS i.pexpressed higher levels of iNOS, TNF-α and IL-12, but lower levels of Arg1, Ym1andFizz1.Taken together, our results demonstrate that CKIP-1is a negative regulator of macrophageproliferation. CKIP-1inhibited M-CSF induced Akt activation through TRAF6.Meanwhile, CKIP-1was phosphorylated by GSK3β, the downstream target of Akt,followed by ubquitin mediated proteome degradation. CKIP-1combined Akt and GSK3βto form a feed-back loop to maintain the steady state of M-CSF induced macrophageproliferation. We also showed that CKIP-1plays a pivotal role in the regulation of macrophage polarization. CKIP-1suppressed M2polarization, but is necessary for M1polarization. CKIP-1-deficient mice were resistant to LPS-induced sepsis.CKIP-1-deficient macrophages were less potential to secret pro-inflammatory cytokines.Under conditions of IL-4/IL-13and Chitin stimulation, CKIP-1-deficient macrophagesexpressed Ym1, a M2marker gene at a higher level.Proliferation in response to cytokines is crucial for macrophages development, and moreimportantly, local proliferation of tissue macrophages dominates inflammatory responseunder certain conditions, such as Th2inflammation. M-CSF signaling plays a key role inregulation of macrophage proliferation. Our studies will contribute to furtherunderstand the underlying mechanism of macrophage population control andM-CSF signaling regulation. Macrophages were involved in many physiopathologic andpathophysiologic processes, and macrophage polarization is tightly connected with theirfunctions. Our research provides new insights into the regulation of macrophagepolarization and will contribute to further understand the relationship betweenmacrophage polarization and inflammation.