The Role And Mechanism Of Integrin CD11b-mediated Microglial Activation And Phenotype Transformation Of M1/M2 In Optic Nerve Injury And Repair

BackgroundFor many years,the methods of promoting central nerve repair ha ve mainly focused on regulating nerve growth factors and neuronal growth inhibitory factors in the central microenvironment,or increasing the intrinsic growth potential of neurons.Especially the combined applications of these methods can achieve a certain degree of regeneration and neuronal functional recovery.However,the effects are still unsatisfactory.In recent years,it has been evidenced that central nervous disease or injury can trigger a variety of repair processes,including neurogenesis,axonal sprouting,synapsis,angiogenesis,axonal regeneration and remyelination,in which microglia plays a very important role.At present,the regulation of microglia mediated neuroinflammation has gradually become a new research direction of central nervous injury and repair.Neuroinflammation in central never system plays a dual role of pro-inflammation（M1）and anti-inflammation（M2）in the process of central nervous injury,and promoting microglia to M2 phenotype polarization can reduce the secondary injur y of central neuroinflammation and promote nerve repair.In recent years,it has been found that the activation of microglia in the process of central nervous disease or injury can present two phenotypes:M1 and M2.M1 type is also termed classical activation type,which plays an important role in promoting inflammation.M1 cells mainly perform host resistance to pathogens by releasing protease and reactive oxygen species,which can also damage healthy neurons.M2 type,also known as alternative activation type,mainly plays an anti-inflammatory role.M2 cells have the functions of neuroprotection,reducing inflammation,clearing tissue debris,promoting tissue repair,angiogenesis and myelin regeneration.CD11b protein is highly expressed on several cell types including macrophages,neutrophils,dendritic cells（DCs）and microglial cells.The function of CD11b signaling is complicated.CD11b signaling on different cells execute different functions.For example,CD11b on peripheral macrophages negatively regulated TLR4-triggered inflammation.Conversely,CD11b on DCs positively regulates TLR4-induced signaling^[1].Intriguingly,CD11b on DCs can also negatively regulates TLR9-triggered DCs cross-priming by upregulating micro RNA-146a.CD11b could negatively regulate TLR4 signaling in peripheral macrophage s,promote cells polarization to m2,alleviate M1 inflammation and promote tissue repair.CD11b can inhibit TLR-triggered inflammation by activating Syk and promoting degradation of My D88 and TRIF via Cbl-b.Degradation of MyD88 and TRIF could reduce activation of NF-κB,thus reduce M1 inflammation.Meanwhile,CD11b can polarize macrophages to M2 type and secrete IL-10 through Src-Akt and Src-STAT6 pathway,so as to promote the healing of experimental enteritis.Remarkably,a recent study of CD11b on brain microglia in CNS revealed that CD11b could negatively regulated TLR4-triggered inflammation in microglia,which then reduce the production of M1 type cytokines IL-6 and TNF-a by activating Src-Akt signal,so as to reduce neuralgia.Many studies,including our previous works,have proved that⑴Loss of TLR4signaling whether by gene knockout or pharmacological blockade significantly reduced retinal inflammation and delayed RGCs loss following ONC.⑵loss of the major adaptor TRIF downstream of TLR4 signaling reduces retinal inflammation,promotes retinal ganglion cell survival and axon regeneration via nuclear factor-κB.In conclusion,（1）inhibition of TLR-4 signal can significantly reduce retinal inflammatory response following optic nerve injury,reduce the degeneration and loss of RGCs,and promote the regeneration of optic nerve.（2）CD11b can negatively regulate TLR-4 signal in macrophages and central microglia,promote M2 phenotype and tissue repair.However,whether CD11b on retinal microglia has the similar function has not been reported.（3）CD11b may perform different functions in different cells.Therefore,we hypothesized that CD11b in retinal microglia may affect TLR-4 signal after optic nerve injury,and then affect subsequent RGCs degeneration and loss and optic nerve regeneration.CD11b on retinal microglia may also have M1/M2 phenotype regulation mechanism.In order to answer these hypotheses,based on the introduction of CD11b gene knockout homozygote and the establishment of optic nerve crush model,the following five aspects of research were carried out:（1）Expression of CD11b protein in retina and optic nerve of adult C57BL/6J mice;（2）The effects of CD11b on the retinal microglial activation and M1/M2 phenotype polarization following optic nerve injury;（3）The effects of CD11b-mediated retinal microglial activation and M1/M2 phenotype polarization on RGCs degeneration and loss following optic nerve crush;（4）The effects of CD11b-mediated retinal microglial activation and M1/M2 phenotype polarization on optic nerve regeneration following optic nerve crush;（5）Exploring the mechanism of CD11b mediated-retinal microglial activation and M1/M2 phenotype polarization following optic nerve crush.The main results and conclusions are as follows:1.Expression of CD11b protein was mainly on retinal microglia,almost undetectable in resting intact retina,significantly increased following optic nerve crush,peaked at 7 days post crush,and remained at a high level till at least 28 days post crush.2.CD11b deficiency significantly exacerbated retinal microglial activation after optic nerve crush.The results showed that the number of total microglia cells,ameboid microglial cells,M1 type microglial cells and the expression levels of microglia marker-Iba1（WB test）in the retinae of CD11b deficient mice groups at each time point following optic nerve crush were significantly higher than those in WT groups,and the difference was most significant at 7 days post crush.3.CD11b deficiency significantly exacerbated retinal M1 type neuroinflammation following optic nerve crush,promoting microglial polarization to M1 phenotype.Compared with WT groups,the number of M1 microglia and the expression of M1 proinflammatoryfactors such as IL-6 and TNF-αincreased while the number of M2 microglia and theexpression of M2 anti-inflammatory factors Arg-1 and IL-10 decreased significantly in retinae from CD11b gene deficient mice.4.CD11b deficiency significantly accelerated RGCs degeneration after optic nerve crush.TUJ1⁺RGCs were gradually decreased and atrophied following ONC.Compared with WT groups at each time point,the number of RGCs in CD11b deficient mice was further reduced.At 28 days post crush,only 13%RGCs remained in retina e of CD11b deficient mice.5.CD11b deficiency significantly reduced GAP-43 expression in retinae after optic nerve crush.GAP-43 expression was almost undetectable in resting intact retina but was strongly up-regulated in RGCs undergoing regeneration.Higher expression levels of GAP-43 are expected to result in a better RGCs regeneration and neuronal survival.We found expression of GAP-43 was very low in resting intact retinae,significantly increased following optic nerve crush.Compared with WT groups at each time poi nt,GAP-43expression significantly decreased in retinae of CD11b gene deficient mice.6.CD11b deficiency significantly reduced the regeneration of optic nerve following optic nerve crush.The quantitative studies of regenerated axons by intravitreal injection of cholera toxin（CTB）and GAP-43 immunofluorescence section showed that the number of regenerated axons in the CD11b gene deficient group was significantly lower than that in the WT group at each time point.Meanwhile,a large number of CTB⁺microglia appeared in the optic nerve from CD11b gene deficient groups,whose source needs further studies.7.The CTB⁺cells were determined as microglia,which indicated that microglia phagocytized the CTB⁺axon fibers.Loss of CD11b gene may cause the disorder of microglia phagocytosis.Microglia phagocytized axon fibers,which may be an important reason for the decrease of optic nerve regeneration in CD11b^-/-mice.8.CD11b deficiency exacerbated the formation of local glial scar following optic nerve crush.The formation of local glial scar is an important cause for the ineffective regeneration of axon following optic nerve crush.We found that the formations of glial scar in the CD11b deficiency groups were more serious than that in the WT groups.9.CD11b deficiency exacerbated the loss of visual function following optic nerve crush.F-VEP experiments of visual electrophysiology confirmed that the functions of optic nerve in CD11b deficiency groups were significantly worse than that in WT groups.10.Expression of TLR-4,My D88 and TRIF,the key molecules of TLR-4 signaling pathway,in retina following optic nerve crush was significantly up-regulated upon CD11b deficiency,suggesting the mechanism that CD11b on retinal microglia can negatively regulates TLR-4 signal probably plays a key role in regulating the retinal microglial activation and balance of pro-inflammatory（M1）and anti-inflammatory（M2）following optic nerve injury.