Investigation Of The Mechanism Of PD-1in Macrophages/microglia Polarization And Its Roles In Spinal Cord Injury

About2.5million people live with spinal cord injury (SCI), with more than130000new cases reported annually [1]. SCI often cause paralysis with complications of pain,ulcer and urethra infection, which result in enormous suffering and burden to the familiesand society [1]. Although great progresses have made in treatment after SCI, there is nonesatisfactory therapy till now. The reason lies in the fact that the very complicatedpathological changes after SCI [2]. So well study and understand every aspect of thepathology after SCI has become the main tasks for neuroscience scientists.With more and more study in this area, the pathological sequelae after SCI aredivided into two phases: primary injury and secondary injury. The primary injury meansthe direct damage caused by the trauma. Caused by the primary injury, the secondaryinjury leads to more seriouse extent than the primary injury. Several interrelated processes are thought to contribute to the secondary injury after SCI, including excitotoxity, freeradical, vascular broken and inflammatory responses et al [3-5]. In which, inflammatoryresponses plays the most important role and determines the recovery consequences. Andthe main inflammatory cells are macrophages/microglia the main inflammatory cells aremacrophages/microglia [6,7]. It is already known that macrophages/microglia can bepolarized into M1or M2based on signals in the lesion microenvironment [8].Polarizaion means macrophages retain different functional phenotypes in differentmicroenvironments [8]. Macrophages can be mainly polarized into two phenotypes:“classic activated” M1phenotype macrophages and “alternative activated” M2phenotypemacrophages. in vitro, Lipopolysaccharide (LPS) and interferon-gamma (IFN-r) inducemacrophages/microglia into “classically activated”M1phenotype and interleukin-4(IL-4)or IL-13induces the cells into “alternatively activated” M2phenotype [9]. The phenotypethat defines M1macrophages/microglia is characterized by increased oxidativemetabolites (such as inducible nitric oxide synthase (iNOS) and nitric oxide synthase2(NOS2)) and high levels of pro-inflammatory cytokines (e.g., IL-12, IL-1β, TNF-, IL-15,IL-18)[10,11], which is cytotoxic to neurons and glia cells [12]. Conversely, in thepresence of IL-4, macrophages/microglia will be polarized into M2phenotype andproduce high levels of Arginase1(Arg1), CD206, IL-10and TGF-β [13,14], and alsosecret kinds of neurotrophic factors (e.g., CNTF, IGF, EGF, NGF), which suppressinflammatory responses and facilitate wound healing [8,15]. In SCI, formacrophages/microglia, to facilitate or inhibit the recovery is determined by the ratio ofM1versus M2during pathological processes [8].Programmed cell death1(PD-1) is a288amino acid type I transmembrane proteinwhich belongs to the CD28superfamily and shares23%amino acid sequence homologywith cytotoxic T-lymphocyte-associated antigen4(CTLA-4)[16]. As a co-inhibitoryreceptor, it has been well studied in T lymphocyte. Ligation of PD-1with its ligandPD-L1could recruit SHP-2or SHP-1/SHP-2to combine with its cytoplasmic domainimmunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM), and then inhibit proliferation and cytokineproduction of T lymphocyte [17]. A relationship between PD-1singnaling pathway andmacrophage activation has been suggested by several studies [19]. The upregulation ofPD-1has been reported in monocytes after HIV infection [20]. PD-1has been proved tonegatively regulate IL-12production by limiting STAT1(signal transducer and activatorof transcription1) phosphorylation in monocytes/macrophages during chronic hepatitis Cvirus infection [21]. While more studies need to be done to explore the roles of PD-1signaling pathway in the polarization of macrophages/microglia, especially its functionduring the process after SCI.In this study, we have introduced PD-1knock out mice based on C57BL/6background mice. Macrophages and microglia were cultured from these two kinds ofmice. The roles and mechanisms of PD-1were detected in the polarization ofmacrophages/microglia in vitro.The results we have obtained are as follows:1. First, macrophages and microglia were cultured from C57BL/6(WT) mice andPD-1KO mice. Then LPS+IFN-γ and IL-4were given toinducedmacrophages/microglia into M1and M2phenotypes respectively. Wefound PD-1paticipated in the regulation of macrophages/microglia polarizationand PD-1KO macrophages/microglia were prone to be polarized into M1phenotype.2. PD-1KO macrophages/microglia secreted more pro-inflammatory factors (IL-1,TNF-, IL-12, IFN-γ, et al) which were the characteristic of M1phenotype. Atthe same time secreted less anti-inflammatory fators (IL-10, IL-4, et al) whichwere the characteristic of M2phenotype3. STAT1and STAT6were reciprocal antagonism in macrophages/microglia, andone of the mechanisms that PD-1regulated macrophages/microglia polarizationis through the regulation of janus kinase-STAT (JAK-STAT) pathway.4. We found there were widely differences in phagocytosis lates beads between bone marrow derived macrophages and microglia.5. In vivo, we detected the expression of PD-1and PD-L1at each time point afterSCI, and we found the roles of PD-1played through the comparation WT andPD-1KO mice after SCI.In summary, PD-1signaling pathway plays important roles in regulation ofmacrophages/microglia polarization, and compromised PD-1signaling pathway,macrophages/microglia are prone to be polarized into M1phenotype in the expense of M2phenotype. There is wide difference between macrophages and microglia in thephagocytosis of latex beads. In vivo, PD-1deficient mice exacerbate inflammatoryresponses and retard functional recovery after SCI compared with WT mice. These resultsprovide new insights into the modulation mechanisms of macrophages/microgliapolarization and shed light on new therapies for SCI through the modulation ofmacrophages/microglia polarization through the PD-1signaling.