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. 2014 Jul;11(3):636-50.
doi: 10.1007/s13311-013-0254-x.

Programmed death 1 deficiency induces the polarization of macrophages/microglia to the M1 phenotype after spinal cord injury in mice

Anhui Yao  1 , Fangfang Liu, Kun Chen, Liang Tang, Ling Liu, Kun Zhang, Caiyong Yu, Ganlan Bian, Hongmin Guo, Jingjing Zheng, Peng Cheng, Gong Ju, Jian Wang
Affiliations

Programmed death 1 deficiency induces the polarization of macrophages/microglia to the M1 phenotype after spinal cord injury in mice

Anhui Yao (VSports最新版本) et al. Neurotherapeutics. 2014 Jul.

Abstract

The inflammatory response following spinal cord injury (SCI) involves the activation of resident microglia and the infiltration of macrophages VSports手机版. Macrophages and microglia can be polarized into the classically activated proinflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype. Programmed cell death 1 (PD-1) is a critical immune inhibitory receptor involved in innate and adaptive immune responses. However, whether PD-1 is involved in the modulation of macrophage/microglial polarization is unknown. In this study, the mRNA levels of pd1 gradually increased after SCI, and PD-1 protein was found in macrophages/microglia in injured spinal cord sections. PD-1 knockout (KO) mice showed poor locomotor recovery after spinal cord crushing compared with wild-type mice. M1-type macrophages/microglia accumulated in greater numbers in the injured spinal cord of PD-1-KO mice. Under polarized stimulation, induced expression of PD-1 occurred in cultured macrophages and microglia. PD-1 suppressed M1 polarization by reducing the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and promoted M2 polarization by increasing STAT6 phosphorylation. In PD-1-KO mice, the M1 response was enhanced via the activation of STAT1 and nuclear factor-kappa B. Furthermore, PD-1 played various roles in phagocytosis in macrophages and microglia. Therefore, our results suggest that PD-1 signaling plays an important role in the regulation of macrophage/microglial polarization. Thus, deregulated PD-1 signaling may induce the polarization of macrophages/microglia toward the M1 phenotype. Overall, our results provide new insights into the modulatory mechanisms of macrophage/microglial polarization, thereby possibly facilitating the development of new therapies for SCI via the regulation of macrophage/microglial polarization through PD-1 signaling. .

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Figures

Fig. 1
Fig. 1
Expression of programmed death-1 (PD-1) in macrophages/microglia after spinal cord injury (SCI) in mice. Quantitative reverse transcription polymerase chain reaction at each timepoint after SCI for (a) Pd1 (n = 3 mice in each group) and (b) Pdl1 (n = 3 mice in each group). Immunohistochemistry (IHC) in macrophages/microglia 14 days postinjury (dpi) in wild-type (WT) mice for (c) PD-1, (d) it’s ligand, PD-L1, and (e) PD-1 in M1 cells [inducible nitric oxide synthase (iNOS)+]; (f) PD-1 in M2 cells [arginase 1 (Arg1+)]; (g) PD-L1 in M1 cells (iNOS+); and (h) PD-L1 in M2 cells (Arg1+). The injury epicenter in (c) and (d) are denoted with an asterisk. (i) Locomotor recovery evaluated by the Basso Mouse Scale (BMS) score acquired at different timepoints after SCI in WT and PD-1–/– mice (n = 8 mice). (j) IHC for iNOS and Arg1 in macrophages/microglia at 14 dpi. (a, b) ### p < 0.001 versus control, ***p < 0.001 versus the previous timepoint. (i) *p < 0.05 versus WT mice. Scale bar = (c–h) 100 μm and (j) 200 μm. KO = knockout; Iba1 = ionized calcium-binding adapter molecule-1; DAPI = 4’,6-diamidino-2-phenylindole
Fig. 2
Fig. 2
Quantification of programmed death-1 (PD-1) and it’s ligand, PD-L1, in cultured macrophages and microglia under polarized stimulation. The presence of PD-1 (a) and PD-L1 (b) in cultured macrophages respectively, under lipopolysaccharide (LPS) + interferon-gamma (IFN-γ) or interleukin (IL)-4 stimulation. (c) Flow cytometry of PD-1 and PD-L1 in cultured microglia under LPS + IFN-γ or IL-4 stimulation. Percentage of (d) PD-1- and (e) PD-L1-positive cells in polarized microglia. Scale bar = 100 μm. n = 3 mice in each group. ***p < 0.001. Con = control; Iba1 = ionized calcium-binding adapter molecule-1; DAPI = 4’,6-diamidino-2-phenylindole
Fig. 3
Fig. 3
Deficiency of programmed death-1 (PD-1) deficiency improves M1 polarization of cultured macrophages. Bone marrow-derived macrophages (BMDMs) from wild-type (WT) and PD-1-knockout (KO) mice were stimulated with lipopolysaccharide (LPS) + interferon-gamma (IFN-γ) or interleukin (IL)-4 for 24 h followed by quantitative reverse transcription polymerase chain reaction for (a) inos, (b) Arg1, and (c) Cd206, and Western blotting (d) for inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1). Densitometric analysis of protein bands for (e) iNOS and (f) Arg1 normalized to β-actin. Enzyme-linked immunosorbent assays for the proinflammatory cytokines: (g) IL-12, (h) tumor necrosis factor-alpha (TNF-α), and (i) IFN-γ, and the anti-inflammatory cytokines: (j) IL-10 and (k) IL-4. Cytoplasmic (l) IFN-γ and (n) IL-4 in BMDMs measured by flow cytometry for (m) IFN-γ and (o) IL-4, respectively. n = 3 mice in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Con = control
Fig. 4
Fig. 4
Deficiency of programmed death-1 (PD-1) deficiency enhances M1 polarization of cultured microglia. (a) Western immunoblot for inducible nitric oxide synthase (iNOS; M1 phenotype) and arginase 1 (Arg1) (M2 phenotype) in primary microglial cells isolated from wild-type (WT) and PD-1-knockout (KO) mice and stimulated with lipopolysaccharide (LPS) + interferon (IFN)-γ or interleukin (IL)-4 for 24 h. Densitometric analysis of (b) iNOS and (c) Arg1 normalized to β-actin. Quantitative reverse transcription polymerase chain reaction of proinflammatory cytokines (d) Il12, (e) Il1b, and (f) Tnfa; anti-inflammatory cytokines (g) Il4, (h) Il10, and (i) Tgfb. n = 3 mice in each group. *p < 0.05, **p < 0.01, ***p < 0.001. Con = control; ns = not significant
Fig. 5
Fig. 5
Deficiency in programmed death-1 (PD-1) deficiency influences the polarization of macrophages and microglia via signal transducer and activator of transcription (STAT)1 and STAT6. (a) Western immunoblot for phosphorylated (p)-STAT1, STAT1, p-STAT6, and STAT6 in macrophages stimulated with lipopolysaccharide (LPS) + interferon (IFN)-γ or interleukin (IL)-4 for 24 h. Band intensities of (b) p-STAT1, (c) STAT1, (d) p-STAT6, and (e) STAT6 normalized to β-actin. (f) Western immunoblot for p-STAT1, STAT1, p-STAT6, and STAT6 in microglial cells stimulated with LPS + IFN-γ or IL-4 for 24 h. Densitometric analysis for (g) p-STAT1, (h) STAT1, (i) p-STAT6, and (j) STAT6 normalized to β-actin. n = 3 mice in each group. *p < 0.05, **p < 0.01. Con = control; ns = not significant
Fig. 6
Fig. 6
Mechanism of programmed death-1 (PD-1) deficiency influences the polarization of macrophages/microglia in vivo after spinal cord injury. (a) Western immunoblot of inducible nitric oxide synthase (iNOS), arginase 1 (Arg1), phosphorylated signal transducer and activator of transcription (p-STAT1), signal transducer and activator of transcription (STAT1), phosphorylated nuclear factor kappa-B (p-NF-κB), and nuclear factor kappa-B (NF-κB) in wild-type (WT) (n = 3) and PD-1-knockout (KO) mice (n = 4) 14 days postinjury (dpi). Densitometric analysis for (b) iNOS, (c) Arg1, (d) p-STAT1, (e) STAT1, (f) p-NF-κB, and (g) NF-κB relative to β-actin. (h) Western immunoblot of iNOS, Arg1, p-STAT1, STAT1, p-NF-κB, and NF-κB in WT mice (n = 3) and PD-1-KO mice (n = 4) 21 dpi. Densitometric analysis for (i) iNOS, (j) Arg1, (k) p-STAT1, (l) STAT1, (m) p-NF-κB, and (n) NF-κB relative to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001. ns = not significant
Fig. 7
Fig. 7
A deficiency in programmed death-1 (PD-1) deficiency promotes opposing phagocytic effects in macrophages and microglia. (a) Representative image under light microscopy showing the morphology of macrophages that did not phagocytize any beads (P0), and phagocytized 1 bead (P1), 2 beads (P2), or numerous beads (>P2). (b) Representative diagram showing macrophages with phagocytized beads by flow cytometric analysis. (c) Phagocytosis of wild-type (WT) macrophages under polarized stimulation analyzed by flow cytometry. Phagocytosis of WT and PD-1-knockout (KO) macrophages with (d) no stimulus, (e) lipopolysaccharide (LPS) + interferon-gamma (IFN-γ), or (f) interleukin (IL)-4. (g) The percentage of phagocytic cells calculated in WT and PD-1-KO macrophages. (h) The phagocytosis index calculated in WT and PD-1-KO macrophages. (i) Phagocytosis of WT microglia under polarized stimulation analyzed by flow cytometry. Phagocytosis of WT and PD-1-KO microglia with (j) no stimulus, (k) LPS + IFN-γ, or (l) IL-4. (m) The percentage of phagocytic cells in WT and PD-1-KO microglia. (n) The phagocytosis index calculated for WT and PD-1-KO microglia. n = 3 mice in each group. # p < 0.05, ## p < 0.01 versus WT, *p < 0.05, **p < 0.01 versus WT and PD-1-KO. Con = control
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