Attenuation of the programmed cell death-1 pathway increases the M1 polarization of macrophages induced by zymosan

Abstract

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals on interaction with its 2 ligands, PD-L1 and PD-L2. We assessed the contribution of the PD-1 pathway to regulating the polarization of macrophages that promote inflammation induced by zymosan. We found that PD-1(-/-) mice developed robust peritonitis with more abundant infiltration of M1 macrophages, accompanied by higher levels of pro-inflammation factors, especially monocyte chemotactic protein-1 (MCP-1) compared with wild-type controls ex vivo and in vitro. Our results indicated that PD-1 deficiency promotes M1 rather than M2 polarization of macrophages by enhancing the expression of p-STAT1/p-NF-κB p65 and downregulating p-STAT6. We found that PD-1 engagement followed by zymosan stimulation might primarily attenuate the phosphorylation of tyrosine residue in PD-1 receptor/ligand and the recruitment of SHP-2 to PD-1 receptor/ligand, leading to the reduction of M1 type cytokine production.

Figure 1

Gating of macrophages/monocytes/neutrophils subsets in peritoneal exudates. Cells collected from the peritoneal cavity were stained with appropriate anti-mouse mAbs, and analyzed by flow cytometry. It showed four subsets in the peritoneal cells gated in CD19⁻ population: CD11b^high F4/80^high CD11c^low in large size (LPMs), CD11b^int F4/80^int LY6C^int CD11c^−/low MHC II ^high in small size (SPMs), CD11b^int LY6C^high MHC II ^low CD11c^−/low (monocytes), CD11b^int F4/80⁻ LY6G^high (neutrophils)

Figure 2

The subsets of macrophages/monocytes/neutrophils in peritoneal exudates. The percentage of LPMs, SPMs, monocytes and neutrophils in the peritoneal exudates from PD-1^−/− (KO) mice were analyzed by flow cytometry compared with that in WT mice after the injection of zymosan for 4, 48 and 72 h. (a) LPMs, SPMs, monocytes and neutrophils were shown with gate of CD11b+ and/or F4/80+. (b) SPMs and monocytes were shown with gate of CD11b+ and/or LY6C+. (c) the numbers of LPMs, SPMs, monocytes and neutrophils in peritoneal exudates. n=3 for each group. Mean±S.D., ^#P<0.05 versus WT naive, ^##P<0.01 versus WT naive,*P<0.05 versus KO naive, **P<0.01 versus KO naive, ^ΔP<0.05 WT versus KO, ^ΔΔP<0.01 WT versus KO

Figure 3

The intracellular staining of cytokines in peritoneal macrophages. Cells were collected from peritoneal cavity after zymosan induction for 4, 48 and 72 h separately. Cells were stained with membrane markers (CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650), then permed with perming buffer, and staining of intracellular cytokines was continued. The mean fluorescence intensity (MFI) of MCP-1 (a) and IL-6 (b) were analyzed in the SPMs and LPMs by gates as previously. n=3 for each group. Mean±S.D., ^#P<0.05 versus WT naive, ^##P<0.01 versus WT naive,*P<0.05 versus KO naive, **P<0.01 versus KO naive, ^ΔP<0.05 WT versus KO, ^ΔΔP<0.01 WT versus KO

Figure 4

Cytokine expression in the peritoneal lavage and macrophages. (a) The levels of cytokines in the peritoneal lavage. The levels of MCP-1, TNF-α, IL-6 and IL-12 in the peritoneal exudates from WT mice or PD-1^−/− (KO) mice were detected by ELISA at 4, 48 and 72 h after zymosan injection. (b) The expression of relative mRNA of M1/M2 markers in the peritoneal macrophages. The expression of genes of cytokines (TNF-α, IL-6, MCP-1, IL-1β, iNOS, CD206, Arg-1 and IL-10) in the peritoneal macrophages from WT mice or PD-1^−/− (KO) mice were sorted by flow cytometry in gate of CD11b⁺F4/80⁺ after zymosan injection for 4, 48 and 72 h, and then analyzed by RT-qPCR. n=3 for each group. Mean±S.D., ^#P<0.05 versus WT naive, ^##P<0.01 versus WT naive,*P<0.05 versus KO naive, **P<0.01 versus KO naive, ^Δ P<0.05 WT versus KO, ^ΔΔ P<0.01 WT versus KO

Figure 5

The expression of PD-1 and cytokine mRNA in BMDMs induced by zymosan. (a) The expression of PD-1 on BMDMs induced by zymosan. BMDMs from WT mice were activated with zymosan for 4, 12, 24 and 48 h separately to stimulate TLR2. Cells were stained with PE-conjugated anti-mouse PD-1 antibody, and analyzed by flow cytometry. Data are presented as the percentage of positive cells. n=3 for each group. Mean±S.D., *P<0.01 versus untreated control. (b) The levels of cytokine genes in BMDMs induced by PD-L1 in the presence of zymosan. BMDMs from WT mice were pretreated with the indicated concentrations of rmB7-H1 or hIgG for 2 h and stimulated with 20 μg/ml zymosan for 12 h. RT-PCR was performed to analyze iNOS, IL-6, TNF-α, MCP-1, Arg-1 and IL-10 gene transcription

Figure 6

(a) The expression of MCP-1 mRNA in BMDMs induced by zymosan via PD-1-specific signaling. MCP-1 mRNA in zymosan-stimulated BMDMs after PD-1 was stimulated with/without antagonistic anti-PD-1 mAb. (b) Effect of PD-1 engagement on signaling pathway responsible for MCP-1 gene expression. BMDMs were pretreated with the indicated inhibitors for 30 min and stimulated with 20 μg/ml zymosan for an additional 12 h in the presence or absence of various signaling inhibitors. Total RNA was prepared and analyzed by RT-qPCR for mouse MCP-1 or GAPDH. LY (PI3K inhibitor); PD (ERK inhibitor); SP (JNK inhibitor); SB (p38 inhibitor). n=3 for each group. Mean±S.D., *P<0.05 versus untreated control, **P<0.01 versus untreated control, #P<0.01 versus zymosan-treated group. (c) PD-1 deficiency influences the polarization of peritoneal macrophages via signal transducer and activator of transcription1 (STAT1), nuclear factor kappa-B (NF-κB) and STAT6. Western immunoblot for phosphorylated p-STAT1, STAT1, p-NF-κB, NF-κB, p-STAT6 and STAT6 in macrophages stimulated with zymosan injection for 4 h, n=3. (d) Densitometry analysis for p-STAT1/STAT1, p-NF-κB/ NF-κB and p-STAT6/ STAT6 relative to β-actin. n=3 for each group. Mean±S.D. (e) BMDMs were cultured with 20 μg/ml zymosan for 12 h and stimulated with 5 μg/ml rmB7-H1 at the indicated time. Cells were lysed in lysis buffer. The cell lysates were immunoprecipitated overnight with 5 μg/ml of anti-mPD-1, and protein A/G-agarose was used for precipitation. The suspended pellets were subjected to western blotting with anti-PD-1, anti-phosphotyrosine, anti-SHP-2 and anti-SHP-1 antibodies. n=3 for each group. Mean±S.D., *P<0.05 versus untreated control, **P<0.01 versus untreated control