CD274 (PD-L1) negatively regulates M1 macrophage polarization in ALI/ARDS

Abstract

Background: Acute lung injury (ALI)/severe acute respiratory distress syndrome (ARDS) is a serious clinical syndrome characterized by a high mortality rate. The pathophysiological mechanisms underlying ALI/ARDS remain incompletely understood. Considering the crucial role of immune infiltration and macrophage polarization in the pathogenesis of ALI/ARDS, this study aims to identify key genes associated with both ALI/ARDS and M1 macrophage polarization, employing a combination of bioinformatics and experimental approaches. The findings could potentially reveal novel biomarkers for the diagnosis and management of ALI/ARDS.

Methods: Gene expression profiles relevant to ALI were retrieved from the GEO database to identify co-upregulated differentially expressed genes (DEGs). GO and KEGG analyses facilitated functional annotation and pathway elucidation. PPI networks were constructed to identify hub genes, and differences in immune cell infiltration were subsequently examined. The expression of hub genes in M1 versus M2 macrophages was evaluated using macrophage polarization datasets. The diagnostic utility of CD274 (PD-L1) for ARDS was assessed by receiver operating characteristic (ROC) analysis in a validation dataset. Experimental confirmation was conducted using two LPS-induced M1 macrophage models and an ALI mouse model. The role of CD274 (PD-L1) in M1 macrophage polarization and associated proinflammatory cytokine production was further investigated by siRNA-mediated silencing.

Results: A total of 99 co-upregulated DEGs were identified in two ALI-linked datasets. Enrichment analysis revealed that these DEGs were mainly involved in immune-inflammatory pathways. The following top 10 hub genes were identified from the PPI network: IL-6, IL-1β, CXCL10, CD274, CCL2, TLR2, CXCL1, CCL3, IFIT1, and IFIT3. Immune infiltration analysis revealed a significantly increased abundance of M1 and M2 macrophages in lung tissue from the ALI group compared to the control group. Subsequent analysis confirmed that CD274 (PD-L1), a key immunological checkpoint molecule, was highly expressed within M1 macrophages. ROC analysis validated CD274 (PD-L1) as a promising biomarker for the diagnosis of ARDS. Both in vitro and in vivo experiments supported the bioinformatics analysis and confirmed that the JAK-STAT3 pathway promotes CD274 (PD-L1) expression on M1 macrophages. Importantly, knockdown of CD274 (PD-L1) expression potentiated M1 macrophage polarization and enhanced proinflammatory cytokines production.

Conclusion: This study demonstrates a significant correlation between CD274 (PD-L1) and M1 macrophages in ALI/ARDS. CD274 (PD-L1) functions as a negative regulator of M1 polarization and the secretion of proinflammatory cytokines in macrophages. These findings suggest potential new targets for the diagnosis and treatment of ALI/ARDS.

Keywords: M1 polarization; PD-L1; biomarker; immune infiltration; inflammation; macrophage; severe acute respiratory distress syndrome; stat3.

Figure 1

Workflow chart for this study.

Figure 2

Identification of DEGs and functional enrichment analyses. (A) Volcano plot of GSE 2411. (B) Volcano plot of GSE 18341. (C) Venn diagrams of co-upregulated DEGs. (D) Bubble plots of GO analysis. (E) Bubble plots of KEGG analysis.

Figure 3

Construction of PPI network and identification of hub genes. (A) PPI network of co-upregulated DEGs. (B) Genes in the module with the highest MCODE value. (C) Top 10 hub genes.

Figure 4

Immune cell infiltration analysis. (A) Immune cell infiltration analysis in GSE2411. (B) Immune cell infiltration analysis in GSE18341.

Figure 5

Validation of hub genes expression in macrophage polarization-related datasets, validation of the diagnostic value of CD274 for ARDS, and GSEA of CD274. (A) Validation of hub genes expression in GSE61298. (B) Validation of hub genes expression in GSE57614. (C) Evaluation of the diagnostic value of CD274 for ARDS using ROC curves in GSE76293. (D–I) GSEA of CD274.

Figure 6

Verification of CD274 (PD-L1) expression in M1 macrophage models through LPS stimulation (500 ng/ml) for 12 hours. (A, B) qPCR analysis of M1 macrophage markers (CD86 and NOS2) in RAW264.7 cells, under control and LPS conditions. (C–E) qPCR analysis of M2 markers (CD206, ARG1, and TGF-β) in RAW264.7 cells under control and LPS conditions. (F) qPCR analysis of CD274 mRNA levels in RAW264.7 cells under control and LPS conditions. (G) Western blotting analysis of PD-L1 and iNOS protein expression in RAW264.7 cells under control and LPS conditions. (H) Flow cytometric analysis of CD86 mean fluorescence intensity (MFI) on RAW264.7 cells under control and LPS conditions. (I) Western blotting of PD-L1 and iNOS protein levels in BMDMs, under control and LPS conditions. (J) Flow cytometry displaying CD86 MFI on F4/80⁺ BMDMs, under control and LPS conditions. (ns, not significant, ***p < 0.001).

Figure 7

Verification of CD274(PD-L1) expression in ALI mice by intraperitoneal LPS (10 mg/kg) for 12 hours. (A) The degree of pulmonary edema was assessed in both the control and ALI groups using the lung Wet/Dry ratio. (B) Pathological changes in lung tissue were detected in both the control and ALI groups using H&E staining. scale bar =100 μm. (C, D) qPCR analysis of CD274 and NOS2 mRNA expression in lung tissues from control and ALI groups. (E) qPCR analysis of CD274 mRNA expression in peripheral blood from control and ALI groups. (F) Western blotting analysis of PD-L1 and iNOS protein expression in lung tissues from control and ALI groups. (G) The expression of PD-L1 in lung tissues from control and ALI groups were analyzed by IHC. scale bar =100 μm. (***p < 0.001).

Figure 8

The JAK-STAT3 pathway upregulates CD274 (PD-L1) expression in LPS-induced M1 macrophages. CD274 (PD-L1) negatively regulates M1 polarization and the production of proinflammatory cytokines in BMDMs induced by LPS. (A) Protein expressions of JAK1,2-STAT3 signaling molecules were determined in BMDMs from both the control and LPS groups by Western blotting. (B) Protein expressions of pSTAT3 and STAT3 were determined in lung tissues from control and ALI groups by Western blotting. (C) Protein expressions of PD-L1, pSTAT3, STAT3, and iNOS were analyzed in BMDMs pretreated with Stattic (2.0 μM) for 1 hour and then stimulated with LPS (500 ng/mL) for 12 hours by Western blotting. (D) Flow cytometry displaying CD86 MFI on F4/80⁺ BMDMs that were pretreated with Stattic and then stimulated with LPS. (E, F) mRNA expression of CD274 and NOS2 was analyzed by qPCR in BMDMs that were transfected with CD274 siRNA (siCD274-1 and siCD274-2) or negative control siRNA (siNC), followed by LPS stimulation for 12 hours. (G) Protein levels of PD-L1 and iNOS were analyzed by Western blotting in BMDMs transfected with CD274 siRNA or siNC, followed by LPS stimulation. (H) Flow cytometry displaying CD86 MFI on F4/80⁺ BMDMs, transfected with CD274 siRNA or siNC, followed by LPS stimulation. (I, J) The levels of IL-6 and TNF-α were analyzed by ELISA in the supernatant of BMDMs transfected with CD274 siRNA or siNC, followed by LPS stimulation (*p < 0.05, ***p < 0.001).