Blockade of PD-1 decreases neutrophilic inflammation and lung damage in experimental COPD

Abstract

Chronic obstructive lung disease (COPD) and lung cancer are both caused by smoking and often occur as comorbidity. The programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis is an important canonic immunoregulatory pathway, and antibodies that specifically block PD-1 or PD-L1 have demonstrated efficacy as therapeutic agents for non-small cell lung cancer. The role of the PD-1/PD-L1 axis in the pathogenesis of COPD is unknown. Here, we analyzed the function of the PD-1/PD-L1 axis in preclinical COPD models and evaluated the concentrations of PD-1 and PD-L1 in human serum and bronchoalveolar lavage (BAL) fluids as biomarkers for COPD. Anti-PD-1 treatment decreased lung damage and neutrophilic inflammation in mice chronically exposed to cigarette smoke (CS) or nontypeable Haemophilus influenzae (NTHi). Ex vivo stimulated macrophages obtained from anti-PD-1-treated mice released reduced amounts of inflammatory cytokines. PD-L1 concentrations correlated positively with PD-1 concentrations in human serum and BAL fluids. Lung sections obtained from patients with COPD stained positive for PD-L1. Our data indicate that the PD-1/PD-L1 axis is involved in developing inflammation and tissue destruction in COPD. Inflammation-induced activation of the PD-1 pathway may contribute to disease progression.

Keywords: COPD; PD-1; PD-L1; inflammation; lung damage; macrophages.

Figure 1.

Anti-PD-1 treatment decreases CS-induced neutrophilic inflammation. C57BL/6 mice were exposed to CS 5 days a week for 5 days and 4 wk and treated with a PD-1-blocking antibody or an isotype antibody two times a week. Numbers of total immune cells (A), neutrophils (B), macrophages (C), and lymphocytes (D), and concentrations of (E) KC and (F) MIP-2 were determined in BAL fluids 24 h after the final exposure to CS; n = 4–8 mice per group. Two independent experiments for each group. Data were compared by two-way ANOVA with Bonferroni posttest and are shown as means ± SE. **P < 0.01 and ***P < 0.001. BAL, bronchoalveolar lavage; CS, cigarette smoke; PD-1, programmed cell death protein 1.

Figure 2.

Anti-PD-1 treatment decreases CS-induced lung damage. C57BL/6 mice were exposed to CS 5 days a week for 12 wk and treated with a PD-1-blocking antibody or an isotype antibody two times a week. A: lung histology (representative hematoxylin-eosin staining, scale bar = 100 µm), B: mean chord length (MCL), C: respiratory resistance, and D: respiratory system compliance were determined 24 h after the final exposure to CS; n = 5 or 6 mice per group. Two independent experiments for each group. Data were compared by two-way ANOVA with Bonferroni posttest and are shown as means ± SE. **P < 0.01 and ***P < 0.001. CS, cigarette smoke; PD-1, programmed cell death protein 1.

Figure 3.

Numbers of PD-L1⁺ and PD-1⁺ cells are increased in the lungs of CS-exposed mice. C57BL/6 mice were exposed to CS 5 days a week for 12 wk and treated with a PD-1-blocking antibody or an isotype antibody two times a week. Immunohistochemical staining (representative histology, scale bar = 50 µm) for (A) PD-L1 and (B) PD-1 and quantification of the PD-L1⁺ and PD-1⁺ cells in lung parenchyma; n = 5 or 6 mice per group. Two independent experiments for each group. Data were compared by two-way ANOVA with Bonferroni posttest and are shown as means ± SE. *P < 0.05. CS, cigarette smoke; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1.

Figure 4.

Numbers of PD-1-expressing CD4⁺ cells and PD-L1-expressing CD68⁺ cells are increased in the lungs of CS-exposed mice. C57BL/6 mice were exposed to CS 5 days a week for 12 wk and treated with a PD-1-blocking antibody or an isotype antibody two times a week. Double immunohistochemical staining (representative histology, scale bar = 20 µm) for (A) CD4 (red)/PD-1 (green), (B) CD8 (red)/PD-1 (green), and (C) CD68 (red)/PD-L1 (green), and quantification of the cells in lung parenchyma; n = 3 or 4 mice per group. Results are presented for each mouse, were compared by two-way ANOVA with Bonferroni posttest and are shown as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001. CS, cigarette smoke; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1.

Figure 5.

Anti-PD-1 treatment decreases NTHi-induced lung damage. C57BL/6 mice were exposed to NTHi 3 days a week for 4 wk and treated with a PD-1-blocking antibody or an isotype antibody three times a week. Immunohistochemical staining (representative histology, scale bar = 50 µm) for (A) PD-L1, (B) PD-1, (C) lung histology (representative hematoxylin-eosin staining, scale bar = 100 µm), and (D) MCL were determined 24 h after the final exposure to NTHi; n = 4 or 5 mice per group. Two independent experiments for each group. Data were compared by two-way ANOVA with Bonferroni posttest and are shown as means ± SE. ***P < 0.001. MCL, mean chord length; NTHi, nontypeable Haemophilus influenza; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1.

Figure 6.

Anti-PD-1 treatment modulates the inflammatory response of alveolar macrophages. C57BL/6 mice were exposed to CS 5 days a week for 4 wk and treated with a PD-1-blocking antibody or an isotype antibody two times a week. Alveolar macrophages were isolated 2 h after the final exposure to CS and stimulated with inactivated NTHi or control media for 24 h. The concentrations of (A) IL-6, (B) TNF-α, (C) G-CSF, (D) KC, and (E) IL-10 were measured in supernatants. Two independent experiments for each group (n = 6 replicates per group). Data were compared by two-way ANOVA with Bonferroni posttest and are shown as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001. CS, cigarette smoke; NTHi, nontypeable Haemophilus influenza; PD-1, programmed cell death protein 1.

Figure 7.

PD-1 and PD-L1 in serum and BAL fluids collected from patients with COPD. A: association of PD-L1 and PD-1 concentrations in serum collected from patients with stable COPD and patients during AECOPD. B: PD-L1 and PD-1 concentrations in serum collected from patients with stable COPD and during AECOPD. C: association of PD-L1 and PD-1 concentrations in BAL fluids collected from stable COPD patients. D: PD-L1 and PD-1 concentrations in BAL fluids collected from stable GOLD I/II and GOLD III/IV patients with COPD. Data were compared by Mann–Whitney and are shown as means ± SE. *P < 0.05. Correlation analysis was performed using the nonparametric Spearman’s correlation test. E: PD-L1 was detected by immunohistochemistry in formalin histological-fixed paraffin-embedded human lung samples from patients with end-stage COPD (representative histology, scale bar = 50 µm). BAL, bronchoalveolar lavage; COPD, chronic obstructive lung disease; GOLD, Global Initiative for Chronic Obstructive Lung Disease; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1.