Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis

Abstract

Background: Influenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood.

Methods: COPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole.

Results: The present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV_20,50/FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1β attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1β/STAT1 signaling via MTs.

Conclusions: These findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1β/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD.

Keywords: Apoptosis; Chronic obstructive pulmonary disease; Influenza virus; Interleukin-1β; Macrophage polarization; Melatonin.

Fig. 1

The regulatory impacts of melatonin on lung function of AECOPD mice induced by IAV infection. The chronic obstructive pulmonary disease (COPD) mouse model was established via cigarette smoke (CS) exposure for consecutive 24 weeks, and AECOPD mouse model was established through 1-week influenza virus A/H3N2 infection, verified through lung function detection. Individual values of total lung capacity (TLC) and functional residual capacity (FRC) (a), volume expired in first 20 and 50 ms of fast expiration (FEV₂₀, FEV₅₀) (b), static lung compliance (Cchord) (c), inspiratory time (Ti) and expiratory time (Te) (d), peak inspiratory flow (PIF) (e), and minute volume (MV) (f) from air group, COPD (smoke) group, AECOPD (smoke + H3N2) group, AECOPD + melatonin (Mel, 30 mg/kg) group. Data expressed as mean ± SD (n = 6). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001

Fig. 2

The protective effects of melatonin on lung damages of AECOPD mice. a Representative lung morphology of mice from air group, COPD (CS exposure) group, AECOPD (smoke + H3N2) group, AECOPD + melatonin (Mel, 30 mg/kg) group. Representative pulmonary alveoli and bronchial photomicrographs of mouse lung tissues in H&E-stained sections (b), individual values of lung injury scores (c), alveolar septal thickening scores (d) and the mean linear intercept (Lm) (e) of alveolar from each group (original magnification × 50, × 250 and × 500). Western blot analysis of the expression of MT1/2 (f), IAV-NP, Caspase1 (g) to GAPDH in lung tissue homogenates from each group. Data expressed as mean ± SD (n ≥ 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001

Fig. 3

The effects of melatonin on abnormal polarization of AMs induced by cigarette exposure combined with IAV/H3N2 infection. a Voltage-gated strategy of flow cytometry analysis to identify alveolar macrophages (AMs) (CD45⁺Siglec-F⁺CD11c⁺) as well as CD86⁺ AMs (M1 type) and CD206⁺ AMs (M2 type) in BALF from from air group, COPD (smoke) group, AECOPD (smoke + H3N2) group, AECOPD + melatonin (Mel, 30 mg/kg) group. Individual percentages of Siglec-F + CD11c + AMs (b), CD86 + AMs (c), CD206 + AMs (d) and CD86 + CD206 + AMs (e) in BALF from each group. Data expressed as mean ± SD (n = 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001

Fig. 4

The effects of melatonin on abnormal polarization of pulmonary macrophages in AECOPD. a, b Mouse lung tissue sections from each group were probed with the specific antibody against pulmonary macrophage marker F4/80 (red) and co-probed with antibodies against M1 marker (iNOS) or M2 marker (CD206), and, representative lung immunofluorescence staining of lung tissue sections were shown (original magnification × 100 and × 500). c, d Western blot analysis of the expression of total-STAT1, phospho-STAT1, total-STAT6, phospho-STAT6, iNOS and Arg1 to β-tublin in lung tissue homogenates from air group, COPD (CS exposure) group, AECOPD (smoke + H3N2) group, AECOPD + melatonin (Mel, 30 mg/kg) group. Data expressed as mean ± SD (n ≥ 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 5

The effects of IAV infection on the polarization of CSE-stimulated Raw264.7 cells. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) measurements of the relative mRNA levels of MCP1 (a), TNF-α (b), IL-1β (c), Arg1 (d), melatonin receptor 1 (MT1) (e) and MT2 (f) in CSE-stimulated Raw264.7 cells infected by influenza A/H3N2 (MOI = 2, infection for 3 h, 6 h, 12 h, 18 h and 24 h). Quantitative RT-PCR measurements of the relative mRNA levels of MCP1 (g), TNF-α (h), IL-1β (i), Arg1 (j), fizz1 (k) and IL-4 (l) in Raw264.7 cells from PBS group, CSE stimulation group, H3N2 infection group, CSE stimulation plus H3N2 infection group. m, n Flow cytometry analysis to the M1 polarized levels (CD86+) of Raw264.7 cells from each group. Data expressed as mean ± SD (n ≥ 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001

Fig. 6

The effects of melatonin abnormal polarization of IAV-infected CSE-stimulated macrophages. a-c Western blot analysis of the expression of total-STAT1, Phospho-STAT1, total-STAT6, Phospho-STAT6 and IAV-NP to GAPDH as well as iNOS, Arg1 and MT1/2 to GAPDH in CSE-stimulated Raw264.7 cells infected by IAV/H3N2 infection (MOI = 2, 12 h) with/without melatonin pretreatment (10 μM, 100 μM and 200 μM, 3 h before H3N2 infection). d Representative Immunofluorescence images of iNOS (green) and Arg1 (red) expression in Raw264.7 cells infected by H3N2 infection (MOI = 2, 12 h) with/without melatonin pretreatment (200 μM, 3 h before H3N2 infection) (bar = 10 μm, original magnification × 630). e Quantitative RT-PCR measurements of the relative mRNA levels of MCP1, TNF-α, Arg1 and Fizz1 in CSE-stimulated Raw264.7 cells. Data expressed as mean ± SD (n = 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001

Fig. 7

The effects of melatonin receptors on the regulatory impacts of melatonin. Representative Immunofluorescence images of IAV-NP (green) (a), iNOS (green) and MT1/2 (red) (b) expression in CSE-stimulated Raw264.7 cells infected by influenza A/H3N2 infection (MOI = 2, 12 h) with/without melatonin pretreatment (200 μM) or combined pretreatment of melatonin and luzindole (10 μM, 3 h before H3N2 infection) (bar = 10 μm, original magnification × 630). c, d Western blot analysis of the expression of total-STAT1, Phospho-STAT1, MT1/2, iNOS and IAV-NP to GAPDH in CSE-stimulated Raw264.7 cells infected by influenza A/H3N2 infection. e Quantitative RT-PCR measurements of the relative mRNA levels of IL-1β, MCP1 and TNF-α in CSE-stimulated Raw264.7 cells. Data expressed as mean ± SD (n ≥ 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001

Fig. 8

The effects of IL-1β on the regulatory impacts of melatonin. a Quantitative RT-PCR measurements of the relative mRNA levels of IL-1β, IL-6, IL-18, MCP1, TNF-α and IFN-γ in CSE-stimulated Raw264.7 cells infected by IAV/H3N2 infection (MOI = 2, 12 h). Representative Immunofluorescence images of IAV-NP (green) (b), iNOS (green) and MT1/2 (red) (c) expression in CSE-stimulated Raw264.7 cells infected by H3N2 infection (MOI = 2, 12 h) with/without melatonin pretreatment (200 μM) or combined pretreatment of melatonin and re-IL-1β (10 μg/ml) or VX765 (50 μM) (bar = 10 μm, original magnification × 630). d, e Western blot analysis of the expression of total-STAT1, phospho-STAT1, iNOS and IAV-NP to GAPDH as well as Caspase1 and Caspase1 p10 to β-Tublin in Raw264.7 cells. f Quantitative RT-PCR measurements of the relative mRNA levels of MCP1, TNF-α and IL-1β in Raw264.7 cells. Data expressed as mean ± SD (n = 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001

Fig. 9

The effects of melatonin on macrophage apoptosis. a Western blot analysis of the expression of Caspase3 to GAPDH in CSE-stimulated Raw264.7 cells infected by IAV/H3N2 (MOI = 2, 12 h) with/without melatonin pretreatment (10 μM, 100 μM and 200 μM, 3 h before H3N2 infection). b, c Western blot analysis of the expression of Caspase3 to β-Tublin in CSE-stimulated Raw264.7 cells infected by H3N2 infection with/without melatonin pretreatment (200 μM) or combined pretreatments of melatonin and luzindole (10 μM) or re-IL-1β (10 μg/ml) or VX765 (50 μM). d, e Flow cytometry analysis of Annexin V⁺ and Propidium Iodide (PI⁺) was done to identify apoptosis in Raw264.7 cells. Data expressed as mean ± SD (n = 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001