Experimental animals

Male C57BL/6 mice (8 weeks old) were housed in the Laboratory Animal Research Center of Anhui Medical University under standard specific pathogen-free condition with 12-h light/12-h dark cycles at 22 ± 2 °C, and free access to the standard laboratory rodent diets and water during modeling experiments. All experimental mice were treated according to the protocols approved by the Animal Care and Ethics Committee of Anhui Medical University (Approval no.20210056, no.LLSC20221233).

Influenza A/H3N2 amplification and plaque assay

Influenza virus A/Anhui/1/2017 (H3N2) was obtained from Prof. Yan Liu (Department of Microbiology, Anhui Medical University, China), and isolated from the patients infected influenza A/H3N2 in 2017, and used in laboratory studies strictly according to standard bio-safety operation practices. The IAV samples were amplified in Madin-Darby canine kidney (MDCK) cells and specific pathogen-free embryonated chicken eggs. Virus titers were assayed via the standard plaque assay on MDCK cells according to previous description [18, 29]. MDCK cells were infected with diluted virus samples 1–2 h at 37 °C. After washed with PBS, the cultivation was proceeded in the plaque medium containing 50% 2 × DMEM, 50% avecil (2.35%) and N-acetyl trypsin (1.5 μg/ml) for 72 h. Then, the cells were stained with Neutral Red stain, and plaques were counted for the calculating virus titers. All experiments involved in viruses were performed according to the bio-safety level two requirements with well-equipped personal protection for all the researchers.

Animal models of AECOPD induced by influenza A/H3N2 virus

Mice were randomly divided into 4 groups (10 mice each group): 1) air group; 2) cigarette smoke (CS) exposure-induced COPD group; 3) IAV/H3N2 infection-induced acute exacerbation of COPD (AECOPD) group; 4) AECOPD + melatonin group. To establish COPD model, mice were exposed whole bodies to CS in a passive smoking chamber (70 cm × 40 cm × 60 cm) with a house directing flow inhalation and CS-exposure system containing in a laminar flow and CS extraction units. Regular CS exposure was proceeded with 10 cigarettes per run, twice/day, 6 days/week for up to 24 weeks. Control mice were exposed to normal air. After 24 h of final CS exposure, mice were anesthetized with 1% sodium pentobarbital (50 mg/kg, ip) free from pain for invasive trachea cannula. To establish AECOPD models, mice were atomized intratracheally with 50 μl IAV/H3N2 stock (100 plaque forming units, PFUs) on day 0 and day 3, while the air mice were atomized intratracheally with 50 μl saline. From day 0, mice were injected intraperitoneally with saline or melatonin (Mel) in 5% DMSO (30 mg/kg) (C13H16N2O2, stated purity ≥ 98%, M5250, Sigma-Aldrich, USA) at daily 18:00 for consecutive 7 days.

Lung function detection

After anesthesia with an intraperitoneal injection of 1% pentobarbital (Injection dose (ml) = mouse body weight (g) × 4/1000), mice were tracheostomized and placed in a whole-body plethysmograph of PFT Pulmonary Maneuvers (DSI Buxco, Minnesota, USA). Total lung capacity (TLC), functional residual capacity (FRC), static lung compliance (chord compliance, Cchord) were measured from the quasi-static pressure-volume (PV) maneuver. Forced vital capacity (FVC), volume expired in first 20 and 50 ms (ms) of fast expiration (FEV20, FEV50) were measured from the fast-flow volume (FV) maneuver. Inspiratory time (Ti), expiratory time (Te), peak inspiratory flow (PIF) and minute volume (MV) were recorded during resistance and compliance (RC) maneuver.

Histological analysis and Lm evaluation

Mice were euthanatized by high-dose 1% sodium pentobarbital (100 mg/kg, ip), the left lung lobes were dissected without proceeding bronchoalveolar lavage, and fixed with 4% paraformaldehyde, and embedded in paraffins. Four micrometers sections were stained with haematoxylin and eosin (H&E) for evaluating the severities of lung injury and emphysema. The indexes of lung injury were double-blindly calculated according to the scoring system including five histological features: a. neutrophils in the alveolar space; b. neutrophils in the interstitial space; c. hyaline membranes formation; d. proteinaceous debris filling the airspace; e. alveolar septal thickening. Each item was scored 0, 1, or 2 based on the severity of lung injury. The final injury scores were figured up according to the following formula [30],

$$\mathrm\hspace=\hspace[(20\hspace\times \hspace})\hspace+\hspace(14\hspace\times \hspace})\hspace+\hspace(7\hspace\times \hspace})\hspace+\hspace(7\hspace\times \hspace})\hspace+\hspace(2\hspace\times \hspace})] / (\mathrm\hspace\times \hspace100).$$

Pulmonary emphysema is evaluated by measuring the mean linear intercept (Lm) which shows interalveolar septal wall distances. The ocular micrometer with 5 lines (each 500 μm long) is utilized to determine Lm of alveolars (avoiding the fields with airways or vessels).

BALF collection and leukocytes counting

After complete anesthesia with 0.2 ml 1% sodium pentobarbital (100 mg/kg, ip), bronchoalveolar lavage was performed with 2 ml sterile PBS via an endotracheal tube. The bronchoalveolar lavage fluids (BALF) were centrifuged at 4 ºC, 1000 rpm for 10 min. Cell pellets were re-suspended in PBS with Red Blood Cell (RBC) Lysis Buffer (C3702, Beyotime technology, Shanghai, China), for total leukocytes counting using a hemocytometer. Then, smeared BAL cells were stained with Wright-Giemsa stain solution (Baso Diagnostics Inc, Zhuhai, China).

Flow cytometry analysis of BALF cells

BAL cells were treated with RBC lysis buffer, and stained with the following fluorochrome-conjugated antibodies to screen alveolar macrophages (AMs): CD45 (APC-CyTM7, 561037, BD Biosciences, USA), CD11c (PerCP-CyTM5.5, 561114, BD), Siglec-F (BV421, 565934, BD). For investigating the effects of melatonin on AMs polarization, BALF cells were stained with M1 macrophage marker CD86 (PE, 561963, BD) and M2 marker CD206 (MR6F3 APC, ThermoFisher Scientific, USA). Specifically, before transmembrane protein CD206 stained, BALF cells were fixed and permeabilized for better intracellular staining. The images and data of flow cytometry were collected using LSRFortessa X30 (BD Biosciences, USA).

Cigarette smoking extract (CSE) preparation

In brief, one burning cigarette (Marlboro Red Label, Longyan Tobacco Industrial co. LTD, Fujian, China) without the filter is sucked at a continuous steady flow-rate (8 ml/s, 40 ml cigarette smoking) into a 50 ml syringe containing 10 ml PBS, and mixing upside down with PBS for 1 min, repeated for 5 times so that the cigarette just finished burning. Next, adjust its pH to 7.4 and filter it with a 0.22 µm filter (Millipore, Bedford, MA). The final concentration is considered as 100%. The obtained CSE was packaged and stored in the -80℃ refrigerator for using in cell experiments.

Cell culture and treatment

Raw264.7 macrophage lines were purchased from Cell Bank of Shanghai Institutes for Biological Sciences (China Academy of Science, Shanghai, China). Raw264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) with 10% foetal bovine serum (FBS) (Excell biology) at 37 °C under saturated humidity conditions containing 5% CO2. When growing to 60–70% confluence, according to previous studies and cell viability analysis [31,32,33], 3% CSE and IAV (Multiplicity of Infection, MOI = 2), melatonin (10 μM, 100 μM and 200 μM), luzindole (10 μM) and VX765 (50 μM) and IL-1β (10 ng/ml) were used in this experiment.

Immunofluorescence staining in vivo and in vitro

The polarization of mouse pulmonary macrophages were detected through Immunofluorescence homologous double-labeling staining of 4 μm paraffin sections. The sections were stained with corresponding anti-rabbit primary antibodies: iNOS (1:1000, ab178945, abcam, USA) and CD206 (1:1000, ab300621, abcam) after antigen retrieval and BSA blocking. After reacted with HRP conjugated Goat Anti-Rabbit IgG, the sections were eluted with tyramide signal amplification (TSA) dye, and processed with microwaves. Next, the sections were stained with the anti-rabbit second antibodies: F4/80 (1:500, GB113373, Servicebio, Wuhan, China), and then stained with Cy3 conjugated goat anti-rabbit IgG, followed by nuclei staining with DAPI. Fluorescent images of the sections were captured by the laser-scanning confocal microscope (Zeiss LSM880, Carl Zeiss AG, Germany).

Raw264.7 cells were fixed with 4% paraformaldehydes, followed by permeabilization and blocking with 5% bull serum albumin (BSA). Then, the cells were incubated with primary antibodies against anti-rabbit Arg1 (1:50, #93668S, Cell signaling technology), anti-rabbit iNOS (1:200, ab178945, abcam), anti-mouse MT-1/2 ((1:50, sc-398788, Santa Cruz, USA) and anti-mouse influenza A nucleoprotein (IAV-NP) (1:100, sc-101352, Santa Cruz) overnight at 4 ℃. After the cells reacting with corresponding second antibodies (1:500, Alexa Fluor® 488 goat anti-rabbit IgG and Alexa Fluor® 594 goat anti-mouse IgG, Abcam), anti-fade Mounting Medium with DAPI (P0131, Beyotime technology) was applied to visualize nuclei. Fluorescent images were gained by the laser-scanning confocal microscope (Zeiss LSM880).

Apoptosis detection

The apoptosis levels of Raw264.7 cells were detected using Annexin V-FITC/PI apoptosis detection kit (40302, Yeasen, Shanghai, China). After digested with trypsin without EDTA, the treated cells were collected and resuscitated with 100 μl 1 × Binding Buffers. And, 5 μl Annexin V-FITC and 10 μl PI Solution were added to stain apoptotic cells. After incubating 10 min avoiding lights, 400 μl 1 × Binding Buffers were added and the ratio of apoptotic cells (Annexin V+/PI+) was detected using LSRFortessa X30 (BD Biosciences).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated with Trizol reagents (Invitrogen, USA). Reverse transcription was conducted using a 5 × Hieff™ One Step RT SuperMix with gDNA Remover (Yeasen, Shanghai, China) according to the manufacturer instruction. RT-PCR was performed using a Hieff™ Universal qPCR SYBR Master Mix (Yeasen). All samples were assayed in triplicates, and the target gene expression was normalized to β-actin. Relative mRNA expression was calculated with the 2−∆∆Ct method. The specific primers for β-actin, melatonin receptor (MT) 1, MT2, IL-1β, Tumor Necrosis Factor α (TNF-α), monocyte chemoattractant protein 1 (MCP1), Arg1, Found in inflammatory zone 1 (Fizz1), IL-6, IL-18 and interferon γ (IFN-γ) were generated by Tsingke Biotech, Beijing, China. The primer sequences were listed in Supplementary Table 1.

Western blot analysis

Total proteins were extracted with RIPA lysis containing protease inhibitors from mouse lung tissues and Raw264.7 cells. And, protein samples were separated through 10–13% SDS-PAGE, transferred to PVDF membranes. The membranes were incubated with primary antibodies: total-STAT1 (1:1000, PTM-5754, PTM Bio, Hangzhou, China), Phospho-STAT1 (1:1000, 340797, ZenBio, Chengdu, China), total-STAT6 (1:1000, 380957, ZenBio), Phospho-STAT6 (1:500, sc-136019, Santa Cruz), iNOS (1:1000, ab178945, Abcam), Arg1 (1:1000, #93668S, Cell signaling technology), Caspase1 (1:500, sc-392736, Santa Cruz), MT1/2 (1:500, sc-398788, Santa Cruz), β-Tublin (1:5000, M20005, Abmart, Shanghai, China) and GAPDH (1:5000, ab181602, Abcam) overnight at 4℃. After incubated with Goat Anti-Rabbit IgG second antibody (1:5000, M21003, Abmart), the membranes were visualized by Odyssey infrared imaging system (Tanon, Shanghai, China).

Statistical analysis

All experiments were randomized and blinded. All results were presented as mean ± SD from at least three independent samples or biological replicates (n ≥ 3). Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA). Student’s t tests were performed for comparisons between two different groups. One-way ANOVA with Bonferroni’s post hoc tests (for equal variance) or Dunnett’s T3 post hoc tests (for unequal variance) were performed for comparisons among multiple groups. *p < 0.05 was considered statistically significant.