Melatonin (purity > 98%) was purchased from Lianshuo biotechnology Co., Ltd. (Shanghai, China). Urethane, LPS and filipin were purchased from Sigma Chemical Co (St. Louis, MO, USA). Anti-F4/80-coated beads were purchased from BioCep (Israel). Antibodies used included anti-mouse-arginase, anti-mouse-iNOS, anti-mouse-F4/80, anti-mouse-CD8, anti-mouse-Foxp3, Cy3-conjugated anti-mouse CD86, PE-conjugated anti-mouse CD163 and FITC-conjugated goat anti-mouse IgG were obtained from BD Pharmingen or Proteintech (Shanghai China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG polyclonal antibody and peroxidase substrate DAB (3, 3′-diaminobenzidine) were obtained from Nichirei Bioscience (Tokyo, Japan).WP1066, DCFH-DA ,MβCD and LY294002 were obtained from Beyotime (Shanghai China). Mouse quantitative ELISA kits (IFN-γ, IL-12, IL-10, TGF-β1, arginase 1 and ROS) were obtained from R&D Systems. L-arginine and nitric oxide assay kits were obtained from Nanjing Jiancheng Bioengineering institute. Standard rodent chow was purchased from Henan Provincial Medical Laboratory Animal Center (Zhengzhou, China), License No. SCXK (YU) 2015-0005, Certificate No. 41000100002406. Liposome-encapsulated clodronate (LEC) was prepared as described previously. 
Cell Culture and Assay
Raw264.7 macrophages and the Lewis lung carcinoma (LLC) cells were from ATCC, purchased from the Chinese Academy of Sciences and grown in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 and 95% air at 37℃. Raw264.7 macrophages were seeded in 24-well plates and stimulated with by 10 ng/ml LPS or 10 ng/ml IL-10 for 24 h to obtain M1-like (M1) and M2-like (M2) macrophages. To collect cell-conditioned media, M1-like or M2-like cells were cultured in serum-free medium for 24 h, centrifugated to remove the cells and further filtered to remove debris for supernatant collection as M1 and M2 cell-conditioned media(M1-CM and M2-CM,respectively). The supernatant levels of IFN-γ, TNF-α, NO, PD-L1, IL-10 and TGF-β1 were determined by ELISA kits, according to the manufacturer’s protocols. The results were calculated from linear curves obtained by using the Quantikine kit standards.
For proliferation analysis, LLC cells at 1 × 105 cells/mL were seeded in a 96-well plate and treated with M1 or M2 cell-conditioned media for 48 h, M1-like or M2-like cells were also treated with LPS or melatonin alone or in combination for 7 d (changing the medium every 2 days), and living cells were examined by MTT reduction assay, according to our previous method. For the morphological assessment, the cells were analyzed by a Laser holographic cell imaging and analysis system (HoloMonitor M4, Phiab, Sweden). For phagocytic ability assessment, neutral red phagocytosis was detected. For autophagy analysis, the cells were stained using PE-conjugated anti-LC3-B or anti-p62 antibodies. For apoptosis analysis, the binding of ANXV-FITC to phosphatidylserine was measured by an automated cell counter and analysis system (Nexcelom Cellometer X2, Nexcelom, USA). For ROS measurement, intracellular fluorescence of DCFH-DA was detected by a fluorescence spectrophotometer(F4600, Hitachi,Japan). For lipid raft detection, cells were then stained with filipin (0.05 mg/ml) for plasma membrane cholesterol, respectively according to conventional methods.
Western blot analysis
M1-like and M2-like macrophages were treated with LPS or melatonin alone or in combination in the presence or absence of MβCD ( a lipid raft inhibitor),WP1066 (a selective JAK2/STAT3 inhibitor) or LY294002 (a PI3k/AKT inhibitor) for 48 h. The protein was extracted in cell lysis buffer, and equal amounts of protein were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose membranes, and probed with antibodies against PD-1, PD-L1, JAK2, pAKT, and Stat3. Antibody binding was detected via enhanced chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL). Band density was quantified using ImageJ software (NIH, Bethesda, MD, USA) and normalized to the corresponding control group.
Ten-week-old female ICR mice were obtained from Henan Provincial Medical Laboratory Animal Center. All mice were housed in individual ventilated cages (lights on 7:00 AM to 7:00 PM). Animals were fed standard rodent chow and water. All animal procedures were approved by the Animal Experimentation Ethics Committee of Henan University (permission number: HUSAM 2016 − 288), and all procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals and the Regulation of the Animal Protection Committee to minimize suffering and injury. Animals were euthanized via carbon dioxide overdose based on experimental needs.
LLC cell immune clearance
LLC cell immune clearance was assessed using a calcein-release assay, according to our previous method. Briefly, NK cells (DX5+) were purified from the ICR mouse spleens using the MACS separation system (Miltenyi Biotec, Bergisch Gladbach, Germany), stimulated with IL-2 (10 ng/ml) for 24 h in the presence of M1-like or M2-like cell-conditioned media and harvested as attacking cells. Mitomycin C-treated LLC cells were labeled with 10 µM calcein-AM as target cells and were placed into a 96-well plate with CD8 + T cells at 100:1, 50:1, and 25:1 (NK cells:LLC cells) ratios for 6 h at 37 °C. The supernatants were transferred from each well to another 96-well plate, and the fluorescence was measured using a Synergy2 multimode microplate reader (BIO-TEK). Maximum release was obtained from detergent-released LLC cells, and spontaneous release was obtained from LLC cells incubated in the absence of CD8 + T cells (n = 5).
Immune clearance was determined as follows:
Immune clearance = (experimental release – spontaneous release) / (maximum release − spontaneous release) × 100%.
To detect how the different macrophage phenotypes affect NK cells, NK cells were cultured in the lower chamber at a concentration of 5 × 106 cells/ml and were stimulated with IL-2, and M1-like or M2-like cells were added in the upper chamber at 2 × 106 cells/ml in the presence or absence of anti-IL-10, anti-TGF-β1, MβCD, WP1066 or LY294002. After coincubation for 24 h at 37 °C, the supernatant was centrifugated for the PD-L1 assay, adherent cells in the upper compartment were removed by a cotton swab, and the filter inserts were incubated in medium supplemented with 5 mg/ml DAPI for 30 min at 37 °C and analyzed for cell migration using an inverted fluorescence microscope. NK cells in the lower chamber were collected for surface PD-1 receptor assay using PE-conjugated anti-PD-1 antibody.
The cytolytic activity of LLC cells was assessed by CFSE-7AAD staining. Briefly, LLC cells were incubated with CFSE -labeled M0, M1-like or M2-like cells at 20:1 and 10:1 (M0, M1 or M2 cells: LLC cells) ratios for 6 h. Then, 7AAD was added to the cell suspensions and incubated on ice for 15 min. The percentages of 7AAD + cells among CFSE + cells were analyzed using an automated cell counter and analysis system.
Urethane-induced lung carcinogenesis model
Urethane (600 mg/kg body weight), alone or in combination with liposome-encapsulated clodronate (LEC, 4 mg/mouse) was injected intraperitoneally (i.p.) into ICR mice once a week for eight weeks, according to our previous protocol. Following the first urethane injection, mice received melatonin (20 mg/kg/day) via intragastric administration once a day or LPS (1 mg/kg/day) via intravenous tail injection once a week alone or in combination for twelve weeks. At thirteen weeks after the first urethane injection, orbital venous blood was collected for serum assays of IFN-γ, IL-2, TNF-α,PD-L1, IL-10 and TGF-β1 using an ELISA kit. The mice were sacrificed, and cell-free alveolar fluid was collected by inserting a cannula into the trachea with three sequential injections of 1 mL PBS, followed by centrifugation, for cytokine assay (IFN-γ, IL-2, TNF-α, ROS, IL-10 and TGF-β1), while the separated cells were resuspended in 0.9% sterile saline for total cell counts. Macrophages in the suspensions were enriched by magnetic cell sorting utilizing anti-F4/80-coated beads, and macrophage immunophenotypes were analyzed by FITC-conjugated anti-mouse CD86 and CD163 staining using an automated cell counter and analysis system (Nexcelom Cellometer X2, Nexcelom, USA).
Spleen NK cells (DX5+) were separated using the autoMACS separation system for assays of surface PD-1 receptor and memory NK cell rate (NKG2c + NKG2a-) using an automated cell counter and analysis system.
The average numbers of lung carcinomas per mouse were calculated. A portion of each lung was preserved in 10% buffered formalin and routinely embedded in paraffin. Lung sections were stained by immunohistochemistry and immunofluorescence according to our previous method. After overnight incubation with the primary antibodies (anti-PD1, anti-iNOS, and anti-CD31), the slides were incubated with the FITC-conjugated goat anti-mouse IgG for 30 minutes. The total immunohistochemical and immunofluorescence scores were calculated by the intensity score and proportion score by excluding the primary antibody and IgG matched serum, respectively, as positive and negative controls.
In addition, the lung vascular integrity was assayed by the Evans blue dye extrabarrier technique according to our previous method.
Tumor allograft model
H22 cells were used for tumor allograft experiments. Two hundred microliters of saline containing 1 × 106 cells were injected subcutaneously into the lateral axilla of mice to establish tumor allografts. One day after tumor inoculation, in vitro LPS-induced M1 or IL-10-induced M2 cells (2 × 106 cells in 200 µL saline) were injected intravenously into mice once a week for three weeks; simultaneously, mice received melatonin (20 mg/kg) via intragastric administration once a day and LPS (1 mg/kg) or LEC (4 mg/mouse) via intravenous tail injection once a week alone or in combination for 3 weeks. Tumor size was monitored twice a week with calipers and calculated as the length x width2 /2. On the twenty-second day after tumor inoculation, orbital venous blood was collected for serum assays of IFN-γ, IL-2, TNF-α,PD-L1, IL-10 and TGF-β1. The mice were euthanized, the tumors were extracted and weighed, peritoneal macrophages were enriched by magnetic cell sorting utilizing anti-F4/80-coated beads, and macrophage immunophenotypes were analyzed. Spleen NK cells were separated using the autoMACS separation system for assays of surface PD-1 receptor and memory NK cell rate. The complete assay procedure was similar to the methods in the urethane-induced lung carcinogenesis model.
In addition, the tumor vascular integrity was assayed with the Evans blue dye extrabarrier technique according to our previous method.
For the immune rechallenge study, the tails of 1/2 of the 40 mice were injected subcutaneously with 5 × 105 H22 cells suspended in 50 µl saline. One day after tumor inoculation, the mice received melatonin (20 mg/kg) via intragastric administration once a day and LPS (1 mg/kg) or LEC (4 mg/mouse) via tail intravenous injection once a week alone or in combination for 2 weeks. Fifteen days after tumor implantation, the tumor-bearing tail was cut off to remove the primary tumor, and the mice were rechallenged with subcutaneous injections of 1 × 106 H22 cells in 200 µl saline in the flanks, while 10 normal mice were challenged with identical H22 cells. Tumor size was monitored twice a week with calipers. At thirty-six days, the same detections as above were carried out.
The regulatory mechanism of melatonin on neutrophils
The gene expression profiles GSE5099 were obtained from the Gene Expression Omnibus (GEO) database, Up- and downregulated genes related to tumor-associated macrophages were identified using GEO2R, and the human structures of these differential proteins were collected from the protein data bank (PDB) for docking analysis. The chemical structure of melatonin was obtained from PubChem, and the docking exercise was conducted using the online software systemsDock with the autoremoval of unspecified protein structures. Docking scores over 5 were regarded as the potential targets for melatonin. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed for the potential targets using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and the online software Omicshare. The protein-protein interactions (PPIs) among these potential targets were constructed using the STRING database, and the hub genes were identified using Cytoscape.
We systematically searched PubMed and Web of Science to identify eligible studies published from Jan 1, 1990 to Apr 1, 2019. OR and hazard ratios (hrs) with 95% confidence intervals (cis) were used to evaluate the risk of bacterial infections in carcinogenesis and cancer survival. Fixed and random-effect meta-analyses were conducted based on the heterogeneity of the included studies. To minimize case selection bias, sensitivity analyses were performed.
The data were statistically analyzed using GraphPad Prism, Version 5.0 (San Diego, CA, USA) and are presented as the mean ± SD. The differences between two groups were evaluated using a t-test. A P value of less than 0.05 was considered statistically significant. Meta-analyses were performed using the fixed-effects inverse-variance method using RevMan 5.3. Heterogeneity was calculated using the I 2 statistics and chi-square Q test, and a P-value of heterogeneity < 0.10 or I 2 >50% indicated significant heterogeneity. Usually, a fixed-effect model was used, and the random-effects model was adopted when the heterogeneity was significant.