We declare that all the data that support the findings of this study are available within the article and its Supplemental Materials.
Whole blots on PVDF membranes are available in the Supplemental Materials.
Rats
Male Sprague–Dawley (SD) rats aged approximately 12 weeks and weighing approximately 400 g were housed in a pathogen-free barrier facility for all experiments. Animals were purchased from the Slacas Animal Centre (Shanghai, China). The animal experiments were performed according to the Guide for the Care of Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, revised 1996), and the research protocol was approved by the Institutional Animal Care and Use Committee of Zhejiang University School of Medicine.
Experimental models of DHCA
DHCA model techniques were performed according to our previously published methods [22, 23]. In brief, the rats were rewarmed after hypothermia and circulatory arrest process, rewarming to a subscalp temperature of 36 °C was recorded at 0 h after DHCA, and the rats were euthanized for sampling at 6 h postoperatively.
Protocol I. To assess the change of monocytes in lung tissue, ten SD rats were randomly assigned and divided into two groups (n = 5). In the sham group (Sham group), all rats underwent the same steps without standard DHCA procedures. The DHCA 6 h group (DHCA group) rats experienced standard DHCA intervention and were sacrificed at 6 h after DHCA. Flow cytometry analysis and immunofluorescence was assessed for each group of rats.
Protocol II. To describe the role of monocytes in lung injury induced by the DHCA procedure, clodronate-liposomes (Clo-Lip, 50 mg/kg, i.v. C-010, Liposoma BV, The Netherlands) or control-liposome (PBS) was administered 24 h prior to DHCA. Flow cytometry analysis and immunofluorescence was assessed for each group of rats. And the extent of lung injury, including lung histology examination, wet/dry lung weight ratio, and inflammatory cytokine quantification, was assessed for each group of rats.
Protocol III. To assess whether suppression (maraviroc, MVC, UK-427857, MedChemExpress, China, jugular vein injection) of CCR5 can modulate the lung injury induced by DHCA. MVC or its corresponding solvents (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline) was injected half an hour prior to DHCA. Flow cytometry analysis, immunofluorescence, and Western blotting was assessed for each group of rats. Lung injury severity was also assessed.
Animal euthanasia and tissue handling
After the rats were anaesthetized via injection of an overdose of sodium pentobarbital (100 mg/kg, i.p.), lung tissue samples were taken after blood sampling from the abdominal aorta. The rats had a total of five lung lobes: the left lung, the anterior lobe of the right lung, the middle lobe of the right lung, the posterior lobe of the right lung, and the accessory lobe of the right lung.
The experimental protocols and uses of the various parts of the lung tissue were as follows. (1) The anterior lobe of the right lung was first ligated, after which the lung tissue was cut at the distal end of the ligature line and fixed in 10% neutral formalin solution (AG2160, ACMEC Biochemical, Shanghai, China). It was used for subsequent preparation of paraffin sections. (2) The middle lobe of the right lung was ligated, and it was weighed (wet weight) after drying its surface moisture for wet/dry weight ratio analysis. The left auricle was clipped, 100 ml of 4 °C saline was instilled from the right ventricle into the pulmonary circulation until the lung tissue became porcelain white, and all remaining lung lobes were harvested. (3) The posterior lobe of the right lung was used to retain bronchoalveolar lavage fluid (BALF). (4) A portion of the left lung tissue was prepared for single-cell suspensions, and the remaining portion of the left lung was immediately stored in liquid nitrogen for Western blotting.
Flow cytometry analysis
Approximately 100 mg of lung tissue was rinsed in a Petri dish containing PBS at pH 7.2 to remove the remaining blood. The lung tissue was transferred into a gentleMACS tube containing approximately 2.5 mL of enzyme mixture (No. 130-095-927; Miltenyi Biotec, Germany), and the sample was subsequently processed via a gentleMACSTM Dissociator (No. 130-093-235; Miltenyi Biotec, Germany). The cell clumps were removed via filtration with a 300-mesh nylon membrane. After centrifugation (2000 rpm, 5 min, 4 °C), the precipitate was mixed with 500 μL of saline by shaking, resulting in a single-cell suspension of lung tissue to be tested. The antibodies used were as follows: CD45 (2202226, Biolegend, America), CD43 (202812, Biolegend, America), CD172α (sc-376884, Santa Cruz, America), and CCR5 (FAB1802A, R&D Systems, America).
Histological assessment of lung injury
The anterior lobe of the right lung was fixed in formalin for 12–72 h and then dehydrated before paraffin embedding. Sections (3 mm thick) were cut and subjected to haematoxylin and eosin (H&E) staining. After staining, the sections were scanned and analysed with 120 P digital section scanning equipment (Tao Han Medical Technology, Shanghai). Histopathological analyses were performed by three uninformed researchers in a blinded fashion and were scored in detail as described previously [24].
Quantification of cytokines in the lung tissue and BALF
After the rats were euthanized, an appropriate amount of left lung tissue was prepared into tissue homogenate and preserved for further analysis. The posterior lobe of the right lung was rinsed with ice-cold saline and then placed in a disposable Petri dish. Two ml of ice-cold saline was perfused into the alveoli, and the lung tissue was gently massaged to obtain lavage fluid. This step was repeated 20 times, and the fluid collected in the Petri dish was BALF (approximately 1.6 ml). The supernatant was retained after centrifugation (400 × g, 5 min at 4 °C) and stored at −80 °C for further analysis. A bicinchoninic acid (BCA) protein assay kit (23227, Thermo Fisher Scientific, Shanghai) was used to determine the total protein concentration in the BALF supernatant.
The protein levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor α (TNF-α) in the lung tissue and BALF supernatants were measured via ELISA kits (Elkbiotech, Wuhan, China).
Lung wet/dry weight ratio
The middle lobe of the right lung was obtained, rolled over absorbent paper 3 times to dry the surface moisture, weighed, and recorded as wet weight (W). Afterwards, it was dried in an oven (at 58 °C) until a stable dry weight (D) was reached. The ratio of wet weight to dry weight (W/D) was calculated to assess the degree of pulmonary oedema.
Western blot
The left lung was stored in liquid nitrogen for protein quantification via Western blotting. The tissues were homogenized and lysed in RIPA lysis buffer. The protein concentration was measured via a BCA assay (23225, Thermo Fisher, Shanghai). Protein samples were subjected to gel electrophoresis via SDS‒PAGE and then transferred to a polyvinylidene fluoride membrane. Nonspecific binding was blocked with either 5% (w/v) nonfat milk in PBS containing 0.1% (v/v) Tween-20 for 1 h at room temperature, after which the membranes were incubated with the indicated primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. Detection was performed using SuperSignal Substrates (Thermo Fisher, 34577 or 34095). The primary antibodies used were as follows: anti-LC3-I/II (4108, Cell Signalling Technology, Shanghai), anti-ATG5 (10181-2-AP, Proteintech, America), anti-Beclin 1 (11306-1-AP, Proteintech, America), anti-SQSTM1/p62 (ab91526, Abcam, Shanghai), and anti-CCR5 (PA5-114965, Thermo Fisher, Shanghai). Western blotting was assessed for Protocol III.
Rat lung single-cell RNA sequencing (scRNA-seq)
Fresh lung tissues were taken from the Sham and DHCA groups for scRNA-seq. In brief, the prepared single-cell suspensions were loaded onto a Chromium Single Cell Controller instrument (10 × Genomics, Pleasanton, CA, USA) to create single-cell gel beads in emulsions for subsequent sequencing and analysis. Single-cell RNA-seq libraries were prepared using a Chromium Single-cell 3’ Reagent Kit, version 2, according to the manufacturer’s protocol. The cells were visualized using a 2-dimensional t-SNE algorithm with the RunTSNE function. GO enrichment and KEGG pathway enrichment analyses of the DEGs were performed using R based on the hypergeometric distribution.
Immunofluorescence staining
First, the paraffin sections were deparaffinised and hydrated, and hydrogen peroxide was added to block endogenous peroxidase activity. Second, the sections were boiled in boiling antigen repair solution (1 mmol Tris-EDTA pH = 9.0) for 15 min and incubated in 5% BSA (B2064, Sigma) blocking solution for 20 min at room temperature. Next, the primary antibody CCR5 (PA5-114965, Thermo Fisher, 1:400, 100 µl, overnight at 4 °C) and the corresponding secondary antibody (red, 30 min at 37 °C) were added sequentially, followed by incubation with Try-488 tyramine converting reagent (Bry-Try488, Runnerbio, 20 min at room temperature). After washing, the following steps were repeated: antigen repair, blocking, incubation with CD172α (sc-376884, Santa Cruz, America), incubation with the corresponding secondary antibody (green), and incubation with Try-Cy3 tyramine conversion reagent (Bry-Try-Cy3, Runnerbio, Shanghai). Finally, the sections were washed and incubated with anti-fade mounting medium containing DAPI (P0131, Beyotime, Shanghai) for 30 min. Images were acquired under a fluorescence microscope (Nikon Eclipse Ci-L, Nikon, Japan).
Statistical analysis
With the exception of the scRNA-seq data, t tests were employed for normally distributed continuous variables between two groups, and one-way ANOVA followed by Tukey’s honest significant difference (HSD) post hoc test was used for comparisons between at least three independent groups. The nonparametric Mann–Whitney U test was used for the lung injury score. The experimental data were analysed using GraphPad Prism software (version 8.0.1; GraphPad Software, Inc., San Diego, CA). The normal distribution of the samples was tested via the Shapiro‒Wilk test. Differences were considered statistically significant when the p value was < 0.05.