T follicular helper cell is essential for M2 macrophage polarization and pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension

Animals and in vivo treatment

Adult male C57BL/6 mice (body weights 25–28 g) were purchased from Hunan Silaikejingda Laboratory Animal Co. Ltd., Changsha, China. IL-21 knockout (KO) mice (IL-21−/−) in C57BL/6 N background were purchased from Cyagen Biosciences (stock no. KOCMP-60505-Il21-B6N-VA; Cyagen Biosciences, Guangzhou, China). All animals were acclimatized for a week and maintained in a temperature and humidity-controlled room with a 12-h light-dark cycle, and were given standard food and purified water ad libitum. All the experiments were performed in accordance with the NIH guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Laboratory Animal Ethics Committee of Hunan Provincial People’s Hospital (XSY-2021-42). All animals were randomly assigned to control or vehicle/treatment groups, which were placed into a chamber containing 10% O2 or under normoxia condition for 4 weeks. For in vivo adoptive transfer experiments, TFH cells were isolated and sorted from mice exposed to hypoxia for 4 weeks. In brief, naive CD4+ T cells were isolated and sorted by using CD4+ T cell isolation kits (Miltenyi Biotec, Germany) from mice spleen according to manufacturer’s protocols. After sorting, purified CD4+ T cells were consecutively incubated with APC-conjugated anti-CXCR5 antibody, PE-conjugated anti-PD-1 antibody (eBiosicience) and anti-APC microbeads (Miltenyi Biotec, Germany) to isolate CD4+CXCR5+PD-1+TFH cells. Experimental mice were administered intravenously through tail vein with 1 × 106 TFH cells on day 1, 8, 15 and 22, and their littermates were injected with normal saline were set as controls. For the TFH cell inhibitor group, Bcl-6 inhibitor 79 − 6 (Millipore, USA) was dissolved in 10% dimethyl sulfoxide (DMSO, Sigma) and intraperitoneally injected (50 mg/kg body weight) every other day from the start of the second week to the end of the experiment. The control group and HPH group were injected with equal volume of vehicle (10% DMSO) every other day. For in vivo silencing ECM1 studies, adeno-associated virus (AAV) vectors containing shECM1 were synthesized and packaged by GenePharma (Shanghai, China). After hypoxia exposure, a total of 150 µl of AAV-shNC or AAV-shECM1 were injected by intratracheal instillation at a daily dose of 50 µl for 3 days. The sequence targeting on ECM1 was 5′-CTTTCAAGATTCAAGAGATCTTGAAAGTGCTCTGGCCTC-3′.

Haemodynamic measurement and histological analysis

After 4 weeks, pulmonary hemodynamics (mean pulmonary artery pressure, mPAP) were measured as we previous described. Briefly, the animals were anesthetized and a polyethylene catheter filled with heparin was inserted into right ventricle (RV) and connected to the Multi-lead Physiological Recorder (BIOPAC System, USA). Then, the catheter was introduced into the pulmonary artery guided by a pressure curve and pulmonary arterial pressure was measured. At the time of sacrifice, animals were anaesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg), the anesthesia depth was monitored with pedal reflex. All animals were anaesthetized by intraperitoneal injection of pentobarbital sodium and then euthanized by exsanguination under anesthesia, the euthanasia method used for all animal procedures. Samples of lung tissues, spleen, heart, peripheral blood and small intrapulmonary arteries were collected for further analysis. The heart was dissected into RV, ventricular septum (VS) and left ventricle (LV), dried and weighed. The right ventricular hypertrophy index was calculated by the ratio of RV to LV plus septum [RV/(LV + S)]. Lung tissues were excised and immersed in 4% paraformaldehyde overnight at 4℃ for fixation. The fixed lung tissues were dehydrated and embedded in paraffin, then cut into 4 μm thick sections, and stained with hematoxylin and eosin (HE). The pathological changes in lung tissue sections were examined with a light microscope (Olympus, Japan). Vascular hypertrophy parameters of pulmonary arteriole media thickness (PAMT) were assessed via the ratio of medial thickness × 2 to external diameter.

Western blot analysis

Western blotting was performed on isolated bronchoalveolar lavage fluid (BALF) to detect Fizz1. BALF protein was extracted by using lysis buffer, and protein concentrations were measured using the Lowry protein assay. Equal amounts of protein from each sample were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% skimmed milk and incubated with the primary antibody Fizz1 (ab39626, Abcam, 1:1000), Bcl-6 (ab272859, Abcam, 1:1000), IL-2 (ab317331, Abcam, 1:1000), p-STAT5 (ab278764, Abcam, 1:1000), STAT5 (ab230670, Abcam, 1:1000) at 4 ℃ overnight. The blots were then incubated with secondary antibody for 2 h at room temperature. Finally, the targeted protein bands were detected and analyzed by an ECL reagent kit and a gel imaging system (Bio-Rad, USA).

Real-time quantitative PCR

Real-time quantitative PCR was detected to quantify mRNA levels of the iNOS, IL-12β, MMP9 and Bcl-6 according to the our previous study. RNA was extracted from the bronchoalveolar lavage fluid (BALF) with the Trizol reagent (Invitrogen, USA) and the purity was analyzed by spectrophotometer. RT-qPCR was performed by using MasterMix (SYBR Green) (Roche, Switzerland). The sequences of the primer pairs used for amplification were listed as follows. All data were normalized to β-actin and expressed as a relative ratio. Primer pairs sequences used as follows.

iNOS: 5′-GCTCATGACATCGACCAGAA-3′, 5′-TGTTGCATTGGAAGTGAAGC-3′;

IL-12β: 5′-AGGTCACACTGGACCAAAGG-3′, 5′-AGGGTACTCCCAGCTGACCT-3′;

MMP9: 5′-CCAGCCGACTTTTGTGGTCT-3′, 5′-CTTCTCTCCCATCATCTGGGC-3′.

Bcl-6: 5′-CGCGAGGCAATTTTTAATCT-3′, 5′-ATTTGCATTGCCCAGTAAGG-3′.

Enzyme-linked immunosorbent assay

The levels of IL-21 in BALF and CX3CL1 and CX3CR1 in M2 macrophages were determined with commercial ELISA kits (BOSTER, Wuhan, China) according to the manufacturer’s protocols.

Flow cytometry analysis

To determine M2 macrophage, GC B cell and TFH cell, individual cell suspensions of BALF and spleen were collected. Then, the individual cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies according to the manufacturer’s protocols. For the TFH cell staining, cells were incubated with FITC-labelled anti-CD4, APC-labelled anti-CXCR5 and PE-labelled anti-PD-1. For B cell analysis, cells were stained with B Cell activation antigen Biotin-conjugated anti-GL-7, PE-conjugated anti-CD95, and PerCP-Cyanine5.5-conjugated anti-B220. To characterize M2 macrophages, cells were stained with FITC-labelled anti-Fizz1, APC-labelled anti-CD11c and PE-labelled anti-F4/80. The cells labelled B220+GL-7+CD95+ represent GC B cells and CD4+CXCR5+PD-1+ represent TFH cells. M2 macrophages were identified as F4/80+CD11c+Fizz1+. All cell surface markers were purchased from eBiosicience, USA. The samples were detected and analyzed by BD FACSCantoTM II flow cytometer and Flowjo software (Tree Star, USA).

Immunofluorescence and immunocytochemistry staining

Paraffin sections from lung and spleen tissues were performed by immunofluorescence (IF) staining. Cultured cells and BALF were performed by immunocytochemistry (ICC) staining. α-SMA (1:500, ab124964, Abcam) and GL-7 (1:200, eBiosicience) were assayed using IF staining. Fizz1 (1:500, ab39626, Abcam), α-SMA (1:200, ab124964, Abcam) and caspase-1 (1:500, ab219935, Abcam) were assayed by ICC staining. Methods for IF and ICC staining were described in our previous study [13]. For IF staining, in brief, paraffin lung and spleen sections were dewaxed in water and subjected to antigen repair. After blocking with 3% BSA at room temperature, the sections were incubated with primary antibodies at 4℃ overnight. Then, the corresponding secondary antibody was incubated. After staining with DAB or DAPI, washed, dehydration and sealing, the sections were examined and pictures were collected by confocal laser-scanning microscope (Nikon, Japan). For ICC staining, the cultured cells were permeabilized by using 0.2% Triton X-100. Then, the cells were washed and incubated with bovine serum albumin. Afterward, the cells were dealt with primary antibodies at 4℃ overnight and incubated with secondary antibodies. After staining with DAPI, the images were observed and captured under a fluorescent microscope.

Isolation and culture of primary alveolar macrophage

Primary alveolar macrophage were isolated and cultured from C57BL/6 mice as previously described [14]. In brief, BALF were collected by serial bronchoalveolar lavage (BAL), rested and filtered for 1 h to exclude contamination of epithelial cells. For cell culture experiment, primary alveolar macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 2% fetal bovine serum (FBS). The cells were seeded at 3.5 × 105 cells per well in 48-well plates containing supplemented DMEM (10% FBS, 100 IU/mL penicillin, 2 mM L-glutamine, and 100 µg/mL streptomycin) and were incubated in a hypoxic work station with 0.5% O2 and stimulated with mouse recombinant IL-21 (10 ng/ml or 100 ng/ml) for 24 h. M2 macrophages of BALF in HPH mice using flow cytometry. Then, M2 macrophages were cultured and stimulated with or without mouse recombinant IL-21 (100 ng/ml) for 24 h.

Isolation, identification and culture of PASMCs

Isolation of primary PASMCs from C57BL/6 mice was performed as previously described [15]. Briefly, the main pulmonary arteries were isolated from anesthetized mice. After removing the intima and adventitia gently, the remaining arteries was cut into small tissue blocks, then transferred to a culture flask with DMEM/F-12 containing 20% FBS, and cultured in a humidified incubator at 37 °C with 5% CO2. Early-passage cells (passage 3 to 6) were used for subsequent experiments and cell purity was determined by immunocytochemistry and immunofluorescence staining with an α-SMA polyclonal antibody (ab124964, Abcam, 1:200).

Cell proliferation assay

PASMCs proliferation was determined using EdU incorporation assay. PASMCs were seeded in 48-well plates with a density of 1 × 105 cells per well in the logarithmic growth phase and placed in cell incubators with 1% O2 and stimulated with mouse recombinant IL-21 or conditioned medium of the alveolar macrophages cultivated with IL-21 for 48 h. Then, PASMCs were incubated with 50 µM EdU solution (Sangon) for 2 h, fixed with PBS containing 4% formaldehyde for 30 min, and subsequently exposed to PBS containing 0.5% Triton X-100 for 10 min. After the PASMCs were washed with PBS, the cells were incubated with 1 × Apollo staining solution for half an hour to protect from light at room temperature. Finally, 1×Hoechst 33,342 was employed to stain the DNA in the dark. The percentage of EdU-positive cells was observed and images were taken with a fluorescence microscope (Nikon, Japan).

Hoechst 33,342/PI fluorescent staining

PASMCs pyroptosis was assessed using by Hoechst 33,342/propidium iodide (PI) double fluorescent staining. PASMCs at the logarithmic growth stage were cultured in 12-well plates at a density of 2 × 105 cells per well, and the cells stimulated with mouse recombinant IL-21 or conditioned medium of the alveolar macrophages cultivated with IL-21 in cell incubators with 1% O2. Then, PASMCs were stained with 10 µl Hoechst 33,342 solution for 10 min in the dark and 5µl PI under dark conditions for 15 min. The stained cells were observed with a fluorescence microscope (Nikon, Japan).

Statistical analysis

SPSS 26.0 software and GraphPad Prism 8.0 were used for statistical analysis and the results were presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) or Bonferroni’s post hoc test were used to assess statistical significance among groups. Test results were reported as two-tailed P values. A value of P < 0.05 was determined statistically significant.

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