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

Respiratory Research volume 25, Article number: 428 (2024) Cite this article

Abstract

Background

Hypoxia-induced pulmonary hypertension (HPH) is a subgroup of type 3 pulmonary hypertension that may cause early right ventricular failure and eventual cardiac failure, which lacks potential therapeutic targets. Our previous research demonstrated that T follicular helper (TFH) cells that produce IL-21 were involved in HPH. However, the molecular mechanisms of TFH/IL-21-mediated pathogenesis of HPH have been elusive. Here we investigate the role of TFH cells and IL-21 in HPH.

Methods

Studies were performed in C57BL/6 mice or IL-21 knockout mice exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in mouse lung and pulmonary arterial smooth muscle cells (PASMCs). M2 signature gene (Fizz1), M1 signature genes (iNos, IL-12β and MMP9), GC B cell and its marker GL-7, caspase-1, M2 macrophages, TFH cells, Bcl-6 and IL-21 level were measured. Proliferation rate of PASMCs was measured by EdU. Pyroptosis was assessed using Hoechst 33,342/PI double fluorescent staining.

Results

In response to chronic hypoxia exposure-induced pulmonary hypertension, IL-21−/− mice or downregulation of TFH cells in WT mice developed blunted pulmonary hypertension, attenuated pulmonary vascular remodelling. Furthermore, chronic hypoxia exposure significantly increased the germinal center (GC) B cell responses, which were not present in IL-21−/− mice or downregulation of TFH cells in WT mice. Importantly, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Consistently, significantly enhanced expression of M2 macrophage marker Fizz1 were detected in the bronchoalveolar lavage fluid of HPH mice. Moreover, alveolar macrophages that had been cultivated with IL-21 promoted PASMCs proliferation and pyroptosis in vitro, while a selective CX3CR1 antagonist, AZD8797 (AZD), significantly attenuated the proliferation and pyroptosis of the PASMCs. Finally, ECM1 knockdown promoted IL-2–STAT5 signaling and inhibited Bcl-6 signaling to inhibit TFH differentiation in HPH.

Conclusions

TFH/IL-21 axis amplified pulmonary vascular remodelling in HPH. This involved M2 macrophage polarization, PASMCs proliferation and pyroptosis. These data suggested that TFH/IL-21 axis may be a novel therapeutic target for the treatment of HPH.

Introduction

Pulmonary hypertension (PH), especially chronic hypoxia-induced pulmonary hypertension (HPH), is a debilitating disease characterized by a sustained and progressive increase in small to medium-sized distal pulmonary arterial pressure with pathological changes involving concentric intimal thickening, arterial muscularization and vascular remodeling, which may lead to right heart failure and ultimately death [1]. Whereas the pathogenesis of PH is multifactorial and complicated, it is known that the main processes lead to pulmonary arterial remodeling, including immune dysregulation, hyperplasia of pulmonary arterial smooth muscle cells (PASMCs) and deposition of the extracellular matrix (ECM) [2]. Immune cells such as T cells and B cells accumulated the plexiform lesions in patients with advanced PH, which recognized as crucial contributors to the pathogenesis of PH [3]. Moreover, proinflammatory cytokines secreted by these immune cells may be responsible for the proliferation of PASMCs in PH [4].

A potential role for immune cells, specifically T cells in PH was noted more than 30 years ago, but we are still far from understanding the crucial immune cell subsets and signal pathways that lead to pulmonary vascular remodeling in PH. In 1990, Duke et al. demonstrated that T and B lymphocytes subpopulations accumulated in the lung lymph and tissue during periods of PH and may participate in endotoxin-induced lung injury [5]. The specific CD4+ T cell subset that provides help to germinal center (GC) reaction is T follicular helper (TFH) cells that mainly produce IL-21 [6]. Many studies have shown that dysregulation of TFH cell was involved in many pathogenic processes of autoimmune diseases, such as Sjögren’s syndrome, multiple sclerosis and systemic lupus erythematosus [7,8,9]. Previously, it was reported that hypoxia inducible factor 1α (HIF-1α) depletion from CD4+ T cells reduced frequency of TFH cell, suggesting that the hypoxia response promoted TFH differentiation [10]. Recently, our laboratory reported the percentage of TFH cells was significantly increased in HPH mice than those of control group, but the underlying mechanisms of TFH cells in HPH remain elusive [11]. In a previous work, M2 macrophage polarization and recruitment was associated with the development of PASMCs proliferation in vitro and pulmonary vascular remodelling in vivo of HPH [12].

However, the role of TFH cells in the development of HPH in association with M2 macrophage polarization and pulmonary vascular remodeling remains unknown. Based on these findings, we hypothesized that TFH cells and cytokine IL-21 may contribute to the development of HPH by promoting the polarization of M2 macrophage. This study identified a crucial role for TFH cells as mediators of pulmonary vascular remodeling in HPH.

Methods

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.

Results

Transfer of TFH cells exacerbated pulmonary vascular remodelling and induced M2 macrophage polarization in HPH

After 28 days of hypoxia exposure, as expected, mean PAP, RV/(LV + S) ratio, PAMT were significantly increased in HPH group compared to control group, while adoptive transfer of TFH cells further exacerbated abnormal hemodynamics, pulmonary vascular remodelling and right ventricle hypertrophy in HPH (Fig. 1a, c-e). We further explored the expression of α-SMA in pulmonary arteries. The results showed that α-SMA was upregulated in HPH group and adoptive transfer of TFH cells significantly increased α-SMA expression (Fig. 1b, f).

Since the main effector function of TFH cells is to promote a GC reaction, our results showed the number and size of germinal centers in spleen were significantly increased in HPH group compared to control group. Additionally, the percentage of GC B cells and expression of GL-7 were significantly increased after exposing to 28 days of chronic hypoxia. Of note, adoptive transfer of TFH cells also significantly increased the number and size of GC (Fig. 2a), percentage of GC B cells (Fig. 2b, c) and expression of GL-7 (Fig. 2d, e).

We next investigated the effect of TFH cells on the macrophage polarization after hypoxia exposure. The results showed that percentages of M2 macrophage in spleen and bronchoalveolar lavage fluid (BALF) of HPH group increased significantly compared to those of control group (Fig. 3a-c). Consistent with these changes, the expression of Fizz1, an M2 signature gene, was significantly increased in the alveolar macrophages, as shown by western blot (Fig. 3e, g) and immunocytochemical (Fig. 3d, f) analyses. Adoptive transfer of TFH cells significantly increased the percentage of M2 macrophage and the expression of Fizz1. In contrast, there were no significant differences in the mRNA levels of M1 signature genes (iNos, IL-12β and MMP9) in HPH group compared to control group. Adoptive transfer of TFH cells also did not affect the expressions of M1 macrophage signature genes (Fig. 3h-j). All the above results suggested the important role of TFH cells in exacerbating pulmonary vascular remodelling and inducing M2 macrophage polarization in HPH.

Fig. 1
figure 1

Adoptive transfer of TFH cells exacerbated pulmonary vascular remodelling in HPH. (a) Representative HE pathological staining of lung section (orginal magnification × 400). (b) The expression of α-SMA in pulmonary arteries was verified by immunofluorescence staining. (c-e) Changes of mean PAP, RV/(LV + S) ratio and PAMT. (f) The positive expression of α-SMA was analyzed by histogram.Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 75 μm. *P < 0.05, ** P < 0.01

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Fig. 2
figure 2

Adoptive transfer of TFH cells promoted germinal center (GC) B cell responses in HPH. (a) The number and size of germinal centers in spleen were analysed by haematoxylin & eosin staining. (b) The percentage of B220+GL-7+CD95+ B cells in the spleen of mice. (c) Percentage of B220+GL-7+CD95+ B cells in each group was analysed by histogram. (d) The expression of GL-7 in spleen was verified by immunofluorescence staining. (e) The positive expression of GL-7 was analyzed by histogram. Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 200 μm. *P < 0.05, ** P < 0.01

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Fig. 3
figure 3

Adoptive transfer of TFH cells induced M2 macrophage polarization in HPH. (a) The percentages of F4/80+CD11c+Fizz1+ M2 macrophages in the spleen and bronchoalveolar lavage of mice. (b, c) Percentages of F4/80+CD11c+Fizz1+ M2 macrophages in the spleen and bronchoalveolar lavage were analysed by histogram. (d) The expression of Fizz1 in bronchoalveolar lavage was verified by immunofluorescence staining. (e) Expression of Fizz1 in bronchoalveolar lavage analysed by western blot. (f) The positive expression of Fizz1 was analyzed by histogram. (g) Quantitative analysis of Fizz1. (h-j) Relative mRNA expression levels of iNOS, IL-12β and MMP9 determined by RT-qPCR in the bronchoalveolar lavage. Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 50 μm. *P < 0.05, ** P < 0.01

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Downregulation of TFH cells ameliorated pulmonary vascular remodelling and suppressed M2 macrophage polarization in HPH

To further evaluate the role of TFH cells in HPH, TFH generation was blocked by 79 − 6, a Bcl-6 specific inhibitor (Bcl-6i). As shown in Fig. 4a, c-e, mean PAP, RV/(LV + S) ratio, PAMT of the HPH group were greater than those of the control group, which remarkably decreased after Bcl-6i treatment. Similarly, Bcl-6i treatment significantly decreased α-SMA expression in HPH (Fig. 4b, f). We next examined the effect of TFH cells blockade on the GC reaction. The results showed that the number and size of germinal centers (Fig. 5a), percentage of GC B cells (Fig. 5b, d) and expression of GL-7 (Fig. 5c, e) in spleen were significantly increased in HPH group, which were inhibited significantly by Bcl-6i treatment. Consistent with this finding, hypoxia exposure significantly increased the percentages of M2 macrophage in spleen (Fig. 6a, b) and BALF (Fig. 6a, c), and the expression of Fizz1 (Fig. 6d, e-g) and IL-21 (Fig. 6h) in BALF, whereas Bcl-6 inhibitor abolished the effect. Notably, treatment with Bcl-6 inhibitor had no effect on M1 macrophage signature gene expression in BALF (Fig. 6i-k). Collectively, these data demonstrated that downregulation of TFH cells in HPH could ameliorate pulmonary vascular remodelling and suppresse M2 macrophage polarization.

Fig. 4
figure 4

Inhibition of TFH cells alleviated pulmonary vascular remodelling in HPH. (a) Representative HE pathological staining of lung section (orginal magnification × 400). (b) The expression of α-SMA in pulmonary arteries was verified by immunofluorescence staining. (c-e) Changes of mean PAP, RV/(LV + S) ratio and PAMT. (f) The positive expression of α-SMA was analyzed by histogram.Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 75 μm. *P < 0.05, ** P < 0.01

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Fig. 5
figure 5

Blockade of Bcl-6 inhibited GC B cell responses in HPH. (a) The number and size of germinal centers in spleen were analysed by haematoxylin & eosin staining. (b) The percentage of B220+GL-7+CD95+ B cells in the spleen of mice. (c) The expression of GL-7 in spleen was verified by immunofluorescence staining. (d) Percentage of B220+GL-7+CD95+ B cells in each group was analysed by histogram. (e) The positive expression of GL-7 was analyzed by histogram. Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 200 μm. *P < 0.05, ** P < 0.01

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Fig. 6
figure 6

Blockade of Bcl-6 inhibited M2 macrophage polarization in HPH. (a) The percentages of F4/80+CD11c+Fizz1+ M2 macrophages in the spleen and bronchoalveolar lavage of mice. (b, c) Percentages of F4/80+CD11c+Fizz1+ M2 macrophages in the spleen and bronchoalveolar lavage were analysed by histogram. (d) The expression of Fizz1 in bronchoalveolar lavage was verified by immunofluorescence staining. (e) Expression of Fizz1 in bronchoalveolar lavage analysed by western blot. (f) The positive expression of Fizz1 was analyzed by histogram. (g) Quantitative analysis of Fizz1. (h) Expression of IL-21 in bronchoalveolar lavage analysed by ELISA. (i-k) Relative mRNA expression levels of iNOS, IL-12β and MMP9 determined by RT-qPCR in the bronchoalveolar lavage. Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 50 μm. *P < 0.05, ** P < 0.01

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Loss of IL-21 protected against hypoxia-induced pulmonary vascular remodeling and suppressed M2 macrophage polarization

IL-21 is produced primarily by TFH cells. To determine the effect of IL-21 deficiency on pulmonary vascular remodeling and M2 macrophage polarization, we exposed WT and IL-21−/− mice to 10% FiO2 for 4 weeks to induce HPH. After 4 weeks of chronic hypoxia exposure, mean PAP, RV/(LV + S) ratio, PAMT increased in WT mice, and this increase was blunted in IL-21−/− mice (Fig. 7a, c-e). Furthermore, we found that the expression of α-SMA increased in pulmonary arteries of WT but not IL-21−/− mice (Fig. 7b, f). Chronic hypoxia exposure significantly increased the number and size of germinal centers (Fig. 7g), percentage of GC B cells (Fig. 7h, j) and expression of GL-7 (Fig. 7i, k) in WT mice, which were not present in IL-21−/− mice. Moreover, WT mice showed increased the percentages of M2 macrophage in spleen (Fig. 8a, e) and BALF (Fig. 8a, f), and the expression of Fizz1 (Fig. 8b, c, g, i) and Bcl-6 (Fig. 8d, h) in BALF. However, these responses to chronic hypoxia exposure were attenuated largely in IL-21−/− mice. Further data showed that M1 macrophage signature gene expression in BALF were similar between WT and IL-21−/− mice (Fig. 8j-l). Thus, these data indicated the IL-21 deficiency protected against pulmonary vascular remodeling and suppressed M2 macrophage polarization in HPH.

Fig. 7
figure 7

IL-21 deficiency protected against chronic hypoxia-induced pulmonary vascular remodelling and inhibited GC B cell responses in HPH. (a) Representative HE pathological staining of lung section (orginal magnification × 400). Scale bar = 75 μm. (b) The expression of α-SMA in pulmonary arteries was verified by immunofluorescence staining. Scale bar = 75 μm. (c-e) Changes of mean PAP, RV/(LV + S) ratio and PAMT. (f) The positive expression of α-SMA was analyzed by histogram. (g) The number and size of germinal centers in spleen were analysed by haematoxylin & eosin staining. Scale bar = 200 μm. (h) The percentage of B220+GL-7+CD95+ B cells in the spleen of mice. (i) The expression of GL-7 in spleen was verified by immunofluorescence staining. Scale bar = 200 μm. (j) Percentage of B220+GL-7+CD95+ B cells in each group was analysed by histogram. (k) The positive expression of GL-7 was analyzed by histogram. Values were expressed as means ± SD and were analyzed by two-tailed Student’s t-test, n = 6. ** P < 0.01

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Fig. 8
figure 8

Blockade of Bcl-6 inhibited M2 macrophage polarization in HPH. (a) The percentages of F4/80+CD11c+Fizz1+ M2 macrophages in the spleen and bronchoalveolar lavage of mice. (b) The expression of Fizz1 in bronchoalveolar lavage was verified by immunofluorescence staining. (c, d) Expression of Fizz1 and Bcl-6 in bronchoalveolar lavage analysed by western blot. (e, f) Percentages of F4/80+CD11c+Fizz1+ M2 macrophages in the spleen and bronchoalveolar lavage were analysed by histogram. (g, h) Quantitative analysis of Fizz1 and Bcl-6. (i) The positive expression of Fizz1 was analyzed by histogram. (j-l) Relative mRNA expression levels of iNOS, IL-12β and MMP9 determined by RT-qPCR in the bronchoalveolar lavage. Values were expressed as means ± SD and were analyzed by two-tailed Student’s t-test, n = 6. Scale bar = 50 μm. ** P < 0.01

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ECM1 knockdown inhibited TFH differentiation and ameliorated pulmonary vascular remodelling in HPH

Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of HPH. We also used specific AAV-shRNA to knock down ECM1 expression in HPH mice. AAV-shECM1 reduced the mean PAP, RV/(LV + S) ratio and PAMT in HPH (Fig. 9a-d). Moreover, AAV-shECM1 reversed TFH cell polarization in BALF (Fig. 9e, f). Consistent with the changes in TFH cells, Bcl-6 expression and IL-21 level in BALF were increased in HPH, these changes were decreased by the AAV-shECM1 treatment given to HPH mice (Fig. 9g, h). Further data showed that AAV-shECM1 treatment enhanced IL-2 and p-STAT5 expression in BALF (Fig. 9i-l). Thus, mechanically, ECM1 knockdown promoted IL-2–STAT5 signaling and inhibited Bcl6 signaling to inhibit TFH differentiation in HPH. These results provided the first evidence that ECM1 regulated TFH cell polarization in HPH.

Fig. 9
figure 9

ECM1 knockdown restrained the TFH cell response and alleviated pulmonary vascular remodelling in HPH. (a) Representative HE pathological staining of lung section (orginal magnification × 400). (b-d) Changes of mean PAP, RV/(LV + S) ratio and PAMT. (e) Percentage of CD4+CXCR5+PD-1+ TFH cells restrained by AAV-shECM1 in bronchoalveolar lavage of mice. (f) Percentage of CD4+CXCR5+PD-1+ TFH cells in bronchoalveolar lavage was analysed by histogram. (g) Relative mRNA expression level of Bcl-6 determined by RT-qPCR in the bronchoalveolar lavage. (h) Expression of IL-21 in bronchoalveolar lavage analysed by ELISA. (i) Expression of IL-2, p-STAT5 and STAT5 in bronchoalveolar lavage analysed by western blot. (j-l) Quantitative analysis of IL-2, p-STAT5 and STAT5. Values were means ± SD, n = 8. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 75 μm. *P < 0.05, ** P < 0.01

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M2 macrophage polarization induced by IL-21 was required for hypoxia-induced proliferation and pyroptosis of PASMCs

To explore whether IL-21 induced M2 macrophage polarization under hypoxic condition in vitro, we cultured alveolar macrophages isolated from BALF. Here, our results showed that IL-21 could remarkably upregulate M2 macrophage polarization (Fig. 10a, b) and the protein level of Fizz1 (Fig. 10c, d) under hypoxic condition in vitro. Moreover, IL-21 significantly promoted the expression of CX3CL1 and CX3CR1 in M2 macrophages (Fig. 10g, h). As shown in Fig. 10e, f, immunocytochemistry and immunofluorescence staining with a-SMA antibody confirmed that the separated cells from pulmonary arteries of mice were PASMCs. Then, we investigated the molecular mechanism by which the IL-21 induces the proliferation and pyroptosis of PASMCs after hypoxia exposure. Treatment with IL-21 did not promote the proliferation of PASMCs (Fig. 11a, b). Similarly, treatment with IL-21 did not increase the pyroptosis of PASMCs (Fig. 11e, f, I, j). However, treatment with the conditioned medium of the alveolar macrophages that had been cultivated with IL-21 promoted PASMCs proliferation and pyroptosis, which indicated that soluble factors secreted by the M2 macrophages may promote PASMCs proliferation and pyroptosis. CX3CL1 is a chemokine that stimulates PASMCs proliferation by binding to its corresponding receptor CX3CR1. Treatment with a selective CX3CR1 antagonist, AZD8797 (AZD), significantly attenuated the proliferation (Fig. 11c, d) and pyroptosis (Fig. 10g, h, k, l) of the PASMCs. Taken together, these data suggested that M2 macrophage polarization induced by IL-21 played a critical role in the hypoxia-induced proliferation and pyroptosis of PASMCs.

Fig. 10
figure 10

IL-21 induced M2 macrophage polarization in primary alveolar macrophage isolated and cultured from mice. (a) The percentage of F4/80+CD11c+Fizz1+ M2 macrophages in bronchoalveolar lavage. (b) Percentage of F4/80+CD11c+Fizz1+ M2 macrophages in bronchoalveolar lavage was analysed by histogram. (c) Expression of Fizz1 in bronchoalveolar lavage analysed by western blot. (d) Quantitative analysis of Fizz1. (e, f) Identification of PASMCs, the expression of α-SMA was verified by immunocytochemistry and immunofluorescence staining. Scale bar = 50 μm. (g, h) CX3CL1 and CX3CR1 concentrations were measured using ELISA. Values were means ± SD, n = 6. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 50 μm. *P < 0.05, ** P < 0.01

Full size image
Fig. 11
figure 11

M2 macrophage polarization induced by IL-21 promoted hypoxia-induced proliferation and pyroptosis of PASMCs. (a) IL-21 did not promote the proliferation of PASMCs assessed using EdU immunofluorescent staining. (b) The percentage of EdU-positive PASMCs. (c) Alveolar macrophages that had been cultivated with IL-21 promoted the proliferation of PASMCs assessed using EdU immunofluorescent staining. (d) The percentage of EdU-positive PASMCs. (e) IL-21 did not promote the expression of caspase-1 in PASMCs assessed using immunofluorescence staining. (f) The positive expression of caspase-1 was analyzed by histogram. (g) Alveolar macrophages that had been cultivated with IL-21 promoted the expression of caspase-1 in PASMCs assessed using immunofluorescence staining. (h) The positive expression of caspase-1 was analyzed by histogram. (i, k) double staining of propidium iodide (PI) and Hoechst 33,342 in PASMCs. (j, l) The percentage of PI-positive PASMCs. Values were means ± SD, n = 6. Comparisons were made using the one-way ANOVA with Bonferroni’s post hoc tests. Scale bar = 100 μm. *P < 0.05, ** P < 0.01

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Discussion

PH is characterized by a progressive and irreversible pulmonary vascular remodeling and the pathological mechanisms remain largely unexplored. HPH due to chronic hypoxia lung diseases is classified as Group 3 PH, with no current proven targeted therapies [16]. Accumulating evidence suggests a functional role of immune cells in the initiation and/or progression of pulmonary vascular remodeling in HPH [17]. TFH cell mainly promote germinal center (GC) reaction, provide help to B cells and primarily produce IL-21, which characterized with the surface molecules C-X-C motif chemokine receptor type 5 (CXCR5) and programmed cell death protein-1 (PD-1) [7]. B cell lymphoma 6 (BCL-6) is identified as a master transcription factor of TFH differentiation. Bcl-6-deficient CD4+ T cells are not able to differentiate into TFH cells [18]. We have previously shown that chronic hypoxia promoted TFH differentiation in HPH [11]. However, the role of TFH cells in HPH is unknown. A previous study demonstrated that TFH cell and IL-21 deficiency protected from chronic Ang II-induced hypertension and vascular dysfunction, which suggested that TFH /IL-21 may be a novel therapeutic target for the treatment of vascular disorders [19].

To determine whether TFH cells would exacerbate pulmonary vascular remodelling in HPH, we adoptively transferred TFH cells from mice exposed to hypoxia for 4 weeks into HPH mice. Our data demonstrated that adoptive transfer of TFH cells remarkably exacerbated abnormal hemodynamics, pulmonary vascular remodelling and right ventricle hypertrophy in HPH, which confirmed TFH cells exhibited the detrimental role in HPH. As a potential regulator, TFH cells participate in stimulating germinal center (GC) formation and supporting B cell differentiation [20]. In our study, we found that the number and size of germinal centers in spleen, percentage of GC B cells and expression of GC B-cell marker GL-7 were significantly increased in HPH group, which were further increased after adoptive transferring of TFH cells. Thus, our study may indicate that GC B cells played an important role in HPH, and TFH cells could exacerbate HPH by promoting GC B cells.

Increasing evidence suggests that perivascular inflammation plays an important role in pulmonary vascular remodeling process [21]. Macrophages are involved and well described in the pathogenesis of PH. Perivascular infiltration by macrophages recognized as a major pathogenic component of PH, suggesting that circulating inflammatory cells can be recruited and activated in affected vessels [22]. More and more evidence showed that macrophages can change tissue remodeling by affecting cell proliferation, survival, migration and adhesion [23]. In the current study, our data showed that percentages of M2 macrophage in spleen and BALF, the expression of Fizz1 of HPH group increased significantly compared to those of control group, which were further increased after adoptive transferring of TFH cells. Therefore, our study indicated that TFH cells specifically induced the M2 macrophage polarization in HPH.

To further evaluate the role of TFH cells in HPH, TFH generation was blocked by 79 − 6, a Bcl-6 specific inhibitor (Bcl-6i), in the HPH mice. 79 − 6 is a commercial and widely used Bcl-6 inhibitor and non-toxic to animals with favorable pharmacokinetics to suppress the generation of TFH cells [24]. In this study, we found that Bcl-6 targeting with the small molecule 79 − 6 resulted in not only the reduction of pulmonary vascular remodelling and right ventricle hypertrophy, but also the decreased number and size of germinal centers, percentage of GC B cells and expression of GL-7. In addition, we also found 79 − 6 exhibited a remarkable effect on suppressing the percentages of M2 macrophage in spleen and BALF, and the expression of Fizz1 and IL-21 in BALF in HPH. Therefore, it was speculated that downregulation of TFH cells protected from chronic hypoxia–induced pulmonary vascular remodelling and M2 macrophage polarization.

Since IL-21 is a key driver of the GC reaction and produced predominantly by TFH cells [25]. Previous work showed that IL-21 may be a potential target for treating pulmonary hypertension [14]. Therefore, we investigated the effect of IL-21 deficiency on pulmonary vascular remodelling and M2 macrophage polarization in HPH. Here, we show that loss of IL-21 abrogated the abnormal hemodynamics, pulmonary vascular remodelling and right ventricle hypertrophy induced by chronic hypoxia. In published data, IL-21 was also the most potent cytokine known to induce GC B cells proliferation [26]. In keeping with this, we found that chronic hypoxia exposure significantly increased the number and size of germinal centers, percentage of GC B cells and expression of GL-7 in WT mice, which were not present in IL-21−/− mice. Finally, we showed that IL-21 deficiency resulted in the reduction of percentages of M2 macrophage in spleen and BALF, and the expression of Fizz1 in BALF. In addition, IL-21 could remarkably upregulate M2 macrophage polarization and the protein level of Fizz1 under hypoxic condition in vitro. However, IL-21 deficiency did not affect the M1 macrophage signature gene expression. Collectively, these findings suggested that IL-21 deficiency protected against pulmonary vascular remodeling and suppressed M2 macrophage polarization in HPH.

Pulmonary hypertension is a condition characterized by change in extracellular matrix deposition, which results in increased vessel wall thickness and lumen occlusion, leading to vessel stiffening [27, 28]. Previous reports have demonstrated that extracellular matrix protein 1 (ECM1) was a biologically important protein with roles in malignant tumors and multiple tissues [29,30,31]. A recent report showed that ECM1 was critical for TFH differentiation and antibody response, ECM1 enhanced TFH differentiation by antagonizing IL-2–STAT5 and promoting Bcl6 signaling. ECM1 regulated the TFH differentiation via the IL-2–STAT5–Bcl6 axis [32]. In the present study, intratracheal instillation delivery of AAV-shECM1 reduced the mean PAP, RV/(LV + S) ratio and PAMT in HPH. Additionally, ECM1 downregulation reversed TFH cell polarization, Bcl-6 expression and IL-21 level in BALF. AAV-shECM1 treatment enhanced IL-2 and p-STAT5 expression in BALF. All these data indicated that ECM1 knockdown promoted IL-2–STAT5 signaling and inhibited Bcl6 signaling to inhibit TFH differentiation in HPH.

The pathogenesis of PH involves aberrant proliferation of pulmonary artery smooth muscle cells, which is the main pathological change causing pulmonary vascular remodelling [33]. Moreover, several novel types of programmed cell death, such as pyroptosis, autophagy and ferroptosis, have been reported to be involved in the development of PH [34,35,36]. Pyroptosis is a pro-inflammatory form of programmed cell death, whose genesis directly depended on caspase-1 activation. Caspase-1 inhibition attenuated the pathogenesis of PH with suppressed pulmonary vascular remodeling and right ventricle hypertrophy [37, 38]. Previous studies have shown that pyroptosis had occurred in hypoxic human pulmonary arterial smooth muscle cells and the media of pulmonary arteries in PH models [39]. Our present study revealed that the expression of α-SMA increased in pulmonary arteries of WT but not IL-21−/− mice. In addition, our results demonstrated that treatment with IL-21 did not promote the proliferation and pyroptosis of PASMCs. Intriguingly, treatment with the conditioned medium of the alveolar macrophages that had been cultivated with IL-21 promoted PASMCs proliferation and pyroptosis. However, treatment with a selective CX3CR1 antagonist, AZD8797 (AZD), significantly attenuated the proliferation and pyroptosis of the PASMCs. Thus, we speculated that IL-21 promoted PASMCs proliferation and pyroptosis in vivo and in vitro by CX3CL1, a soluble factor secreted by the M2 macrophages.

In conclusion, we showed that pharmacological inhibition of TFH cells ameliorated pulmonary vascular remodelling and suppressed M2 macrophage polarization. Mice deficient in IL-21 exhibited reduced M2 macrophage polarization and blunted pulmonary hypertension. Furthermore, M2 macrophage polarization induced by IL-21 played a critical role in the hypoxia-induced proliferation and pyroptosis of PASMCs. To our knowledge, this is the first study suggested that inhibition of TFH cells or specific depletion of IL-21 may be a novel therapeutic strategy for the treatment of HPH.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

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Funding

This work was supported by grant from the Doctoral Fund Project of Hunan Provincial People’s Hospital (No. BSJJ202210).

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Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University, Changsha, Hunan, 410005, People’s Republic of China

    Cheng Li, Hao Zhu, Huan Yang, Shaoze Zhang, Jin Huang, Guang Li, Gang Jiang & Yongliang Jiang

  2. Department of Emergency, Hunan Province Key Laboratory of Pediatric Emergency Medicine, Hunan Children’s Hospital, Changsha, Hunan, People’s Republic of China

    Pingping Liu

  3. Department of Respiratory and Critical Care Medicine, Changsha Central Hospital, University of South China, Changsha, Hunan, People’s Republic of China

    Jun Zha

  4. Department of General Medicine, Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University, Changsha, Hunan, People’s Republic of China

    Huiling Yao

  5. Department of Respiratory Diseases, Medical School, Hunan University of Chinese Medicine, Changsha, Hunan, 410208, People’s Republic of China

    Aiguo Dai

  6. Hunan Province Key Laboratory of Vascular Biology and Translational Medicine, Changsha, Hunan, People’s Republic of China

    Aiguo Dai

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Contributions

Conception and design: Cheng Li, Yongliang Jiang and Aiguo Dai. Collection and assembly of data: Pingping Liu, Huiling Yao, Hao Zhu and Shaoze Zhang. Data analysis and interpretation: Cheng Li, Huan Yang, Jun Zha, Guang Li, and Jin Huang. Manuscript writing: Cheng Li. Manuscript revision: Cheng Li, Gang Jiang, Yongliang Jiang and Aiguo Dai. All authors read and approved the final manuscript.

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Li, C., Liu, P., Zhu, H. et al. T follicular helper cell is essential for M2 macrophage polarization and pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension. Respir Res 25, 428 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03058-9

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