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STAT6 deficiency mitigates the severity of pulmonary arterial hypertension caused by chronic intermittent hypoxia by suppressing Th2-inducing cytokines

Respiratory Research volume 26, Article number: 13 (2025) Cite this article

Abstract

Background

Obstructive sleep apnea (OSA) is frequently associated with increased incidence and mortality of pulmonary hypertension (PH). The immune response contributes to pulmonary artery remodeling and OSA-related diseases. The immunologic factors linked to OSA-induced PH are not well understood. STAT6 is crucial in the signaling pathway that modulates immune response. However, the status of phosphorylated STAT6 (p-STAT6) in an OSA-induced PH mouse model remains largely unexplored.

Methods

Chronic intermittent hypoxia (CIH) plays a crucial role in the progression of OSA. This study utilized a in vivo CIH model to examine the role of STAT6 in CIH-induced PH.

Results

CIH mice exhibited pulmonary artery remodeling and pulmonary hypertension, indicated by increased right ventricular systolic pressure (RVSP), higher right ventricular to left ventricular plus septum (RV/LV + S) ratios, and significant morphological alterations compared to normoxic (Nor) mice. Increased p-STAT6 in the lungs and elevated p-STAT6 + IL-4 + producing T cells in CIH mice. STAT6 deficiency (STAT6-/-) improved PH and PA remodeling in CIH-induced PH mouse models.STAT6 deficiency impaired the T helper 2 (Th2) immune response, affecting IL-4 and IL-13 secretion. IL-4, rather than IL-13, activated STAT6 in human pulmonary artery smooth muscle cells (hPASMCs). STAT6 knockdown decreased the proliferation in IL-4 treated hPASMCs.

Conclusion

These findings exhibit the critical role of STAT6 in the pathogenesis of CIH induced PH by regulating Th2 immune response.STAT6 could be a significant therapeutic target for OSA-related PH.

Introduction

OSA is a prevalent public health issue with significant mid- and long-term consequences, including metabolic, cognitive, cancer-related, and cardiovascular complications [1,2,3]. A potentially serious complication is pulmonary hypertension (PH), marked by pulmonary artery remodeling, elevated pulmonary vascular resistance, right ventricular hypertrophy, and eventual right heart failure [4]. PH, a serious lung disease, frequently occurs in patients with OSA. If OSA-induced PH is not treated, it leads to poorer outcomes and increased mortality [5, 6]. Meanwhile, CIH is the hallmark of OSA [7, 8]. Exploring the pathophysiology of CIH-induced PH and pinpointing crucial molecules or signaling pathways in PA remodeling may hold significant clinical relevance. OSA is prevalent and involves recurring upper airway blockages, causing hypoxemia and sleep disruption [9]. Chronic, low-grade systemic inflammation is likely the main factor contributing to cardiovascular issues in OSA patients, despite the multifactorial and not fully understood underlying mechanisms [10].

Elevated levels of IL-4 and IL-13 (Th2 cytokines), have been observed in both OSA patients and CIH animals [11, 12]. Emerging evidence indicates that perivascular immune and inflammatory responses are crucial in regulating vascular remodeling and PH processing [13]. Patients with PH exhibit significant CD4 + T cell infiltration around PAs and elevated serum Th2 cytokines, including IL-4, IL-5, and IL-13 [14, 15]. Moreover, the Th2 immune response contributes to pulmonary artery remodeling in the progression of PH in animal models induced by soluble antigens or hypoxia [16, 17]. Evidence indicates that Th2 cytokines are pro-oxidative and proangiogenic, influencing the cardiovascular system by inducing inflammatory responses. This includes VEGF, VCAM-1, MCP-1, and P-selectin accumulation in vascular endothelial cells, as well as directly stimulating PASMC proliferation under hypoxic conditions, partially mediated by STAT6 [14, 18, 19].

STAT6 regulates lung inflammatory responses, crucially influencing smooth muscle alterations, B cell IgE synthesis, airway eosinophilia, Th2 cell differentiation, and epithelial mucus production in vivo [20, 21]. STAT6 is downstream of IL-4 and IL-13, which are elevated in human pulmonary inflammatory diseases and are thought to be crucial in the progression of PH [16, 22, 23].

The role of STAT6 and Th2 response in CIH induced PH has not yet been characterized in CIH-induced PH. We analyzed the expression of STAT6 and p-STAT6 in lungs of mice with CIH induced PH. Furthermore, Th2 immune response and STAT6 activation in immune subsets from the lungs of CIH-treated mice were detected by flow cytometry. STAT6-KO mice were utilized to create a CIH-induced PH model to examine the impact of STAT6 deficiency on Th2 activation and vascular remodeling. In vitro, the effect of CIH, Th2 cytokines and STAT6 on the proliferation of hPASMCs were detected.CIH-treated mice exhibited elevated p-STAT6 levels in both lungs and Th2 cells compared to Normoxic mice. STAT6 deficiency attenuated PH, decreased Th2 immune response and reduced the proliferation of hPASMCs.

Methods

Animal model of CIH

WT control and STAT6-KO mice on a C57BL/6 background were sourced from the Model Animal Research Center of Nanjing University and housed at the Experimental Animal Center of Fudan University. In vivo CIH research was conducted as outlined in [24, 25]. Eight-week-old male animals underwent a 1-minute intermittent hypoxia cycle in a specialized chamber, with oxygen levels alternating between 4% and 21% (CIH group; n = 6).The control group (Nor group; n = 6) was exposed to room air under identical environmental conditions.Nitrogen was introduced into the chambers to achieve and maintain a 4–7% fraction of inspired oxygen (FiO2) within 30 s for a duration of 10 s. Oxygen was given to reach an FiO2 of 20–21% within 30 s.This intermittent hypoxia (IH), simulating 60 apneas per hour typical of OSA [24, 25], was applied for 8 h daily (8:00–16:00) over 6 weeks.

Echocardiography

Echocardiography was performed under anesthesia after 12 h following the final IH cycle. Isoflurane was administered via inhalation at 3% for induction and maintained at 1-1.5%. Following anesthetization, each mouse was positioned supine on a temperature-controlled pad. Hair was removed from the anterior chest using a depilatory agent. The Vivid S5 echocardiography system probe (GE Healthcare, Chicago, IL) was carefully tilted laterally to visualize the pulmonary artery crossing the aorta.Subsequently, the ultrasound was set to color Doppler mode.Positioning the ultrasound pulse wave line parallel to the blood flow in the PA allows for the acquisition of a flow waveform. This procedure can be replicated to assess flow through the pulmonary valve and right ventricular outflow tract. The saved views were utilized to test the PA’s peak flow velocity using Vevo LAB 2.1.0 software.

PA pressure assessment

After echocardiographic measurement, a longitudinal incision was made on the right neck, followed by layer-by-layer blunt dissection to expose the right external jugular vein. A Mikro-Tip catheter with transducer (Millar, Inc, USA) was inserted into the PA via an incision in the mouse’s right external jugular vein to record RV systolic pressure. After the experiment, mice were anesthetized with 3% sodium pentobarbital through intraperitoneal injection, and organs were collected. Right ventricular hypertrophy was evaluated by determining the ratio of the right ventricular wall weight to the total weight of the left ventricle and septum.

Immunohistochemistry

Following flushing, the upper lung lobe tissue was preserved at -80 °C for RNA and proteomic analysis.The left lung’s lower lobe was perfusion-fixed using 3.8% paraformaldehyde and subsequently embedded in paraffin.For standard histological examination, 4 μm tissue sections underwent hematoxylin and eosin (H&E) or Masson staining.

Flow cytometry

Lungs were separated into individual lobes and digested using an enzyme mixture comprising buffer S, enzyme D, and enzyme A (MiltenyiBiotec, Bergisch Gladbach, Germany). Cell suspensions were filtered through a 70-µm nylon mesh, and red blood cells were lysed with a cell lysing solution (BD Biosciences PharMingen, San Diego, CA, USA). Cells were incubated on ice for 10 min with saturating doses of purified anti-mouse Fc receptor (CD16/32, clone 2.4G2, BD Bioscience) in 1 ml PBS containing 5% FCS to block Fc receptor binding. The following antibodies were used (eBioscience): BV510-labelled anti‐CD3, FITC‐labelled anti‐CD4, HorizonTM BV605 labelled anti-CD4. We stimulated cytokine expression in the lung using Leuko Act Cktl With GolgiPlug (BD Pharmingen) for 6 h to detect Th1, Th2, and p-STAT6 + cells. After surface staining, cells were fixed and permeabilized using Fix/Perm Reagent (BD Biosciences), followed by intracellular staining with FITC‐labelled anti‐IL‐INF-γ, BV421‐labelled anti‐IL-4, and APC‐labelled anti‐p-STAT6 antibodies.Isotype controls for IL-4, IL-INF-γ, and p-STAT6 antibodies were employed to adjust compensation and verify antibody specificity.

ELISA analysis

We instilled 500 µl of ice-cold PBS into the lungs to collect bronchoalveolar lavage fluid (BALF) from the mice.This process was repeated three times to ensure adequate retrieval of the lavage fluid. BALF was centrifuged at 1500 g for 10 min at 4 °C, and the supernatant was stored at -80 °C for cytokine analysis. Cytokine concentrations in mouse serum and BALF were determined using ELISA kits (Multisciences, Hangzhou, China) according to the manufacturer’s guidelines.The optical density (OD) of the tested samples was compared to values from serial dilutions of the corresponding recombinant cytokine.

Western blot analysis

Western blot analysis on cell and tissue lysates was conducted as previously described [13]. Membranes were blocked with 5% non-fat dry milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies targeting total STAT6 (Millipore), p-STAT6 (Cell Signaling), α-SMA (Abcam), or GADPH (Sigma-Aldrich). Subsequently, membranes were treated with secondary antibodies (anti-rabbit or anti-mouse horseradish peroxidase, Cell Signaling), and protein bands were visualized using chemiluminescence with Western Lightning (Perkin Elmer, Waltham, MA).

LV-shSTAT6 transfection

Cells were seeded in 24-well plates at 1.0 × 10^4 cells per well or in six-well plates at 1.0 × 10^6 cells per well and incubated overnight before transfection.Cells at 30%-40% confluence were transfected with LV-shSTAT6 (5’-GCCACATTAACGCGCAGAT-3’) or LV-NC according to the manufacturer’s instructions (Biolink, Shanghai, China). Transduction was performed using LV-shSTAT6 or LV-shNC at an MOI of 100 in 300 µL of DMEM with 10% FBS for 24-well plates or 1 mL for six-well plates.After 12 h, the cells were washed with PBS to remove lentiviral genomic RNA, and fresh medium was added.

Quantitative real-time PCR

Cells were exposed to CIH or normoxia for different periods 48 h post-transfection before collection for further experiments. Total RNA was extracted from lung tissue samples and cultured cells using TRIzol (Takara Bio, Japan). cDNA was synthesized from isolated RNA and subjected to quantitative PCR using an SYBR QPCR kit (Toyobo, Osaka, Japan).

Adenovirus construction and infection

Mouse STAT6 was cloned from mouse cDNA into the RedTrack-CMV vector (Addgene, MA, USA) using KpnI and XbaI restriction sites. The AdTrack-CMV-STAT6 plasmid was linearized using PmeI. Subsequently, it was cotransformed into Escherichia coli BJ5183 cells alongside the pAdEasy-1 plasmid. Clones that underwent AdTrack-Adeasy recombination were selected using kanamycin and verified through enzyme digestion. The recombinant plasmid was linearized with PacI and transfected into the Adeno-X cell line using Lipofectamine 3000 Transfection Reagent to package it into active virus particles. The adenoviral-RFP-STAT6 (Ad-STAT6) and adenoviral-RFP-empty (Ad-Ctrl) viruses were further amplified through serial passage to achieve concentration.Adeno-X cells at 70% confluence were infected with the virus and cultured for 72–96 h. For adenovirus infection, 1 × 10^6 cells were plated and exposed to either a STAT6 vector (Ad-STAT6) or an empty vector (Ad-Ctrl) at the same infectious unit concentration for 6 h.

Culture of hPASMCs

hPASMCs were obtained from iCell Bioscience, Inc., Shanghai.hPASMCs were cultured at 37 °C in a humidified incubator with 5% CO2 using PriMed-iCell-004-LS medium (iCell Bioscience, China).Cells at passages 2–5 were used for further investigation.

CIH model in vitro

In this study, we employed a IH or normoxia model in vitro using a custom computer-controlled incubator chamber connected to an external O2-CO2 servo controller (Biospherix, Lacona, NY), following the method outlined by Almendros et al. [26]. hPASMCs were maintained at 37 °C, with O2 levels adjusted between 0% and 21% every 30 min by injecting N2 or O2, alongside 5% CO2. The dissolved O2 in the culture medium was monitored using a laser O2 probe (Biospherix), ensuring hypoxic conditions at 5% O2 and normoxic conditions at 21% O2, based on cellular sensing. Normal air conditions were defined as 21% O2 and 5% CO2.

hPASMCs proliferation measurement

The proliferation of hPASMCs was assessed using a CCK-8 Kit (Bestbio, Shanghai, China) based on the manufacturer’s instructions.Measurements were taken at 450 nm using a Tecan multimode reader (Männedorf, Switzerland).

Statistical analyses

All statistical analyses were conducted using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as mean ± standard deviation.Student’s t-test was used for two-group comparisons, and ANOVA with Dunnett’s test was applied for comparisons involving multiple groups.Significance was determined at p < 0.05. All results are based on a minimum of three independent replicates.All experiments were conducted under blind conditions.

Results

Increased STAT6 activation in the lung of PH mouse model induced by CIH

STAT6 activation is crucial in pulmonary diseases. We established a mouse model to examine the role of STAT6 activation in CIH-induced PH pathogenesis by analyzing changes in STAT6 expression. After 6 weeks of exposure, mice subjected to CIH exhibited significantly higher RV pressure (RVP) compared to those exposed to Nor (22.33 ± 2.60 mmHg vs. 47.33 ± 2.36 mmHg, p < 0.001, n = 6; Fig. 1A). The RV hypertrophy index indicated an increased thickness of the RV wall in CIH mice compared to the normal group (Nor vs. CIH: 22.17 ± 2.84% vs. 32.32 ± 1.42%, p < 0.01, n = 6; Fig. 1B). Echocardiography revealed a reduced velocity in the CIH group compared to the control group (Nor: 630 ± 11.53 mm/s vs. CIH: 574.5 ± 9.32 mm/s, p < 0.05, n = 6; Fig. 1C). Meanwhile, vascular wall thickness (Fig. 1D-E), was changed in CIH mouse. These results above indicated the pulmonary arterial remodeling and the successful establishment of the PH mice model. We evaluated pulmonary artery p-STAT6 induction in a CIH mice model by analyzing total and p-STAT6 expression in lung tissues from WT and CIH mice using Western blot. Figure 1F-G demonstrates a significant increase in pulmonary p-STAT6 in WT mice exposed to CIH compared to normoxic controls. Moreover, with the immunofluorescence staining of p-STAT6, we found that CIH exposure induced p-STAT6 expression in pulmonary arteries (Fig. 1H-I).

Fig. 1
figure 1

CIH induced pulmonary hypertension in mice and p-STAT6 was upregulated in CIH-treated mice. Mice were exposed to Nor or CIH conditions for 6 weeks. PVSP (A), RV/(LV + S) (B) and pulmonary blood flow velocity (C) were measured. (D) The respective H&E and Masson stain of pulmonary arterioles in Nor and CIH groups were presented. (E) The percentage wall thickness of pulmonary arterioles was defined as the area occupied by the vessel wall divided by the total cross-sectional area of the arteriole. (F-G) Western blot analysis of p-STAT6, total-STAT6 and α‐SMA protein in the mice lungs from the two groups. (H) Immunohistochemistry analysis of p-STAT6 expression in mice lungs from the two groups. (I) The relative fluorescence density of p-STAT6 were measured. Data are presented as the mean ± SD (n = 6 per group). No significant difference is indicated by ns. Significant differences are presented as *(p < 0.05), **(p < 0.01), ***(p < 0.001) and determined by Student’s t‐test unless specified. Nor: normoxia; CIH: chronic intermittent hypoxia; RVSP: Right ventricular systolic pressure; H&E: hematoxylin and eosin

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Th2 immune response was enhanced in CIH mice model

Prior studies indicate an elevation of the Th2 immune response and associated cytokines in OSA patients and CIH animals, highlighting the significant role of STAT6 in lymphocytes. To investigate if enhanced Th2 immune response and lymphocyte STAT6 activation are involved in CIH induced PH, we performed flowcytometry on single cell of digested lungs with staining for CD3, CD4, IL-4, INF-γ (Th1 and Th2 signature cytokines, respectively) and p-STAT6. CIH mice exhibited an increased number of CD3 + CD4 + T and Th2 cells compared to the control group (Fig. 2A-B). Since STAT6 have been proposed as a key mediator in Th2 differentiation, we interrogated lung cells for p-STAT6 + Th2 cells (IL-4 + CD4 + CD3+) and p-STAT6 + Th1 cells (INF-γ + CD4 + CD3+) using flow cytometry (Fig. 2C). We found increased expression of p-STAT6 in Th2 cells instead of Th1 cells in CIH group compared to control (Fig. 2D-E). These data indicate that CIH enhances Th2 immune response and activates STAT6, potentially contributing to PH development in a mouse model.

Fig. 2
figure 2

Th2 inmmune response was enhanced in CIH-treated mice. (A) The gating strategy for Th1 and Th2 cells. (B) The percentage of CD3+CD4+ T cells in lung, IL-4+ cells in CD3+CD4+ T cells, Th2 in lung and Th1/Th2 were statistically analyzed in Nor and CIH groups. (C) The gating strategy for p-STAT6 in Th2 cells. (D) The representative flow cytometric analysis of p-STAT6 in Th1 and Th2. (E) The percentage of p-STAT6+ cell in Th1 and Th2 were measured and analyzed. Data are presented as the mean ± SD (n = 3 per group). No significant difference is indicated by ns. Significant differences are presented as *(p < 0.05), **(p < 0.01), ***(p < 0.001). Nor: normoxia; CIH: chronic intermittent hypoxia

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CIH-induced PH is attenuated in STAT6-KO mice

To determine the role of STAT6 in vivo, we induced PH by CIH exposure in WT and STAT6-/- mice.STAT6-/- CIH mice exhibited attenuated PH as evidenced by decreased RVP values compared with those of WT CIH mice (all p<0.001, n=6; Fig. 3A-B). CIH mice exhibited reduced pulmonary artery acceleration time (PAAT), whereas STAT6-/-CIH mice demonstrated improved PAAT compared to the CIH control group (Fig. 3C-D). The decreased PH was further determined in STAT6-/- CIH mice by RV/(LV + S) (Fig. 3E-F). STAT6 deletion mitigated CIH-induced pulmonary vascular remodeling, reducing vascular wall thickness by decreasing media and significantly loweringα-SMA-expressing cells, thereby improving pulmonary vascular muscularization (Fig. 4A-B). STAT6 deficiency also inhibited the collagen deposition in lungs (Fig. 4C). The data showed that WT CIH mice exhibited histologic features akin to human PH, including PA remodeling and collagen deposition, which were less pronounced in STAT6-/- CIH mice.

Fig. 3
figure 3

Deficiency in STAT6 ameliorates CIH-induced pulmonary hypertension. (A) Right ventricular systolic pressure (RVSP) in STAT6-/- and WT mice after six weeks of Nor or CIH exposure. (B) The representative photomicrographs are presented. (C) Pulmonary artery acceleration time were measured in four groups, and the representative echocardiogram are presented (D). (E) RV/(LV + S) weight ratio in STAT6-/- and WT mice after 6 weeks of normoxia or CIH exposure. (F) Representative images of H&E staining of heart sections of STAT6-/- and WT mice after 6 weeks of Nor or CIH exposure. Scale bar: 1 mm. Data are presented as the mean ± SD (n = 6 per group). No significant difference is indicated by ns. Significant differences are presented as (p < 0.05), **(p < 0.01), ***(p < 0.001) and determined by Student’s t-test unless specified. Nor: normoxia; CIH: chronic intermittent hypoxia; WT: wide type; H&E: hematoxylin and eosin

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Fig. 4
figure 4

STAT6 deficiency ameliorates CIH-induced pulmonary remodeling. (A) Representative images of H&E staining, α-SMA immunostaining and Masson staining of lung sections of STAT6-/- and WT mice after 6 weeks of normoxia or CIH exposure. (B) The percentage wall thickness of pulmonary arterioles was defined as the area occupied by the vessel wall divided by the total cross-sectional area of the arteriole. (C) The vascular collagen area was evaluated by quantitative image analysis of Masson’s trichrome staining. Scale bar: 20 μm. The results are expressed as the mean ± SD of 20 vessels from 6 mice per group. *p < 0.05, **P < 0.01, and ***P < 0.001 compared with the WT group. Nor: normoxic; CIH: chronic intermittent hypoxia; H&E: hematoxylin and eosin; WT: wild type

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STAT6 deficiency suppressed Th2 response in CIH-induced PH mouse models

To test whether STAT6 deficiency reduces Th2 response, the CD4 + T cells and the subsets in the lungs of STAT6-/- mice were detected by immunohistochemical analysis and flow cytometry. Consistent with expectations, CD4 + T cell infiltration around the PAs in the lungs of STAT6-/- CIH mice was diminished (Fig. 5A-B and D-E). Flow cytometry analysis revealed that both the percentage and total count of Th2 cells in lung tissues were reduced in STAT6-/- CIH groups compared to WT CIH groups (Fig. 5C and F). IL-4 and IL-13, the primary cytokines secreted by Th2 cells, were significantly inhibited in both serum and BALF of STAT6-/- mice following CIH treatment compared to WT mice (Fig. 6A-D).

Fig. 5
figure 5

STAT6 deficiency decreases Th2 immune response induced by CIH. (A) Representative images of CD4 staining of lung sections of STAT6-/- and WT mice after 6 weeks of normoxia or CIH exposure. (B) Statistics analysis of the number of CD4+ cells in the cross-section of lungs from the four groups. (C) Representative flow cytometry of CD3+CD4+ T cells and Th1 and Th2 cells from the four gourps. (D) The percentage of CD3+CD4+ T cells and Th2 in CD3+CD4+ T cells were measured and analyzed. Data are presented as the mean ± SD (n = 6 per group). No significant difference is indicated by ns. Significant differences are presented as *(p < 0.05), **(p < 0.01), ***(p < 0.001) and determined by Student’s t-test unless specified. Nor: normoxic; CIH: chronic intermittent hypoxia; H&E: hematoxylin and eosin; WT: wild type

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Fig. 6
figure 6

IL-4 promotes the proliferation of hPASMCs. The concentration of IL-4 (A) and IL-13 (B) in serum from STAT6-/- and WT mice after 6 weeks of normoxia or CIH exposure (n = 6 per group). The concentration of IL-4 (C) and IL-13 (D) in BALF from STAT6-/- and WT mice after 6 weeks of normoxia or CIH exposure (n = 6 per group). (E) CCK8 analysis of hPASMCs with IL-4 treatment under concentration of 0, 0.5, 1,10, and 100 ng/ml. (F) CCK8 analysis of hPASMCs with IL-13 treatment under concentration of 0, 0.5, 1,10, and 100 ng/ml. (G) The percentage of Ki67-positive cells in 10ng/ml IL-13 or IL-4 treated hPASMCs. Data are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *(p < 0.05), **(p < 0.01), ***(p < 0.001) and determined by Student’s t-test unless specified. hPASMCs: human pulmonary artery smooth muscle cells; Nor: normoxic; CIH: chronic intermittent hypoxia; WT: wide type; BALF: bronchoalveolar lavage fluid

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STAT6 deficiency suppressed IL-4 induced proliferation of hPASMCs under CIH

Our earlier study showed that intermittent hypoxia stimulates PASMC proliferation both in vivo and in vitro [27]. To determine if the proliferative phenotype of vascular smooth muscle cells is directly influenced by STAT6 or by a combined effect of IL-4 and IL-13, we utilized an in vitro system. We cultured hPASMCs under CIH conditions as described in methods.CCK8 investigated the roles of STAT6 and Th2 cytokines in the proliferation of hPASMCs under CIH conditions.The results of CCK8 and flow cytometry results indicate that IL-4, rather than IL-13, promotes the proliferation of PASMCs (Fig. 6E-G). We further investigated the effect of CIH on the activation of STAT6 in hPASMCs. The results showed that CIH did not directly cause the phosphorylation of STAT6.However, when combined with IL-4, CIH significantly increased the content of p-STAT6 in hPASMCs, while IL-13 has no obvious effect on p-STAT6 (Fig. 7A-D). Subsequently, we examined the involvement of STAT6 in IL-4-induced hPASMC proliferation. We transfected hPASMCs with lentivirus, knocked down or overexpressed STAT6, and then treated the cells with or without IL-4.Knocking down STAT6 reduced hPASMC proliferation and weakened the proliferative effect of IL-4 on hPASMCs. The proliferation of hPASMCs was not increased with overexpression of STAT6, while treated with IL-4, the proliferation of hPASMCs increased (Fig. 7E-H). The findings indicate that IL-4 enhances hPASMC proliferation through STAT6 activation.

Fig. 7
figure 7

IL-4 increased p-STAT6 expression in hPASMCs. (A) Western blotting was performed to assess the expression of the STAT6 and p-STAT6 in hPASMCs treated with 10ng/ml IL-4 or IL-13 for 24 h. (B) Quantitative analysis of relative expression analysis of STAT6 and p-STAT6 using ImageJ. (C) Western blotting was used to detect the expression of STAT6 and p-STAT6 in hPASMCs. (D-E) Quantitative analysis of STAT6 and p-STAT6 in hPASMCs using ImageJ. (F) Under Nor or IH conditions, hPASMCs were treated with 10 ng/ml IL-4 for 48 h. Cell proliferation was assessed using the CCK8 assay. (G-H) Knockdown or overexpression of STAT6 expression in hPASMCs, treat with 10 ng/ml IL-4 and culture for 48 h, then measure hPASMCs proliferation under different conditions using the CCK8 assay. Data are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *(p < 0.05), **(p < 0.01), ***(p < 0.001) and determined by Student’s t-test unless specified. Nor normoxia, IH intermittent hypoxia

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Discussion

Accumulating evidence have reported the key role of STAT6 and Th2 in the progression of pulmonary diseases.Our study extends this concept to the CIH model, indicating that STAT6 deficiency alleviates CIH-induced PH through mechanisms involving the Th2 immune response, reduced IL-4 and IL-13 expression, and decreased PASMC proliferation.Our research identifies STAT6 as a target for treating CIH-induced PH.

Here, we concluded the activation of STAT6 was triggered in mice lungs and Th2 cells with CIH treatment. Otherwise, we observed that Th2 immune response was enhanced in CIH-treated mice. To further validate the role of STAT6 and investigate potential mechanisms, we established a CIH model using WT and STAT6-/- mice. We compared pulmonary arterial pressure, pulmonary small artery remodeling, and Th2 immune responses between the two types of mice. Our findings confirm that the absence of STAT6 can alleviate CIH-induced pulmonary hypertension by reducing Th2 immune responses. In various animal models, such as those sensitized with OVA or schistosome antigens [28], persistent hypoxia animals [29], and COPD models, the infiltration of inflammatory cells around remodeled pulmonary vessels has been observed. Previous studies have reported that hypoxia creates a inflammatory environment in the pulmonary arteries, and the accumulation of lymphocytes in the lungs can lead to vascular remodeling [30]. Notably, Th2 cells can cause muscularization of pulmonary small arteries and are involved in the onset and progression of PH [14, 31, 32]. These observations are consistent with our findings. In the CIH mouse model, we observed increased infiltration of CD4+ T cells around the pulmonary vasculature and enhanced Th2 immune responses in the lungs.

The influence of CIH on the T cells in adult OSA patients has been rarely explored, and the available research presents inconsistent findings. For instance, Dyugovskaya et al. [33] reported that T cells in OSA patients exhibited both phenotypic and functional changes, with CD4+ and CD8+ T cells shifting toward a type 2 cytokine profile, as evidenced by elevated IL-4 expression. However, Elias et al.’s study [34] showed that OSA patients had an increased percentage of CD4 + T cells, with no significant difference in serum IL-4 levels. In contrast, IFN-γ levels were significantly lower in OSA patients. Ye et al. [35]. reported an elevated level of Th17 cells in the peripheral blood of OSA patients, along with an increased Th17/Treg ratio that correlated with the severity of the condition. In contrast, Polasky et al.’s study [36] found no significant change in the concentration of Th1/Th17 cells. Despite these conflicting findings, our study observed enhanced Th2 immune responses in the lungs of CIH mice, supporting a tendency for Th2 immune responses in OSA patients.

STAT6 is known to be involved in regulating Th2 differentiation [21]. Literature shows that STAT6-/- mice are unresponsive to IL-4 and IL-13-mediated signaling pathways. T lymphocytes lacking STAT6 also fail to differentiate into Th2 cells when stimulated with IL-4 or IL-13, despite Th2 cells being a major source of these cytokines [37,38,39]. In our study, we observed that Th2 responses in STAT6-/- mice were significantly reduced, and these mice could not mount a Th2 response to CIH stimulation. Immune cells can cause vascular damage through cytokine secretion, with IL-4 being associated with PH. Our study found that CIH increased IL-4 and IL-13 levels in mouse serum and BALF, whereas STAT6-/- affected Th2 cell differentiation and reduced IL-4 and IL-13 expression. Some studies suggest that antagonizing STAT6 can alleviate or reverse the development of PH [16, 40]. However, other research has found no significant difference in the response to hypoxia between STAT6-/- and WT mice, while IL-4-/- mice showed significant reduction in pulmonary small artery changes induced by hypoxia. This discrepancy may be due to differences in experimental conditions, such as continuous hypoxia exposure for 4 days [41].

IL-4 is reported to result in the development of PH [42, 43]. A study by Kazuyo et al. [44] found that under sustained hypoxic conditions, IL-4 indirectly promotes the activation of PASMCs by inducing fibroblasts to secrete VCAM-1 and VEGF. Additionally, studies have demonstrated that Th2-derived IL-4 and IL-13 promote vascular smooth muscle cell proliferation in a STAT6-dependent manner [14, 45]. Our study observed increased p-STAT6 in pulmonary arteries of CIH mice and increased IL-4 and IL-13 levels in BALF and serum. In vitro studies of human PASMCs showed that IL-4 promotes cell proliferation and activates STAT6, unlike IL-13. While CIH alone does not directly activate STAT6 in hPASMCs, it enhances p-STAT6 levels when combined with IL-4. The combination of IL-4 and CIH increases cell proliferation. Knockdown of STAT6 reduces the effect of IL-4, while STAT6 overexpression enhances IL-4-induced proliferation.

IL-4 and IL-13 engage their respective receptors (IL-4Rα, IL-13Rα1, or IL-13Rα2) to activate shared signaling pathways, including p-STAT6 [46]. The dual deficiency of IL-4 and IL-13 attenuates the development of experimental PH in mice [16]. Furthermore, Th2 activation via IL-4 stimulates IgE antibody production by B cells, which in turn activates mast cells and eosinophils [47]. These immune cells, particularly eosinophils and mast cells, play a crucial role in the pathogenesis of certain forms of pulmonary arterial hypertension, such as IL-33- or allergy-induced PAH [48, 49]. The role of IL-13 in PASMCs remains controversial [23]; some studies suggest that IL-13 promotes PASMC proliferation [50], while others disagree [51]. In endothelial cells, responses to IL-13 and IL-4 vary by location. IL-13 can activate STAT6 in pulmonary microvascular endothelial cells, promoting cellular transformation, though other studies show conflicting effects on angiogenesis [52, 53]. In this study, IL-13 did not significantly promote hPASMC proliferation or STAT6 activation compared to IL-4. Differences in the effects of IL-4 and IL-13 on PH suggest that IL-13 may involve other mechanisms in PH development. Further investigation is needed on IL-13’s other impacts on hPASMCs and the role of endothelial cells in PH formation. Additionally, STAT6 can be activated by other ligands like PDGF, angiotensin II, and leptin, which are related to cardiovascular diseases and OSA [38, 54]. Further validation of these factors’ effects on STAT6 in the CIH model is required.

Our study has several limitations. First, we did not analyze changes in p-STAT6 expression in lung tissues from OSA patients with PH. Additionally, the role of STAT6 in macrophage subset differentiation requires further investigation. Furthermore, while animal models of OSA have contributed significantly to our understanding of its pathogenesis and consequences, it is important to recognize the limitations of CIH models. Specifically, in CIH animal models, hypoxic exposure is introduced abruptly, whereas in clinical OSA, the severity of the condition and associated hypoxic events generally develop gradually over time. Moreover, both sleep deprivation and sleep fragmentation can exert distinct effects on the immune system. Due to substantial physiological differences between animals and humans, it remains unlikely that any animal model can fully replicate the anatomical and pathophysiological features of human OSA.

Conclusions

In summary, our study demonstrates that IL-4/STAT6 participates in CIH-induced PH by regulating Th2 immune responses. IL-4 promotes cell proliferation in hPASMCs by activating STAT6. Additionally, the combined effects of CIH and IL-4 significantly increase the levels of p-STAT6 in the cells and enhance cell proliferation. Therefore, STAT6 could be a potential therapeutic target for CIH-related PH.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We would like to thank Prof. Xiangdong Yang from Zhongshan Hospital, Fudan University, for sharing the experimental methods and his invaluable guidance.

Funding

Our research was sponsored by the Shanghai Sailing Program (22YF1407700 and 21YF1440300), the National Science Fund for Young Scholars (82200061) and the National Natural Science Foundation of China (No. 82370088, 82070094, 82470089).

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Author notes

  1. Pan Jiang and Huai Huang contributed equally to this work.

Authors and Affiliations

  1. Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Pan Jiang, Huai Huang, Zilong Liu, Guiling Xiang, Xiaodan Wu, Shengyu Hao & Shanqun Li

  2. Clinical Center for Sleep Breathing Disorder and Snoring, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Xiaodan Wu & Shanqun Li

  3. The Critical Care Medicine Department at Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Guiling Xiang & Shengyu Hao

  4. The Nutrition Department at Zhongshan Hospital, Fudan University, Shanghai, China

    Pan Jiang

  5. The Nutrition Department, QingPu District Central Hospital, Shanghai, 200032, China

    Pan Jiang

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Contributions

Conceptualization: Pan Jiang and Huai Huang; Formal analysis: Shengyu Hao and Guiling Xiang; Funding acquisition: Pan Jiang, Shengyu Hao, Xiaodan Wu and Shanqun Li; Methodology: Zilong Liu; Project administration: Pan Jiang, Shengyu Hao, Xiaodan Wu and Shanqun Li; Software: Zilong Liu; Supervision: Xiaodan Wu and Shanqun Li.; Validation: Pan Jiang and Huai Huang; Visualization: Pan Jiang and Shengyu Hao; Writing-original draft, Pan Jiang and Shengyu Hao; Writing-review and editing: Pan Jiang and Shengyu Hao. All authors reviewed the manuscript.

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Correspondence to Xiaodan Wu, Shengyu Hao or Shanqun Li.

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Jiang, P., Huang, H., Liu, Z. et al. STAT6 deficiency mitigates the severity of pulmonary arterial hypertension caused by chronic intermittent hypoxia by suppressing Th2-inducing cytokines. Respir Res 26, 13 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03062-z

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