OX40, which belongs to the tumour necrosis factor (TNF)-receptor family, is a costimulatory receptor that can potentiate T-cell receptor signalling on the surface of T-lymphocytes. The role of OX40 in non-immune systems, particularly the cardiovascular system, has not been defined. In the present study, we observed a noticeable increase in OX40 expression during cardiac remodelling in rodent heart. In the present study, cardiac hypertrophy was induced by aortic banding (AB) in OX40 knockout (KO) mice and wild-type (WT) mice. After 8 weeks, the OX40 KO mice showed significantly attenuated cardiac hypertrophy, fibrosis and inflammation as well as preserved cardiac function compared with the WT mice. Follow-up in vitro studies suggested that CD4+ T-lymphocyte proliferation and pro-inflammatory cytokine release were significantly decreased, whereas anti-inflammatory cytokine release was considerably increased in OX40 KO mice compared with WT mice as assessed by Cell Counting Kit-8 (CCK-8) assay and ELISA. Co-culturing neonatal rat cardiomyocytes with the activated supernatant of CD4+ T-lymphocytes from OX40 KO mice reduced the hypertrophy response. Interestingly, OX40 KO mice with reconstituted CD4+ T-lymphocytes presented deteriorated cardiac remodelling. Collectively, our data indicate that OX40 regulates cardiac remodelling via the modulation of CD4+ T-lymphocytes.

CLINICAL PERSPECTIVES

  • The present study provides in vivo and in vitro evidence that OX40 deficiency ameliorates cardiac remodelling by altering CD4+ T-cell function.

  • These results confirm a role for OX40 in cardiac remodelling in response to pressure overload and the association between OX40 and T-lymphocyte function in cardiac remodelling.

  • These observations are crucial to providing new insights into the mechanisms of cardiac remodelling and to developing novel strategies for the treatment of cardiac remodelling by targeting T-lymphocytes.

INTRODUCTION

Heart failure (HF) is a complex syndrome that results from acute injury, such as myocardial infarction or from more long-standing diseases such as pressure and volume overload [1]. The pathophysiological substrate of HF during the overall process of heart tissue remodelling [2] includes not only cardiomyocytes but also interstitial tissue, fibroblasts, inflammatory cells and endothelial cells [3]. Cardiac remodelling subsequently leads to diminished cardiac function. The mechanisms responsible for the progression of cardiac remodelling are complex and have not been clearly elucidated.

More recently, several lines of evidence have indicated the involvement of T-lymphocytes in cardiac remodelling. One report showed that adoptive Treg transfer could attenuate MI-induced cardiac remodelling through alterations to interferon-gamma (IFN-γ) and Foxp3 [4]. T-lymphocytes also regulate cardiac extracellular matrix (ECM) composition through modulation of collagen synthesis, degradation and cross-linking in cardiac remodelling in response to pressure overload [5]. Moreover, mice lacking CD4+ T-cells did not develop ventricular dilation and dysfunction after transverse aortic constriction (TAC) [6]. These results suggest the significance of the contribution of T-cells to cardiovascular remodelling.

OX40 (CD134) belongs to the tumour necrosis factor (TNF)-receptor family, also named TNF-receptor super family-4 (TNFrsf-4) [7]. OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signalling on the surface of CD4+ and CD8+ T-lymphocytes, leading to their activation by a specifically recognized antigen. Interaction between OX40 and its ligand, OX40L, dramatically increases the proliferation, survival and differentiation of and cytokine production by T-cells [8]. OX40/OX40L interactions have been found to play vital roles in the pathogenesis of multiple inflammatory and autoimmune diseases, including asthma [9], graft-versus-host disease [10] and autoimmune encephalomyelitis [11], as well as in anti-tumour therapy [12]. OX40 is mainly present on activated T-cells, whereas OX40L is primarily expressed on antigen presenting cells and on vascular endothelial cells [13]. OX40/OX40L interactions up-regulate intracellular levels of reactive oxygen species (ROS) and increase the secretion of cyclophilin A by lymphocytes, serving an important function in atherosclerotic plaque formation [14]. OX40/OX40L interactions also affect the development of acute coronary syndrome (ACS) [15], and blocking the OX40 costimulatory receptor may improve the survival of patients with ACS [16]. In humans, single nucleotide polymorphisms (SNPs) in both the OX40 and OX40L genes affect the incidence of cardiovascular disease [17,18]. These findings suggest that OX40 plays a role in cardiovascular disease. However, the role of OX40 in cardiac remodelling has not yet been defined.

In the present study, we used mouse models to investigate the role of OX40 in cardiac remodelling. Our results showed the key role of OX40 in CD4+ T-lymphocyte function in the progression of cardiac remodelling to HF.

MATERIALS AND METHODS

The animal protocol was approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. An expanded Materials and Methods section is available in the online-only Data Supplement, and includes the following detailed subsections: animals and animal models, echocardiography and haemodynamics, histological analysis and immunohistochemistry, quantitative real-time PCR (RT-PCR), western blot analysis, CD4+ T-lymphocyte isolation and culture, reconstitution of CD4+ T-cell populations into OX40 knockout (KO) recipient mice, cardiac immune cell isolation, flow cytometry, T-cell proliferation, ELISA, neonatal rat cardiomyocyte (NRCM) culture and immunofluorescence staining, and statistical analysis.

RESULTS

OX40 expression in hypertrophic hearts and cardiomyocytes

To explore the potential role of OX40 in the development of cardiac remodelling and HF, we first examined the OX40 expression levels in pathological hearts and cardiomyocytes. From an experimental mouse model of aortic banding (AB)-induced cardiac hypertrophy [as evidenced by elevated levels of β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP)], western blot and PCR analyses revealed that the OX40 expression level increased in mouse hearts at week 1 after AB, peaked at week 2 after AB and decreased to the baseline at week 4 after AB compared with sham-operated hearts (Figure 1A). Using in vitro cultured neonatal rat cardiomyocytes (NRCMs) treated with angiotensin II (Ang II, 1 μmol/l) for 48 h to induce hypertrophy, we found that OX40 expression was not significantly different compared with the PBS group (Figure 1B). The immunohistochemistry results showed that OX40 was mainly expressed on cardiac interstitial cells and that its expression was up-regulated in hypertrophic hearts. The expression of OX40 was consistent with that of CD45, which marks the area of infiltration of inflammatory cells. No notable increase in collagen III deposition was observed in mouse heart at week 1 after AB, although collagen III deposition clearly increased at weeks 2–4 after AB surgery, as shown in Figure 1(C). Because OX40 is a costimulatory receptor on T-cells, we performed double immunostaining with CD3 and OX40. Our data revealed that CD3+ T-cell infiltration increased at week 2 following AB surgery, which is consistent with the expression trend of OX40, and that OX40 expression overlapped with CD3 expression, as shown in Figure 1(D). Thus, OX40 expression significantly increased in pressure overload-induced hypertrophic murine hearts and was mainly expressed on T-lymphocytes infiltrating the heart. In contrast, OX40 expression was not altered in Ang II-treated cardiomyocytes in vitro.

OX40 expression in hypertrophic hearts and cardiomyocytes

Figure 1
OX40 expression in hypertrophic hearts and cardiomyocytes

(A) Analysis of myocardial OX40 expression by western blot and RT-PCR. Top, representative western blots; middle, quantitative results of ANP, β-MHC and OX40 protein and mRNA expression in a WT mouse model of cardiac hypertrophy induced by AB for the indicated time (n=6 mice per experimental group). *P<0.05 compared with sham. (B) OX40 expression in NRCMs treated with Ang II (1 μmol/l) for 48 h (n=6 samples per experimental group). Top, representative western blots; middle, quantitative results of ANP, β-MHC and OX40 protein and mRNA expression. *P<0.05 compared with PBS. (C) Immunohistochemical detection of cardiac OX40, CD45 and collagen III in WT mice after AB. (D) Immunofluorescence detection of CD3 and OX40 in WT and KO mouse hearts after AB. W represent week.

Figure 1
OX40 expression in hypertrophic hearts and cardiomyocytes

(A) Analysis of myocardial OX40 expression by western blot and RT-PCR. Top, representative western blots; middle, quantitative results of ANP, β-MHC and OX40 protein and mRNA expression in a WT mouse model of cardiac hypertrophy induced by AB for the indicated time (n=6 mice per experimental group). *P<0.05 compared with sham. (B) OX40 expression in NRCMs treated with Ang II (1 μmol/l) for 48 h (n=6 samples per experimental group). Top, representative western blots; middle, quantitative results of ANP, β-MHC and OX40 protein and mRNA expression. *P<0.05 compared with PBS. (C) Immunohistochemical detection of cardiac OX40, CD45 and collagen III in WT mice after AB. (D) Immunofluorescence detection of CD3 and OX40 in WT and KO mouse hearts after AB. W represent week.

OX40 deletion attenuates AB-induced hypertrophy

To evaluate the effects of OX40 in vivo, we first utilized a mouse model with global KO of OX40 (OX40−/−), in which OX40 expression was absent (Figure 2A). Cardiac function (Table 1) and the ratios of heart weight (HW) to body weight (BW) or tibia length (TL) (Figure 2C), did not differ between wild-type (WT) and OX40-deficient mice. In addition, hearts from OX40-deficient mice were macroscopically indistinguishable from those from WT mice (Figure 2B). However, after 8 weeks of AB, OX40−/− mice exhibited remarkably attenuated cardiac hypertrophy compared with WT mice, as indicated by decreased ratios of HW/BW and HW/TL. In addition, the development of pulmonary congestion (the ratio of LW to BW or TL) as an indirect indicator of HF significantly decreased in OX40−/− mice (Figure 2B). Histological examination revealed decreased cross-sectional area (CSA) of cardiomyocytes in OX40−/− mice compared with those in WT mice after an 8-week course of AB. Consistently, the induction of hypertrophic markers, including ANP, β-type natriuretic peptide (BNP) and β-MHC, was strikingly suppressed in OX40−/− mice after pressure overload and was accompanied by the up-regulation of α-MHC and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2α) (Figure 2C). In addition, cardiac dilation and dysfunction induced by AB surgery were ameliorated in OX40-deficient mice (Table 1).

OX40 deficiency attenuated pressure overload-induced cardiac remodelling

Figure 2
OX40 deficiency attenuated pressure overload-induced cardiac remodelling

(A) Deletion of OX40 in WT and OX40 KO mouse hearts and T-lymphocytes was confirmed by western blot analysis. (B) Histological analyses of the haematoxylin–eosin (HE) staining and wheat germ agglutinin (WGA) staining of WT and OX40 KO mice 8 weeks after AB surgery (n=8 mice per experimental group; upper, HE staining; lower, WGA staining). (C) Statistical results for the CSA (n=100+ cells per group), ratios of HW/BW, HW/TL, LW/BW and LW/TL in the indicated groups (n=12). (D) RT-PCR analyses of hypertrophic markers (ANP, BNP, β-MHC, α-MHC and SERCA2α) induced by AB in the indicated mice (n=6 per experimental group). (E) PSR staining of histological sections of the LV in the indicated groups 8 weeks after AB (n=8 per experimental group). Top, representative image; bottom, quantification of the total collagen volume in WT and OX40 KO mice after AB (n=25+ fields per experimental group). (F) Real-time PCR analyses of the fibrotic markers (collagen I, collagen III, CTGF and fibronectin) in the indicated groups (n=6). *P<0.05 compared with WT/sham; #P<0.05 compared with WT/AB.

Figure 2
OX40 deficiency attenuated pressure overload-induced cardiac remodelling

(A) Deletion of OX40 in WT and OX40 KO mouse hearts and T-lymphocytes was confirmed by western blot analysis. (B) Histological analyses of the haematoxylin–eosin (HE) staining and wheat germ agglutinin (WGA) staining of WT and OX40 KO mice 8 weeks after AB surgery (n=8 mice per experimental group; upper, HE staining; lower, WGA staining). (C) Statistical results for the CSA (n=100+ cells per group), ratios of HW/BW, HW/TL, LW/BW and LW/TL in the indicated groups (n=12). (D) RT-PCR analyses of hypertrophic markers (ANP, BNP, β-MHC, α-MHC and SERCA2α) induced by AB in the indicated mice (n=6 per experimental group). (E) PSR staining of histological sections of the LV in the indicated groups 8 weeks after AB (n=8 per experimental group). Top, representative image; bottom, quantification of the total collagen volume in WT and OX40 KO mice after AB (n=25+ fields per experimental group). (F) Real-time PCR analyses of the fibrotic markers (collagen I, collagen III, CTGF and fibronectin) in the indicated groups (n=6). *P<0.05 compared with WT/sham; #P<0.05 compared with WT/AB.

Table 1
Echocardiographic and haemodynamic parameters in OX40 KO mice after 8 weeks of AB surgery

LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; EF, ejection fraction; FS, fractional shortening; ESP, end-systolic pressure; EDP, end-diastolic pressure; CO, cardiac output; dp/dtmin, maximal rate of pressure decay. *P<0.05 for difference from corresponding sham group. #P<0.05 compared with WT AB group after AB.

 Sham AB 
Parameter WT n=8 KO n=8 WT n=8 KO n=8 
LVEDD (mm) 4.21±0.07 4.10±0.09 5.35±0.14* 4.60±0.13*# 
LVESD (mm) 2.65±0.06 2.58±0.10 4.23±0.17* 2.80±0.09# 
EF (%) 73.13±0.81 73.88±1.49 48.88±3.02* 73.38±1.70# 
FS (%) 37.00±0.71 37.38±1.24 21.38±1.80* 37.38±1.33# 
ESP (mmHg) 103.10±3.30 95.86±3.75 155.37±1.50* 153.66±1.62* 
EDP (mmHg) 5.00±0.40 3.75±1.52 26.28±0.89* 19.31±0.22*# 
CO (ul/min) 6501±548 6855±370 3551±406* 6811±614# 
dp/dtmax (mmHg/s) 9420±113 9120±293 5735±184* 8724±502# 
dp/dtmin (mmHg/s) −9565±133 −9659±212 −5438±248* −9278±618# 
 Sham AB 
Parameter WT n=8 KO n=8 WT n=8 KO n=8 
LVEDD (mm) 4.21±0.07 4.10±0.09 5.35±0.14* 4.60±0.13*# 
LVESD (mm) 2.65±0.06 2.58±0.10 4.23±0.17* 2.80±0.09# 
EF (%) 73.13±0.81 73.88±1.49 48.88±3.02* 73.38±1.70# 
FS (%) 37.00±0.71 37.38±1.24 21.38±1.80* 37.38±1.33# 
ESP (mmHg) 103.10±3.30 95.86±3.75 155.37±1.50* 153.66±1.62* 
EDP (mmHg) 5.00±0.40 3.75±1.52 26.28±0.89* 19.31±0.22*# 
CO (ul/min) 6501±548 6855±370 3551±406* 6811±614# 
dp/dtmax (mmHg/s) 9420±113 9120±293 5735±184* 8724±502# 
dp/dtmin (mmHg/s) −9565±133 −9659±212 −5438±248* −9278±618# 

Fibrosis, a classical feature of the development of pathological cardiac hypertrophy to HF, was also assessed in AB-induced cardiac remodelling. Heart sections were stained with Picro-Sirius red (PSR). Striking perivascular and interstitial fibrosis was observed in the WT mice in response to AB, and the extent of cardiac fibrosis was remarkably decreased in the OX40 KO mice. No significant difference in fibrosis was detected between the WT and KO mice after the sham operation (Figure 2E). Cardiac fibrosis was further quantified by measuring the left ventricular (LV) collagen volume and the expression of the fibrotic markers collagen Iα, collagen III, fibronectin and connective tissue growth factor (CTGF) and revealed that the AB-induced fibrotic response was significantly inhibited in OX40 KO mice compared with WT mice (Figure 2F). Collectively, these global loss-of-function data indicate that OX40 deficiency does not cause baseline cardiac abnormalities but remarkably renders the heart more defensive to stress-induced pathological cardiac remodelling.

Effect of OX40 on heart inflammation induced by pressure overload

Inflammation plays an important role in the pathogenesis of cardiac remodelling. To investigate the mechanism underlying the effect of OX40 on cardiac remodelling, we assessed the expression levels of myocardial pro-inflammatory cytokines and the infiltration of inflammatory cells. First, the mRNA expression levels of TNF-α, interleukin (IL)-1β and IL-6 were assessed in WT mice after 2, 4 and 8 weeks of AB. The result indicated that the expression levels of these pro-inflammatory cytokines increased, peaked at 2 weeks after AB, and then gradually decreased (Figure 3A). Thus, hearts of OX40 KO mice were harvested after 2 weeks of AB to evaluate cardiac inflammation. The mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly decreased in OX40 KO mouse hearts compared with WT mouse hearts (Figure 3B). Immunohistochemical staining for CD68 and CD45, the molecular markers of macrophages and leucocytes, respectively, revealed that inflammatory cell infiltration was suppressed in OX40 KO mouse hearts compared with WT mouse hearts (Figure 3C). The infiltration of CD4+ and CD8+ T-lymphocytes was also reduced in OX40 KO mouse hearts compared with WT mouse hearts (Figures 3D and 3E). Taken together, these data suggest that OX40 deficiency can mitigate cardiac hypertrophy, fibrosis and inflammation induced by pressure overload.

Effect of OX40 on heart inflammation induced by pressure overload

Figure 3
Effect of OX40 on heart inflammation induced by pressure overload

(A) RT-PCR analysis of TNF-α, IL-1α and IL-6 mRNA expression in WT mouse heart induced by AB for the indicated time (n=6). *P<0.05 compared with sham. (B) RT-PCR analysis of TNF-α, IL-1α and IL-6 mRNA expression induced by 2 weeks of AB in the indicated mice (n=6). *P<0.05 compared with WT/sham; #P<0.05 compared with WT/AB. (C) Detection of CD68 and CD45 by immunohistochemistry in the indicated mice after 2 weeks of AB. (D) Detection of CD4 and CD8 by immunofluorescence in the indicated mice after 2 weeks of AB. (E) Representative flow cytometry dot plots and quantification of CD4+ (CD3+ CD4+) and CD8+ (CD3+ CD8+) T-cell subsets isolated from cardiac tissue of sham- and AB-operated mice (n=5–6).

Figure 3
Effect of OX40 on heart inflammation induced by pressure overload

(A) RT-PCR analysis of TNF-α, IL-1α and IL-6 mRNA expression in WT mouse heart induced by AB for the indicated time (n=6). *P<0.05 compared with sham. (B) RT-PCR analysis of TNF-α, IL-1α and IL-6 mRNA expression induced by 2 weeks of AB in the indicated mice (n=6). *P<0.05 compared with WT/sham; #P<0.05 compared with WT/AB. (C) Detection of CD68 and CD45 by immunohistochemistry in the indicated mice after 2 weeks of AB. (D) Detection of CD4 and CD8 by immunofluorescence in the indicated mice after 2 weeks of AB. (E) Representative flow cytometry dot plots and quantification of CD4+ (CD3+ CD4+) and CD8+ (CD3+ CD8+) T-cell subsets isolated from cardiac tissue of sham- and AB-operated mice (n=5–6).

OX40 deficiency impaired CD4+ T-lymphocyte function and subsequently attenuated Ang II-induced cardiomyocyte hypertrophy in vitro

OX40 has been shown to regulate the proliferation, survival and differentiation of and cytokine production by T-cells [4,5,10]. Therefore, we next sought to determine whether OX40 would affect T-cell function and consequently regulate cardiac remodelling. To this end, CD4+ T-lymphocytes were isolated from OX40 KO mouse spleen and WT mouse spleen. Our initial results showed that OX40-deficient CD4+ T-lymphocytes presented reduced proliferative abilities, decreased pro-inflammatory cytokine (TNF-α, IFN-γ and IL-2) release and enhanced anti-inflammatory cytokine (IL-10) release after activation by anti-CD3ε (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 48 h (Figure 4B). To validate our hypothesis further, NRCMs were co-cultured with activated CD4+ T-lymphocyte supernatant from either OX40 KO or WT mouse spleen and stimulated with Ang II for 48 h. These experiments clearly demonstrated that cell surface area and hypertrophic marker (ANP, β-MHC) levels were significantly increased by the CD4+ T-cell supernatant from WT mouse spleen but were limited after co-culture with the CD4+ T-cell supernatant from OX40 KO mouse spleen (Figures 4C–4E). To gain insight into the mechanisms responsible for the anti-hypertrophy effect of OX40-deficient CD4+ T-lymphocytes, IL-10 neutralizing antibody (R&D, 0.8 μg/ml) was used. Our results showed that the anti-hypertrophy effect of supernatant from OX40-deficient CD4+ T-lymphocytes was almost abolished by IL-10 neutralizing antibody as evidenced by increased cell surface area and expression levels of hypertrophic markers (Figures 4F–4H). The above findings suggest that OX40 regulates cardiac remodelling via CD4+ T-cells and that modulation of CD4+ T-cell function by OX40 KO can attenuate the hypertrophic response. The protective effect of OX40 KO T-cells supernatant on Ang II-induced cardiomyocyte hypertrophy is at least partially dependent on IL-10.

OX40 deficiency impaired CD4+ T-lymphocyte function, which was associated with attenuated cardiomyocyte hypertrophy in vitro

Figure 4
OX40 deficiency impaired CD4+ T-lymphocyte function, which was associated with attenuated cardiomyocyte hypertrophy in vitro

(A) Flow cytometric analysis of the ratio of CD4+ T-lymphocytes isolated from mouse spleen. (B) Upper left, Cell Counting Kit-8 (CCK-8) analysis of the proliferation of CD4+ T-lymphocytes from WT and OX40 KO mouse spleens after treatment with anti-CD3ε (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 48 h (n=5). *P<0.05 compared with WT; upper right and bottom, ELISA analysis of the production of the inflammatory cytokines TNF-α, IFN-γ, IL-2 and IL-10 in CD4+ T-lymphocytes from the indicated mouse spleens in response to anti-CD3h and anti-CD28 antibodies (n=6). *P<0.05 compared with WT/PBS; #P<0.05 compared with WT/CD3+CD28. (C and D) Cardiomyocytes were co-cultured with activated CD4+ T-lymphocyte supernatant from either OX40 KO or WT mouse spleen or RPMI-1640 medium as negative control (NC) and stimulated with Ang II (1 μmol/l) for 48 h. (C) Representative images (upper) and quantitative results (bottom) of the cell surface area (n=50+ cells per group). (D) Western blot analysis of the protein levels of ANP and β-MHC (n=6). Upper, representative western blots; bottom, quantitative results. (E) RT-PCR analysis of ANP and β-MHC mRNA expressions (n=6) in each group. *P<0.05 compared with the indicated PBS group; #P<0.05 compared with NC/Ang II. (FH) Cardiomyocytes were co-cultured with activated CD4+ T-lymphocyte supernatant from OX40 KO CD4+ T-lymphocytes and IL-10 neutralizing antibody (R&D, 0.8 μg/ml) and stimulated with Ang II (1 μmol/l) for 48 h. (F) Quantitative results (upper) and representative images (bottom) of the cell surface area (n=50+ cells per group). (G) Western blot analysis of the expression levels of ANP and β-MHC (n=6). Upper, representative quantitative results; bottom, western blots. (H) RT-PCR analysis of ANP and β-MHC mRNA expressions (n=6) in each group. *P<0.05 compared with the PBS group; #P<0.05 compared with Ang II.

Figure 4
OX40 deficiency impaired CD4+ T-lymphocyte function, which was associated with attenuated cardiomyocyte hypertrophy in vitro

(A) Flow cytometric analysis of the ratio of CD4+ T-lymphocytes isolated from mouse spleen. (B) Upper left, Cell Counting Kit-8 (CCK-8) analysis of the proliferation of CD4+ T-lymphocytes from WT and OX40 KO mouse spleens after treatment with anti-CD3ε (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 48 h (n=5). *P<0.05 compared with WT; upper right and bottom, ELISA analysis of the production of the inflammatory cytokines TNF-α, IFN-γ, IL-2 and IL-10 in CD4+ T-lymphocytes from the indicated mouse spleens in response to anti-CD3h and anti-CD28 antibodies (n=6). *P<0.05 compared with WT/PBS; #P<0.05 compared with WT/CD3+CD28. (C and D) Cardiomyocytes were co-cultured with activated CD4+ T-lymphocyte supernatant from either OX40 KO or WT mouse spleen or RPMI-1640 medium as negative control (NC) and stimulated with Ang II (1 μmol/l) for 48 h. (C) Representative images (upper) and quantitative results (bottom) of the cell surface area (n=50+ cells per group). (D) Western blot analysis of the protein levels of ANP and β-MHC (n=6). Upper, representative western blots; bottom, quantitative results. (E) RT-PCR analysis of ANP and β-MHC mRNA expressions (n=6) in each group. *P<0.05 compared with the indicated PBS group; #P<0.05 compared with NC/Ang II. (FH) Cardiomyocytes were co-cultured with activated CD4+ T-lymphocyte supernatant from OX40 KO CD4+ T-lymphocytes and IL-10 neutralizing antibody (R&D, 0.8 μg/ml) and stimulated with Ang II (1 μmol/l) for 48 h. (F) Quantitative results (upper) and representative images (bottom) of the cell surface area (n=50+ cells per group). (G) Western blot analysis of the expression levels of ANP and β-MHC (n=6). Upper, representative quantitative results; bottom, western blots. (H) RT-PCR analysis of ANP and β-MHC mRNA expressions (n=6) in each group. *P<0.05 compared with the PBS group; #P<0.05 compared with Ang II.

Reconstitution of T-lymphocytes promoted pressure overload-induced cardiac remodelling

To confirm that OX40 modulates cardiac hypertrophy and function through T-cells, we transferred purified CD4+ T-cells from donor WT mouse spleens into OX40 KO mice. After the T-cells were reconstituted, OX40 KO+CD4 and OX40 KO mice injected with PBS (OX40 KO+PBS) were subjected to AB. As expected, strong infiltration of CD3+ T-cells, as detected by immunofluorescence, was observed in the LV tissue of OX40 KO+CD4 mice after AB; OX40 expression was also observed (Figure 5A). T-cell reconstitution in OX40 KO mice after surgery resulted in significantly deteriorated cardiac hypertrophy and pulmonary congestion as revealed by increased ratios of HW/BW, HW/TL, LW/TL and LW/BW, and by increased CSAs of cardiomyocytes in OX40 KO+CD4 compared with control OX40 KO+PBS mice (Figures 5B–5D). According to the deterioration of cardiac dilation and dysfunction (Table 2), the expression levels of the hypertrophic markers ANP and β-MHC were significantly increased, whereas the expression level of α-MHC decreased in OX40 KO+CD4 mice compared with OX40 KO+PBS mice (Figure 5D).

Reconstitution of T-lymphocytes promoted pressure overload-induced cardiac remodelling

Figure 5
Reconstitution of T-lymphocytes promoted pressure overload-induced cardiac remodelling

(A) Immunofluorescence detection of CD3 and OX40 in OX40 KO+CD4 and OX40 KO+PBS mice after 2 weeks of AB. (B) Ratios of HW/BW, HW/TL, LW/BW and LW/TL in the indicated groups (n=6–10) in OX40 KO+CD4 and OX40 KO+PBS mice after 4 weeks of AB. (C) Histological analyses of HE staining of OX40 KO+CD4 and OX40 KO+PBS mice at 4 weeks after AB surgery (n=8 mice per experimental group). (D) Statistical results for the CSA (n=100+ cells per group) and RT-PCR analyses of hypertrophic markers (ANP, β-MHC and α-MHC) induced by AB in the indicated mice (n=6 per experimental group). (E) PSR staining of histological sections of the LV in the indicated groups at 4 weeks after AB (n=8 per experimental group). Top, representative image. (F) Quantification of the total collagen volume in OX40 KO+CD4 and OX40 KO+PBS mice after AB (n=25+ fields per experimental group) and real-time PCR analyses of the fibrotic markers (collagen I, collagen III and CTGF) in the indicated groups (n=6). *P<0.05 compared with OX40 KO+PBS/sham; #P<0.05 compared with OX40 KO+PBS /AB. (G) Immunohistochemical staining of CD68 and CD45 in OX40 KO+CD4 and OX40 KO+PBS mouse hearts after 2 weeks of AB.

Figure 5
Reconstitution of T-lymphocytes promoted pressure overload-induced cardiac remodelling

(A) Immunofluorescence detection of CD3 and OX40 in OX40 KO+CD4 and OX40 KO+PBS mice after 2 weeks of AB. (B) Ratios of HW/BW, HW/TL, LW/BW and LW/TL in the indicated groups (n=6–10) in OX40 KO+CD4 and OX40 KO+PBS mice after 4 weeks of AB. (C) Histological analyses of HE staining of OX40 KO+CD4 and OX40 KO+PBS mice at 4 weeks after AB surgery (n=8 mice per experimental group). (D) Statistical results for the CSA (n=100+ cells per group) and RT-PCR analyses of hypertrophic markers (ANP, β-MHC and α-MHC) induced by AB in the indicated mice (n=6 per experimental group). (E) PSR staining of histological sections of the LV in the indicated groups at 4 weeks after AB (n=8 per experimental group). Top, representative image. (F) Quantification of the total collagen volume in OX40 KO+CD4 and OX40 KO+PBS mice after AB (n=25+ fields per experimental group) and real-time PCR analyses of the fibrotic markers (collagen I, collagen III and CTGF) in the indicated groups (n=6). *P<0.05 compared with OX40 KO+PBS/sham; #P<0.05 compared with OX40 KO+PBS /AB. (G) Immunohistochemical staining of CD68 and CD45 in OX40 KO+CD4 and OX40 KO+PBS mouse hearts after 2 weeks of AB.

Table 2
Echocardiographic and haemodynamic parameters in CD4+ T-cell reconstituted mice after 4 weeks of AB surgery

LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; EF, ejection fraction; FS, fractional shortening; ESP, end-systolic pressure; EDP, end-diastolic pressure; CO, cardiac output; dp/dtmin, maximal rate of pressure decay. *P<0.05 for difference from corresponding sham group. #P<0.05 compared with WT AB group after AB.

 Sham AB 
Parameter OX40 KO+PBS n=8 OX40 KO+CD4 n=8 OX40 KO+PBS n=8 OX40 KO+CD4 n=8 
LVEDD (mm) 2.98±0.08 3.13±0.10 4.16±0.04* 4.70±0.1*# 
LVESD (mm) 2.28±0.04 2.34±0.13 3.32±0.06* 3.51±0.13* 
EF (%) 73.00±1.23 76.00±3.37 53.25±1.29* 45.50±1.29*# 
FS (%) 37.38±0.69 37.25±0.89 33.13±0.63* 28.25±0.57*# 
ESP (mmHg) 102.01±4.62 101.21±2.46 153.59±1.46* 146.14±4.88* 
EDP (mmHg) 3.81±0.64 4.64±0.56 16.52±0.52* 25.02±0.93*# 
CO (ul/min) 6951±532 6565±155 5127±148* 4242±142*# 
dp/dtmax (mmHg/s) 9843±491 9627±246 7393±129* 6166±314*# 
dp/dtmin (mmHg/s) −9472±297 −9605±214 −7151±202* −5645±335*# 
 Sham AB 
Parameter OX40 KO+PBS n=8 OX40 KO+CD4 n=8 OX40 KO+PBS n=8 OX40 KO+CD4 n=8 
LVEDD (mm) 2.98±0.08 3.13±0.10 4.16±0.04* 4.70±0.1*# 
LVESD (mm) 2.28±0.04 2.34±0.13 3.32±0.06* 3.51±0.13* 
EF (%) 73.00±1.23 76.00±3.37 53.25±1.29* 45.50±1.29*# 
FS (%) 37.38±0.69 37.25±0.89 33.13±0.63* 28.25±0.57*# 
ESP (mmHg) 102.01±4.62 101.21±2.46 153.59±1.46* 146.14±4.88* 
EDP (mmHg) 3.81±0.64 4.64±0.56 16.52±0.52* 25.02±0.93*# 
CO (ul/min) 6951±532 6565±155 5127±148* 4242±142*# 
dp/dtmax (mmHg/s) 9843±491 9627±246 7393±129* 6166±314*# 
dp/dtmin (mmHg/s) −9472±297 −9605±214 −7151±202* −5645±335*# 

To gain insight into the ventricular fibrotic response induced by CD4+ T-cell replenishment, we determined the levels of collagen deposition in cardiac tissue by PSR staining. As shown in Figures 5(E) and 5(F), OX40 KO mice who received reconstituted CD4+ T-lymphocytes presented significantly excessive perivascular and interstitial fibrosis compared with control mice. The transcription levels of the three main procollagens, collagen I, collagen III and CTGF, were also up-regulated in OX40 KO+CD4 mouse heart (Figure 5F). In addition, we observed that AB-induced cardiac remodelling was associated with greater macrophage accumulation and leucocyte infiltration in OX40 KO+CD4 mice compared with OX40 KO+PBS mice (Figure 5G). Taken together, these results show that CD4+ T-cells contribute to the effect of OX40 on cardiac remodelling.

DISCUSSION

An increasing number of studies have demonstrated that OX40/OX40L interactions affect the development of ACS and that blocking the OX40 costimulatory receptor can improve the survival of patients with ACS [16,17]. Moreover, in humans, SNPs in both the OX40 and OX40L genes affect the incidence of cardiovascular disease [18]. These findings suggested that OX40 plays a role in cardiovascular disease. However, the role of OX40 in cardiac remodelling has not been defined. In the present study, we identified that OX40, a membrane receptor known to be expressed in immune cells, was also highly expressed in mouse heart CD3+ T-lymphocytes but not in cardiomyocytes after hypertrophic stimulation, suggesting that OX40 may be involved in the processes of cardiac hypertrophy and HF through T-lymphocytes. We utilized global loss-of-function approaches to decipher thoroughly the role of OX40 in chronic pressure overload-induced cardiac remodelling. We discovered that the global loss of OX40 is instrumental for hearts to resist pressure overload-induced hypertrophy, fibrosis, dysfunction and inflammation. These findings indicate that OX40 participates in the progression of cardiac remodelling to HF.

Notably, numerous studies have strongly implicated immune cells, particularly T-lymphocytes, in cardiac remodelling observed in heart diseases [6]. Various pathological stimuli can lead to the formation of T-cells and accumulation of monocytes/macrophages, which release cytokines, including IFN-γ, TNF-α and IL-6, and which eventually contribute to heart damage and dysfunction [19]. Mice with cardiac-specific TNF-α overexpression were shown to develop severe cardiomyopathy, and neutralization of T-cells with anti-CD3 antibody was shown to reduce inflammatory cell recruitment and to block hypertrophy [20]. In pressure overload-induced hypertrophy, CD4+ T-cell depletion using MHC II deficient mice reduced myocardial fibrosis, decreased macrophage infiltration and attenuated cardiac dysfunction, although the CD8 KO mice did not present alleviated cardiac remodelling [1], suggesting that T-cells, particularly CD4+ T-cells, play a crucial role in the progression of cardiac remodelling to HF. The activated CD4+ T-cells in mediastinal draining lymph nodes after 6 weeks of TAC were shown to have a preferential Th1-type polarization [1]. The pro-inflammatory factors released by Th1 cells, such as TNF-α and IL-1β, can enhance the synthesis of matrix metalloproteinases (MMPs) and enhance cardiomyocyte apoptosis. Additionally, pro-inflammatory cytokine signalling can suppress contractility, accentuating ventricular systolic dysfunction [4]. CD4+ T-cells also modulate the recruitment of monocytes/macrophages to heart tissue undergoing remodelling [21]. OX40, a costimulatory receptor on the T-cell surface, is mainly present on activated T-cells; blocking OX40 and OX40L interactions could inhibit the production of Th1- and Th2-type cytokines [22]. Studies of OX40 have also demonstrated that OX40 signalling can block naïve T-cells from differentiating into Foxp3+ iTreg cells and IL-10+ iTreg cells, contributing to the clonal expansion and development of effector and memory T-cells [23,24]. Thus, we hypothesized that OX40 may regulate the activation and differentiation of, as well as cytokine release by, CD4+ T-cells in the process of cardiac remodelling. Our in vitro experiments indicated that OX40 KO decreased the proliferation of CD4+ T-cells and the release of the pro-inflammatory cytokines TNF-α and IFN-γ, but enhanced the release of the anti-inflammatory cytokine IL-10. Previous studies have demonstrated the crucial effect of TNF-α on the key signalling molecules that contribute to cardiac hypertrophy, such as NF-κB and MAPK as well as ROS [25,26]. IL-10 is also a multi-functional cytokine with potent anti-inflammatory properties; IL-10 can limit Ang II-induced increases in blood pressure [27]. IL-10 also inhibits Ang II-mediated pathological autophagy and improves cardiac function [28]. These functional alterations of CD4+ T-cells may contribute to the improved cardiac remodelling in OX40-deficient mice. To further confirm our hypothesis, we performed in vitro experiments in which NRCMs were treated with Ang II and with supernatants from CD4+ T-cells in each group. Our data demonstrated that Ang II-induced cardiomyocyte hypertrophy was attenuated since cardiomyocytes were co-cultured with the supernatant from OX40-deficient CD4+ T-cells. Additionally, this supernatant contained lower levels of the pro-inflammatory cytokine TNF-α and higher levels of the anti-inflammatory cytokine IL-10, which can strongly inhibit Ang II-induced cardiomyocyte hypertrophy [28,29]. By using IL-10 neutralizing antibody in vitro, the anti-hypertrophy effect of OX40 deficiency was almost abolished, suggesting that IL-10 is a key factor that mediates the protective effect of OX40 KO. Consistently, reconstitution of CD4+ T-lymphocytes in OX40 KO mice produced aggravated cardiac remodelling and dysfunction. Therefore, the functional alteration of CD4+ T-cells by OX40 deficiency retarded the processes of cardiac hypertrophy and remodelling.

In summary, the findings of the present study reveal that global loss of OX40 is helpful for hearts to resist pressure overload-induced hypertrophy, fibrosis, dysfunction and inflammation. Moreover, by modulating CD4+ T-cell function, OX40 alters the pathology of cardiac remodelling.

LIMITATIONS

Several limitations of the present study should be considered. First, NRCMs were used instead of neonatal mouse cardiomyocytes essentially due to the poor survival of neonatal mouse cardiomyocytes. Although NRCMs are widely employed together with mouse models to explore the pathogenesis and prevention of cardiovascular disease [30,31], caution should be taken in interpreting results due to species differences. Second, hypertrophic mechanisms induced by Ang II, an agonist for activating the G-alpha(q) protein-coupled receptor (GPCR) signalling pathway, are distinct in pressure overload-induced cardiac hypertrophy. The Ang II experiments in the present study may shed light on the interplay between OX40 and cardiac remodelling induced by various stimuli. Further work is needed to confirm the role of OX40 in cardiac remodelling.

AUTHOR CONTRIBUTION

Qing-Qing Wu and Qi-Zhu Tang contributed to the conception of the work and designed experiments; Qing-Qing Wu and Yuan Yuan, Xiao-Han Jiang and Yang Xiao carried out experiments; Zheng Yang, Zhen-Guo Ma and Hai-Han Liao analysed experimental results and revised the manuscript; Qing-Qing Wu and Wei Chang wrote and revised the manuscript; and Yuan Liu and Zhou-Yan Bian participated in the analysis and interpretation of the data.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers 81470516, 81270303 and 81530012]; the Hubei Province's Outstanding Medical Academic Leader program; and the Central Universities of China [grant number 2014302020202].

Abbreviations

     
  • AB

    aortic banding

  •  
  • ACS

    acute coronary syndrome

  •  
  • Ang II

    angiotensin II

  •  
  • ANP

    atrial natriuretic peptide

  •  
  • BNP

    β-type natriuretic peptide

  •  
  • BW

    body weight

  •  
  • CCK

    Cell Counting Kit

  •  
  • CSA

    cross-sectional area

  •  
  • CTGF

    connective tissue growth factor

  •  
  • ECM

    cardiac extracellular matrix

  •  
  • GPCR

    G-alpha(q) protein-coupled receptor

  •  
  • HE

    haematoxylin–eosin

  •  
  • HF

    heart failure

  •  
  • HW

    heart weight

  •  
  • IFN-γ

    interferon-gamma

  •  
  • IL

    interleukin

  •  
  • KO

    knockout

  •  
  • LV

    left ventricular

  •  
  • MHC

    myosin heavy chain

  •  
  • NRCM

    neonatal rat cardiomyocyte

  •  
  • PSR

    Picro-Sirius red

  •  
  • ROS

    reactive oxygen species

  •  
  • RT-PCR

    quantitative real-time polymerase chain reaction

  •  
  • SERCA2α

    sarcoendoplasmic reticulum Ca2+-ATPase

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • TAC

    transverse aortic constriction

  •  
  • TL

    tibia length

  •  
  • TNF

    tumour necrosis factor

  •  
  • WGA

    wheat germ agglutinin

  •  
  • WT

    wild-type

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