Abstract
Increasing evidence suggests that CD4+ T cells contribute to neovascularization in ischaemic tissue. However, the T cell subset responsible for neovascularization after ischaemia remains to be determined. Here, we investigated the role of Th17 cells secreting interleukin (IL)-17, a newly identified subset of CD4+ T cells, in the neovascularization after murine hindlimb ischaemia.
Unilateral hindlimb ischaemia was produced in wild-type (WT) C57BL/6 mice. Depletion of CD4+ T cells resulted in significantly reduced blood flow perfusion in the ischaemic limbs. The expression of IL-17 and retinoic acid receptor-related orphan receptor γt (RORγt) was up-regulated in the ischaemic limbs. IL-17-deficient mice showed a significant reduction in blood flow perfusion, inflammatory cell infiltration, and production of angiogenic cytokines in the ischaemic limbs compared with WT mice. In bone marrow transplantation experiments, the absence of IL-17 specifically in bone marrow cells diminished the neovascularization after ischaemia. Furthermore, IL-17-deficient CD4+ T cells transferred into the ischaemic limbs of T cell-deficient athymic nude mice evoked a significantly limited neovascularization compared with WT CD4+ T cells.
These findings identify Th17 cells as a new angiogenic T cell subset and provide new insight into the mechanism by which T cells promote neovascularization after ischaemia.
1. Introduction
Post-ischaemic neovascularization contributes to the tissue repair and remodelling that occurs after acute and chronic ischaemic vascular diseases. Increasing evidence indicates that inflammatory responses, such as chemokine expression and subsequent inflammatory cell infiltration, play a pivotal role in modulating the neovascularization after ischaemia.1 In particular, previous studies revealed the central role played by monocytes/macrophages and their chemokine, monocyte chemoattractant protein-1 (MCP-1).2 In addition to monocytes/macrophages, recent studies also showed the potential role of T cells in this process. For instance, athymic nude mice, which lack a T cell-mediated immune system, exhibited a marked reduction in post-ischaemic neovascularization.3 Moreover, the arteriogenic response to hindlimb ischaemia was significantly impaired in mice specifically deficient in CD4+ T cells.4 Mice deficient in CD8+ T cells also showed an impaired arteriogenic response, owing to the inefficient recruitment of CD4+ T cells.5 These observations highlight the importance of CD4+ T cells in the neovascularization after ischaemia.
It is well established that CD4+ T cells develop into discrete subsets with unique cytokine profiles and effector functions. To clarify the role of CD4+ T cells in neovascularization after ischaemia, it is therefore important to know which CD4+ T cell subset is responsible for these responses. Among several CD4+ T cell subsets, Th1 and Th2 cells are generally identified by their signature cytokines—interferon-γ (IFN-γ) and interleukin (IL)-4, respectively.6 Recently, Th17, another subset of effector CD4+ T cells that produces IL-17 and IL-22, has been identified and implicated in multiple forms of inflammatory and autoimmune diseases.6,7 On the other hand, much attention has been focused recently on yet another CD4+ T cell subset termed the regulatory T cells (Treg), which suppresses adaptive immune responses and prevents autoimmunity.8 Treg and Th17 cells have been shown to require the transcription factors FoxP3 and retinoic acid receptor-related orphan receptor γt (RORγt) for their development, respectively, and these transcription factors may be used to detect these cells. Our initial observations that IL-17, but neither IL-4 nor IFN-γ, were produced in CD4+ T cells isolated from ischaemic limbs and that these ischaemic tissues expressed RORγt, but not FoxP3, prompted us to directly examine the involvement of Th17 cells in the neovascularization after ischaemia by using IL-17-deficient (IL-17−/−) mice (Supplementary material online, Figure S1). In this study, we found that both IL-17−/− mice and T cell-deficient mice reconstituted with IL-17-deficient CD4+ T cells mounted only a limited neovascularization. These findings thus showed the critical role played by Th17 cells in the angiogenic responses associated with ischaemic tissue damage, thereby providing a new insight into the mechanism by which T cells promote neovascularization. Modulation of Th17 cells could possibly be a neovascularization strategy for the treatment of ischaemic cardiovascular diseases.
2. Methods
For an expanded Methods sections, please refer to the Supplementary material online.
2.1 Experimental animals
C57BL/6J mice [wild-type (WT), male, 8 weeks old] and BALB/c-nu/nu mice (nude mice, male, 8 weeks old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). IL-17−/− mice (C57BL/6 background) were generated as described previously.9 The mice were fed a standard diet and water and were maintained on a 12-h light and dark cycle. All experiments in this study were performed in accordance with the Shinshu University Guide for Laboratory Animals that conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996).
2.2 Isolation of naïve T cells and culture conditions
Naïve T cells were purified from the spleens of the mice by using the CD4+ T cell isolation kit and CD62L microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purified naïve T cells were stimulated with anti-CD3/CD28 beads in the presence of IL-6 and TGF-β to develop Th17 cells, as described previously.10 For implantation experiments, CD4+ T cells were purified from spleens by using an automated magnetic cell sorter (Auto MACS; Miltenyi Biotech) with >95% purity in all experiments.
2.3 Murine hindlimb ischaemia model
A murine hindlimb ischaemia model was produced as described previously.11,12 The blood perfusion was measured using a laser Doppler perfusion imaging system (Moor LDI; Moor Instruments, Wilmington, DE, USA). To deplete CD4+ T cells, the CD4 T+ cell-selective depleting GK1.5 monoclonal antibody (mAb; 100 µL ascitic fluid of nu/nu mice) or phosphate-buffered saline (PBS; control) was injected intraperitoneally into the WT mice once a week, starting 7 days before the ischaemia induction. For cell implantation experiments, hindlimb ischaemia was produced in athymic nude mice. Splenocytes isolated from the WT or IL-17−/− mice (5 × 106 cells in 200 μL PBS, 50 μL × 4 sites) or the same volume of PBS was injected into the ischaemic muscle 24 h after the induction of ischaemia. The blood perfusion in the ischaemic and contralateral limbs was measured using a laser Doppler perfusion imaging system (Moor LDI; Moor Instruments). The perfusion ratio was calculated as the flux ratio between the ischaemic and non-ischaemic limbs. All measurements were performed in a double-blind manner by two independent researchers.
2.4 Real-time reverse transcription-polymerase chain reaction (RT–PCR)
RNA extraction and real-time reverse transcription-polymerase chain reaction (RT–PCR) analysis were performed to detect the mRNA expression of IFN-γ, IL-4, IL-6, IL-17, IL-22, RORγt, FoxP3, and β-actin, as described previously (detailed methods available in the Supplementary material online).12
2.5 Immunohistochemistry
Immunohistochemical analysis for CD31, F4/80, Gr-1, CD4, IL-1β, and vascular endothelial growth factor-A (VEGF-A) was performed as described previously (detailed methods available in the Supplementary material online).12
2.6 Flow cytometric analysis
Blood samples were collected from the mice and analysed using flow cytometry as described previously.12,13 Circulating cells were identified using the nucleated cell fraction. The nucleated cells were stained with antibodies against CD4, CD8, CD45, Gr-1, and F4/80. To identify intracellular IL-17 expression, the cells were collected and permeabilized with an intracellular antigen detection kit (Cytofix/Cytoperm; BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. The cells were examined by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA) and analysed using CellQUEST software version 3.3 (Becton Dickinson). Details are described in the Supplementary material online.
2.7 Measurement of cytokine and growth factor concentration
The samples were prepared from the ischaemic tissues of the WT and IL-17−/− mice. The levels of IL-1β, VEGF-A, MCP-1, IL-6, IL-10, hepatocyte growth factor (HGF), and basic fibroblast growth factor (bFGF) in the ischaemic limbs were assessed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Inc., Minneapolis, MN, USA; RayBiotech, Inc., Norcross, GA, USA) and Cytometric Beads Assay Mouse Inflammation Kit (BD Biosciences), according to the manufacturer's instructions.
2.8 Bone marrow transplantation (BMT)
Bone marrow-transplanted mice were developed as described previously.13,14 By using this protocol, we produced three types of bone marrow-transplanted mice: WT to WT (BMTWT→WT), WT to IL-17−/− (BMTWT→IL−17−/−), and IL-17−/− to WT (BMTIL−17−/−→WT). Details are described in the Supplementary material online.
2.9 Statistical analysis
Data are expressed as mean ± SEM. The unpaired two-tailed t-test was used to compare the two groups. The analyses for the perfusion ratio were performed by repeated-measures analysis of variance. When any significant difference (P< 0.05) was seen in the main effect (group differences), the comparison at each time point was analysed by the unpaired two-tailed t-test or Dunnett's test. For comparisons between multiple groups, we determined the significance of difference between the group means by the Turkey–Kramer test. Analyses were performed using the EXSAS software (version 5.00, Arm Co., Ltd., Osaka, Japan) or StatView software (Abacus Concepts, Inc., Berkeley, CA, USA). Differences with values of P< 0.05 were considered to be statistically significant.
3. Results
3.1 Depletion of CD4+ T cells impairs the neovascularization after hindlimb ischaemia
We first examined whether CD4+ T cells are required for the neovascularization after hindlimb ischaemia. To deplete CD4+ T cells in vivo, mice were injected intraperitoneally with a neutralizing antibody against CD4, and hindlimb ischaemia was induced 7 days after the injection. Flow cytometry analysis showed that circulating CD4+ T cells were almost completely depleted at 7 and 21 days after the injection (Figure 1A and B). Compared with the control mice, the recovery of blood flow perfusion (determined using a laser Doppler imaging system) and capillary density (determined by CD31 staining) in response to ischaemia was significantly impaired at 14 and 21 days after ischaemia induction (Figure 1C and D, Supplementary material online, Figure S2). These results are consistent with a previous report that involved CD4−/− mice4 and indicate the substantial role of CD4+ T cells in the neovascularization after ischaemia.
Depletion of CD4+ T cells impairs the neovascularization after hindlimb ischaemia. Mice were treated intraperitoneally with a neutralizing antibody against CD4. (A and B) Blood samples were collected from vehicle (PBS)-treated or anti-CD4-antibody-treated (αCD4) mice after the induction of hindlimb ischaemia. The percentage of CD4+ and CD8+ cells was assessed by flow cytometric analysis. Data are expressed as mean ± SEM (n = 4–5). (C and D) Blood flow perfusion was measured using a laser Doppler perfusion imaging system. (C) Representative photographs of blood flow after the surgery are shown. (D) The blood flow ratio was quantitatively analysed. Data are expressed as mean ± SEM (n = 4–5). **P< 0.01 vs. control.
3.2 Cytokine expression in the ischaemic limbs
In addition to the Th1 and Th2 lineages, recent investigations have revealed other lineages of CD4+ T cells, particularly the Treg and Th17 cells, those are developed by distinct inflammatory cytokine signals.6–8 Among the inflammatory cytokines, IFN-γ, IL-4, and IL-6 have been shown to be responsible for the development of Th1, Th2, and Th17 cells, respectively. Therefore, we prepared samples from the ischaemic and contralateral limbs and assessed the protein levels of these inflammatory cytokines. Intriguingly, IL-6 production was increased prominently in the ischaemic limbs, when compared with the non-ischaemic limbs (Supplementary material online, Figure S1). On the other hand, there was no significant difference in IFN- γ and IL-4 production between the ischaemic and contralateral limbs (data not shown). These results suggest that Th17 cells might be involved in the neovascularization after ischaemia.
We therefore investigated the expression of IL-17, RORγt (a master regulator of Th17 differentiation), and FoxP3 (a master regulator of Treg differentiation) by using real-time RT–PCR analysis. The expression of IL-17 and RORγt was predictably up-regulated in the ischaemic limbs, when compared with the sham-operated and contralateral limbs (Supplementary material online, Figure S1). In contrast, the expression of FoxP3 was observed in the non-ischaemic limbs, and this expression was markedly decreased in the ischaemic limbs. Furthermore, although IL-22 expression was also increased in the ischaemic limbs, the expression level was relatively low (Supplementary material online, Figure S1).
3.3 Role of IL-17 in the neovascularization after hindlimb ischaemia
To investigate the role of Th17 cells, we used IL-17−/− mice and examined the effect of IL-17 deficiency on the neovascularization after hindlimb ischaemia. A significant reduction in the blood flow perfusion after ischaemia was observed in IL-17−/− mice; however, in contrast to the WT mice, blood flow recovery was significantly impaired (day 7, P = 0.041; day 14, P = 0.0052; day 21, P = 0.0037; Figure 2A and B). Capillary density, as determined by CD31 staining, was also significantly diminished at 21 days after the ischaemia induction (Figure 2C and D).
Role of IL-17 in neovascularization after hindlimb ischaemia. Hindlimb ischaemia was produced in WT and IL-17−/− mice. Blood flow perfusion was measured using a laser Doppler perfusion imaging system. The adductor muscles were excised 21 days after ischaemia induction and immunohistochemically stained with an antibody against CD31. (A) Representative photographs of blood flow after the surgery are shown. (B) The blood flow ratio was quantitatively analysed. Data are expressed as mean ± SEM (n = 12–13). *P< 0.05, **P< 0.01 vs. WT. (C) Representative photographs of capillary density are shown. (D) Capillary density was quantitatively analysed. Data are expressed as mean ± SEM (n = 4). ***P< 0.001.
Recent investigations have revealed the contribution of circulating endothelial progenitor cells (EPCs) in the process of generating an angiogenic response.15 Therefore, we determined the number of circulating CD34+/Flk-1+ (ordinary EPC marker) after ischaemia induction in WT and IL17−/− mice by using flow cytometry. The number of CD34+/Flk-1+ cells was significantly increased after the induction of ischaemia in both the WT and IL-17−/− mice; however, no significant difference in the circulating CD34+/Flk-1+ cells was observed between these mice (Supplementary material online, Figure S3).
3.4 Infiltration of monocytes, neutrophils, and CD4+ T cells
Because infiltrated inflammatory cells have been shown to be implicated in the neovascularization after ischaemia,16,17 we investigated the infiltration of monocytes/macrophages and neutrophils in the ischaemic limbs of WT and IL-17−/− mice. Immunohistochemical analysis showed that the numbers of infiltrated macrophages and neutrophils—determined by F4/80 and Gr-1 staining, respectively—were clearly reduced in the ischaemic limbs of L-17−/− mice, when compared with the WT mice (Figure 3A). To further explore these findings, we performed flow cytometry analysis. Consistent with the immunohistochemical findings, we found that the percentage of CD45+ cells (pan-leucocytes), macrophages, and neutrophils was significantly decreased in the ischaemic limbs of L-17−/− mice (CD45, P = 0.023; Gr-1, P = 0.0048; F4/80, P = 0.0103; Figure 3C–E, Supplementary material online, Figure S4). A marked reduction in CD4+ T cells was also observed in the ischaemic limbs of L-17−/− mice by using immunohistochemical and flow cytometry analyses (Figure 3B and F, Supplementary material online, Figure S4).
Infiltration of monocytes, neutrophils, and CD4+ T cells. Hindlimb ischaemia was produced in WT and IL-17−/− mice. After this, the adductor muscles were excised and immunohistochemically stained with antibodies against Gr-1, F4/80, and CD4. (A and B) Representative photographs of infiltrated Gr-1+, F4/80+ cells (3 days), and CD4+ cells (7 days) after ischaemia induction are shown. (C–E) The adductor muscles of the ischaemic limbs were excised after ischaemia induction and digested using collagenase. The percentage of CD45+, Gr-1+, and F4/80+ cells was assessed by flow cytometric analysis. Quantitative analysis was performed. Data are expressed as mean ± SEM (n = 6). (F) Quantitative analysis of infiltrated CD4+ cells in the ischaemic limbs was performed. Data are expressed as mean ± SEM (n = 6). *P< 0.05, **P< 0.01, ***P< 0.001.
3.5 Production of cytokines and growth factors
Because IL-1β and VEGF-A have been shown to be key factors in the process of generating neovascularization after hindlimb ischaemia,12,18 we examined the expression of these factors in the ischaemic limbs of WT and IL-17−/− mice. The production of IL-1β and VEGF-A was significantly increased in the ischaemic limbs of WT mice, when compared with the non-ischaemic limbs, on day 2 after ischaemia induction (Figure 4A and B). Interestingly, the increased production of IL-1β and VEGF-A was significantly inhibited in IL-17−/− mice, when compared with WT mice (IL-1β, P = 0.0002; VEGF-A, P = 0.0154). However, there was no significant difference of HGF and bFGF in the ischaemic limbs between these mice (Supplementary material online, Figure S5). On day 5 after ischaemia induction, although IL-1β production was already deceased in the ischaemic limbs, such an inhibition was still observed (Supplementary material online, Figure S5). We also assessed the production of other inflammatory cytokines known to be involved in the process of angiogenesis and found that the production of MCP-1 and IL-6, but not IL-10, was increased in the ischaemic limbs of WT mice, and this increased production tended to be less prominent in the limbs of IL-17−/− mice (Figure 4C–E).
Production of cytokines and growth factors. Hindlimb ischaemia was produced in WT and IL-17−/− mice. The adductor muscles of the ischaemic and contralateral limbs were excised at 2 days after ischaemia induction. The protein levels of IL-1β (A), VEGF-A (B), MCP-1 (C), IL-6 (D), and IL-10 (E) were measured. Data are expressed as mean ± SEM (n = 5–6). *P< 0.05, **P< 0.01, ***P< 0.001.
We and other investigators have shown that IL-1β and VEGF-A were secreted by the regenerating skeletal muscles in the later phase after ischaemia induction,12,18 At 14 days after ischaemia induction, the expression of IL-1β and VEGF-A was clearly visible in the skeletal muscles of ischaemic limbs, and this increased expression was also reduced in the ischaemic limbs of IL-17−/− mice (data not shown).
3.6 Contribution of bone marrow-derived cells to the neovascularization
As it has recently been shown that IL-17 could be produced by non-haematopoietic cells such as endothelial cells and smooth muscle cells in addition to the principal IL-17 producer Th17 cells, we generated a series of reciprocal bone marrow chimeric mice (BMTWT→WT mice, BMTWT→IL-17−/− mice, and BMTIL-17−/−→WT mice) using WT and IL-17−/− mice to determine relative contribution of haematopoietic and non-haematopoietic-derived IL-17. We have previously confirmed >90% bone marrow reconstitution using green fluorescent protein transgenic mice at 8 weeks after bone marrow transplantation (BMT).13 To further verify the reconstitution, Th17 cells were generated from spleen cells and analysed using flow cytometry. As expected, the percentage of Th17 cells was markedly decreased in BMTIL-17−/−→WT mice, when compared with that the percentage in BMTWT→WT mice. In addition, this percentage was also reduced in BMTWT→IL-17−/− mice (Figure 5A and B). The recovery of blood flow perfusion was markedly impaired in BMTIL-17−/−→WT mice, when compared with that in BMTWT→WT mice (day 7, P = 0.0001; day 14, P = 0.0003; day 21, P< 0.0001; Figure 5C and D). Perfusion recovery was also significantly, yet less prominently, decreased in BMTWT→IL-17−/− mice (day 7, P = 0.0148; day 14, P = 0.0498; day 21, P< 0.0076). These results suggest that although non-bone marrow-derived IL-17 could also be involved in the angiogenic response to ischaemia, the major source of IL-17 was bone marrow-derived haematopoietic cells, most likely the Th17 cell.
Contribution of bone marrow-derived cells. (A–D) Hindlimb ischaemia was produced in BMT mice (BMTWT→WT, BMTWT→IL-17−/−, and BMTIL-17−/−→WT mice) 8 weeks after transplantation. Blood flow perfusion was measured using a laser Doppler perfusion imaging system. (A) Splenocytes were isolated and treated with CD3/CD28 antibodies for 6 h. The expression of IL-17 and IFN-γ was assessed by flow cytometric analysis. (B) The percentage of Th17 cells in BMT mice was quantitatively analysed. (C) Representative photographs of blood flow on days 14 and 21 after the surgery are shown. (D) The blood flow ratio was quantitatively analysed. Data are expressed as mean ± SEM (n = 10). *P< 0.05, **P< 0.01, ***P< 0.001 vs. BMTWT→WT.
3.7 Contribution of Th17 cells to the neovascularization
In addition to being produced by Th17 cells, IL-17 has recently been shown to be produced by several other types of cells, including monocytes/macrophages, endothelial cells, and vascular smooth muscle cells.19,20 To exclude the effect of other IL-17-producing cells, CD4+ T cells purified from the spleens of WT and IL-17−/− mice were implanted into the ischaemic limbs of athymic nude mice. This experiment clarifies the role of Th17 cells in the neovascularization after ischaemia because athymic nude mice completely lack a T cell system. There was no difference in the percentage of purified CD4+ T cells between WT and IL-17−/− mice (Figure 6A). The perfusion of blood flow was significantly higher in mice implanted with WT-derived CD4+ T cells (Th17-containing) at 7 days after ischaemia induction than in the control mice (P = 0.0006; Figure 6B and C). This blood flow recovery was significantly reduced in mice implanted with IL-17−/−-derived CD4+ T cells (Th17-depleted) (P = 0.0342), indicating that Th17 cells are as angiogenic T cell subsets. Finally, we confirmed that IL-17 induced the expression of angiogenic cytokines, such as VEGF, IL-1β, IL-6, and MCP-1 in bone marrow cells in vitro (Supplementary material online, Figure S7).
Effect of Th17 cell implantation. (A) CD4+ T cells were purified from the spleens of WT and IL-17−/− mice with >95% purity, using an MACS. (B and C) The cells were injected into the ischaemic limbs 24 h after the ischaemia. Blood flow perfusion was measured using a laser Doppler perfusion imaging system. (B) Representative photographs of blood flow on days 0 and 7 after the surgery are shown. (C) The blood flow ratio on day 7 was quantitatively analysed. Data are expressed as mean ± SEM (n = 8). *P< 0.05 and ***P< 0.001.
4. Discussion
The major findings of this study are as follows: (i) the depletion of CD4+ T cells impairs the neovascularization after murine hindlimb ischaemia; (ii) the expression of IL-17 and RORγt (a regulator of Th17 differentiation) is up-regulated in the ischaemic limbs, whereas that of FoxP3 (a regulator of Treg differentiation) is down-regulated; (iii) when compared with WT mice, IL-17-deficient (IL-17−/−) mice show a significant reduction in blood flow perfusion, inflammatory cell infiltration, and production of angiogenic cytokines such as IL-1β and VEGF in the ischaemic limbs; (iv) the absence of IL-17 specifically in the bone marrow diminishes the neovascularization; and (v) when compared with the implantation of WT-derived CD4+ T cells (Th17-containing), the implantation of IL-17−/−-derived CD4+ T cells (Th17-depleted) results in a significant reduction in the neovascularization. These findings show that Th17 cells are a new angiogenic T cell subset and also provide new insight into the mechanism by which T cells promote neovascularization. Furthermore, these findings suggest that the modulation of Th17 cells can potentially be used for therapeutic angiogenesis in ischaemic cardiovascular diseases.
Increasing evidence suggests a link between the cellular components of the immune system and the neovascularization after ischaemia.16,17 In particular, it is now widely accepted that monocytes/macrophages play a key role in this process as potent sources of angiogenic factors such as VEGF and IL-1β.12,18 In contrast to monocytes/macrophages, little information is available on the role of T cells in the process of neovascularization despite the fact that a substantial number of T cells are found at the ischaemic site. Several lines of evidence have recently suggested the importance of T cells in neovascularization after ischaemia. The specific role of T cells in the angiogenic process was suggested by a previous report describing that athymic nude mice completely lacking T cells show impaired collateral vessel development in response to ischaemia.3 In the present study, we demonstrated that the depletion of CD4+ T cells markedly impaired blood flow perfusion recovery and capillary formation in response to hindlimb ischaemia. Consistent with this finding, Stabile et al.4 also showed that CD4−/− mice exhibited a significant reduction in post-ischaemic neovascularization. They further demonstrated that CD8+ T cells also contribute to the early phase of neovascularization after ischaemia, and the angiogenic effect of CD8+ T cells is mediated through the IL-16-dependent recruitment of CD4+ T cells at the ischaemic site.5 These findings suggest the importance of CD4+ T cells in the process of neovascularization after ischaemia.
On the basis of the function of CD4+ T cells in cytokine production and specific transcription factor expression, CD4+ T cells are now classified into four subsets: Th1, Th2, Treg, and Th17.6,7 However, the specific CD4+ T cell subset that promotes neovascularization has not been identified. To investigate which subset(s) is responsible for the angiogenic response, we determined the expression of IFN-γ, IL-4, and IL-6 as cytokines in the development of Th1, Th2, and Th17, respectively, and we found that IL-6 production increased prominently in the ischaemic limbs. On the basis of this observation, we predicted that Th17 cells play a role in the neovascularization after ischaemia. The up-regulation of RORγt, a master regulator of Th17 differentiation, in the ischaemic limbs further supports the role of Th17 cells in this process. On the other hand, the down-regulation of FoxP3, a master regulator of Treg differentiation, was also observed in the ischaemic limbs. This finding is consistent with recent evidence showing the reciprocal regulation of Th17 cells and Tregs.6,21 Moreover, Zouggari et al.22 recently demonstrated that Tregs induces anti-angiogenic activity by down-modulating the effector immune cell response.
Recent investigations have implicated Th17 cells in a variety of diseases, including infectious diseases, autoimmune conditions, transplantation reactions, allergies, and tumours.6,7 However, the role of Th17 in angiogenesis is not clearly understood. In the present study, we demonstrated that in the inflammatory response and neovascularization after ischaemia are impaired in IL-17−/− mice and that angiogenic cytokines, such as IL-1β and VEGF, are involved in this process. Consistent with our findings, several studies have shown that IL-17 stimulates VEGF production in fibroblasts.23,24 Very recently, Pickens et al.25 demonstrated that IL-17 enhances migration and tube formation of human lung microvascular endothelial cells and these effects are mediated through IL-17 receptor C (IL-17RC), suggesting the direct effect of IL-17 in angiogenesis. Based on the findings of our study, we postulate that ischaemic insults recruit inflammatory cells, including monocytes/macrophages and CD4+ T cells, at the site of ischaemia, and these inflammatory cells, in turn, produce angiogenic cytokines and factors, thereby resulting in the acceleration of neovascularization. At the site of ischaemia, IL-6 may promote generation of Th17 cells. IL-17 produced by Th17 cells enhances the neovascularization after ischaemia through acting on endothelial cells and/or indirectly via further promoting local inflammation by recruiting inflammatory cells.
As for the cellular source of IL-17, the BMT experiments showed that IL-17 deficiency in bone marrow cells considerably impaired the neovascularization after ischaemia, indicating the major source of IL-17 was bone marrow-derived cells. Since WT but not IL-17-deficient CD4+ cells evoked ischaemia-induced neovascularization in athymic nude mice, Th17 cells were among the bone marrow-derived cells. However, we also observed that IL-17 deficiency in non-bone marrow-derived cells partially but significantly impaired blood flow recovery after ischaemia induction, suggesting that the IL-17 produced by non-Th17 cells may also contribute to neovascularization, yet to a lesser extent than T cell-derived IL-17. Supporting this finding is the recent report that IL-17 is produced by several types of cells, including monocytes/macrophages, endothelial cells, and vascular smooth muscle cells.19,20 Nevertheless, given the greater contribution of bone marrow-derived cells, most likely Th17 cells, as a source of the angiogenic cytokine IL-17, we infer that Th17 cells play an important role in ischaemia-induced neovascularization and would be a potential source for therapeutic neovascularization.
In conclusion, we have demonstrated the contribution of Th17 cells to the inflammatory response and neovascularization after ischaemia and identified Th17 cells as a new angiogenic T cell subset. As proposed mechanisms, ischaemia induces accumulation of inflammatory cells, such as monocytes/macrophages and neutrophils, that produce inflammatory cytokines. Among these cytokines, IL-6 can promote differentiation from CD4+ T cells into Th17 cells. IL-17 produced by Th17 cells may stimulate the production of IL-1β and VEGF-A, and induce the subsequent angiogenic responses (Supplementary material online, Figure S8). Although Th17 cells have been shown to play an important role in the pathophysiology of various diseases, their role in angiogenesis provide a novel aspect of the function of Th17 cells. This study suggests that the modulation of IL-17 cells may be useful for promoting therapeutic neovascularization in ischaemic cardiovascular diseases. Furthermore, a possibility may also be suggested that ex vivo generated Th17 cells are used for adoptive angiogenic therapies of these diseases.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Funding
This study was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology (to M.T.), the Ministry of Health, Labor and Welfare (to M.T. and U.I.), and the Vehicle Racing Commemorative Foundation (to M.T.).
Acknowledgments
We thank Junko Nakayama, Yuka Ichihara, and Kazuko Misawa for excellent technical assistance.
Conflict of interest: none declared.
