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Circulation. Author manuscript; available in PMC Jul 7, 2010.
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PMCID: PMC2898514
NIHMSID: NIHMS126514

Interleukin-17 and Interferon-γ are Produced Concomitantly by Human Coronary Artery-Infiltrating T Cells and Act Synergistically on Vascular Smooth Muscle Cells

Raymond E. Eid, MD,* Deepak A. Rao, MS, Jing Zhou, MD, PhD,* Sheng-fu L. Lo, BA,* Hooman Ranjbaran, MD,* Amy Gallo, MD,* Seth I. Sokol, MD,‡,1 Steven Pfau, MD, Jordan S. Pober, MD, PhD, and George Tellides, MD, PhD*,2

Abstract

Background

Atherosclerosis is an inflammatory disease in which interferon (IFN)-γ, the signature cytokine of Th1 cells, plays a central role. We investigated whether interleukin (IL)-17, the signature cytokine of Th17 cells, is also associated with human coronary atherosclerosis.

Methods and Results

Circulating IL-17 and IFN-γ were detected in a subset of patients with coronary atherosclerosis as well as in referent outpatients of similar age without cardiac disease, but not in young healthy individuals. IL-17 plasma levels correlated closely with those of the IL-12/IFN-γ/CXCL10 cytokine axis, but not with known Th17 inducers, such as IL-1β, IL-6, and IL-23. Both IL-17 and IFN-γ were produced at higher levels by T cells within cultured atherosclerotic coronary arteries after polyclonal activation than within non-diseased vessels. Combinations of pro-inflammatory cytokines induced IFN-γ, but not IL-17 secretion. Blockade of IFN-γ signaling increased IL-17 synthesis, whereas neutralization of IL-17 responses decreased IFN-γ synthesis; production of both cytokines was inhibited by transforming growth factor-β1. Approximately ten-fold fewer coronary artery-infiltrating T helper cells were IL-17 producers than IFN-γ producers, and unexpectedly IL-17/IFN-γ double producers were readily detectable within the artery wall. Although IL-17 did not modulate the growth or survival of cultured vascular smooth muscle cells, IL-17 interacted cooperatively with IFN-γ to enhance IL-6, CXCL8, and CXCL10 secretion.

Conclusions

Our findings demonstrate that IL-17 is produced concomitantly with IFN-γ by coronary artery-infiltrating T cells and these cytokines act synergistically to induce pro-inflammatory responses in vascular smooth muscle cells.

Keywords: coronary disease, inflammation, interleukins, lymphocytes, muscle, smooth

Atherosclerosis, the leading cause of death and disability worldwide, is recognized as an inflammatory disease exacerbated by dysregulation of T cell-derived cytokines within the vessel wall (1). CD4+ effector T cells may differentiate into different lineages characterized by the polarization of secreted cytokines. Interferon (IFN)-γ, the signature cytokine of Th1 cells, is present in coronary arteries of patients with atherosclerosis (2-4) and plays a non-redundant role in T cell-mediated injury and remodeling of human coronary arteries in vivo (5). Th2 cells, in contrast, produce mostly interleukin (IL)-4, IL-5, and IL-13 and are associated with eosinophilic inflammatory responses to helminths, ticks, and allergens. Th2 cells inhibit the formation and differentiation of Th1 cells and vice versa. More recently, a new lineage of IL-17-producing effector CD4+ T lymphocytes, termed Th17 cells, has been described (6). Thus far, six IL-17 family ligands (IL-17A-F) and five receptors (IL-17RA-E) have been identified, of which the founding member IL-17A (hereafter referred to as IL-17) and its receptor IL-17RA are the most widely studied. Th17 cells also secrete IL-21 and IL-22; the former may promote Th17 lineage commitment and the latter has pro- and anti-inflammatory functions. Studies in mice have identified the combination of transforming growth factor (TGF)-β1 plus IL-6 as essential for the development of naïve T cells into Th17 cells (7), although it has been recently described that TGF-β1 may suppress Th17 polarization in humans under certain conditions (8,9). In addition, IL-1β may combine with IL-6 to promote Th17 differentiation and IL-23 is required for maintaining Th17 cells (7-10). On the other hand, the Th1 cytokine, IFN-γ and the Th1-inducing factor, IL-12 suppress the differentiation of Th17 cells (7-10). Evidence that the transcription factor, ROR-γt specifically controls Th17 cell differentiation supports the concept of a distinct T cell lineage (11). Based on studies in mice, the differentiation of Th17 and Th1 cells is generally thought to be mutually exclusive. The paradigm is more complex in humans where single effector T helper cells may produce both IL-17 and IFN-γ (8,9). Even in mice, double producers of IL-17 and IFN-γ have been noted under certain circumstances, such as infection with extracellular bacteria (7).

Th17 cells have an antimicrobial role through the production of pro-inflammatory factors from diverse cell types by IL-17, including the cytokine, IL-6 and the chemokine, CXCL8 (6). Defining features of IL-17-dependent immune responses include the recruitment of neutrophils and protection against extracellular bacterial and fungal infection. Beyond their function in host defense, Th17 cells have been linked to the pathogenesis of several inflammatory and autoimmune diseases. In mice, IL-17-producing T cells are responsible for pathologic outcomes in experimental models of arthritis, autoimmune encephalitis, and colitis (12-14). In humans, several studies have described a correlation between plasma or tissue concentrations of IL-17 and the severity of rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease (15-17). However, the role of Th17 cells and that of IL-17 in atherosclerotic arterial disease remains undefined. Here, we report the concomitant presence of IL-17 and IFN-γ in patients and clinical specimens of coronary atherosclerosis, the presence of IL-17/IFN-γ dual producing T cells within coronary plaques, and a synergistic effect of IL-17 and IFN-γ on elicitation of pro-inflammatory cytokine and chemokine production by cultured human vascular smooth muscle cells (VSMCs).

Methods

Patients and Artery Donors

Research protocols were approved by the institutional review board of the West Haven VA Hospital, Yale University, and the New England Organ Bank. Clinical characteristics are detailed in the Supplemental Methods and Supplemental Table 1. Briefly, there were 108 patients with symptomatic coronary atherosclerosis documented by cardiac catheterization (63.7±10.5 years of age, 99.1% male, and 88.0% Caucasian), 59 referent outpatients treated for diverse medical conditions, but without a diagnosis of coronary atherosclerosis (66.4±9.7 years of age, 94.9% male, and 89.8% Caucasian), and 18 healthy laboratory personnel and students (32.4±4.8 years of age, 66.1% male, and 44.4% Caucasian). The two patient cohorts with and without a diagnosis of coronary atherosclerosis had similar demographics, whereas the healthy control group were younger (P<0.0001), had fewer males (P<0.001), and fewer Caucasians (P<0.001). Human coronary arteries and aortas were obtained from explanted hearts of transplant recipients or cadaver organ donors.

Artery and Cell Culture

Epicardial coronary arteries were divided into 3 mm rings and cultured in M-199 medium supplemented with 20% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). The medium was changed after 6 hr prior to treatment with agonistic antibodies to CD3 (eBiosciences, San Diego, CA) and CD28 (BD Biosciences, San Jose, CA), with PMA and ionomycin (Sigma-Aldrich, St. Louis, MO), and/or with human cytokines.

Human aortic and coronary artery VSMCs were isolated by explant outgrowth, serially cultured in M199 medium supplemented with 20% FBS, and used at passage 3 to 4. VSMCs were serum-deprived in 0.5% FBS for 48 hr prior to cytokine treatment.

Circulating CD4+ T cells were isolated by positive selection with magnetic beads (Dynal Biotech, Oslo, Norway). Human coronary artery-infiltrating leukocytes were isolated from enzyme-digested, minced artery segments as described in the Supplemental Methods followed by magnetic bead selection which yielded a uniformly positive cell population with >90% CD4 expression.

Cytokines and Antibodies

Human coronary arteries and VSMCs were treated with recombinant human IFN-γ, IL-1α, IL-6, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, and TGF-β1 as well as neutralizing antibodies to IL-17RA, IFN-γR1, or irrelevant, isotype-matched antibody (R&D Systems, Minneapolis, MN).

Cytokine Array and ELISA

Cytokine arrays were performed using soluble biotinylated detection antibodies and immobilized capture antibodies on nitrocellulose membranes, and bound cytokine- or chemokine-antibody complexes were detected by chemiluminescent reagents according to the manufacturer's instructions (R&D Systems). The spot pixel density was measured by image analysis software in arbitrary units.

Plasma and supernatant levels of cytokines were determined using sandwich ELISA kits (R&D Systems). The lower limit of detection (or lowest concentration of standard sample) were 7.8 pg/mL for IL-17, IFN-γ, CCL20, CXCL8, and IL-12p70, 1.9 pg/mL for IL-1β, 4.6 pg/mL for IL-6, 31.2 pg/mL for IL-12p40 and IL-23, and 15.6 pg/mL for CXCL10. High-sensitivity C-reactive protein assays were performed by the clinical laboratory of Yale-New Haven Hospital.

Flow Cytometry and Immunoblotting

Details of the techniques are provided in the Supplemental Methods. In brief, for flow cytometric analysis the cells were labeled with mouse anti-human CD4 (Beckman, Miami, FL), IL-17, and IFN-γ (eBioscience) monoclonal antibodies or isotype-matched, irrelevant IgG, and analyzed using a FACSort (BD Biosciences). For immunoblotting analysis, protein lysates from VSMCs were blotted with rabbit antibodies to IκBα, phospho-p38 MAPK, and phospho-STAT1 (Cell Signaling Technology, Beverly, MA), or with mouse monoclonal antibodies to β-actin (Sigma-Aldrich), followed by horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA).

Statistical Analysis

Results were analyzed from three different projects: a patient-related serologic study, an organ culture model, and a cell culture model. In the serologic study, plasma cytokine levels were undetectable in a substantial proportion of subjects and the distribution was skewed, even after log transformation. The two groups of patients with and without coronary atherosclerosis and a third group of healthy individuals were compared for a binary outcome of detectable vs. undetectable cytokine plasma levels using Fisher's exact test. Comparisons between individual groups were performed after establishing significance with a global test, i.e. with two degrees of freedom for three groups using the Chi-square test. Correlations between cytokine plasma levels were examined by the nonparametric Spearman test. Continuous clinical variables were compared between groups using the two-sample t test and categorical clinical variables were analyzed by Fisher's exact test. In the organ culture and cell culture models, the distribution of supernatant cytokine levels were Gaussian and parametric tests were performed. For in vitro assays with two experimental arms comparisons were by t test, for those with more than two groups by one-way analysis of variance (ANOVA), and for assays over time by Repeated Measures ANOVA. All P values were two-tailed and values <0.05 were considered to indicate statistical significance. Statistical analyses were performed using Prism 4 (GraphPad Software, San Diego, CA).

Statement of Responsibility

The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

Results

IL-17 is Detected Concomitantly with IFN-γ in the Plasma of Patients with Coronary Atherosclerosis

Plasma cytokine levels were measured in patients presenting with symptomatic coronary atherosclerosis (n=108), in referent outpatients without cardiac diseases of similar age, gender, and race (n=59), and in healthy young adults (n=18). Circulating IL-17 and IFN-γ was detected in a subpopulation of both groups of patients, but not in healthy younger subjects (Fig. 1A,B). The presence of IL-17 did not discriminate between patients with or without coronary atherosclerosis. Plasma levels of IL-17 did not differ between coronary atherosclerosis patients with higher vs. lower angiographic extent of disease score (Fig. 1C) or with presenting symptoms of chronic stable angina vs. acute coronary syndrome (Fig. 1D), similar to the findings previously reported for IFN-γ (4). Also, differences in IL-17 plasma levels did not reach statistical significance in referent outpatients with no current medical illnesses vs. those with current non-cardiac medical illnesses (Fig. 1E). The results provide systemic evidence of adaptive immune responses in certain patients with coronary atherosclerosis, but did not reveal a relationship of cytokine plasma levels with particular clinical manifestations in this relatively small number of subjects.

Figure 1
Serologic evidence for Th17 and Th1 immune responses in patients with coronary atherosclerosis. (A) IL-17 and (B) IFN-γ plasma levels were measured in patients with symptomatic coronary atherosclerosis (n=108), in referent outpatients without ...

We examined for an association between circulating cytokines. Surprisingly, there was a strong correlation between IL-17 and IFN-γ plasma levels (in particular at higher concentrations) in patients with coronary atherosclerosis (Fig. 2A) and somewhat less so in referent patients (r=0.76, P<0.0001). IL-17 also correlated with other markers of the IFN-γ cytokine axis in patients with coronary atherosclerosis, such as the IFN-γ-inducer, IL-12 and the IFN-γ-inducible chemokine, CXCL10 (Fig. 2B,C). In contrast, IL-17 concentrations did not correlate with the inflammatory marker most frequently described in patients with coronary atherosclerosis, C-reactive protein (r=-0.13, P=0.19), nor with levels of cytokines known to promote human Th17 immune responses, such as IL-1β, IL-6, and IL-23 (Fig. 2D-F). Independently, measurements of the common p40 subunit for IL-12 and IL-23 revealed an intermediate association with IL-17 (r=0.41, P<0.0001). The close correlation between circulating IL-17 and IFN-γ suggested at least a partially shared, common trigger for production of these cytokines in coronary atherosclerosis.

Figure 2
Circulating IL-17 correlated with the systemic IFN-γ cytokine axis in patients with coronary atherosclerosis. (A) IFN-γ, (B) IL-12, (C) CXCL10, (D) IL-1β, (E) IL-6, and (F) IL-23 plasma levels were correlated to those of IL-17 ...

Both IL-17 and IFN-γ are Produced by T Cells within Atherosclerotic Coronary Arteries

We used an organ culture system to determine stimuli that can increase IL-17 production within human coronary arteries. We first characterized the cytokine polarization of artery-infiltrating T cells in response to polyclonal T cell receptor activation with agonistic antibodies to CD3 and CD28. Optimal concentrations of the T cell-stimulating antibodies elicited IL-17 and even greater IFN-γ secretion from atherosclerotic coronary artery rings in a time-dependent fashion (Fig. 3A,B). A lesser secretion of cytokines in response to anti-CD3/CD28 was detected from coronary arteries of donors without gross signs of atherosclerosis. In the absence of cognate stimuli, inflammatory factors known to promote the differentiation of Th17 cells or maintain Th17 immune responses, such as IL-1, IL-6, IL-15, IL-21, IL-23, and TGF-β1 did not result in detectable IL-17 production either alone or in various combinations (Fig. 3C). These data demonstrated that antigen, but not inflammatory, pathways effectively activated Th17 immune responses in human coronary arteries.

Figure 3
Coronary artery-infiltrating T cells produced both IL-17 and IFN-γ. Equal length segments of atherosclerotic or macroscopically normal coronary arteries in organ culture were treated with agonistic antibodies (α) to CD3 (0.1 μg/mL) ...

We further investigated cytokine cross-regulation of receptor-activated T cells present within atherosclerotic coronary arteries. T cells resident in plaques were polyclonally stimulated in situ under pathologically relevant conditions in the presence of various treatment conditions. Inhibiting IL-17 responses with neutralizing antibody to IL-17RA decreased IFN-γ secretion without modulating IL-17 secretion. In contrast, inhibiting IFN-γ responses with neutralizing antibody to IFN-γR1 increased IL-17 secretion, but decreased IFN-γ production (Fig. 4A). TGF-β1 inhibited the production of both IL-17 and IFN-γ after T cell receptor engagement (Fig. 4B). Finally, the combination of IL-12/IL-18, known to induce IFN-γ production, did not stimulate IL-17 secretion either with or without CD3/CD28 antibody treatment (Fig. 4C). The results indicated distinct regulation of Th17 and Th1 cytokine production in atherosclerotic coronary arteries.

Figure 4
Distinct regulation of coronary artery-infiltrating Th17 and Th1 cells. (A) T cells infiltrating atherosclerotic coronary arteries were stimulated with agonistic antibodies (α) to CD3 (0.1 μg/mL) and CD28 (1 μg/mL) for 48 hr in ...

A Subset of Coronary Artery-Infiltrating CD4+ T Cells are IL-17/IFN-γ Double Producers

To extend the characterization of artery-infiltrating IL-17-producing T cells, we isolated CD4+ T cells from enzyme-digested atherosclerotic coronary vessels using antibody-coated magnetic beads. An analysis of one of the purified preparations is presented in Fig. 5A and the resultant population appears uniformly positive for CD4 expression. We analyzed these artery-infiltrating T helper cells by flow cytometry to assess cytokine production following polyclonal stimulation. Analysis at the single-cell level demonstrated that a major population of T helper cells produced only IFN-γ, whereas minor populations produced only IL-17 or both IFN-γ and IL-17 (Fig. 5B,C). The corresponding fractions of cytokine-producing CD4+ T cells were not significantly different in the peripheral blood of healthy donors, except for fewer IL-17/IFN-γ dual producers (Fig. 5B,D). These findings confirmed that artery-infiltrating T helper cells were a source of IL-17 and the readily detectable double producers of IL-17 and IFN-γ may explain, at least in part, the parallel elevations in Th1 and Th17 cytokines in patients with coronary atherosclerosis.

Figure 5
A subset of coronary artery-infiltrating T helper cells were IL-17 and IFN-γ double producers. CD4+ T cells from atherosclerotic coronary arteries or from peripheral blood of healthy subjects were isolated using antibody-conjugated magnetic beads ...

IL-17 does not Modulate VSMC Growth or Survival

To determine if IL-17 produced by artery-infiltrating T cells may have direct effects on VSMCs, we first investigated if it modulated cell growth and survival, as VSMC proliferation and apoptosis are known to cause disease progression or complications of atherosclerosis. IL-17 had no effect on the number of VSMCs cultured under optimal (20%) or low (0.5%) serum conditions over a period of 8 days (Supplemental Fig. 1A). Similarly, IL-17 treatment did not alter the frequency of dying (subdiploid population), non-dividing (GO/G1 phases), or dividing (S/G2/M phases) VSMCs as determined by flow cytometric analysis of DNA content using propidium iodide staining (Supplemental Fig. 1B). IL-17 also had no effect on VSMC death as assessed by flow cytometric analysis of phosphatidylserine membrane translocation using Annexin-V labeling and loss of membrane integrity using 7-amino-actinomycin D uptake (Supplemental Fig. 1C). Furthermore, IL-17 did not interact with IFN-γ in modulating VSMC growth or survival (Supplemental Fig. 1D,E). Collectively, these data indicated that IL-17 did not have significant mitogenic or pro-apoptotic effects on cultured VSMCs.

IL-17 Acts Synergistically with IFN-γ to Induce Inflammatory Responses in VSMCs

We then investigated if IL-17 may interact with IFN-γ in the induction of pro-inflammatory responses in VSMCs using a cytokine array. The production of several cytokines and chemokines was elicited by IL-17 or IFN-γ, and the detection of IL-1 receptor antagonist, IL-6, IL-8, CCL5, CXCL1, CXCL10, C5a, and soluble ICAM-1 was more enhanced in the presence of both factors compared to twice higher concentrations of either IL-17 or IFN-γ alone (Supplemental Table 2 and Supplemental Fig. 2). The induction of other cytokines and chemokines, such as IL-13 and CCL2, was not augmented by the combination treatment, and the variable detection of IL-17 and IFN-γ in the supernatant reflected the different concentrations of exogenous cytokines added to the cell cultures. More detailed dose response experiments using ELISA confirmed synergistic interactions of IL-17 with IFN-γ in the production of IL-6, CXCL8, and CXCL10 (Fig. 6). These results suggested that IL-17 production within the vessel wall may contribute to the pro-inflammatory milieu of atherosclerosis even in the presence of dominant Th1 immune responses.

Figure 6
IL-17 and IFN-γ synergistically induced pro-inflammatory cytokine and chemokine production by VSMCs. (A) IL-6, (B) CXCL8, and (C) CXCL10 levels were measured in the supernatant of VSMCs treated with IL-17±IFN-γ at 5, 10, or 20 ...

To gain insight into the mechanisms for synergy of IL-17 and IFN-γ effects in VSMCs, we assessed their signaling responses (Fig. 7). IL-17 activated NFκB and p38 MAPK signaling pathways as evidenced by rapid degradation (followed by resynthesis) of IκBα and phosphorylation of p38 MAPK, respectively. However, IL-17 did not induce phosphorylation of STAT1. In contrast, IFN-γ activated STAT1, but not NFκB or p38 MAPK. Combined cytokine treatment demonstrated that IFN-γ only modestly enhanced NF-κB and p38 MAPK activation by IL-17 at early time points and at high doses, whereas IL-17 had minimal, if any, effect on phospho-STAT1 expression by IFN-γ. Thus, the interactions between the pro-inflammatory effects of IL-17 and IFN-γ likely occurs at transcriptional and/or translational responses by VSMCs, a subject of future studies, rather than at the level of signaling pathways.

Figure 7
Interactions between IL-17 and IFN-γ signaling. VSMCs were treated with IL-17±IFN-γ for various times and at different doses. Signaling activity was compared to that of TNF-α at 10 ng/mL for 15 min. Cell lysates were prepared ...

Discussion

There is a great deal of interest currently in defining the pathogenetic consequences of aberrant Th17 immune responses. IL-17 has been hypothesized to potentially play a role in the development and complications of atherosclerosis (18-20), although there is little direct evidence to date to support this position. We investigated the production and effects of IL-17 in patient-related studies and experimental systems of human coronary atherosclerosis. We anticipated that IL-17 production would be minimal due to the described antagonism between the Th1 and Th17 subsets of T helper cells (8-10), but we unexpectedly found a positive correlation between IL-17 and IFN-γ plasma levels. Furthermore, a significant population of artery-infiltrating T helper cells produced both IL-17 and IFN-γ, and we observed a synergy between IL-17 and IFN-γ pro-inflammatory effects on VSMCs.

Circulating IL-17 and IFN-γ was not detected in the healthy subjects who were also much younger, and the differences in inflammatory markers may be due to either age or state of health. Our data from 185 subjects shows no difference between elevated plasma levels of IL-17 in patients with coronary atherosclerosis vs. referent patients without coronary atherosclerosis, perhaps due to Th17 immune responses in other chronic disease that may obscure biomarkers of cardiovascular disease. In contrast, subgroup analyses in a previously published study of 78 individuals found that circulating IL-17 was modestly higher in patients with acute coronary syndrome vs. referent patients, but not between patients with stable angina vs. referent patients (21). We do not find a correlation between plasma levels of IL-17 and those of cytokines known to be inducers (e.g. IL-1β, IL-6, and IL-23) or products (IL-1β, IL-6, and C-reactive protein) of Th17 immune responses. However, there is a strong correlation between circulating IL-17 and the IFN-γ cytokine axis (4). Our results do not provide evidence for a serologic IL-17 cytokine axis in patients with coronary atherosclerosis. Instead, our findings are consistent with broad activation of T cells with systemically detectable lymphokines in a subgroup of patients that is unrelated to the onset of acute coronary syndrome. The putative antigen(s) for such immune responses is unknown, but may also reflect antigen-independent activation of bystander T cells within the artery wall by circulating or local pro-inflammatory factors (4,22). Caveats to our conclusions include the relatively small number of patients we studied, which require verification in large epidemiologic clinical trials, and that serologic measurements may not reflect local vascular events.

Our in vitro studies on the production of IL-17 are in keeping with our observations in patients. IL-17 was secreted synchronously with IFN-γ in response to polyclonal T cell activators suggesting that either Th1 and Th17 cells or dual IL-17/IFN-γ producers infiltrated atherosclerotic coronary arteries. This response was also seen in arteries without macroscopic disease, confirming our previous observations of at least minimal T cell accumulation in human arteries regardless of disease (4,23). We did not find that cytokines, such as IL-23, induced detectable IL-17 production by effector T cells resident in atherosclerotic coronary arteries as previously reported for human peripheral blood memory T helper cells (9). This may reflect differences in T cell phenotype, cell numbers, or donor variation. Our inability to determine antigen-independent inflammatory stimuli for IL-17 production in human T cells was not an exhaustive investigation and does not exclude other cytokines which may fulfill these criteria. Our results also do not contradict critical roles for these cytokines in the differentiation or maintenance of Th17 cells. The combination of IL-12 and IL-18 robustly and specifically induced IFN-γ, not IL-17, secretion, which may reflect activation of distinct signaling pathways or the selective upregulation of IL-12Rβ2 in naïve T cells under Th1-, but not Th17-polarizing conditions (10,12). This suggests that the expression of IL-12Rβ2 may differentiate human Th1 from IL-17/IFN-γ double producers, similar to the reported distinctions in CCR4, CCR6, and CXCR3 expression between these T cell subtypes (24).

We have adapted a method of extracting leukocytes from mouse aorta (25) and applied it to atherosclerotic human coronary arteries with sufficient yield of CD4+ T cells to enables flow cytometric intracellular cytokine staining analysis. Th1 cells predominated and were around 10- to 20-fold more frequent than IL-17 and IL-17/IFN-γ producing T helper cells. The latter double producers have been described for human effector T cells, although their lineage identity is unclear. Peripheral blood CCR6+/CXCR3+ memory T helper cells producing both IL-17 and IFN-γ express more abundant TBX21, the human ortholog of the murine Th1 transcription factor T-bet, than RORC, the human ortholog of the murine Th17 transcription factor RORγt (8). On the other hand, IL-23 stimulates both IL-17 and IFN-γ secretion from peripheral blood CD4+/CD45RO+ T cells (9). The production of IL-17 by Th1 cells or IFN-γ by Th17 cells may represent plasticity in cytokine production by T cell lineages that are not always distinct, particularly in humans. The increased frequency of IL-17/IFN-γ double producers to true Th17 cells in atherosclerotic coronary arteries may also reflect the predominance of Th1 immune responses in this disease process.

Similar to studies on endothelial cells (26), we did not find mitogenic or cytopathic effects of IL-17 on cultured VSMCs, arguing against direct pathogenetic roles in intimal expansion or plaque rupture. Rather, IL-17, alone and cooperatively with IFN-γ, induced the production of pro-inflammatory cytokines and chemokines in VSMCs which may promote secondary pro-arteriosclerotic vascular changes (1). The production of cytokines and chemokines in response to IL-17 has been reported in other cell types, including endothelial cells (6), and we have recently described pro-inflammatory effects of IL-17 on VSMCs that were considerably greater than in endothelial cells (27). The only other report of IL-17 effects on VSMCs described the induction of the acute phase reactant, C-reactive protein (28), a biomarker that may also contribute to the pathogenesis of atherosclerosis. Synergy between IL-17 and tumor necrosis factor-α or IL-1β, but not IFN-γ, has also been reported in cell types other than VSMCs (6). The basis for enhanced production of pro-inflammatory factors by two cytokines may reflect the activation of distinct signaling pathways and transcription factors with positive interactions on transcription rates as first described for IFN-γ and tumor necrosis factor-α (29). Our observation that blockade of IL-17 signaling reduces IFN-γ production in atherosclerotic arteries indicates that IL-17 may promote IFN-γ production, in addition to IFN-γ responses, suggesting additional layers of interaction between these two cytokines.

Our findings may be of relevance regarding recommendations for anti-IFN-γ biologic therapy in coronary atherosclerosis and graft arteriosclerosis (4,30). Neutralizing IFN-γ activity within the vessel wall may be expected to greatly increase IL-17 production due to a reversal of its anti-Th17 property, although this may be offset from loss of synergy between IFN-γ and IL-17 for pro-inflammatory effects on VSMCs. Anti-inflammatory therapeutic strategies need to consider a network of interactive cytokines and chemokines rather than individual pro-inflammatory factors in isolation.

Supplementary Material

Supplementary material

Acknowledgments

Funding Sources: This work was supported by the NIH (PO1 HL70295).

Footnotes

Short Commentary regarding the potential clinical impact of the paper: Coronary atherosclerosis is considered an inflammatory disease in which T cells play a key role. Different lineages of T cells are characterized by the production of specific cytokines. Interferon (IFN)-γ, the signature cytokine of Th1 cells, is detected in clinical specimens of coronary atherosclerosis and has a pro-arteriosclerotic effect in experimental models. More recently, a new lineage of interleukin (IL)-17-producing Th17 cells has been described. There is currently a great deal of interest in defining the pathogenetic consequences of IL-17 in diverse inflammatory disease processes, although the role of IL-17 in atherosclerosis remains unknown. We investigated the production and effects of IL-17 in patient studies and experimental models of human coronary atherosclerosis. While we anticipated that IL-17 production would be minimal owing to the described antagonism between Th1 and Th17 cells, we unexpectedly found a positive correlation between IL-17 and IFN-γ plasma levels. A distinct population of coronary artery-infiltrating CD4+ T helper cells produced both IL-17 and IFN-γ that differed from classical Th1 and Th17 cells. Finally, we observed a synergy between IL-17 and IFN-γ pro-inflammatory effects on vascular smooth muscle cells. Our findings underscore the interactions between a network of cytokines present in coronary atherosclerosis and may be of relevance regarding proposals for anti-cytokine biologic therapy.

Disclosures: The authors have no financial conflict of interest.

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