IL-2 Modulates Th2 cell Responses to Glucocorticoid: A Cause of Persistent Type 2 Inflammation?

Background Inhaled glucocorticosteroids (GCs) are the main treatment for asthma as they reduce type 2 cytokine (IL-4, IL-5 and IL-13) expression and induce apoptosis. Asthma severity is associated with GC insensitivity, increased type 2 inflammation and circulating Th2 cells. Since IL-2 is a T cell survival factor, we assessed whether IL-2 levels associate with the proportion of Th2 cells and/or correlate with clinical features of asthma severity. Methods Peripheral blood from asthma patients (n=18) was obtained and Th2 cell numbers determined by flow cytometry. Peripheral blood cells were activated with mitogen (24hrs) and supernatant levels of IL-2 and IL-13 measured by ELISA. In vitro differentiated Th2 cells were treated with dexamethasone and IL-2 and assessed for apoptosis by flow cytometry staining of Annexin V. Level of mRNA for anti-apoptotic (BCL-2) and pro-apoptotic (BIM) genes as well as IL-13 were determined by qRT-PCR. Results IL-2 produced by activated peripheral blood cells correlated negatively with lung function (FEV1) and positively with daily dose of inhaled GC. When patients were stratified based on IL-2 level, high IL-2 producers made more IL-13 and had more circulating Th2 cells. In vitro, increasing the level of IL-2 in the culture media was associated with resistance to DEX-induced apoptosis, more BCL-2 and less BIM mRNA. Th2 cells cultured with higher IL-2 also had more IL-13 mRNA and required higher concentrations of DEX for cytokine suppression. Conclusions and Clinical Relevance IL-2 modulates Th2 cell responses to GC, supporting both their survival and pro-inflammatory capacity, suggesting that a patient’s potential to produce IL-2 may be a determinant in asthma severity.


INTRODUCTION
Asthma is a syndrome characterized by symptoms of diverse pathogenesis (1, 2). Type 2 cytokines promote many of the processes responsible for the development of asthma and symptom manifestation. IL-4 is essential for Th2 cell differentiation and drives B cell isotype switching to IgE; IL-5 is a differentiation factor for eosinophils and mediates their egress from the bone marrow during allergic responses; IL-13 mediates airway hyperresponsiveness through inflammatory cell infiltration, smooth muscle contraction and epithelial secretions [reviewed in (3)]. These cytokines are produced by both type 2 CD4 + helper T cells (Th2 cells) (4,5) and group 2 innate lymphoid cells (ILC2s) (6). ILC2s are innate immune cells found mainly within tissues (7,8), activated by the epithelial-derived cytokines IL-25 and IL-33 (9). Th2 cells are allergen-specific memory T cells that circulate between the lymph nodes and circulation, infiltrating tissues upon allergen exposure (10)(11)(12).
While both cell types contribute to overall type 2 cytokine levels, Th2 cells are considered the principle cell population responsible for their rapid release upon allergen re-exposure as well as maintenance of chronic allergic inflammation (11,13).
Glucocorticoids (GCs) are the main treatment for asthma (14). Their efficacy is considered, in large part, to be due to their ability to suppress type 2 cytokines, in vivo (15), ex vivo (16) and in vitro (17). In most cases inhaled GC therapy is sufficient to achieve asthma control, though the amount required varies greatly amongst patients (18). In severe asthma, however, even high doses of inhaled GC fail to control symptoms and adequately improve poor lung function (18). Studies show that some moderate/severe asthmatics experience improved exacerbation rates, lung function and eosinophilia after using anti-Th2 therapies that block IL-2 drives persistence of type 2 inflammation 4 IL-5 and IL-13 (19)(20)(21)(22), revealing that persistent type 2 inflammation was a factor in their disease severity.
Another anti-inflammatory effect of GC is their ability to induce apoptosis (23). This has been well demonstrated for primary human eosinophils (24, 25), though for T cells the effects vary based on subset examined. For instance, GCs effectively induce apoptosis of thymocytes (26), while memory T cells are less sensitive (27,28). Studies with murine T cells indicate Th2 cells are less sensitive than Th1 cells to GC-induced apoptosis (29). Recently, we reported higher levels of serum IL-13 as well as circulating Th2 cells in severe compared to mild/moderate asthmatics (30). Others have shown Th2 are also higher in bronchoalveolar lavage of severe compared to non-severe asthmatics (31). As such, asthma severity is related not only to persistent type 2 cytokine expression but also to the inability of GC therapy to eliminate Th2 cells.
One mechanism that could regulate the sensitivity of Th2 cells to GC is the IL-2 pathway.
IL-2 is a T cell growth and survival factor that promotes differentiation of the memory T cell phenotype (32). It signals through the IL-2 receptor (IL-2R) which couples with Janus kinases (JAK)s to activate STAT5 transcription factors (33). Studies in T cell lines have shown that IL-2 interferes with GC receptor (GR) nuclear translocation, reducing signaling (34) and inhibiting GC-induced apoptosis (35). Single nucleotide polymorphisms in IL-2 and the IL-2R have been associated with asthma severity (36), suggesting the strength of the IL-2 pathway could be a factor in T cell resistance to GC. To investigate this, we examined IL-IL-2 drives persistence of type 2 inflammation 5 2 production from peripheral blood cells and its relationship with clinical features of asthma, type 2 inflammation and the effect of this cytokine on GC responses of human Th2 cells.
IL-2 drives persistence of type 2 inflammation 6

Subjects
This study was approved by the institutional review board of the University of Alberta (approval number PRO1784). All subjects gave informed consent. Patients were recruited from the tertiary care Asthma Clinic at the University of Alberta, where all clinical measures were obtained. Severe asthma was defined as patients on high dose inhaled corticosteroids (ICS, ≥ 1000 µg/day fluticasone equivalent), a 2 nd line controller (long-acting beta agonist, leukotriene modifier/or theophylline) and/or oral corticosteroid (OCS) therapy for ≥ 50% of the previous year who remain uncontrolled despite this therapy (18).

Flow Cytometry
Profiling of peripheral blood cells The proportion of helper T cells and Th2 cells in peripheral blood were identified by whole blood staining as in (30). In brief, antibodies against CD4 (Clone 1F6; Serotec, Oxford, UK) and CRTh2 (clone BM16; Miltenyi Biotech) were used to determine the proportion of cells exhibiting positive staining for IL-2 drives persistence of type 2 inflammation 7 these markers, as assessed by flow cytometry. The proportion of helper T cells was identified by low side scatter (SSC low ) and high CD4 (high) . Th2 cells were (SSC low ), CD4 (high) and CRTh2 positive and reported as a proportion of total white blood cells (WBC). Flow cytometry data were collected on BD LSR Fortessa (BD, CA, USA) using FACS Diva software. Gates were set in accordance with the profiles of the isotype control and/or negative control beads.

Statistical analysis
Patient data stratified by IL-2 levels were analyzed for statistical significance using Pearson's Chi-Square test for categorical data and Independent t-test for continuous variables. For cell culture experiments, statistical significance for apoptosis and gene expression were determined by ANOVA with post hoc analysis (Student-Newman-Keuls method) or Student's t-test. Correlations were determined using Pearson's Correlation. Data was analyzed using SigmaPlot Version 12.5 and considered significant with p < 0.05.

Peripheral blood cell production of IL-2 associates with asthma severity
Patients were recruited (n=18) from new referrals to our Asthma Center and clinically characterized to assess asthma severity. The amount of IL-2 produced by activated peripheral blood cells from these patients was highly variable, ranging from 14 -102,933 pg/mL.
Patients stratified based on median IL-2 production (42,600 pg/ml) showed no difference in age, body mass index, IgE levels or smoking status. However, those with high IL-2 (> median) did have lower FEV1 and were taking a higher daily dose of inhaled corticosteroid (Table 1). Analyzing the whole population together, IL-2 production was inversely correlated with FEV1 (r = -0.558, p = 0.0162; Fig. 1A) and positively correlated with total daily dose of inhaled corticosteroid (r = 0.561, p = 0.0155; Fig. 1B), suggesting its association with clinical features of asthma severity. While this study population consisted of only 4 severe asthmatics, our analysis does suggest this group is characterized by higher IL-2 production (56,380 pg/ml) compared to non-severe asthmatics (32,133 pg/ml, p = 0.07).

Peripheral blood cell production of IL-2 associates with type 2 inflammation
The propensity for high IL-2 production was also related to the degree of type 2 inflammation. Supernatants from patients with high IL-2 following activation of their peripheral blood cells contained more IL-13 (Table 1) and flow cytometry staining of whole blood showed these patients had a higher proportion of CD4 + T cells and Th2 cells (CD4 + CRTh2 + T cells as a proportion of total white blood cells; Table 1). While the IL-2 drives persistence of type 2 inflammation 11 association between Th2 cells and IL-2 production was not significant (r = 0.430, p = 0.0752), Th2 cells were correlated with total daily dose of inhaled corticosteroid (Fig. 1C, r = 0.583,

The IL-2-GC axis regulates survival of Th2 cells
Patients with a high proportion of circulating Th2 cells were also taking more inhaled GC Interestingly, we failed to see any increase in Annexin V + cells ( Fig. 2A), indicating there was no effect on apoptosis and/or cell death. This was surprising since this level of DEX was able to induce apoptosis in an immortalized T cell line (CCRF CEM, Fig. 2B). A major difference between these two experiments was that the primary Th2 cells were cultured in IL-2 (5 ng/ml). Considering that patients with high IL-2 production had more Th2 cells (Table 1), we next assessed whether titrating the level of this growth factor would alter Th2 cell sensitivity to GC-induced apoptosis. When Th2 cells were cultured with DEX and lower concentrations of IL-2 a significant increase in Annexin V + cells was observed (Fig. 2C).
Apoptosis is regulated by the expression of pro-and anti-apoptotic genes of the BCL-2 family, with the balance between these referred to as the BCL-2 rheostat (38, 39). Since IL-IL-2 drives persistence of type 2 inflammation 12 2 induces the anti-apoptotic gene BCL-2 (40) and GC induces the pro-apoptotic gene BIM (41), we assessed expression of these factors. Th2 cells cultured in high IL-2 had more BCL-2 and less BIM mRNA compared to Th2 cells cultured in low IL-2 (Fig. 3A). The ratio of BCL-2 to BIM (BCL-2:BIM) was significantly higher in Th2 cells cultured in high vs low IL-2 (Fig. 3B) and remained higher following DEX treatment (Fig. 3C), suggesting its involvement in the observed resistance to GC-induced apoptosis (Fig. 2C). Indeed, when the data for all three DEX concentrations were combined (10 -8 -10 -6 M), the BCL-2:BIM ratio of Th2 cells cultured with high vs low IL-2 was significantly higher (Fig. 3D).

The IL-2-GC axis regulates suppression of type 2 cytokines
Since patients exhibiting high IL-2 production from peripheral blood cells also produced more IL-13 (Table 1), we assessed whether IL-2 directly regulates IL-13 expression. We found that Th2 cells cultured with high IL-2 (24 hours) expressed significantly more IL-13 mRNA compared to those cultured with low IL-2 (Fig. 4A), despite there being no difference in Th2 cell numbers (Fig. 4B).
To assess the effect of IL-2 on the ability of GC to suppress type 2 cytokines, we cultured Th2 cells with varying concentrations of IL-2 and DEX. When Th2 cells were cultured in high IL-2 the ability of 10 -8 M DEX to suppress IL-13 mRNA was significantly less than when cells were cultured with low IL-2 (Fig. 4C), though this difference was not observed at higher DEX concentrations. 13 To assess the durability of GC suppression following activation, Th2 cells were pretreated with DEX (24hours), washed and activated with mitogen in the presence or absence of DEX (24hours) and high IL-2. Th2 cells receiving DEX as both a pre-treatment and during activation exhibited almost complete suppression of IL-13 mRNA (88 and 96%, respectively), while cells receiving DEX only as a pre-treatment had substantially more IL-13 mRNA following activation (Fig. 4D).

DISCUSSION
Our study reveals that peripheral blood cell production of IL-2 associates with poor lung function, increased GC usage and heightened type 2 inflammation. In vitro experiments showed that maintaining Th2 cells in high IL-2 shifted them toward GC resistance, both at the level of apoptosis and cytokine production. Taken together, these results suggest that an environment of high IL-2 may mediate asthma severity by driving persistence of Th2 cells and their ability to produce type 2 cytokines. Our findings support and extend previous reports indicating a role for IL-2 in GC responsiveness, including Leung et. al., which detected more IL-2 mRNA in bronchial alveolar lavage cells of steroid resistant than sensitive asthmatics (42) and Mercado et. al. which showed that PBMCs from severe asthmatics produced more IL-2 that non-severe asthmatics (43). Though we did not study this per se, severe asthmatics in our population did appear to produce more IL-2. Indeed, the level of IL-2 varied widely and was positively correlated with total daily dose of inhaled steroid, suggesting it may reflect the continuum of GC responsiveness across our population.
IL-2 is a growth factor known to drive proliferation of CD4 + T cells (44) and so the higher proportion of Th2 cells in those exhibiting high IL-2 production could be due to having more total CD4 + T cells. However, our analyses failed to show a significant difference in the proportion of total CD4 + T cells between the high and low IL-2 group, a linear relationship between CD4 + T cells and IL-2 (r = 0.354, p = 0.150) or differences in growth when Th2 cells were cultured in varying concentrations of IL-2 (1.25 -10 ng/ml). On the other hand, our in vitro data did show that Th2 cells cultured in high IL-2 exhibited less GC-induced apoptosis and had more BCL-2 and less BIM mRNA. Furthermore, the BCL-2:BIM ratio remained above 1 (i.e., more BCL-2 than BIM) in Th2 cells cultured in high (but not low) IL-2, corresponding with the observed resistance to GC-induced apoptosis. IL-2 induction of BCL-2 is well documented (40, 45). To our knowledge we are the first to report IL-2 supressing BIM expression, though the opposite has been shownthat IL-2 starvation of murine cytotoxic T lymphocytes up-regulated BIM (46). Similarly, Banuelos et.al. showed that Th17 cell resistance to GC-induced apoptosis was related to higher expression of BCL-2 (47). Our data highlight the ability of IL-2 to modulate the rheostat, increasing BCL-2 and reducing BIM, to shift Th2 cells toward an anti-apoptotic phenotype. This could suggest that in vivo the higher proportion of circulating Th2 cells may be related to the ability of IL-2 to modulate their sensitivity to GC.
Level of IL-2 in the culture media also influenced IL-13 expression, despite no differences in Th2 cell growth, similar to a previous report that IL-2 induced IL-13 expression from antigen-specific Th clones (48). This may be through IL-2 induction of c-Fos (49), a member of the AP-1 complex shown to upregulate IL-13 (50). IL-2 also dampened the capacity of GC to suppress IL-13 transcription. In low IL-2, IL-13 mRNA levels were > 90% suppressed in response to all DEX concentrations, whereas in high IL-2 only a 61% suppression was observed with low DEX. This suggests IL-2 increases GC requirements to achieve cytokine suppression, though simply increasing GC dose may not be effective. Indeed, McKeever et.
al. reported that asthmatics receiving quadruple their GC dose still exhibited a 45% exacerbation rate in the following year compared to 52% in those that maintained usual GC dose (51). In light of our study, whether this approach could be improved by focusing on 'low IL-2 producers', i.e. those with a potentially better chance of responding to increased GC, should be assessed.
There are several potential mechanisms underlying the ability to IL-2 to suppress the effects of GC and this heterogeneity contributes to the difficulty of solving the clinical problem of GC resistance (14). For apoptosis, induction of BIM by GC is known to be a direct transcriptional effect as GR binding to regulatory elements within the BIM locus has been shown (52). Therefore, the ability of IL-2 to downregulate BIM could be through IL-2-STAT5 signaling, previously shown to impede GR nuclear translocation (34). In the case of IL-13, less GC-induced inhibition may also be due to IL-2 driving expression of factors that mediate IL-13 transcription such as cFos, a member of the AP-1 complex (49) known to be expressed at higher levels in PBMCs of steroid insensitive asthmatics and to impede GC signaling (53). IL-13 is also regulated by NFB (54) and IL-2 reverses the GC-mediated induction of IkB, an NFB inhibitor (54,55). Yet another mechanism could be through increased activity of the p38MAPK pathway, which can also be activated by IL-2. Indeed, induced GC resistance which could be reversed with an inhibitor of p38MAPKSB203580) (56). Interestingly, this study also identified two populations of severe asthmatics, based on responsiveness to the p38MAPK inhibitor (56), supporting the view that in vivo several mechanisms mediate GC resistance. IL-2 upregulation of GR, an inactive form of GR, is another mechanism by which GR signaling may be reduced (57), though the effect was more pronounced in airway than circulating T cells (58) and so some of the complexity surrounding GC resistance may be the result of tissue specific differences.
The strong, super-physiologic activation with PMA and ionomycin resulted in very high levels of IL-2 from patients' peripheral blood cells, showing their maximal potential for production. In our in vitro system we empirically developed a model of high and low IL-2 based on typical concentrations used for Th2 cell cultures (13,59) as well as functional readouts of resistance/susceptibility to GC-induced apoptosis. Concentrations were lower than observed after ex vivo mitogenic activation of patients' cells, however, are similar to levels found to be produced from in vivo antigen-activated T cells (60) and BAL lymphocytes activated using beads coated with CD2/CD3/CD28 (16) and so may be more physiologically relevant. The source of IL-2 within whole blood could be naïve and/or memory CD4 + T cells (60), Th1 cells (4) or even dendritic cells previously exposure to gram negative bacteria (61). Dendritic cell production would suggest prior infections may influence one's propensity to produce IL-2 and could even occur in utero, as IL-2 promoter methylation at birth was associated with asthma severity in childhood (62). IL-2 genetic variants are also associated with asthma (63, 64) as well as inflammatory bowel disease, type 1 diabetes and multiple sclerosis (63, 65,66). Together, these data suggest that control of IL-2, whether genetic or epigenetic, may be fundamental to the development of immune disease.
Our finding that suppression of IL-13 required continual exposure to GC, since mRNA levels were double if DEX was received only as a pre-treatment and not also during activation.
IL-2 drives persistence of type 2 inflammation 18 These data suggest that unless a) the dose of GC is sufficient to induce Th2 cell apoptosis and b) exposure to GC is continual, suppression of IL-13 is temporary. This could be important clinically, since asthma phenotyping efforts recently proposed a classification based on type 2 cytokine expression (67). However, if a patient is taking a GC dose sufficient to suppress cytokines, but insufficient to eliminate Th2 cells, this could result in misclassification of type 2-low asthma. Using a cell based approach, such as eosinophils and/or Th2 cells, to classify type 2-high asthma may improve identification of the phenotype as well as response to therapies directly target type-2 cells, i.e. CRTh2 antagonist (2,68).
Alternative approaches such as BCL-2 inhibitors, which evoke apoptosis and are in use for leukemia (69), also demonstrate promise. In mouse models of both eosinophilic and neutrophilic asthma they were found to be more efficient than steroids to induce granulocyte apoptosis ex vivo from patients with severe asthma (70).
In summary, our study shows that IL-2 modulates GC responsiveness of human Th2 cells, supporting both their survival and pro-inflammatory capacity, and suggest that a patient's potential to produce IL-2 may be a determinant in asthma severity.