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Differentiation and activation of CD4+ T cells is controlled by various cytokines produced by innate immune cells. We have shown that eosinophils (EOS) have the potential to influence Th1 and Th2 cytokine generation by CD4+ cells, but their influence on IL-17A (IL-17) has not been established.
The purpose of the study is to determine the effect of EOS on IL-17 production by lymphocytes.
Preactivated CD4+ T cells were cultured in the presence of either autologous EOS or EOS culture supernatants. Expression of IL-17 was determined by real-time quantitative PCR (qPCR) after 5 h and protein level was measured after 48 h. To determine the effect of allergen-induced airway EOS on IL-17, subjects with mild allergic asthma underwent bronchoscopic segmental bronchoprovocation with allergen (SBP-Ag) after a treatment with an anti-IL-5 neutralizing antibody (mepolizumab) to reduce airway eosinophilia. IL-17 mRNA was measured in bronchoalveolar lavage (BAL) cells by qPCR.
In vitro, EOS significantly increased IL-17 production by CD4+ T cells. Addition of exogenous IL-1β increased expression of IL-17 mRNA by CD4+ T cells. EOS expressed and released IL-1β. Furthermore, levels of IL-1β in EOS supernatants highly correlated with their ability to increase IL-17 expression by CD4+ T cells, and neutralizing antibody to IL-1β reduced expression of IL-17 mRNA. In vivo, reduction of EOS in the airway using mepolizumab was associated with diminished IL-17 expression after SBP-Ag.
Our data demonstrate that EOS can promote IL-17 production through the release of IL-1β. Enhanced IL-17 cytokine production is another mechanism by which EOS may participate in pathogenesis of allergic airway inflammation in asthma.
While expression of IL-4 and IL-13 or IFN-γ characterizes either Th2 or Th1 lymphocytes, the expression of IL-17A (IL-17) identifies the Th17 subset. Th17 cells are paramount to autoimmune diseases [1;2], but there is growing excitement for the potential role of Th17 cells in asthma [3–5]. There is evidence that IL-17 is elevated in bronchoalveolar lavage (BAL) fluid of asthmatic subjects . In many studies, IL-17+ T cells and IL-17 levels have been associated with a more severe asthma phenotype, characterized by airway neutrophilia [7–9]. In other studies, IL-17 correlates with eosinophils (EOS)  or IL-5 mRNA . IL-17 likely has many functions in asthma including activation of stromal cells to release cytokines, chemokines, and profibrotic mediators, and to increase the generation of extracellular matrix proteins and the expression of the dominant negative isoform of the glucocorticoid receptor (GRβ) [6;12–14]. In addition, IL-17 upregulates angiogenesis and endothelial cell invasion . In animal models, IL-17 has been associated with increased mucus production by goblet cells and airway smooth muscle proliferation [16;17].
Eosinophilia is typically associated with atopic asthma; however, EOS are also increased in autoimmune diseases, such as eosinophilic fasciitis, autoimmune pancreatitis, systemic sclerosis, and bullous pemphigoid [18–21]. EOS can influence type 1 and 2 immune responses directly [22–24] and by producing T cell chemoattractants [25;26]. In a mouse model, EOS have been associated with increased IL-17 expression due to their effect on recruitment of dendritic cells to the lung draining lymph nodes . However, a direct effect of human EOS on IL-17 expression has not been reported. Therefore, to evaluate the ability of EOS to regulate IL-17 during an inflammatory response in the airway, we developed a model in which CD4+ T cells with a memory/effector phenotype were cultured in the presence of EOS or EOS supernatants. In addition, the effect of EOS on IL-17 expression was further analyzed in vivo in cells from BAL fluid after segmental bronchoprovocation with allergen (SBP-Ag) and pretreatment with an anti-IL-5 neutralizing antibody (mepolizumab) which decreases EOS numbers in the airways .
Studies were approved by the University of Wisconsin-Madison Center for Health Sciences Human Subjects Committee. Informed written consent was obtained from subjects prior to participation. All subjects were atopic and were skin prick test positive. For the mepolizumab (in vivo) study, subjects had a history of mild asthma with airway reversibility to albuterol. None of the subjects were using inhaled or oral corticoids.
EOS and CD4+ T cells were purified by negative selection as previously described . Heparinized blood was centrifuged through Percoll (1.090 g/ml), PBMCs at the interface were used for isolation of T cells, and EOS were purified from the granulocyte-containing pellet. After lysis of RBCs, EOS were purified by negative selection using anti-CD16, anti-CD3, and anti-CD14 immunomagnetic beads (AutoMac system, Miltenyi Biotec Inc., Auburn, CA, USA). The EOS purity was >99%. CD4+ T cells were prepared by negative selection using the Miltenyi Biotec CD4+ T Cell Isolation Kit II. Purity was >98% as determined by flow cytometry.
CD4+ T cells (2×106/ml) were cultured in 1 ml of complete medium (RPMI plus 10% fetal bovine serum (FBS)) alone (resting) or were activated with either 1 μg/ml of plate-bound anti-CD3 or anti-CD3 plus 1 μg/ml of soluble anti-CD28 (clones 37407 and UCHT1, respectively; R&D Systems, Minneapolis, MN, USA) in a 24-well plate (Corning Costar, Lowell, MA, USA). After 48 h activation, CD4+ T cells were harvested and cultured for an additional 24 h in complete medium in the absence of anti-CD3. Simultaneously, autologous EOS (1×106/ml) were maintained in complete medium with GM-CSF (100 pg/ml) added at the beginning of the culture and after 24 h to sustain viability. At 72 h, EOS supernatants were collected and EOS and CD4+ T cells were harvested. EOS and lymphocyte viability was 99%. CD4+ T cells were plated (105 cells in 100 μl/well) in a 96-well plate pre-coated with anti-CD3 (1 μg/ml). EOS (105 cells in 100 μl/well) or 100 μl of EOS supernatant were added to CD4+ T cells. As a negative control, CD4+ T cells were cultured alone in the absence of anti-CD3. For transwell experiments, 6×105 CD4+ T cells were added to the bottom chamber-well coated with anti-CD3 and 6×105 EOS were placed in the upper chamber of a 0.4 μm pore polycarbonate filter 24-well transwell plate (Costar). The recombinant human (rh) IL-1β was purchased from Endogen (Endogen/Pierce/Thermo Scientific, Rockford, IL, USA) while rhIL-6 and rhTGF-β1 were provided by R&D Systems (Minneapolis, MN, USA).
Detailed methods for bronchoscopy, SBP-Ag, and BAL cell preparation have previously been described . The allergen dose for SBP-Ag was 20% of the AgPD20 (the provocative dose of Ag leading to a 20% fall in FEV1). A baseline bronchoscopy with BAL (4 × 40 ml aliquots of sterile 0.9% NaCl) was performed followed by administration of Ag into the lavaged segment. Bronchoscopy with BAL was repeated 48 h later. One month after the first SBP-Ag, a single dose of anti-IL-5 (750 mg, mepolizumab) was administered intravenously. One month after mepolizumab treatment, the participant underwent a second SBP-Ag and BAL was performed immediately before and 48 h later. WBC counts and differentials were performed by the hospital clinical laboratory. On the day of the bronchoscopies, WBC counts were determined in the research laboratory using a coulter counter and EOS were enumerated by hemacytometer using phyloxin staining. BAL cells were assessed by hemacytometer using Turk’s counting solution containing acetic acid and methylene blue and cell differentials were determined on cytospin preparations stained with the Wright-Giemsa-based Hema-3 (ThermoFisher/Thermo Scientific, Rockford, IL, USA). Airway EOS were purified from BAL cells using a two-step Percoll gradient as previously described . EOS were collected from the 1.085/1.100 g/ml interface. IL-17 mRNA levels were measured in EOS when purity was >99%.
Before co-culture, activated or resting CD4+ T cells were assessed using anti-CD25/PE (BD Biosciences, San Jose, CA, USA), -CD69/PE (eBioscience, San Diego, CA, USA), or -CD45RO/PE (BD Biosciences). Cells were acquired on a LSR II (BD Biosciences). Positive staining and gating strategy were determined by comparison to isotype control antibodies. Flow cytometry data were analyzed using the FlowJo software (Treestar Inc., Ashland, OR, USA).
Total RNA was extracted from BAL cells or from cells co-cultured for 5 h using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The reverse transcription reaction was performed using the Superscript III system (Invitrogen/Life Technologies, Grand Island, NY, USA). mRNA expression was determined by qPCR using SYBR Green Master Mix (SABiosciences, Frederick, MD, USA) and human IL-17A (forward: cgatccacctcaccttgga, reverse: tcccagatcacagagggatatctctc), CD3ε (forward: gatgcagtcgggcactcactgg, reverse: cttgcccccaaacgccaactgat), and IL-1β (forward: tggaccccttggtaaaagaca, reverse: gaagaaatcagtagagctatgaaacaaataag) specific primers were designed using Primer Express 3.0 (Applied Biosystems, Carlsbad, CA, USA) and blasted against the human genome to determine specificity using http://www.ncbi.nlm.nih.gov/tools/primer-blast. The reference gene, β-glucuronidase ((GUSB), forward: caggacctgcgcacaagag, reverse: tcgcacagctggggtaag), was used to normalize the samples. Standard curves were performed and efficiencies were determined for each set of primers. Efficiencies ranged between 91 and 96%. Data are expressed as fold change using the comparative cycle threshold (ΔΔCT) method. For the in vitro study, ΔCt = (Ct target gene (IL-17 or IL-1β) - Ct reference gene (GUSB)); ΔΔCt = (ΔCt of anti-CD3 activated cells with or without EOS or EOS supernatants − ΔCt of unstimulated cells); for the in vivo (mepolizumab) study, ΔCt = (Ct IL-17 − Ct CD3ε); ΔΔCt = (ΔCt of BAL cells after allergen challenge with or without mepolizumab treatment − ΔCt of BAL cells before allergen challenge and mepolizumab). For both the in vitro and in vivo studies, the values presented are fold change = (2−ΔΔCt).
Cytokines were measured in the 72 h EOS supernatants used on CD4+ cells or in the supernatants 48 h after the beginning of the co-culture of EOS and CD4+ T cells. IL-17, IL-1β, and GM-CSF were measured utilizing “in-house” sandwich ELISA . Coating antibodies included anti-human IL-17A (clone 41809.111, R&D Systems), IL-1β (clone IL1B-H67, Endogen/Pierce/Thermo Scientific), and GM-CSF (clone 6804.11, R&D Systems). Detection antibodies included biotinylated polyclonal goat anti-human IL-17A (R&D Systems), IL-1β (clone ILB1-H6, Endogen), and GM-CSF (clone 3209.1, R&D Systems). The ELISA assay sensitivities were <3 pg/ml.
To compare expression of IL-17 in CD4+ T cells with or without EOS or EOS supernatants, and to compare IL-17 expression in vivo in BAL cells after SBP-Ag with or without mepolizumab treatment, data were log transformed and analyzed using a Student’s paired t-test. The Mann-Whitney Rank Sum test was used to test the effect of the recombinant proteins on IL-17 expression by CD4+ T cells. The correlations were analyzed using the Spearman Rank Order Correlation. Statistical analyses were performed using the SigmaPlot 11.0 software package and significance was reached for p<0.05.
To analyze the effect of EOS on the expression of IL-17 by T lymphocytes, we developed an in vitro model where CD4+ T cells were cultured for 48 h with anti-CD3 and anti-CD28, harvested, and “rested” for 24 h. In parallel, part of the CD4+ T cells (resting) was cultured in medium for 72 h. After 72 h, activated CD4+ T cells expressed greater levels of CD69 compared to resting CD4+ T cells, and nearly 100% of the activated T cells were CD25+ and CD45RO+ (Figure 1). Activated CD4+ cells were then added to fresh anti-CD3-coated plates and co-cultured with EOS. These data imply that the activation step induces CD4+ T cell differentiation to a memory/effector phenotype.
CD4+ T cells prepared as described above using anti-CD3 and anti-CD28 and activated again on anti-CD3 expressed 2- to 5-fold more IL-17 mRNA when co-cultured with EOS (Figure 2A). The requirement of cell-cell contact and/or soluble mediators was evaluated by separation of EOS and CD4+ T cells using transwell plates or by addition of EOS-conditioned medium to T cells. CD4+ T lymphocytes expressed increased levels of IL-17 when co-cultured with EOS in transwell (Figure 2B). Furthermore, conditioned medium from EOS cultured for 72 h augmented IL-17 mRNA expression by CD4+ T cells (Figure 2C). Importantly, EOS supernatants did not contain detectable levels of IL-17, and IL-17 mRNA was not expressed by EOS cultured with CD4+ T cells on transwell plates, indicating that CD4+ T cells are the source of IL-17. In accordance with mRNA levels, more IL-17 protein accumulated in the supernatants of the co-cultures of CD4+ T cells plus EOS compared to CD4+ T cells alone (Figure 2D). These data suggest that EOS promote IL-17 expression in activated memory/effector-like CD4+ T cells through a soluble(s) mediator(s).
In human T cells, expression of IL-17 is controlled by IL-1β, IL-6, and TGF-β1 [30;31], which are all potentially released from EOS. To determine if these cytokines regulate IL-17 in our model, preactivated CD4+ T cells were stimulated with anti-CD3 in the presence of rhIL-1β, rhIL-6, or rhTGF-β1. rhIL-1β increased IL-17 mRNA expression by more than 3-fold, whereas IL-6 and TGF-β1, used alone or in combination, did not affect IL-17 expression (Figure 3).
Because IL-1β is a potent inducer of IL-17 in CD4+ T cells, we sought to determine the expression of IL-1β by EOS. IL-1β mRNA expression in EOS was compared to autologous CD4+ T cells when both were cultured for 5 h on immobilized anti-CD3 (Figure 4A). CD4+ T cells expressed little IL-1β, whereas high levels were expressed in EOS.
Protein amounts of IL-1β in supernatants of EOS cultured for 72 h highly correlated with the ability of the supernatants to increase IL-17 mRNA by activated CD4+ T cells (Figure 4B, r=0.964, p<0.0001, n=7). Furthermore, addition of neutralizing anti-IL-1β antibody to EOS supernatants inhibited 70% of the increase in IL-17 (Figure 4C). Enhanced IL-17 expression did not correlate with levels of GM-CSF present in the conditioned media of EOS (3–168 pg/ml; r=−0.0714, p=0.843, n=7). Altogether, these data demonstrate that active IL-1β released from EOS enhances IL-17 expression by CD4+ T cells.
To evaluate our in vitro data in an in vivo model, IL-17 and CD3ε expression was measured by qPCR in BAL cells from 9 subjects before and 48 h after SBP-Ag with or without a mepolizumab pretreatment. At baseline, BAL cells are typically composed of macrophages. SBP-Ag leads to accumulation of EOS and activated lymphocytes after 48 h. To account for the variation of the % of T lymphocytes in the BAL cells, levels of IL-17 mRNA were related to CD3ε mRNA. Treatment with mepolizumab significantly reduced IL-17 levels after SBP-Ag (Figure 5). The reduction of IL-17/CD3ε was more pronounced (~2.5 fold) for the 4 subjects displaying higher expression of IL-17 after SBP-Ag and before mepolizumab treatment. While percentages of EOS were reduced by mepolizumab (~57%; Table 1), purified BAL EOS did not express any detectable IL-17 mRNA (Ct values >40) and cannot account for the loss of IL-17. These data suggest that, in individuals with allergic asthma, EOS may be associated with airway IL-17 upregulation in T lymphocytes.
Our study demonstrates that EOS can enhance the production of IL-17 in CD4+ T lymphocytes through the release of IL-1β. Our data suggest that EOS interaction with effector/memory CD4+ T cells can contribute to inflammation and remodeling via IL-17 production during an adaptive immune response. Because IL-17 is often associated with neutrophilia or neutrophil activation, eosinophilia might be a prelude to a neutrophilic inflammation, at least in some individuals with allergic asthma.
The potential impact of EOS on lymphocyte phenotypes has been studied in the context of Th1/Th2-type responses. We previously reported that EOS co-cultured with staphylococcal enterotoxin B-activated CD4+ T cells enhance production of type 1 and type 2 cytokines . Using a co-culture model whereby CD4+ T cells are “primed” with anti-CD3/CD28 and then activated again with anti-CD3 in the presence of EOS, we now show that EOS can augment the expression of the type 17 cytokine, IL-17, by CD4+ T lymphocytes. In this model, CD4+ T cells displayed a similar phenotype as activated memory/effector T cells (CD25+CD45RO+) that are predominant in the airways of asthmatics . The enhancement of IL-17 correlated with EOS release of IL-1β. The release of IL-1β is consistent with reports that EOS are a source of IL-1β [33;34] and express a constitutive, inducible, active IL-1β converting enzyme protease(s) [35;36].
In previous studies, IL-1β release was due to EOS activation with either toll-like receptor ligands or uric acid [33;34]. In our co-culture model, the mediators used by CD4+ T cells to rapidly activate EOS remain unknown but might involve a variety of cytokines. Spencer and colleagues demonstrated that EOS can quickly respond to TNF-7agr;, IFN-γ, IL-10, or IL-4 to release multiple cytokines . Surprisingly enough, in our study, EOS polarization by lymphocytes required CD4+ T cells to be activated again on anti-CD3 (not shown). This suggests that a secondary T cell activation leads to a fast (within several hours) release of mediators that cannot involve newly transcribed proteins but might rather implicate post-transcriptional or post-translational mechanisms.
Our data are consistent with results by Liu et al. demonstrating that IL-1β increases IL-17 production by activated Th17+ memory T cells . In that study and others, IL-23 and IL-6 amplified IL-1β-induced IL-17. In our study, IL-23 was never detected in EOS at the mRNA level or as protein in EOS supernatant. IL-6 is also known to be released by EOS ; however, rhIL-6 alone did not increase IL-17 in our activated CD4+ T cells. We have not ruled out the possibility that EOS release of IL-6 enhances IL-17 induction by other factor(s). Also, in accordance with published studies of differentiated human Th17 cells [38;39], TGF-β1 was not required for IL-17 production in our model. It is important to note that our complete medium contained 10% FBS in which exogenous TGF-β1 may be present and could thus interfere with the effects of rhTGF-β1.
The heterogeneity and plasticity of Th17 is an area of active research and controversy. IL-17 can be expressed in Forkhead box P3+ T cells  and co-expressed with both IFN-γ (Th17/Th1) and IL-4 (Th17/Th2) . In addition to IL-17, our CD4+ T cells that were activated again on anti-CD3 also produced high levels of Th2 (IL-13) and Th1 (IFN-γ) (data not shown). Future studies are required to determine the effect of EOS on various “subsets” of IL-17+ T cells.
The biological significance of EOS regulation of IL-17+ cells during asthma remains speculative. We demonstrated that despite high levels of BAL EOS, antigen-induced increases in IL-17 mRNA only occurred in a subset of subjects. Notably, antigen-induced IL-17 was mitigated after administration of mepolizumab and subsequent reduction in Ag-induced BAL EOS. It is not clear why EOS would increase IL-17 expression in only a subset of asthmatics. Our in vitro study suggests that the amount of IL-1β produced, stored, and released by EOS could explain the expression and the heterogeneity of IL-17 expression. The cause for elevated IL-1β in EOS and its association with IL-17 expression remain unknown. Importantly, it is plausible that EOS-induced IL-17 is an important link to a more severe asthma phenotype that is observed in only 5–10% of the asthmatic population. As an important inducer of neutrophil chemoattractants, IL-17 is associated with neutrophilia in the airway . While mild allergic asthma is often associated with eosinophilia, more severe asthma can be characterized by the presence of both EOS and neutrophils .
Patients with nocturnal asthma, have elevated levels of EOS, IL-1β, and CD4+ T lymphocytes in the airway at nighttime [44–47], suggesting a link between eosinophilia and IL-1β in symptomatic asthma. Other reports have demonstrated elevated levels of IL-1β in BAL and sputum of asthmatics [48;49] and that IL-1R1 is a potential therapeutic target for asthma . Also importantly, IL-1β and IL-17 have synergistic functions on epithelial cells and airway smooth muscle cells by enhancing mucin production and the neutrophil chemoattractant CXCL-8 and therefore participate in airway obstruction and inflammation [51;52].
In conclusion, we demonstrated that EOS augment IL-17 during CD4+ T lymphocyte activation via IL-1β release. The increase of pathogenic cytokines such as IL-1β and IL-17 during an immune response could have dramatic effects in vivo. Understanding IL-1β processing in EOS could further elucidate EOS function in pathologies such as asthma.
The authors thank our coordinators, Mary Jo Jackson, RN, BSN, Holly Eversoll, RN, BSN, Michele Wolff, RN, MSN, and Evelyn Falibene for patient recruitment and screening; Sameer Mathur, MD, PhD, for oversight of the Laboratory core that recruited and screened subjects and purified blood eosinophils; Elizabeth Schwantes, BS, and Paul Fichtinger, BS, for blood eosinophil and lymphocyte purification; Jami Hauer, BS, Larissa DeLain, BS, and Moneeb Akhtar for their technical support; Lou Rosenthal, PhD, and Ruedi Braun, PhD, for editorial comments; the UW-Asthma Program Project Grant group for helpful suggestions; and Mike Evans for statistical analyses.
This work was supported in part by a Program Project Grant (NIH HL088594), the University of Wisconsin General Clinical Research Center grant (NIH M01RR03186), and the University of Wisconsin Institute for Clinical and Translational Research (NCRR/NIH 1UL1RR025011).
Author contributionsAll the authors contributed to the design and/or acquisition and analysis of the data. All the authors contributed to the drafting of the article or critically reviewed the manuscript. All authors approved the final version of the manuscript for submission.
The authors have no financial conflict of interest.