Increased Proportion of Dual-Positive Th2–Th17 Cells Promotes a More Severe Subtype of Asthma

Asthma is a heterogeneous disease, and abnormal activation of T cells is the driving link of asthma's pathophysiological changes. Dual-positive Th2–Th17 cells, as newly discovered T-helper cells, have the functions of Th2 and Th17 cells and can coproduce Th2 and Th17 cytokines. Previous studies have shown that dual-positive Th2–Th17 cells increase the chances of asthma and correlate with asthma severity. However, the exact role of dual-positive Th2–Th17 cells in asthma is not known. Since there is no mature differentiation method for dual-positive Th2–Th17 cells, the present study aimed to clarify the strict differentiation conditions and reveal how dual-positive Th2–Th17 cells regulate asthma phenotypes. In this study, we confirmed that IL-1β, IL-6, anti-IFN-γ, and IL-21 promoted biphenotypic cell differentiation. Moreover, more proportion of dual-positive Th2–Th17 cells can be obtained by conditioned differentiation of mouse CD4+ T cells after classical allergic asthma modeling. Before asthma modeling, adoptive dual-positive Th2–Th17 cells promoted T cells to differentiate into the same biphenotype cells and exacerbated the severity of asthma. Together, our results clarify the differentiation conditions of dual-positive Th2–Th17 cells and further confirm that it stimulates asthma T cells to differentiate into the same biphenotype cells, leading to exacerbation of asthma.


Introduction
Bronchial asthma is a chronic inflammatory disease of the airways [1,2]. Different subtypes can be seen in asthma patients due to their heterogeneity [3][4][5]. As an important part of the pathogenesis of asthma, the abnormal activation of T cells is the initiating factor leading to a series of pathophysiological changes [6,7]. At present, a large number of studies have proved that T-helper cells have plastic deformability and transform each other under different stimuli [8][9][10].
e presence of dual-positive 2-17 cells was first discovered in 2010 [11]. Further studies have shown that the proportion of dual-positive 2-17 cells in asthmatic patients is significantly higher than that of healthy people [12]. As a newly discovered T-helper cell, however, no further study on dual-positive 2-17 cells has been made. Dual-positive 2-17 cells have the functions of 2 and 17 cells and can secrete the characteristic cytokines of 2 and 17 simultaneously [11]. Some researchers have confirmed the existence of the 2-17 cell population which can coexpress transcription factors GATA binding protein 3 (GATA3) and retinoic acid receptor-related orphan receptor gamma (RORct) [13][14][15][16] and secrete both IL-4 and IL-17 in a mouse model of acute asthma [17]. is further proves that CD4 + T cells have the plasticity to transform into different T-cell subpopulations under appropriate environmental stimuli. But the differentiation conditions of dual-positive 2-17 cells are not clear. It has also been clinically found that 2/ 17 predominant asthma (dual-positive 2-17 cells predominate in asthma) patients are more severe and have a poor response to inhaled corticosteroids [18][19][20]. e above studies show that the dual-positive 2-17 cells may be an important cell subgroup in the occurrence and development of asthma. However, its mechanism in the pathogenesis of asthma is not very clear.
us, elucidating the effect of dual-positive 2-17 cells on the development and phenotype of asthma may provide new insight for the pathogenesis and prevention of asthma.

Mouse and Asthma
Model. Female C57BL/6 mice were provided by the animal center of the Second Xiangya Hospital of Central South University (Changsha, China), weighing 18-20 g, aged 6-7 weeks. e asthma model was established as previously reported [21,22]. Mice (n � 6/ group) were given an intraperitoneal sensitization injection with 25 μg Ovalbumin (OVA, Grade V, Sigma-Aldrich, USA) with 2 mg aluminum hydroxide (Sigma-Aldrich) on days 0 and 7. en, mice were challenged with OVA solution atomized for 30 minutes on days 14, 15, 16, 17, 18, 19, and 20. Mice were sacrificed on day 21. All studies were performed in compliance with the Second Xiangya Hospital and Central South University Animal Care and Use Committee guidelines.

Flow Cytometry.
After CD4 + T-cell differentiation, cells were stimulated with 3 μg/mL of monensin, 1 μg/mL of ionomycin, and 50 ng/mL of phorbol-12-myristate-13-acetate (PMA) for 5 h. en, cells were fixed and permeabilized with fixation and permeabilization buffer for 15 min. Reagents used above were purchased from Multi Sciences Co. (Multi Sciences, China). After that, cells were washed with permeabilization buffer and then stained with intracellular markers PE-anti-IL-4 cytokine antibodies and APC-anti-IL-17 cytokine antibodies (BioLegend, USA) in the permeabilization buffer for 20 min. Cell surface marker FITCanti-CD4 (Multi Sciences Company, China) cytokine antibodies were stained without fixed and permeabilized. Flow cytometry was conducted, and the data were analyzed using the FACSCalibur and FlowJo V10 software.

Enzyme-Linked Immunosorbent Assay (ELISA).
CD4 + T-cell culture supernatants after cytokine treatment and blood serum of mice after asthma modeling were collected for ELISA. IL-4 and IL-17 ELISA kits were used following the manufacturer's instructions (R&D, USA) to detect the cytokine secretion.

Airway Hyper-Responsiveness (AHR) Assessment.
On the 21 st day of the asthma model, methacholine-(Mch-) induced airway resistance was measured by direct plethysmography (Buxco Electronics, RC System, Wilmington, NC, USA) according to published methods [24,25]. After tracheal intubation, we first measured mice baseline lung resistance for 1 min. en, mice were given 10 μL of atomized saline and 10 μL of Mch at the following doses (0.39, 0.78, 1.56, and 3.12 mg/mL) to stimulate the airway and record RL for analyzing.

Bronchoalveolar Lavage Fluid (BALF) Processing.
BALF was collected after 3 injections of 0.5 mL saline (37°C) into the lungs. e red blood cells were removed. e BALF cells after centrifugation (1500 rpm, 5 min, 4°C, Eppendorf centrifuge, Hamburg, Germany) were resuspended in phosphate buffer saline. BALF cells were counted using a hemocytometer. To obtain a BALF cell differential count, the cells were fixed and stained with Wright-Giemsa stain, and 200 cells were counted under a light microscope for statistical analysis.

Histopathology.
e lungs were first slowly instilled in the trachea with 10% formalin, then removed, and stored in 10% formalin for fixation. e fixed lung tissue was embedded in paraffin and cut into thin slices (5 μm) and then stained with hematoxylin and eosin (H&E). Stained sections were selected from each group and evaluated individually under a light microscope.
2.9. Statistics Analysis. All quantitative data were recorded as means ± SD. Data were analyzed using a t-test (two-group comparison) or one-way ANOVA (multigroup comparison). And, all the data were analyzed using SPSS software version 22.0. Differences between groups were considered statistically significant with a p value <0.05.

Exploration of Dual-Positive 2-17 Cells Differentiation Conditions.
So far, there is no mature differentiation method for dual-positive 2-17 cells. e current controversy is whether IL-21 or IL-23 is the necessary cytokines for the differentiation of dual-positive 2-17 cells, in the presence of cytokines IL-1β, IL-6, and anti-IFN-c [11,12]. We first explored the differentiation conditions. We performed the following experiments: CD4 + T cells were isolated 3 days after OVA intervention (Figure 1(a)), 20 ng/ mL IL-1β, 20 ng/mL IL-6, 20 ng/mL anti-IFN-c, and 10 ng/ mL IL-21 or 10 ng/mL IL-23 were added for differentiation intervention, and flow cytometry was used to detect the effects of different intervention cytokines and intervention time on the differentiation of cells in each group. After 24 h, no significant differentiation trend was observed in the cells of each group (Figure 1(b)). At the 2 nd , 4 th , and 6 th days, we found a dramatical increased in dual-positive 2-17 cells in the IL-1β/IL-6/anti-IFN-c/IL-21 group, comparing with the IL-1β/IL-6/anti-IFN-c/IL-23 group (Figure 1(b)). Among them, the best differentiation effect is at the 2 nd and 4 th days (Figure 1(b)). ese data indicate that cytokines IL-1β/IL-6/anti-IFN-c/IL-21 is the differentiation conditions for dual-positive 2-17 cells. Particularly, IL-21 is necessary to promote the differentiation of dual-positive 2-17 cells.

Increased Dual-Positive 2-17 Cells after Differentiation Based on Asthma
Modeling. Based on the differentiation conditions we have obtained, we next test whether differentiation based on asthma modeling can obtain more biphenotypic cells. C57BL/6 mice were used to build the asthma model (Figure 2(a)). Twenty-four hours after the last challenge, CD4 + T cells were isolated for differentiation. Forty-eight hours after cytokine stimuli, cells were collected for detection. In the asthma/differentiation group, there was an increased proportion of dual-positive 2-17 cells between the differentiation group (Figure 2(b)). ELISA confirmed that IL-4/IL-17 was overexpressed in asthma/ differentiation cells (Figure 2(c)). ese results show that differentiation after asthma modeling stimulates more dualpositive 2-17 cell differentiation.

Adoptive Dual-Positive 2-17 Cells Promote T-Cell Differentiation in an Asthma Model.
e abnormal activation of T cells is the initiating factor leading to a series of pathophysiological changes in the pathogenesis of asthma. We hypothesized that dual-positive 2-17 cells may alter T-cell differentiation and function in asthma. To this end, 2-17 biphenotypic cells obtained from asthma/ differentiation mice were transferred intravenously into C57BL/6 mice to build the asthma model. In adoption/ asthma mice, we found that dual-positive 2-17 cells proportion was shown to be increased compared to asthmatic mice (Figure 3(a)). Similarly, real-time PCR confirmed a substantial increase in the expression of key transcription factors GATA3 and RORct in the adoption/asthma group (Figure 3(b)). Meanwhile, IL-4 and IL-17 show the same trend as GATA3 and RORct (Figure 3(c)). Together, these data suggest that dual-positive 2-17 cells promote further differentiation and function of T cells into the same biphenotypic cells.

Adoptive Dual-Positive 2-17 Cells Cause More Severe Asthma Subtype in an Asthma
Model. Clinically, patients with 2/ 17 predominant asthma have severe symptoms and poor hormone treatment. We speculated that dual-positive 2-17 cells may be involved in affecting the severity of asthma. After adoption and asthma modeling, mice were sacrificed for detection. Lung resistance and BALF cell count were measured as previously published [24,25]. As shown in Figure 4(a), there was a significant increase in lung resistance in the adoption/asthma group compared with the asthma group. Also, BALF cell count suggested a significantly higher total number and as well as eosinophils and neutrophils number in adoption/asthma group compared with the asthma group (Figure 4(b)). Besides, compared with the asthma group, the pathological analysis showed that the adoption/asthma group had more inflammatory cell infiltration (Figure 4(c)). Taken together, these data indicate that dual-positive 2-17 cells interventions could exacerbate AHR and airway inflammation, resulting in a more severe asthma subtype.

Discussion
As a newly discovered T-helper cell, dual-positive 2-17 cells do participate in the occurrence and development of asthma [19,20]. However, little research has been done on dual-positive 2-17 cells. e reason is that there are no mature conditions for their differentiation.
eoretically, naïve CD4 + T cells, 2 cells, or 17 cells may have the potential to become dual-positive 2-17 cells. Cosmi et al. confirmed that the differentiation of naïve CD4 + T cells into dual-positive 2-17 cells in response to stimulation with IL-1β, IL-4 with IL-23 [11]. Notably, 17 cells have the plasticity to become dual-positive 2-17 cells in the presence of IL-4 [11]. While other researchers showed 2polarizing stimuli (IL-4) could induce 17 cells to produce IL-4, but shut down their IL-17 production. Moreover, it was not IL-23, but IL-21 accompanied by IL-1β, IL-6, and    anti-IFN-c, which had the potential to produce dual-positive 2-17 cells, especially when stimulating 2 cells [12]. Study showed that IL-6 induced expression of IL-21 that amplified an autocrine loop to induce more IL-21 and IL-23 receptor in naïve CD4 + Tcells. Both IL-21 and IL-23 can induce IL-17 expression [26]. At present, the controversy is mainly focused on whether IL-21 or IL-23 is the necessary cytokines for the differentiation of dual-positive 2-17 cells. In our study, we tried to change the key cytokine to confirm that IL-21 can promote biphenotypic cell differentiation, and the optimal intervention time is 2-4 days. Not only this but we also found for the first time that more dual-positive 2-17 cells can be obtained by conditioned differentiation of mouse CD4 + T cells after classical allergic asthma modeling.
is laid a solid foundation for the subsequent research of dual-positive 2-17 cells. Future studies are warranted to reveal the underlying mechanisms that drive the induction of dualpositive 2-17 cells during allergic inflammation.
Dual-positive 2-17 cells make up a very small portion among circulating CD4 + T cells in healthy subjects. eir numbers are increased in donors with asthma [11]. Other researchers have also found dual-positive 2-17 cells in the peripheral blood of allergic asthma and further confirmed the existence of this cell subtype by constructing asthma models [27]. Subsequent studies on this type of cell mostly focused on the relationship between the characteristics of the cell and its proportion and the severity of asthma [19,20]. Wang et al.'s experiment designed the effect of injection of dual-positive 2-17 cells on asthma, but asthma modeling was received only 2 days, which was not enough to simulate asthma phenotype [12,28,29]. Beyond that, there are no studies on the use of dual-positive 2-17 cells in the asthmatic model. For the first time, we constructed a classic allergic asthma model based on adoptive dual-positive 2-17 cells and studied its effect on asthma subtypes. Our study found that dual-positive 2-17 cells can not only promote further differentiation and differentiation of T cells into the same biphenotype cells but also exacerbate airway hyperresponsiveness and airway inflammation, leading to more severe asthma subtypes. ese results suggest a significant role for dual-positive 2-17 cells in asthma pathogenesis. Mechanism of this type of cell subsets regulating asthma subtype changes needs to be investigated further. In conclusion, our data have clarified the differentiation conditions of dual-positive 2-17 cells and further confirmed that it stimulates asthma T-cell differentiation and function, leading to exacerbation of asthma. is study could lead to new therapeutic prospects for the treatment of patients with more severe asthma.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors delclare no conflicts of interest.