DPP4 Regulates the Th17/IL-17 Axis and Accelerates Epithelial Mesenchymal Transition to Promote Ovalbumin-Induced Asthma in Female C57BL/6J Mice

Background : Dipeptidyl peptidase-4 (DPP4) is a transmembrane glycoprotein, prevalent across a variety of tissues and cells and can be foundin a solubilised in peripheral blood. This paper aims at determining the role of sCD26/sDPP4 in Th17 cell polarization and airway epithelial cell to epithelial mesenchymal transition (EMT) in asthma. Methods : Female C57BL/6J mice were treated with ovalbumin to constructed asthma mice. The CD4 + T cell, and bronchial epithelial cells (BECs) were purified from the spleens and bronchus of mice. The KRT8 expression in BECs were identified by immunofluorescence (IF). Th17 cells were differentiated from a CD4 + T cell. Flow cytometry was usewd to identify and calculate the Th17 and Treg cells. Mice woth asthma were treated by DPP4 overexpressing lentivirus or DPP4 inhibitor. Histopathological modifications were assessed by hematoxylin-eosin (HE), periodic acid Schiff (PAS), and Masson staining. The total number of leucocytes was detected using a hemocytometer. For detection, quantitative Real-time PCR (qRT-PCR), western blotting (WB), and IF were used to evaluate the expression of E-cadherin and alpha-smooth muscle actin ( α -SMA). Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the DPP4, IL-4, IL-5, IL-13 and IL-17 levels. Results : The findings suggest that sCD26/sDPP4 promote CD4 + T cells differentiation into Th17 cells in a depending on the applied dose. sCD26/sDPP4 up-regulated the expression of α -SMA and down-regulated the expression of E-cadherin in TGF-β 1-induced mouse BECs, which was reversed by DPP4 inhibitor. Co-culture induced a synergic effect between Th17 cells and sCD26/sDPP4 on the formation of airway EMT in BECs. Furthermore, DPP4 inhibitor prevented lung-bronchial inflammatory infiltration, mucus secretion, goblet cell hyperplasia and collagen deposition in asthma mice. Meanwhile, DPP4 inhibitor decreased the levels of DPP4, IL-4, IL-5, IL-13, IL-17 and increased the total number of leukocytes in bronchoalveolar lavage fluid of asthma mice. In addition, DPP4 inhibitor also inhibited airway EMT and Th17 cell polarization in asthma mice. Conclusions : The results in this paper show that up-regulation of DPP4 enabled airway inflammation and airway remodeling in asthmatic mice by modulating the Th17/IL-17 axis and accelerating the airway EMT, which isa therapeutic target in asthma.


Introduction
Asthma is a chronic inflammatory disease of the respiratory system that causes repeated bronchoconstriction and airflow limitation [1].The CD4 + T helper (Th) cells contribute significantly in the pathogenesis of asthma [2].Clinically, asthma was generally divided into two categories: allergic asthma and non-allergic asthma [3].In recent years, with the in-depth research on the pathogenesis of severe asthma and refractory asthma.It has been found that neutrophilic inflammation in the airways and airway remodeling are essential factors contributing to the occurrence of severe asthma and refractory asthma [4,5].IL-17, mainly produced by Th17 cells, stimulate the neutrophilic airway inflammation [6] and airway hyperresponsiveness that induces the pathogenesis of asthma [7][8][9].Given the central role of IL-17 in the pathogenesis of asthma, the exploration of the factors regulating Th17 cell polarization and affecting the secretion of IL-17 by Th17 cells is crucial.
Dipeptidyl Peptidase-4 (DPP4) is a soluble glycoprotein with serine protease activity, expressed as CD26 on the cell surface in immune cells [1].In asthma, CD26 is an activation marker, up-regulatedin lymphocytes, particulary in the CD4 + T cells [2].Studies have confirmed that the expression of CD26 in lymphocytes of asthmatic patients was significantly increased [10].Furhermore, the high expression of CD26 was associated by the differentiation of T lymphocytes into Th1 and Th17 [11,12].Further research is needed to investigate the potential mediation of airway neutrophil inflammation by DPP4 in asthma, by affecting Th17/IL-17 signaling.
Airway remodeling is a common pathological feature of severe asthma, resulting in permanent airway obstruc-tion in up to 50% of cases and respiratory dysfunction [13].Airway epithelial mesenchymal transition (EMT) is a vital mechanism of airway remodeling in asthmatic patients [14].Previous studies have shown that IL-17 enhanced TGF-β1-induced EMT in bronchial epithelial cells (BECs) [15].In addition, DPP4 could promote the EMT of BECs induced by TGF-β1 and the IL-17 had a synergistic effect on TGF-β1-induced EMT [16].Based on the latter, the central hypothesis in this paper was that DPP4 could be related to airway inflammation and airway remodeling in asthma by promoting TGF-β1-induced airway EMT and modulating the Th17/IL-17 axis.Therefore, the effect of DPP4 on Th17 cell polarization initially investigated in vitro.Subsequently, the TGF-β1-induced bronchial epithelial cells (BECs) model and Ovalbumin (OVA)-induced mouse asthma model were established to assess the effect of DPP4 on airway EMT and remodeling in asthma.

Isolation and Characterization of Mouse BECs
Female C57BL/6J mice (5 mice) were anesthetized by intraperitoneal injection of overdose 2% phenobarbital, then the bronchus was extracted.A moderate amount of 0.05% pronase pre-cooled in 4 °C was injected into the bronchi to cleanse the inner wall of the trachea.The entire trachea was filled with digestive enzymes and infiltrated in DMEM/F12 after being ligated.After incubating at 4 °C for overnight, the digestion was collected.The red blood cells were lysed by red blood cell lysis buffer (R1010, Solarbio, Beijing, China).BECs were obtained by removing the adherent fibroblasts after 1 h of incubation in complete medium.

Experimental Grouping and EStablishment of a Mouse Asthma Model
Forty female C57BL/6J mice were divided into four groups: control group, OVA group, OVA + DPP4 group, and OVA + DPP4 inhibitor group.No-loaded overexpressing lentivirus (oe-NC) and DPP4 overexpressing lentivirus (oe-DPP4, NM_010074, HG-LV010074, Honor-Gene, China) were purchased from HonorGene.The mice in control group were treated with normal saline and oe-NC.The mice in OVA group were intraperitoneal-injected with lentivirus 30 min before OVA (A5503, Sigma-Aldrich, Darmstadt, Germany) ultrasonic nebulization.Moreover, the mice in OVA + DPP4 group were intraperitonealinjected of oe-DPP4 30 min before OVA ultrasonic nebulization.The mice in OVA+DPP4 inhibitor group were intraperitoneal injected with 200 mg/kg DPP4 inhibitor (B3941, APEXBIO, USA) 30 min before OVA ultrasonic nebulization.On days 0 and 12, mice were sensitized by intraperitoneal injection of 0.2 mL of aluminum hydroxide gel, containing 10 µg of OVA.Mice in the control group received the same volume of normal saline.Subsequently, from day 18 to day 23, all groups of mice received 5% OVA for 30 min daily, through the airway.Afterwards, mice were exposed to 5% OVA once every two days for 30 min until operated on day 56.

Bronchoalveolar Lavage
The airways of the mice were lavaged three times with 0.4 mL of PBS by tracheal intubation.Subsequently, the bronchoalveolar lavage fluid (BALF) was centrifuged at 2000 g for 5 min (4 °C) and the supernatant was collected for subsequent experiments.The pellets were resuspended in 50 µL pre-cold PBS and the cells were calculated by a hemocytometer.

Quantitative Real-Time PCR (qRT-PCR)
TRIzol reagent (15596026, Thermo Fisher Scientific, Waltham, MA, USA) was utilised to extract the total RNA from cells and lung tissues.RNA samples were subsequently applied to generate the cDNA by mRNA Reverse Transcription Kit (CW2569, CWBIO, Beijing, China).The expression of specific RNAs was quantified by UltraSYBR Mixture (CW2601, CWBIO, Beijing, China) in a QuantStu-dio1 Real-Time PCR System (ABI, Fosters, CA, USA).βactin was used as reference gene and the primer sequences are listed in Table 1.

Histological Staining
Lung tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into sections.Sections were stained with hematoxylin-eosin (H&E), periodic acid Schiff (PAS) and Masson to evaluate the inflammation, epithelial injury, and degree of collagen deposition, respectively.Images were collected using a microscope (BA210T, Motic, Xiamen, China).

Statistical Analysis
The data was analyzed using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA).The results from the data presented were the form of mean ± standard deviation.The normality and homogeneity of variance were tested to confirm the normality of the data distribution and homogeneity of variance.Statistical differences were assessed by unpaired two-tailed Student's t test between two groups.
One-way analysis of variance (ANOVA) was applied to statistical differences among multiple groups.Tukey's test was used for pairwise comparison after one-way ANOVA.Statistical significance was defined at p < 0.05.

sCD26/sDPP4 Promoted Th17 Cell Polarization and IL-17 Secretion
CD4 + T cells were successfully isolated and (Supplementary Fig. 1A), treated with sCD26/sDPP4 to explore Th17 cells polarization and IL-17 secretion.Flow cytometry detection of Th17 cells revealed that 10-100 ng/mL sCD26/sDPP4 significantly promoted the Th17 polarization.The higher concentrations of sCD26/sDPP4 were accompanied by a higher degree of polarization of Th17 cells.After the addition of the DPP4 inhibitor, the effect of sCD26/sDPP4 was inhibited (Fig. 1A,B).In addition, the change trend of IL-17 level in cell supernatant was consistent with the Th17 cells (Fig. 1C).Therefore, sCD26/sDPP4 could promote Th17 cell polarization and IL-17 secretion in a dose-dependent manner.

sCD26/sDPP4 Promoted EMT in TGF-β1-Induced BECs
The influence of sCD26/sDPP4 on EMT in TGF-β1induced BECs was further analysed.BECs were identified by immunocytochemical staining and the expression of KRT8 was positive (Supplementary Fig. 1B).As shown in Fig. 2A, after TGF-β1 treatment, cell-to-cell contact was reduced, and cells became spindle-forming fibroblasts.IF (Fig. 2B,C, Supplementary Fig. 1C,D) and WB (Fig. 2F-H) were used to evaluate the expression of E-cadherin and α-SMA in BECs.Compared with the normal group, the protein level of E-cadherin decreased in the TGF-β1 group, while the protein level of α-SMA increased.sCD26/sDPP4 enhanced TGF-β1-induced down-regulation of E-cadherin and up-regulation of α-SMA, while DPP4 inhibitors alleviated the effects of sCD26/sDPP4.In addition, the mRNA level of E-cadherin decreased in the TGF-β1 group and a further decrease following sCD26/sDPP4 treatment, while the level of α-SMA was reversed.DPP4 inhibitor reversed the effect of sCD26/sDPP4 as expected (Fig. 2D,E).The above results indicated that sCD26/sDPP4 promoted TGF-β1-induced EMT in BECs.

sCD26/sDPP4 Regulated EMT in Mouse BECs by Modulating the Th17/IL-17 Axis
In order to explore the interaction between BECs and Th17 cells, the latter were successfully induced from CD4 + T cells (Supplementary Fig. 1A).BECs were co-cultured with Th17 at the ratio of 1:1, 1:5, 1:10 for 24 h, 48 h, and 72 h.The EMT degree of BECs was gradually deepened by the increase of the cell ratio and the prolongation of culture time (Fig. 3A).Moreover,the level of IL-17 in the cell supernatant was also gradually increased (Fig. 3B).There-  fore, the BECs and Th17 were co-culture at the ratio of 1:10 for 72 h for subsequent experiments.The protein levels of E-cadherin and α-SMA in BECs were detected by IF (Fig. 3C,D, Supplementary Fig. 2A,B) and WB (Fig. 3G-I).Compared with the Th17+BEC group, the protein level of E-cadherin was down-regulated and protein level of α-SMA was up-regulated in the Th17+DPP4 group.However, the addition of the DPP4 inhibitor could reverse this process.The mRNA expression of E-cadherin and α-SMA was accordant with the protein expression (Fig. 3E,F).In addition, compared with the Th17+BEC group, the level of IL-17 in the Th17+DPP4 group was significantly increased (Fig. 3J), suggesting that sCD26/sDPP4 could promote the secretion of IL-17.The increase of IL-17 level was accompanied by the deepening of EMT in BEC cells, indicating that sCD26/sDPP4 and Th17 cells had a synergistic effect on the formation of EMT.The combination of the above results suggest that sCD26/sDPP4 promoted EMT in BECs by modulating the Th17/IL-17 axis.

Overexpression of DPP4 Promoted Airway Inflammation in OVA-Induced Asthmatic Mice
The role of DPP4 in OVA-induced asthmatic mice was further investigated.An OVA-induced asthma mouse model was established to evaluate the effects of DPP4 on asthma.Compared with the control group, the concentration of DPP4 in the BALF of asthmatic mice increased, and the total number of leukocytes decreased (Fig. 4A,B).After oe-DPP4 treatment, the concentration of DPP4 in the BALF of asthmatic mice was further increased, while the total number of leukocytes decreased (Fig. 4A,B).DPP4 inhibitors has the opposite effect in OVA-induced asthma mouse (Fig. 4A,B).HE staining indicated that oe-DPP4 could significantly promote OVAinduced BECs shedding and inflammatory cell infiltration in lungs-bronchi (Fig. 4C).Similarly, PAS staining demonstrated that oe-DPP4 enhanced OVA-mediated bronchial goblet cells proliferated and increased mucus secretion in lungs-bronchi (Fig. 4D).In addition, compared with the OVA group, the levels of IL-4, IL-5, and IL-13 in the BALF of mice in the DPP4 group were distinctly increased (Fig. 4E-G).DPP4 inhibitor could alleviate OVA-induced bronchial inflammation and reduce the levels of IL-4, IL-5, and IL-13 in the BALF of asthmatic mice (Fig. 4C-G).These results confirmed that oe-DPP4 promoted airway inflammation in asthmatic mice.

Overexpression of DPP4 Promoted Airway EMT in OVA-Induced Asthmatic Mice
The collagen fibers in the bronchus of the mice in the OVA group were significantly proliferated compared with the control group (Fig. 5A).Overexpression of enhanced OVA-induced bronchus collagen fibril deposition in lungsbronchi, which was significantly improved by DPP4 inhibitor (Fig. 5A).The protein levels of E-cadherin and α-SMA in the bronchial-lung tissue of mice were assessed by IF (Fig. 5B,C, Supplementary Fig. 3A,B) and WB (Fig. 5F).Compared with the OVA group, the mRNA and protein expressions of E-cadherin in the bronchial-lung tissue of mice in the DPP4 group were down-regulated, while α-SMA was up-regulated.Overexpression of DPP4 significantly promoted airway EMT, whereas DPP4 inhibitors relieved airway EMT in asthmatic mice.Evolutions of the mRNA levels of E-cadherin and α-SMA were consistent with the proteins levels (Fig. 5D,E).In addition, oe-DPP4 significantly increased the concentration of IL-17, TGF-β1, and MMP9 in the BALF of OVA-induced asthmatic mice (Fig. 5G-I).Similarly, oe-DPP4 enhanced the OVA-mediated reduction of Treg cells while contributing to an increase in the ratio of Th17 cells.In contrast, DPP4 inhibitors reversed the OVA-mediated increases of Th17 and IL-17 in asthma mice (Fig. 5J,K, Supplementary Fig. 3C,D).The above results demonstrated that oe-DPP4 increased the levels of TGF-β1 in airway and regulated the Th17/IL-17 axis, thereby mediating airway EMT and causing airway remodeling.

Discussion
CD26/DPP4 is a multifunctional glycoprotein with broad distribution, which can exist both in the form of homodimers on the surface of immune cells and in a solubilized form in body fluids [17].Studies have shown that CD26 was highly expressed on the surface of Th17 cells and participated in coordinating the immune response of Th17 cells in human inflammatory diseases [11].Zhao et al. [12] confirmed that the expression of CD26 was conducive to the differentiation of CD4 + T cells to Th17 cells.Meanwhile, there other studies have demonstrated that Th17/IL-17A could induce the accumulation of neutrophils in the airways and eventually participate in the pathogenesis of neutrophilic asthma [5,13,18].Therefore, the impacts of DPP4 on Th17 cell polarization in vitro was initially investigated, and the results suggested that sCD26/sDPP4 promoted Th17 cell polarization in a dose-dependent manner, while DPP4 inhibitors could inhibit Th17 cell polarization.The change in IL-17 concentration was consistent with the change in Th17 cell count.In conclusion, sCD26/sDPP4 could promote the secretion of IL-17 by promoting the polarization of Th17 cells.
EMT is a dynamic process in which epithelial cells gradually lose their epithelial characteristics and acquire mesenchymal characteristics [19].During EMT, polarized bronchial epithelial markers such as cytokeratin and E-cadherin are down-regulated, and mesenchymalspecific markers such as α-SMA and vimentin are upregulated [20].Studies have indicated that BECs could be transformed into myofibroblasts after EMT, thereby promoting asthma airway remodeling [21].In this paper, BECs of mice were stimulated with TGF-β1 for 72 h, and the cell morphology changed from goose-warm stone-like to spindle-like and fibroblast-like morhologies.sCD26/sDPP4 significantly promoted the EMT process induced by TGF-β1.Compared with the TGF-β1 group, the expression of E-cadherin was down-regulated while the expression of α-SMA was up-regulated in the DPP4 group, and DPP4 inhibitor reversed the effects of DPP4.In addition, one of our previous studies showed that the chronic inflammatory environment provided by IL-17 was beneficial   to the TGF-β1-induced EMT in BECs [15].And another study has shown that IL-17 and DPP4 had a synergistic effect on the formation of EMT [16].The findings in this paper indicated that sCD26/sDPP4 promoted Th17 cells to secrete IL-17 to further promote EMT in BECs.
OVA, one of the most abundant glycoprotein allergens, induces IgE production and Th2 immune responses in asthmatic patients [22].To further explore the role of DPP4 in asthma, an OVA-induced asthmatic mouse model was established.We observed that DPP4 significantly aggravated airway inflammation in asthmatic mice, while promoting mucus secretion, goblet cell hyperplasia, and collagen deposition.Additionally, DPP4 increased the levels of Th2 cell-derived cytokines IL-4, IL-5 and IL-13 in the BALF of asthmatic mice.These cytokines not only contribute to airway inflammation and airway hyperresponsiveness, but also induce subepithelial fibrosis [23][24][25].TGF-β1 is a central mediator is involved in tissue repair and fibrosis progression, and induces EMT in multiple organs [26].In our study, the concentration of TGF-β1 in the BALF of DPP4 group mice was increased compared with the OVA group.Meanwhile, oe-DPP4 significantly down-regulated the Ecadherin expression and up-regulated the α-SMA expression in bronchial of asthmatic mice.These results suggest that oe-DPP4 could promote the asthmatic airway EMT, subsequently promoting airway remodeling in asthmatic mice.
In addition, researches have revealed that the decreased expression of E-cadherin in the lung tissue of asthmatic patients results in the loss of airway barrier function, which further promotes the occurrence of airway remodeling [27].A Th17/Treg imbalance has been reported in acute OVA challenge or house dust mite-induced asthmtic mouse models [28,29].Similar to the previous findings, Th17/Treg imbalance was also confirmed in our OVAinduced asthmatic mouse model.The number of Th17 cells in the airways of asthmatic mice increased, while the number of Treg cellsdecreased.Overexpression of DPP4 promoted the differentiation of CD4 + T cells to Th17 cells but not Treg cells, therefore further accelerated this imbalance.Meanwhile, compared with the OVA group, the level of IL-17 in the BALF of the DPP4 group mice were also significantly increased.High levels of IL-17 were related to the airway inflammation, responsiveness, and remodeling in asthmatics [30,31].Therefore, oe-DPP4 promote airway inflammation and remodeling in asthmatic mice by promoting airway EMT and Th17 cell polarization.

Conclusions
Our in vitro results have shown that sCD26/sDPP4 could promote the Th17 cell polarization and IL-17 secretion, promote the TGF-β1-induced airway EMT.In addition, Th-17 and DPP4 exhibited a synergistic effect on the formation of EMT.Compatible with the results of our in vitro experiments, oe-DPP4 also promoted airway EMT and Th17 cell polarization in OVA-induced asthmatic mouse models.In summary, the findings in this paper show that DPP4 promotes airway inflammation and airway remodeling by modulating the Th17/IL-17 axis and accelerating the airway EMT.

Fig. 3 .
Fig. 3. sCD26/sDPP4 promoted EMT in BECs by modulating the Th17/IL-17 axis.In (A,B), the BECs were co-cultured with Th17 at the different ratio, while in (C-J), the BECs were co-cultured with Th17 at the ratio of 1:10 and treated with sCD26/sDPP4 or DPP4 inhibitor.(A) Microscope estimation of the cell morphology changes of BECs.(B) The IL-17 level in the cell supernatant of BECs co-cultured with Th17 was analyzed by ELISA.(C,D) The fluorescence intensity of E-cad and α-SMA in BECs.(E,F) The mRNA expression of E-cad and α-SMA in BECs was assessed by qRT-PCR.(G-I) The protein levels of E-cad and α-SMA in BECs were measured by WB. (J) The level of IL-17 in the cell supernatant of BECs co-cultured with Th17 at the ratio of 1:10 and treated with sCD26/sDPP4 or DPP4 inhibitor was analyzed by ELISA.Data was showed as the mean ± SD, in Fig. 3B, *p < 0.05 vs 1/1, #p < 0.05 vs 1/5; and in Fig. 3C-J, *p < 0.05 vs Th17+BEC group, #p < 0.05 vs Th17+DPP4 group.E-cad, E-cadherin.

Fig. 4 .
Fig. 4. Overexpression of DPP4 promoted airway inflammation in asthmatic mice.(A) The level of DPP4 in the bronchoalveolar lavage fluid (BALF) was analyzed by ELISA.(B) The total number of leukocytes in the BALF was calculated by a hemocytometer.(C) Hematoxylin-eosin (HE) staining.(D) Periodic acid Schiff (PAS) staining.(E-G) The levels of IL-4, IL-5 and IL-13 in the BALF were assessed by ELISA.Data was showed as the mean ± SD, *p < 0.05 vs control group, #p < 0.05 vs OVA group, &p < 0.05 vs OVA+DPP4 group.

Fig. 5 .
Fig. 5. Overexpression of DPP4 promoted airway epithelial mesenchymal transition (EMT) in OVA-induced asthmatic mice.(A) Masson staining.(B,C) The fluorescence intensity of E-cad and α-SMA in lungs-bronchi of mice.(D,E) The mRNA expression of E-cad and α-SMA in lungs-bronchi of mice were evaluated by quantitative Real-Time PCR (qRT-PCR).(F) The protein levels of E-cad and α-SMA in lungs-bronchi of mice were estimated by WB. (G-I) The levels of IL-17, TGF-β1 and MMP9 in BALF were analyzed by ELISA.(J,K) The ratio of Th17 and Treg cells in the airways of mice was detected by flow cytometry.Data was showed as the mean ± SD, *p < 0.05 vs control, #p < 0.05 vs OVA group, &p < 0.05 vs OVA+DPP4 group.E-cad, E-cadherin.