IL‐19 induced by IL‐13/IL‐17A in the nasal epithelium of patients with chronic rhinosinusitis upregulates MMP‐9 expression via ERK/NF‐κB signaling pathway

Abstract Background Tissue remodeling is a crucial characteristic of chronic rhinosinusitis (CRS). Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is crucial for the pathologic tissue remodeling in CRS. Elevation of interleukin (IL)‐19 or MMP‐9 levels in patients with CRS had been proven in previous studies. Here, we aimed to investigate the role of IL‐19 in mediating MMP‐9 expression in CRS. Methods Nasal tissue samples were collected from 45 individuals having chronic rhinosinusitis with nasal polyps (CRSwNP), 24 CRS without nasal polyps (CRSsNP), and 17 controls. Expression of IL‐19, its receptors (IL‐20R1/IL‐20R2), and MMP‐9 were investigated using RT‐qPCR and Immunofluorescence (IF). Human nasal epithelial cells (HNECs) were stimulated by IL‐19; ERK phosphorylation, nuclear factor‐κB (NF‐κB) pathway activation, and MMP‐9 level were detected by RT‐qPCR, enzyme‐linked immunosorbent assay, western blot, and IF. We also explored the effect of type1/2/3 cytokines on IL‐19 production by RT‐qPCR, and western blot. Results Expression levels of IL‐19, its receptors (IL‐20R1/IL‐20R2), and MMP‐9 were increased in nasal tissues from individuals with CRSwNP compared to those with CRSsNP as well as the controls. IL‐19 significantly elevated the production of MMP‐9 in HNECs. Furthermore, IL‐19 could activate the ERK and NF‐κB pathways, accompanied by increased MMP‐9 production in HNECs. Conversely, both ERK and NF‐κB inhibitors significantly attenuated the role of IL‐19 in MMP‐9 production. siRNA knockdown of IL‐20R1 suppressed ERK and NF‐κB pathway activation, thereby decreasing MMP‐9 expression. IL‐13 and IL‐17A were found to stimulate IL‐19 production in HNECs. Conclusion IL‐19, promoted by IL‐13 and IL‐17A, contributes to the upregulation of secretion of the tissue remodeling factor MMP‐9 in patients with CRS.


| INTRODUCTION
Chronic sinusitis (CRS) is a common disease, with prevalence of 8% in China and 12% in the United States. [1][2][3] It is a considerable public health concern as well as a socioeconomic burden. 4 Clinically, CRS is categorized into two types: CRS with (CRSwNP) or without nasal polyps (CRSsNP). Histologically, CRSsNP is predominantly characterized by basement membrane thickening and interstitial fibrosis.
On the contrary, CRSwNP is characterized by stromal tissue edema with albumin deposition and pseudocyst formation. 5 Moreover, CRSwNP is considered a heterogeneous disease, and based on the expression of different inflammatory cytokines in CRSwNP, it can be divided into different clusters. Clinical characteristics, such as treatment outcomes, asthma prevalence, and postoperative recurrence, are significantly different across the clusters. 6,7 Despite the variation across subtypes of CRSwNP, manifestation of tissue remodeling is similar, hence indicating different inflammatory cytokines to eventually lead to similar tissue remodeling pattern through common downstream factors or pathways in nasal polyps.
Tissue remodeling in CRS involves transformation of the tissue structure and extracellular matrix (ECM). Several elements involved in tissue remodeling include matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and transforming growth factor (TGF)-β. Imbalance between MMPs and TIMPs is considered to be the key factor for pathological tissue remodeling in CRS. 8,9 As a family of zinc-dependent proteolytic enzyme, MMPs can degrade a variety of constituents of ECM and regulate remodeling. 10 However, MMP-1, MMP-2, and MMP-3 expression in patients with CRS is controversial, whereas several studies have reported MMP-7 and MMP-9 levels to be higher in cases with CRSwNP. [11][12][13][14] Overall, the detailed upstream modulation of MMPs promoting tissue remodeling in chronic sinusitis remains unclear.
IL-19 is one of the members of IL-20 cytokine subfamily, which also includes IL-20, IL-22, IL-24, and IL-26. 15 The structures of IL-20 subfamily receptor heterodimers are somewhat similar. IL-19 needs to bind to a functional heterodimeric receptor IL-20R1/IL-20R2 for mediating its signal transduction. 16 Studies concerning IL-20 subfamily have shown IL-20 to induce MMP-3 in primarily cultured human disc cells 17 ; IL-20 had also been reported to significantly promote MMP-9 production in bladder cancer cells. 18

| Human nasal epithelial cells culture
Human nasal tissues derived from patients with CRSwNP and CRSsNP were cut into 1 � 1-mm fragments, digested in dispase II (50 mg/mL, Sigma-Aldrich) overnight at 4°C, followed by further digestion in trypsin for 15 min at 37°C; the digestion was terminated by adding complete Dulbecco's modified eagle medium (DMEM; 90% DMEM containing 10% fetal bovine serum). The digested nasal tissue pieces were then filtered by a 100-μm cell strainer to obtained cells.
Cells were then suspended in complete DMEM and cultured in an incubator for 30 min to remove fibroblasts. Then the obtained HNECs were cultured in bronchial epithelial growth medium (BEGM; Lonza) added with 1% antibiotic-antimycotic (Invitrogen) at a density of (3-5) � 10 5 cells/cm 2 at 37°C in an atmosphere of 5% CO 2 and 95% relative humidity. BEGM was refreshed every 2 days, after 10-14 days incubation, HNECs were stimulated and collected for subsequent experiments.

| Real-time quantitative polymerase chain reaction
Real-time quantitative polymerase chain reaction (RT-qPCR) was performed, as described previously, to detect the mRNA level of target genes. 24 More details can be found in Supporting Information Material.

| Enzyme-linked immunosorbent assay
Supernatant of HNECs was collected and examined by MMP-9 ELISA kits (CUSABIO). All procedures followed the manufacturers' instructions.

| Western blotting
Western blotting was performed as reported previously. 13 The

| Immunofluorescence staining and confocal microscopy
The frozen sections from human nasal tissue were blocked with 10% goat serum for 30 min and stained first with IL-19 (1:50; Abcam) and

| Statistical analysis
IBM SPSS 20 (SPSS) was used for statistical analyses. When data were normally distributed, they were presented as mean ± SEM; otherwise, they were presented as median (25-75 percentiles). Oneway analysis of variance or Kruskal-Wallis test was applied to analyze significance across groups, for comparative study, Student's t or Mann-Whitney U test (two-tailed) was then applied for comparisons across groups. Paired Student's t or Wilcoxon matchedpair signed rank test was used when suitable. p < 0.05 was regarded as significant.

| Increased expression of IL-19, its receptors (IL-20R1/IL-20R2), and MMP-9 in nasal mucosa of patients with CRSwNP
Tissues collected from patients with CRSwNP and CRSsNP, and from controls, were examined for IL-19, its receptors, and MMP-9 expression by RT-qPCR. As expected, IL-19, its receptors (IL-20R1/ IL-20R2), and MMP-9 mRNA expression levels were significantly higher in patients with CRSwNP than in CRSsNP and control subjects. Besides, IL-20R2 and MMP-9 mRNA expression was increased in patients with CRSsNP than those in control subjects ( Figure 1A). IF double staining was used to further evaluate the probable interaction between IL-19 and MMP-9. IL-19 was mainly found co-localized with MMP-9 in the epithelial cells of CRS mucosa ( Figure 1B). The above results suggested functional correlation of IL-19 with MMP-9 in the mucosal tissue of patients with CRS.

| IL-19 induced MMP-9 expression in HNECs
Considering the colocalization of IL-19 and MMP-9 in the mucosa of patients with CRS, we next explored whether IL-19 could elevate MMP-9 secretion in HNECs. HNECs were treated with IL-19 LI ET AL. (0-200 ng/mL) for 24 h. Thereafter, RT-qPCR was used to assess mRNA levels while enzyme-linked immunosorbent assay, western blotting, and immunofluorescence were applied to evaluate protein expression. As expected, both mRNA and protein expression of MMP-9 were elevated in response to IL-19 (Figure 2A-D); the optimal concentration of IL-19 was 100 ng/mL.

| IL-19 promoted MMP-9 expression via NF-κB pathway in HNECs
The MMP-9 gene promoter includes several transcription factors, and NF-κB was verified as a binding site. 25 To confirm the signaling pathways that induce up-regulated MMP-9 production in HNECs in response to IL-19, first we evaluated whether IL-19 could promote the activation of NF-κB in HNECs. HNECs were treated with IL-19 at optimal concentrations (100 ng/mL), and phosphorylation level of IκBα protein was measured by western blot while p65 nuclear translocation was detected by confocal microscopy. Results suggested treatment with IL-19 to elevated the phosphorylation of IκBα effectively in HNECs ( Figure 3A).

| IL-19 upregulated MMP-9 expression via ERK pathway in HNECs
Since earlier studies had demonstrated the NF-κB pathway to be regulated by ERK signaling, 10
These findings together demonstrated IL-19 to possibly mediate ERK and NF-κB pathway activation, and MMP-9 production in HNECs by binding to IL-20R1.

| DISCUSSION
One of the main events in tissue remodeling is the degradation of ECM components by proteolytic enzymes. 27 Insufficient suppression of MMP-9 by TIMP-1 in polyp could lead to degradation of ECM, and hence, pseudocyst formation. 28 However, TGF-β1 can promote TIMP-1 expression, and elevated levels of TGF-β1 and TIMP-1, relative to MMP-9, may account for the principal fibrosis observed in CRSsNP. 29 In our previous study 12 and the current one, we demonstrated MMP-7 and MMP-9 expression in CRSwNP to be elevated compared to that in CRSsNP and the control group, which was consistent with other previous reports. 11,14,29 MMPs could be an important treatment target for chronic rhinosinusitis. As one of the MMPs inhibitors, doxycycline has been demonstrated to effectually F I G U R E 5 IL-19 stimulated MMP-9 production by binding to receptor in HNECs. (A, B) HNECs were transfected with IL-20R1 siRNA for 48 h and subsequently incubated with IL-19 for 24 h. They were then collected and western blotting performed to measure the phosphorylation of ERK1/2 (n = 3) and IκBα (n = 3). (C-E) MMP-9 expression was measured by RT-qPCR (n = 3), western blot (n = 3), and immunofluorescence. *p < 0.05, **p < 0.01, ***p < 0.001, mean ± SEM. HNEC, human nasal epithelial cell; IL, interleukin; MMP, matrix metalloproteinase; RT-qPCR, real-time quantitative polymerase chain reaction F I G U R E 6 IL-13 and IL-17A stimulated IL-19 production in HNECs. HNECs were incubated with Type 1 cytokines (IFN-γ, IL-β), Type 2 cytokines (IL-4, IL-5, and IL-13), Type 3 cytokines (IL-17A), and IL-25 at a concentration of 20 ng/mL. (A) After 6-h incubation, HNECs were collected to measure transcription levels of MMP-9 (n = 4). (B) After 12-h stimulation, proteins were extracted and measured by western blotting (n = 3). (C, D) HNECs were transfected with IL-20R1 siRNA for 48 h and subsequently stimulated with IL-13 or IL-17A for 24 h. HNECs were collected to measure MMP-9 expression, and analyzed using quantitative western blot (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, mean ± SEM. HNEC, human nasal epithelial cell; IL, interleukin; MMP, matrix metalloproteinase; RT-qPCR, real-time quantitative polymerase chain reaction. diminish MMP-9 expression in nasal secretions, eventually shrinking the size of polyps. 30 Studies have also shown high concentrations of MMP-9 after functional endoscopic sinus surgery (FESS) to be related to bad treatment outcome. 31 Doxycycline-releasing sinus stents have been shown to effectively reduce nasal MMP-9 concentrations and improve therapeutic effect in patients post FESS. 32 Therefore, the upstream regulators of MMP-9 in CRS would be worth studying.
Recent reports have indicated IL-20 subfamily to participate in many inflammatory diseases, liver fibrosis, rheumatoid arthritis, psoriasis, and asthma. [33][34][35][36] Our previous studies had shown IL-19 to be involved in mucin production, a participant in tissue remodeling in CRS. 37 In the present study, mRNA levels of IL-19, its receptors (IL-20R1/IL-20R2), and MMP-9 were found elevated in CRSwNP.
Although smoking and atopy may be considered to affect results, the multiple linear regression proved that only grouping variable was responsible for change in IL-19 mRNA expression in human nasal tissues in our study (Table S4). Additionally, IL-19 was mainly seen to be colocalized with MMP-9 in the epithelium of CRS mucosa. According to the above results, we speculated IL-19 to possibly participate in the development and progression of tissue remodeling in CRS. Thereafter, we aimed to investigate how IL-19 could conduce to the modulation of MMP-9 production in HNECs. As expected, IL-19 stimulation was observed to promote MMP-9 production in HNECs at an optimal concentration of 100 ng/mL. Additionally, transfection of IL-20R1 siRNA into HNECs effectively attenuated IL-19-promoted MMP-9 production. The function of IL-19 might manifest after combining with IL-20R1 receptor, thus resulting in MMP-9 overexpression and ECM destruction. This finding was in line with studies concerning the function of IL-19 depending on its binding to a functional heterodimeric receptor IL-20R1/IL-20R2. 26 In contrast, IL-19 had no effect on the expression of MMP-7 in HNECs ( Figure S1).
Till date, only a few studies have been reported about the specific mechanism of MMP-9 modulation in CRS. Previous studies had demonstrated IL-20 subfamily members to activate the signal transducer and activator of transcription (STAT), Janus kinase, and mitogen-activated protein kinase (MAPK) signaling pathways. 15 As a subclass of MAPK pathways, the ERK signaling pathway is also activated by a sequence of phosphorylation events in response to extracellular stimuli, which then transmits extracellular signals that induce cellular proliferation, differentiation, and survival. After being activated, RAF kinases (MAPKKK) phosphorylate and activate the elements of MAPKK module MEK1/2, which then activate the MAPK protein kinase, ERK1/2. Once activated, ERK1/2 phosphorylates and connects various extracellular signals to cytoplasm or nucleus. 38 In our current study, we demonstrated effective induction of phosphorylation of MEK1/2 and ERK1/2, modules of ERK pathway, and expression of MMP-9 in HNECs by IL-19. We also identified the participation of transcription factor NF-κB in ERK-regulated MMP-9 production in IL-19-stimulated HNECs. These findings were similar to that reported in studies concerning atherosclerosis and bladder cancer. In reports regarding the pathogenesis of atherosclerosis, CD147 had been demonstrated to mediate MMP-9 induction through ERK and the nuclear translocation of NF-κB pathway in macrophage. 39 IL-20 could also induce bladder cancer cell migration by activating the ERK-mediated NF-κB pathway, and promoting MMP-9 production subsequently. 18 Regulatory components that exist in the MMP-9 gene contain the binding site for NF-κB, which has been proved to drive MMP-9 production in macrophages, neutrophils, cardiomyocytes, fibroblasts, and bladder cancer cells. 18,25,[40][41][42] As a dimeric transcription factor (p50/p65), the classical NF-κB acts as a pivotal inflammatory regulator and modulates various genes. Upon activation, NF-κB translocates to the nucleus, after which the active subunit p65 promotes transcription of chemokines, cytokines, and adhesion molecules. 43 We demonstrated the promotion of NF-κB activation by IL-19, which generated phos-