IL-19 Promotes Nasal Mucosal Tissue Remodeling in Chronic Rhinosinusitis Patients By Upregulating Fibronectin and Collagen Ι Expressions in Fibroblasts Via NF-κB-Smad2/3 Signaling Pathways

Chronic sinusitis without nasal polyps (CRSsNP), a subtype of chronic rhinosinusitis, is characterized by tissue remodeling, mainly by interstitial brosis. Fibroblasts are essential effectors of tissue remodeling, mainly expressing bronectin (FN), collagen Ι (Col Ι), and other extracellular matrices. Our previous study found that IL-19 can promote the aberrant expression of extracellular matrix, and pre-experiments revealed that the Smad2/3 pathway plays a crucial role in tissue remodeling. However, the exact mechanism how IL-19 participants in tissue remodeling remains unclear. This study aimed to determine the roles of IL-19 in regulating the expression of FN and Col Ι, and the roles of Smad2/3 and NF-κB signaling pathways in broblasts.


Background
Chronic rhinosinusitis (CRS) is a heterogeneous, chronic in ammatory disease of the nasal cavity and sinuses. Nearly 10% of the global population suffers from this disease, and its incidence is increasing annually [1]. In China, the prevalence of this disease is approximately 8% [2].
Tissue remodeling is a prominent pathological feature of chronic in ammation of the upper and lower airways and is a crucial determinant of the nature and prognosis of CRS. Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by interstitial edema, accompanied by albumin deposition and decreased collagen synthesis in the extracellular matrix, which eventually leads to polyp formation. On the other hand, chronic sinusitis without nasal polyps (CRSsNP) is characterized by interstitial brosis and massive deposition of the extracellular matrix, including collagen, basement membrane thickening, and Goblet cell proliferation, accompanied by massive in ltration of neutrophils, which ultimately leads to mucosal thickening [3,4]. Fibroblasts play an essential role in maintaining tissue morphology and inducing extracellular matrix production. However, its overexpression leads to increased deposition of extracellular matrix components, such as bronectin (FN) and collagen Ι (Col Ι), thus promoting brosis.
TGF-β plays a central role in forming chronic brosis by regulating the function of broblasts, inducing broblasts to produce extracellular matrix, and promoting the accumulation of extracellular matrix [5,6].
Meanwhile, TGF-β, as an essential switch, plays a crucial role in the brosis process of the CRS subtype.
It is precisely regulated during tissue remodeling by secreting various activating and inhibiting factors, such as LAP, LTBP, FN, integrin, and protease [7,8].Smad2/3 protein is a substrate for type 1 TGF-β receptors and mediates intracellular TGF-β signaling [9,10]. It has been demonstrated that Smad proteins participate in tissue remodeling by activating TGF-β [11,12]. Studies have con rmed that blocking the TGF-β-mediated Smad2/3 classic pathway can reduce the deposition of nasal submucosal collagen, thereby inhibiting the brosis progression of chronic sinusitis tissues [13,14]. The NF-κB pathway is another signaling pathway that can in uence broblast differentiation and signi cantly affects the expression of tissue remodeling associated proteins [15,16].
The IL-20 subfamily, an essential class of pre-in ammatory factors, is considered to participate in tissue remodeling regulation by regulating extracellular matrix homeostasis, mucin deposition, and in ammatory cell in ltration in liver brosis, psoriasis, and other diseases [17][18][19]. Studies have found that IL-22 and IL-24, members of the IL-20 subfamily, play an essential role in liver brosis by enhancing TGF-β activation signals and increasing the liver susceptibility to tumor in ammation [20].IL-20 levels were much higher in hepatocytes and hepatic stellate cells (HSCs) in liver biopsies of patients with brosis, cirrhosis, and liver cancer. It can also increase the expression of TGF, TNF, and Col1 and promotes the proliferation and migration of activated HSCs [21]. In psoriasis, IL-20 stimulates monocytes to produce pro-in ammatory cytokines and indirectly exerts its effect on keratinocyte proliferation through immune cells in the skin [22].In renal brosis disease, the pro-brotic effects of IL-19, IL-22, and IL-24 on the kidney was con rmed in a mouse renal disease model [23]. As a crucial member of the IL-20 subfamily, IL-19 has been found to be highly expressed in many chronic in ammatory diseases, such as psoriasis and asthma. It has been reported that LPS stimulation induces the upregulation of IL-19 and further increases the proliferation of nasal epithelial cells. Our previous study found higher expression of IL-19 and its receptors in nasal mucosal epithelial cells of CRS patients, and it promotes matrix metalloproteinase (MMP) expression, which plays critical roles in extracellular matrix balance [24][25][26]. However, whether a higher level of IL-19 plays a vital role in the brosis of CRS and the exact mechanism remains unclear.
This study aimed to determine the correlation between IL-19 upregulation and nasal mucosal interstitial brosis in CRS patients and to investigate the effect and mechanism of IL-19 in regulating the expression of FN and Col Ι via the NF-κB and Smad2/3 signaling pathways.

Materials And Methods
Patient and tissue samples CRS patients were diagnosed and classi ed, according to EPOS2012 [27]. Nasal polyps of CRSwNP patients, sinus mucosa of CRSsNP patients, and inferior turbinate tissue of control patients without sinusitis were collected. A portion of the specimens was stored at -80 °C and reserved in biobank of clinical resources of the third a liated hospital of Sun Yat-sen university for immunohistochemistry and immuno uorescence testing of frozen sections. The remaining fresh specimens were soaked in PBS containing 1% antibiotic-antimicrobial agent and used for broblast cell culture. This study was approved by the ethics committee of the Third A liated Hospital of Sun Yat-sen University (NO. [2016]2-16). All subjects were included in the study only after informed consent was obtained.
Human nasal mucosa primary broblast cell culture Human nasal mucosal tissues were cut into 1 × 1-mm fragments and digested with dispase II (50 mg/mL, Sigma-Aldrich, St. Louis, USA) overnight at 4 °C and further digested with trypsin at 37 °C for 15 min. DMEM containing 10% fetal bovine serum was added to stop the digestion. The digested nasal tissues were then ltered through a 70-μm cell lter to obtain a cell suspension.

Real-time quantitative PCR (RT-qPCR)
RT-qPCR was used to detect the mRNA levels of the target genes coding for FN and Col Ι in the Control, CRSwNP, and CRSsNP groups, as previously described. Total RNA was extracted from cells using RNAiso Plus and was reverse transcribed to cDNA using the PrimeScript RT kit (TaKaRa Biotechnology). Realtime quantitative PCR was conducted using SYBR premix Ex Taq kit (TaKaRa Biotechnology) and the corresponding product primers (Invitrogen, Carlsbad, CA, USA). β2 microglobulin (β2M) was used as a housekeeping gene for normalization. Relevant gene expression was analyzed using the comparative CT value method.

Immunohistochemistry
Tissues of surgical specimens from control, CRSwNP, and CRSsNP patients were xed with 4% paraformaldehyde, para n-embedded, and cut into 4μm-thick sections. The sections were dewaxed, rehydrated, and subjected to heat retrieval following routine procedures. The sections were covered with 10% goat serum (Boster Biotechnology) to block non-speci c binding for 30 min and then incubated with

Statistical analysis
Statistical analysis was performed using IBM SPSS 20 (SPSS, Chicago, IL, USA). Three or more groups were analyzed for signi cance using one-way analysis of variance (ANOVA) or the Kruskal-Wallis test for comparative study. The t-test or Mann-Whitney U-test (2-tailed) was applied for comparison between groups. The paired t-test or Wilcoxon's paired with the signed-rank test was used to compare the two groups. Signi cance was set at P < 0.05.

Results
Fibroblasts, FN and Col Ι, were highly expressed in CRSsNP tissues Immunohistochemistry showed the number and location of vimentin (VIM), suggesting that broblasts were mainly located in the basement membrane (located between the epithelial and lamina propria, as shown by the arrows in Figure 1). The expression of VIM in the CRSsNP group was signi cantly higher than that in the control and CRSwNP groups (P<0.05 for both, Figure 1A, 1B). Both CRSwNP and CRSsNP demonstrated higher expression of FN and Col Ι when compared with the control group (P<0.05 for both, Figure 1A, 1B). Moreover, the CRSsNP patients showed the highest levels of FN and Col Ι in CRSsNP patients than in CRSwNP patients (P<0.05, Figure 1A, 1B).

Co-localization of IL-19 with FN and Col Ι in mucosal tissues of CRSsNP patients
Immuno uorescence showed that IL-19 co-localized with FN and Col Ι in the cytoplasm of the nasal mucosal epithelium. The expressions of FN and Col Ι in the CRSsNP group were higher than those in the control and CRSwNP groups ( Figure 2).

IL-19 promoted the expressions of FN and Col Ι in broblasts
To further con rm the relationships between IL-19, FN, and Col Ι in broblasts, primary broblasts were cultured in vitro and pretreated with human recombinant IL-19 (hIL- 19) or not. The protein levels of FN and Col Ι in broblasts were detected by western blotting after pretreatment with different concentrations of IL-19 for 24 hours. As shown in Figure 3A, the expression of FN and Col was signi cantly increased in broblasts treated with hIL-19-treated compared to the control. The optimum concentration of hIL-19 was 200 ng/mL (P=0.01, for both). We further veri ed the immuno uorescence results, which also showed that the expression of FN and Col Ι was increased in the IL-19-treated group ( Figure 3B). Furthermore, RT-qPCR analysis showed that IL-19 promoted the mRNA expression of FN and Col Ι in broblasts at an optimum concentration of 200 ng/mL (P=0.001 for FN, P=0.01 for Col Ι, Figure 3C). These results suggest that IL-19 promotes the expression of FN and Col Ι in broblasts at both the protein and transcript levels.

IL-19 promoted FN and Col Ι expressions in broblasts via Smad2/3 pathway
The role of TGF-induced brosis has been widely recognized, and Smad2/3, as a switch of TGF signaling, also plays an essential role in the development of tissue brosis [11,12]. Therefore, to further explore the mechanism by which IL-19 promotes the expression of FN and Col Ι in broblasts and to verify the role of the Smad2/3 pathway further, we treated primary broblasts with IL-19 stimulation combined with Smad2/3 pathway inhibitor. It was found that hIL-19 could promote p-Smad2/3 and Smad2/3 protein expression levels ( Figure 4A). Furthermore, western blotting demonstrated that hIL-19 induced upregulation of p-Smad, FN and Col Ι were eliminated by the Smad pathway inhibitor (Indoxinmod) pretreatment (Fig. 4B). This was further con rmed by immuno uorescence (Fig. 4C). The results indicated that IL-19 promoted FN and Col Ι expression in broblasts through the Smad2/3 pathway.
IL-19 promotes FN and Col Ι expression in broblasts via NF-κB-Smad2/3 pathway Studies have reported that the NF-κB pathway is a class of classical pathways associated with in ammation in human liver and lung broblasts [28]. To further verify the NF-κB pathway's role, primary broblasts were treated with different doses of hIL-19, western blot analysis showed that IL-19 could promote p-IκBa protein expression ( Figure 5A). Furthermore, we treated broblasts with hIL-19 combined with NF-κB pathway inhibitors (Bay11-7082) and found that the FN and Col Ι protein expression in the IL-19 combined with Bay11-7082 treatment group was lower than that in the IL-19 treatment alone (P=0.01, Fig. 5B). It was further con rmed by immuno uorescence that both NF-κB activator (EVP4953) and hIL-19 treatment could increase the FN and Col Ι expression in broblasts, while the Bay combined with hIL-19 treatment decreased FN and Col Ι expression ( Figure 5C). The results indicated that IL-19 promoted FN and Col Ι expression in broblasts through the NF-κB pathway.
We further con rmed the upstream and downstream relationships between the NF-κB and Smad2/3 pathways. As shown in Figure 5D, after adding the Smad2/3 activator, the expression of FN, Col Ι, and Smad2/3 increased signi cantly, but the increase in p-IκBa was not signi cant. After adding Smad2/3 inhibitor, the expression of Smad2/3 protein and FN, Col-1 decreased signi cantly, and the expression of p-IκBa did not change much compared with the control group. NF-κB agonist treatment signi cantly increased the expression of FN, Col Ι, and p-Smad2/3 protein signi cantly. The expression of FN, Col Ι, and p-Smad2/3 protein decreased signi cantly after the NF-κB inhibitor's addition compared with the IL-19 stimulation group. This was corroborated by the confocal assay and microscopic observation of FN, Col Ι and p-Smad2/3, and p-65 expression (Figure 5e). It is inferred that NF-κB may act as an upstream pathway of the Smad2/3 pathway and act as a bridge in the process of tissue brosis.

Discussion
Tissue remodeling is the main pathological feature of chronic in ammation of the upper and lower airways and is an essential factor in determining the nature and prognosis of CRS lesions [4]. In CRSwNP, extracellular connective tissue matrix degradation, markedly less collagen deposition might be a critical pathological step and feature, while, in CRSsNP, basement membrane thickening ( brosis), collagen deposition, and goblet cell hyperplasia are the main characteristics, with extracellular matrix composition [29,30]. Extracellular matrices (ECMs) are vital external environments that regulate cell and tissue homeostasis and play a critical role in tissue remodeling, a class of complex network structures consisting of a large variety of matrix macromolecules whose precise composition and specific structures vary from tissue to tissue. [31]. In the heart, muscle cells can produce collagen 4, 6, laminin (LN), and proteoglycans. Endothelial cells can produce collagen I, III,IV, LN, and bronectin (FN), while broblasts can produce Col I, III, and FN to form ECMs [32,33]. It has been reported that in CRSwNP tissues, the major components of ECMs are collagen I, III, and FN; they are most highly expressed in non-eosinophilic CRSwNP. Collagen IV did not differ signi cantly among groups and was mainly distributed around blood vessels. In eosinophilic CRSwNP, the loss of matrix deposition may be due to decomposition or failure to produce the necessary components, with lower levels of extracellular matrix expression [34]. It was also reported that signi cant mucinous gland hyperplasia and collagen deposition were seen in the extracellular matrix of CRSsNP [35]. The cup cell number and basement membrane thickness of CRSsNP were signi cantly increased, and the collagen deposited in the ECMs consisted mainly of collagen I, with signi cantly less collagen III and IV [36]. Our experiments revealed that Col Ι and FN are highly expressed in the intrinsic layer of CRS nasal mucosal tissues and as their primary source of production from broblasts, and the expression of the broblast marker waveform protein was found to be increased in the CRSsNP patients. Also, the expression levels of FN and Col Ι were high in the mucosa of CRSsNP patients. Therefore, two proteins, Col Ι and FN, were selected as extracellular matrix markers for detecting different subgroups of chronic sinusitis. However, the mechanism underlying the higher FN and Col deposition in the mucosa of CRSsNP was unclear.
Our previous study reported that IL-19 was highly expressed in CRSsNP, especially in the epithelium of nasal mucosa, and correlated with MMPs, which are the primary regulators in manipulating ECM balance. It also contributes to mucus overproduction by promoting MUC5AC expression in CRS epithelial cells [25,37]. This indicates that IL-19 plays a critical role in the tissue remodeling and pathological process of CRS. In this study, we found that FN and Col Ι co-localized with IL-19 in the nasal mucosa of CRS patients, and the expression levels of these two factors were found to be higher in the nasal mucosa of patients with CRSsNP than in those with CRSsNP. Moreover, stimulation of nasal broblasts with suitable concentrations of IL-19 promoted high expression of FN and Col Ι. This was consistent with the tissue remodeling characteristics that CRSsNP predominantly shows mesenchymal brosis, collagen deposition, and higher ECM composition, while CRSwNP presents edema and decreased ECM [29]. When stimulation with higher concentrations (>200 ng/mL) of hIL-19, the expression levels of FN and Col Ι in broblasts decreased, which was speculated to be related to the toxic effect of IL-19 on cells. Similar to the existing experiments, the study also con rmed that IL-24 in the IL-10 superfamily could be cytotoxic to tumor cells at a certain concentration level [38].
As an inhibitory effect on the extracellular matrix, IL-19 was reported to decrease the expression of TGF-β and connective tissue growth factor (CTGF) in rat broblasts at day 7, and further signi cantly inhibited the generation of extracellular α-SMA, Col-1, and FN at 28 days, via the Erk and p38 pathways [39]. In this study, IL-19 was found to activate the Smad2/3 pathway, a substrate for type 1 TGF-β receptors, which plays a crucial role in transferring TGF-β signals from cell surface receptors to the nucleus and mediates TGF-β intracellular signaling. Currently, Smad proteins are divided into three categories based on their different functions in signal transduction in the TGF family: public mediator Smad proteins, inhibitory Smad proteins, and pathway-restricted Smad proteins, including Smad2 and Smad3. Activated TGF-β receptor-induced pathway-restricted (speci c) Smad 2 and Smad 3 undergo phosphorylation via cytosolspeci c serine/threonine kinase receptor activation and bind to common Smad 4 for oligomeric translocation to the nucleus, where they directly respond to the transcriptional function of TGF-β. This structure serves as a switch for intracellular signaling by Smad. The role of protein-activated TGF in tissue remodeling brosis has also been demonstrated [5,6,14,35].
This study found that the NF-κB pathway can in uence the IL-19 induced overproduction of FN and Col Ι in broblasts. Inhibition of NF-κB activity decreased the speci city of p-Smad2/3 expression. Also, the present experiment, in which the expression of FN and Col Ι was detected by two-way validation by adding NF-κB and Smad2/3 pathway activators and inhibitors, con rmed that IL-19 promotes the production of extracellular matrix including FN and Col Ι through the NF-κB-Smad2/3 pathway. This was consistent with a previous study showing that TGF stimulated the expression of extracellular matrix including FN, Col Ι, and α-SMA in broblasts, decreased when combined with NF-κB pathway inhibitors [16,40]. This study also shows that the NF-κB pathway can in uence the differentiation of myo broblasts and signi cantly affect tissue remodeling and related protein expression.
In this study, we used IL-19 stimulation of broblasts to investigate the effects of FN and Col Ι upon activation of the NF-κB-Smad2/3 pathway. It was found that IL-19 affects the process of CRS tissue brosis by activating the NF-κB signaling pathway, which in turn activates the downstream Smad2/3 pathway and stimulates broblasts to produce Col and FN, leading to alterations in CRSsNP tissue remodeling. This study investigates the feasibility of preventing and reducing CRSsNP tissue brosis and provides potential therapeutic targets for the inhibition of pathological nasal tissue remodeling by investigating pathway activation and related protein expression in broblasts in CRSsNP. However, this study did not explore the speci c process of Smad2/3 protein activation by IL-19 in detail and only demonstrated that IL-19 promotes the activation of the Smad2/3 pathway in broblasts, and the speci c mechanism of Smad2/3 activation remains to be further explored.

Declarations Ethics approval and consent to participate
The studies involving human participants were reviewed and approved by Third A liated Hospital, Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.

Consent for publication
Consent to publish was obtained from all authors.

Availability of data and materials
All data in our study are available upon request.

Competing interests
There is no con ict of interest.

Author Contributions
Gehua Zhang conceived the study design, reviewed manuscript and provided comments. Lihong Chang supervised the experiments and data analysis, reviewed manuscript. Hongwei Bao drafted the manuscript and nished the majority of experiments, and acquired the data. Xia Li and Xiaoping Lai design experiments, data analysis and revised manuscript. Xiaohong Chen analyzed and interpreted the data, collected samples and revised manuscript. Zizhen Huang helped for the data analysis and recruited patients and collected samples. Yue Li and Zhouzhou Yao helped for the data analysis and sample collection. Jiancong Huang participant in the experiment design and supervision, and recruited patients and collected samples.  Table   Table 1