Targeting KDM4B attenuates IL-13-mediated fibrosis in bronchial fibroblasts of severe asthmatics

Khuloud Bajbouj; Rakhee K Ramakrishnan; Huda Alketbi; Lina Sahnoon; Jasmin Shafarin; Mahmood Y Hachim; Ronald Olivenstein; Qutayba Hamid

doi:10.4103/abhs.abhs_42_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 2 | Issue : 1 | Page : 13-22

Targeting KDM4B attenuates IL-13-mediated fibrosis in bronchial fibroblasts of severe asthmatics

Khuloud Bajbouj¹, Rakhee K Ramakrishnan¹, Huda Alketbi², Lina Sahnoon², Jasmin Shafarin², Mahmood Y Hachim³, Ronald Olivenstein⁴, Qutayba Hamid⁵
¹ Basic Medical Sciences, College of Medicine; Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
² Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
³ Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates
⁴ Department of Medicine, Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada
⁵ Basic Medical Sciences, College of Medicine; Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates; Department of Medicine, Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada

Date of Submission	26-Jun-2022
Date of Decision	13-Nov-2022
Date of Acceptance	22-Nov-2022
Date of Web Publication	14-Dec-2022

Correspondence Address:
Prof. Qutayba Hamid
Clinical Sciences Department, College of Medicine, University of Sharjah, Sharjah

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abhs.abhs_42_22

Abstract

Background: Asthma is a heterogeneous disorder characterized by chronic inflammation and remodeling of the airways. Asthma is mainly driven by type 2 immune responses, where interleukin-13 (IL-13) plays a key role in asthma pathogenesis. KDM4B/JMJD2B is an IL-13-regulated epigenetic modifier in asthmatic airway fibroblasts. Therefore, this study aimed to target KDM4B to potentially alleviate IL-13-mediated fibrosis in asthma.
Methods: Bronchial fibroblasts isolated from asthmatic individuals were stimulated with IL-13 and treated with JIB-04, a pan-selective inhibitor of histone demethylase(s). The expression of extracellular matrix (ECM) markers was assessed using quantitative real-time polymerase chain reaction, Western blotting, and matrix metalloproteinase (MMP) activity assay. Chromatin immunoprecipitation assay was used to determine the binding of KDM4B and H3K36me3 to promoter region of tissue inhibitor of metalloproteinase-2 (TIMP-2). KDM4B knockdown was performed to confirm its direct role on TIMP/MMP regulation.
Results: JIB-04 inhibited KDM4B activity by reducing the demethylation of its downstream target, H3K36me3, in asthmatic fibroblasts. Inhibition of KDM4B significantly affected the viability of the bronchial fibroblasts at 48 h. KDM4B inhibition was further associated with the downregulation of ECM proteins such as MMP-2, MMP-9, collagen-1, and fibronectin, and upregulation of TIMP-2, at both the gene and protein levels. This was accompanied by the inhibition of IL-13-mediated fibrotic response. JIB-04 further prevented KDM4B association and enhanced H3K36 binding with promoter region of TIMP-2 leading to its increased transcription. KDM4B knockdown further resulted in inducing TIMP-2 expression and inhibited MMP-9 activation.
Conclusion: Therapeutic targeting of KDM4B using JIB-04 is a promising candidate to alleviate IL-13-mediated responses in chronic disorders such as asthma.

Keywords: Asthma, fibrosis, interleukin-13, JIB-04, KDM4B

How to cite this article:
Bajbouj K, Ramakrishnan RK, Alketbi H, Sahnoon L, Shafarin J, Hachim MY, Olivenstein R, Hamid Q. Targeting KDM4B attenuates IL-13-mediated fibrosis in bronchial fibroblasts of severe asthmatics. Adv Biomed Health Sci 2023;2:13-22

How to cite this URL:
Bajbouj K, Ramakrishnan RK, Alketbi H, Sahnoon L, Shafarin J, Hachim MY, Olivenstein R, Hamid Q. Targeting KDM4B attenuates IL-13-mediated fibrosis in bronchial fibroblasts of severe asthmatics. Adv Biomed Health Sci [serial online] 2023 [cited 2023 Jun 9];2:13-22. Available from: http://www.abhsjournal.net/text.asp?2023/2/1/13/363792

Background

Asthma remains one of the most important noncommunicable diseases that affect people of all ages, ethnic groups, and regions. According to the World Health Organization 2019 estimates, asthma caused more than 400,000 deaths globally and over 80% of these asthma-related deaths occurred in low- and lower-middle-income countries.[1] There is no comprehensive data on the prevalence of asthma in the United Arab Emirates (UAE) to date. However, some cross-sectional studies have reported asthma prevalence in the UAE to range from 2.79% to 8%.[2]

The Global Initiative for Asthma defined asthma as a heterogeneous chronic inflammatory disease, characterized by a history of respiratory symptoms such as shortness of breath, wheeze, chest tightness, and cough that vary over time and in intensity, together with variable expiratory airflow limitation.[3] Asthma is mainly driven by type 2 immune responses, which account for about 90% of the cases in asthmatic children and 50% of those in asthmatic adults.[4] It includes an increase in airway eosinophils, T helper 2 (Th2) cells, and the secretion of interleukin-4 (IL-4), IL-5, and IL-13. Among these cytokines, IL-13, in particular, is a central mediator for the development of allergic airway disease. Initial studies in asthmatic animal models provided evidence that IL-13 is important and sufficient to induce all characteristics of allergic asthma.[5] In accordance, administration of IL-13 to nonimmunized T cell-deficient mice produced an asthma-like phenotype through an IL-4Rα-dependent pathway.[6] Furthermore, selective neutralization of IL-13 in a murine model of allergic asthma led to the reversal of airway hyperresponsiveness and inflammation.[7]

Subepithelial fibrosis in asthma is a characteristic of airway remodeling. Key proteases such as matrix metalloproteinases (MMPs) and their regulators, tissue inhibitor of metalloproteinases (TIMPs), play an important role in extracellular matrix (ECM) remodeling. MMPs are a large family of zinc-dependent peptidases produced by many inflammatory and epithelial cells, and TIMPs are a family of secreted proteases that inhibit MMP activity. These enzymes contribute to the regulation of progressive tissue repair, tissue remodeling, and morphological changes, seen in normal biological processes such as bone remodeling, and in disease conditions such as cancer.

Both MMPs and TIMPs work together to repair, replace, and restore normal tissue structure after injury. Upsetting this matrix homeostatic balance can affect the whole system leading to serious disease conditions. Indeed, fibrosis formation in the lung tissue is suggested as a consequence of an imbalance between the synthesis and degradation of ECM molecules due to an imbalance of MMPs and TIMPs.[8] An increase in MMP expression was associated with lung tissue fibrosis in idiopathic pulmonary fibrosis.[9] Furthermore, the absence of TIMP regulatory activity on MMPs leads to fibrosis. Among the MMPs, MMP-9 is largely implicated in asthma[10] and TIMP-1 is the major inhibitor of MMP-9. While MMP-9 and TIMP-1 levels are elevated in the sputum of patients with asthma,[11] an excess of TIMP-1 over MMP-9 was found to be associated with chronic airflow obstruction, possibly through airway fibrosis [11,12] and airway wall thickening[13] in asthmatic patients. Similarly, MMP-2 levels were elevated in asthmatics, particularly in those with severe asthma.[14] TIMP-2 complexes with MMP-2 in fibroblasts and is known to inhibit the activity of MMP-2.[15]

There are several evidence suggesting the importance of epigenetics in the regulation of immune response and inflammatory gene expression in allergic diseases such as asthma. In fact, Th2 cells are strongly regulated by DNA methylation and histone modifications at the Th2 locus control region.[16] Moreover, resting CD4 + T lymphocytes have methylated IL-4 locus and INF-γ locus, and when stimulated by allergen challenge, demethylation at these loci correlates to IL-4 expression in sensitized host.[17] In support, single exposure of primary airway epithelial cell cultures to IL-13 selectively induced long-lasting DNA methylation changes.[18] From a potential therapeutic viewpoint, targeting these epigenetic changes may lead to new therapeutic approaches, even in cases of corticosteroid-resistant asthma.

Using Gene Expression Omnibus, KDM4B/JMJD2B (Jumonji C domain containing), an epigenetic regulator, was found to be differentially expressed upon IL-13 treatment.[19] JMJD proteins are a family of transcription factors that cause histone demethylation at H3K4, H3K9, H3K27, H3K36, or H4K20, by hydroxylation of a lysine methyl group. JMJDs have an important role in biological processes such as proliferation, apoptosis, and inflammation. In support, overexpression of different JMJDs was found to be associated with multiple cancers, such as breast, colorectal, lung, and prostate cancer.

The relationship between JMJDs and MMPs that are involved in fibrosis is not clear or fully understood. The aim of the current study was to examine the association between KDM4B and MMPs in asthmatic bronchial fibroblasts as well as to investigate whether targeting the epigenetic modulator KDM4B may serve as a novel therapeutic approach in IL-13-mediated fibrosis in asthma.

Materials and Methods

Fibroblast cell culture

Human primary bronchial fibroblasts derived from endobronchial tissue specimens from nonsmoking subjects with severe asthma, where subjects were females with a mean age of 39.5 ± 6, were used in this study. These cell lines were provided by Quebec Respiratory Health Research Network (McGill University Health Centre/Meakins-Christie Laboratories Tissue Bank, Montreal, Canada). Cells were maintained in Dulbecco's Modified Eagle's Medium-high glucose (Sigma-Aldrich: with 4500 mg/L glucose, L-glutamine, and sodium bicarbonate) supplemented with 10% fetal bovine serum (FBS, Gibco®), 1% sodium pyruvate (Invitrogen) and 1% penicillin/streptomycin (Gibco®). In stimulation and treatment experiments, serum-starved media was used (DMEM media with 0.05% FBS). Cells were grown in a 37°C humidified incubator (Thermo Scientific HERAcell 150i Carbon Dioxide Incubator) containing 5% CO₂ and 21% oxygen. For stimulation experiments, the asthmatic fibroblasts were seeded in 6-well plate. At ~ 75% confluency, the cells were serum starved and treated with 10 ng/ml of IL-13 (ProSpec). These conditions were continued for 4 h, 24 h, and 48 h. In order to test the effect of JIB-04 drug treatment, the asthmatic fibroblasts were treated with 10 mg/ml JIB-04 solution (Tocris Bioscience) as a single dose (as a control) or in combination with IL-13. These conditions were present for 24 h.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay

Cell viability was determined in cells treated with JIB-04 by using the colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT], Sigma-Aldrich). 10³ cells were grown in 96-well plates with 0.2 mL culture media and were cultured in the presence or absence of JIB-04 for 24, 48, and 72 h. After that, MTT salt was added and kept for 2-h incubation in a humidified incubator at 37°C and 5% CO₂. The MTT formazan product was dissolved in dimethyl sulfoxide and absorbance reading was taken at 570 nm using a microplate reader.

Western blot

Western blotting was used to assess the protein expression of markers associated with ECM remodeling in asthma, such as MMP2, MMP9, TIMP1, TIMP2, collagen type 1, and fibronectin. Cells were lysed in ice-cold RIPA buffer (Abcam) containing protease inhibitor cocktail tablets (Sigma). Whole-cell lysates were quantified using the standard Bradford method (Bio-Rad). Lysate aliquots containing 30–50 μg of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was then blocked with 5% skimmed milk powder for 1 h at room temperature, washed with Tris-buffered saline with 0.1% Tween 20 Detergent (TBST), and reacted with primary immunoglobulin G (IgG) unlabeled primary antibodies; KDM4B (catalogue number, 8639, Cell Signaling), H3K36me3 (catalogue number, 4909, Cell Signaling) histones and ECM sampler kit (catalogue number, 9783 and 33437T, Cell Signaling), Matrix Remodeling Sampler Kit (catalogue number, 73959T, Cell Signaling), Tubulin (catalogue number, ab7291, Abcam), at 1:1000 dilution overnight at 4°C. The secondary (anti-mouse and anti-rabbit) antibodies (Cell Signaling) were then reacted with the membrane at 1:1000 dilution for 1 h at room temperature. Chemiluminescence was detected using the ECL kit (Thermo Scientific Pierce). Protein band quantification was carried out using the Bio-Rad Image Lab software (ChemiDoc™ Touch Gel and Western Blot Imaging System; Bio-Rad). Tubulin was used as a normalization control.

Quantitative real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the mRNA expression of markers associated with ECM remodeling in asthma, such as MMP2, MMP9, TIMP1, TIMP2, collagen type 1, and fibronectin. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's protocol. qPCR was performed using 1:1 of cDNA, specific primers for each gene as listed in [Table 1], SYBR^® Green I, and an iCycler Thermal Cycler. Expression levels of target human genes were normalized to GAPDH expression.

Table 1: List and sequence of primers used for quantitative real-time polymerase chain reaction

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Matrix metalloproteinase activity assay

Total MMP activity was measured using SensoLyte™ Generic MMP Assay Kit (AnaSpec) and specific MMP activity using MMP-2 and MMP-9 kits (Abcam). These colorimetric assays measure the amount of substrate degradation by MMP enzymatic activity. Briefly, to measure the MMP activity levels, supernatants of cells were collected after 24 h and 48 h. According to the manufacturer protocol, 10 μg/ml of Trypsin was added and incubated for 1.5 h. Trypsin inhibitor was then added at 100 μg/ml concentration for 15 min. MMP substrate solution with the test components was added to the microplate along with the controls. The plate was read at room temperature at 412 nm absorbance, and the data were recorded and analyzed.

Chromatin immunoprecipitation assay and real-time polymerase chain reaction

Chromatin immunoprecipitation (ChIP) assay was performed using SimpleChIP Plus Enzymatic Chromatin IP Kit according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA, USA). Briefly, cells were cross-linked with 1% formaldehyde for 15 min at room temperature and then treated with micrococcal nuclease to obtain fragments with a length of approximately 150–1000 base pairs. Fragmented chromatin was incubated with ChIP-grade antibodies (anti-KDM4B [cat # 8639] and H3K36me3 [cat #4909], Cell Signaling). Normal rabbit IgG was used as an internal negative control. Purified immune-precipitated chromatin, input chromatin, and control-IP (IgG) were subjected to PCR amplification for the candidate genes. The binding patterns were examined by qPCR as described above using the primer sequences [Table 1]. Precipitated DNA was analyzed by PCR for TIMP-2 promoter region-specific primers (F: 5′-TCATATGCCTGGGTCTTTCC-3′, R: 5′-GGGGGTGTGGTTACTGTGAA-3′),[20] p21 promoter which served as positive enrichment control (F: 5'-CGCGTTTGGATTCCGTG-3', R: 5'-CCAGTTATAATCTGCCTTCCCTATTATC-3'),[21] and GAPDH promoter which served as negative enrichment control (F: 5'-CGCGAGGATGCGTGTTC-3', R: 5'-CATTCACCTGCCGCAGAAA-3'). Immunoprecipitated chromatin binding affinity was normalized to that of input chromatin using the formula '2 – Ct _IP – Ct _input.' Binding affinity enrichment for the gene of interest or known positive control region was normalized using the formula: enrichment = gene of interest or positive control/negative control region.

KDM4B knockdown

Knockdown of KDM4B was performed using small interfering RNA (siRNA) following the manufacture's protocol (Santa Cruz Biotechnology). Briefly, 1 μl of KDM4B siRNA was mixed with 100 μl of transfection medium (solution A) and 6 μl of transfection reagent was diluted into another 100 μl of transfection medium (solution B). After mixing both solutions, the mixture was incubated for 50 min at RT. The mixture was then incubated with 0.8 ml of transfection medium, added to the cells, and incubated at 37°C for 24 h. Cells were afterward treated with the above-indicated conditions.

Statistical analysis

Each experiment was performed at least two times. The results are expressed as mean ± standard error of the mean. Unpaired two-tailed Student's t-test, one-way analysis of variance (ANOVA), or two-way ANOVA with Tukey's multiple comparison test was used to identify the significant differences between the groups. P < 0.05 was considered statistically significant. Data fitting and graphs were presented using the GraphPad Prism 8 software (San Diego, USA).

Results

Treatment of asthmatic fibroblasts with JIB-04, a KDM4B inhibitor

JIB-04 is a small-molecule inhibitor that blocks the demethylase activity of Jumonji histone demethylases (KDMs).[22] The effective dose of JIB-04 on asthmatic fibroblasts was tested using MTT viability assay in the absence and presence of IL-13. As shown in [Figure 1]a, treatment of fibroblasts with JIB-04 at different concentrations (0.5 μM, 1 μM, 5 μM, and 10 μM) had more effect on cell viability at 48 h of treatment and significantly at 0.5 μM concentration in comparison to the control. Cell viability was significantly reduced at 0.5 μM upon IL-13 induction at 48 h. Moreover, JIB-04 treatment hindered fibroblast adhesion capability, as shown in [Figure 1]b. Western blot was then performed to observe the methylation status of H3K36me3 histone protein upon JIB-04 treatment. KDM4B was stimulated upon IL-13 treatment and found to be associated with reduced methylation of H3K36me3 [Figure 1]c and [Figure 1]d, as reported previously.[19] However, after JIB-04 treatment, KDM4B protein levels were slightly altered when compared to the control; however, there was an increased methylation of H3K36me3 histone protein, which indicates that KDM4B activity was hindered rather than its expression following JIB-04 treatment, as observed in [Figure 1]c and [Figure 1]d.

Figure 1: (a) Assessment of fibroblast cell viability using MTT assay following treatment with different concentrations of JIB-04 in the absence or presence of IL-13 stimulation at 24, 48, and 72 h. (b) Asthma fibroblast morphology. Asthma fibroblasts were left untreated, stimulated with IL-13, treated with JIB-04 alone, or treated with JIB-04 and IL-13 for 48h. (c) Representative immunoblots depicting protein expression of KDM4B and H3K36me3 by Western blot on asthmatic fibroblasts with and without IL-13 induction where tubulin was used as a loading control. (d) Graphical representation of fold change in protein expression levels comparing fibroblasts with or without IL-13 stimulation in asthma fibroblasts based on two separate experiments. Graphical data are represented as mean ± SEM. *Statistically significant change (P < 0.05), **Statistically significant change (P < 0.01), ***Statistically significant change (P < 0.001) determined using two-way ANOVA between treatment groups versus untreated control. IL: Interleukin, SEM: Standard error of the mean, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

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Interleukin -13 regulates matrix metalloproteinase/tissue inhibitor of metalloproteinase balance in asthmatic fibroblasts and JIB-04 treatment alters matrix metalloproteinase/tissue inhibitor of metalloproteinase expression in interleukin-13-stimulated fibroblasts

Since IL-13 potentiated airway remodeling in a MMP-dependent manner in asthmatic airway fibroblasts,[23] we next assessed the expression of different MMPs and TIMPs in these fibroblasts upon IL-13 stimulation. In asthmatic fibroblasts, mRNA expression of MMP-2 and MMP-9 was significantly upregulated post-IL-13 stimulation [Figure 2]a and [Figure 2]b. While a mild increase in MMP-2 expression was noted upon IL-13 stimulation, a more significant upregulation of MMP-9 was observed after IL-13 induction [Figure 2]b. With regard to TIMP expression, while TIMP-2 was significantly downregulated after IL-13 stimulation [Figure 2]d, no reduction was observed for TIMP-1 following IL-13 stimulation [Figure 2]c.

Figure 2: Relative expression of mRNA (a-d) encoding MMP-2, MMP-9, TIMP-1, and TIMP-2, in asthmatic fibroblasts upon IL-13 induction, using qPCR. Expression levels of target human genes were normalized to GAPDH expression. Graphical data are presented as mean ± SEM based on two separate experiments. **Statistically significant change (P < 0.01), ***Statistically significant change (P < 0.001) determined using unpaired two-tailed Student's t-test between IL-13 treatment versus untreated control. IL: Interleukin, SEM: Standard error of the mean, MMP: Matrix metalloproteinase, TIMP: Tissue inhibitor of metalloproteinase.

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We next investigated the effects of JIB-04 on the mRNA expression of MMP-9 and TIMP-2 in asthmatic fibroblasts as they were most significantly affected by IL-13 treatment [Figure 2]. mRNA expression of MMP-9 was found to be highly upregulated in IL-13-stimulated asthmatic fibroblasts. With administration of JIB-04, MMP-9 level was significantly downregulated and reached a null value and a similar pattern was observed with IL-13 stimulation [Figure 3]a. Moreover, JIB-04 showed an opposite effect on TIMP-2 mRNA expression where IL-13 stimulated cells exhibited reduced TIMP-2 levels, and with JIB-04 treatment, it was upregulated, more so with combination of IL-13 stimulation [Figure 3]a.

Figure 3: Relative expression of mRNA encoding (a) MMP-9, (b) TIMP-2, (c) COL1, and (d) fibronectin in asthmatic fibroblasts, in the presence of IL-13 and JIB-04 (0.5 μM) treatments, using qPCR after normalization against GAPDH. (b) Relative protein expression of TIMP-1, TIMP-2, MMP-2, MMP-9, COL-1, and fibronectin by Western blot on asthmatic fibroblasts, in the presence of IL-13 and JIB-04 treatment. (c) Graphical data are represented as mean ± SEM. (d) Association between MMP-2/TIMP-1 or 2 and MMP-9/TIMP-1 or 2 ratios in asthmatic fibroblasts, in the presence of IL-13 and JIB-04 treatment. Enzymatic activity of MMPs using ELISA assay of total MMP activity (e), individual assay of MMP-9 activity (f), and individual assay of MM-P2 activity (g). Values represent the mean ± SEM, *Statistically significant change (P < 0.05), **Statistically significant change (P < 0.01), ***Statistically significant change (P < 0.001) determined using one-way ANOVA between treatment groups vs. untreated control based on three separate experiments. IL: Interleukin, SEM: Standard error of the mean, MMP: Matrix metalloproteinase, TIMP: Tissue inhibitor of metalloproteinase, qPCR: Quantitative real-time polymerase chain reaction.

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The effect of JIB-04 treatment was also examined on the mRNA expression of collagen type 1 (COL1) and fibronectin as asthmatic airways have increased deposition of the various collagen subtypes and fibronectin.[24] While IL-13 stimulated the expression of COL1 and fibronectin, an overall reduction in their expression was induced with JIB-04 treatment compared with their control counterpart [Figure 3]a.

It has been reported that patients with severe asthma had increased MMP-9 levels and activity in addition to disruption in the MMP-9/TIMP ratio. [11,25-29] Western blot was performed to detect the protein expression of MMPs, TIMPs, collagen-1, and fibronectin [Figure 3]b and [Figure 3]c. In IL-13-stimulated asthmatic fibroblasts, protein expression of TIMP-2 was found to be significantly reduced in comparison to the control. However, their protein expression was increased after JIB-04 treatment, even in IL-13 stimulated cells when compared to JIB-04 alone [Figure 3]b and [Figure 3]c. In addition, relative protein expression levels of MMP-9 and MMP-2 active forms were found to be significantly increased in IL-13-stimulated asthmatic fibroblasts and they decreased with JIB-04 treatment [Figure 3]b and [Figure 3]c. Moreover, protein expression of collagen-1 and fibronectin also significantly increased in IL-13-stimulated fibroblasts, and with JIB-04 treatment, the protein levels were highly reduced to almost no expression in the case of fibronectin [Figure 3]b and [Figure 3]c. In addition, IL-13 stimulation induced the most significant increase in MMP-9/TIMP-2 ratio among MMP-9/TIMP-1, MMP-2/TIMP-2, and MMP-2/TIMP-1 ratios in comparison to unstimulated cells. The IL-13-stimulated increase was reduced after treating the asthma fibroblasts with JIB-04 across all the tested ratios [Figure 3]d.

MMP activity was next measured by ELISA in order to assess the effect of JIB-04 in IL-13-stimulated asthmatic cells. We found that total MMP activity was enhanced in IL-13-stimulated cells compared to the control, and with the addition of JIB-04, total MMP activity reduced to below baseline level [Figure 3]e. Furthermore, JIB-04 treatment showed a more significant reduction of MMP-9 activity in IL-13-stimulated cells when compared to JIB-04 alone [Figure 3]g. In addition, MMP-2 activity also showed a significant reduction in unstimulated and IL-13-stimulated cells treated with JIB-04 [Figure 3]f. We, therefore, investigated further the impact of KDM4B inhibition in the regulation of MMP-9 and TIMP-2 expression.

JIB-04 inhibits KDM4B association and increases H3K36me3 binding at tissue inhibitor of metalloproteinase-2

Asthmatic fibroblasts stimulated with IL-13 were associated with significantly decreased expression of TIMP-2 [Figure 2]d, whereas JIB-04 treatment resulted in induction of TIMP-2 gene expression, especially after IL-13 stimulation [Figure 3]a. This effect was accompanied by an alteration in KDM4B activity evidently shown by reduced demethylation of its downstream target, H3K36me3 [Figure 1]b, which raised the probability that KDM4B might be recruited to mediate histone demethylation events that silence TIMP-2 gene. To this end, ChIP assay was performed to pull down KDM4B and H3K36me3 proteins and analyze TIMP-2 gene enrichment. IL-13 stimulation of asthmatic fibroblasts for 6 h resulted in increased binding of KDM4B and decreased HK36 enrichment at TIMP-2 promoter as compared to controls [Figure 4]a and [Figure 4]b. Contrarily, JIB-04 treatment resulted in detachment of KDM4B and increased H3K36me3 binding on TIMP-2 promoter, as shown in [Figure 4]a and [Figure 4]b. GAPDH promoter was utilized as a negative enrichment control and p21 promoter was included as a positive enrichment control [Figure 4]c in this assay.

Figure 4: TIMP-2 promoter activity and ChIP assay. (a) ChIP assay showing the interaction of (a) KDM4B and (b) H3K36me3 on TIMP-2 gene in asthmatic fibroblasts, in the presence of IL-13 and JIB-04 treatment. Fibroblasts were immunoprecipitated using antibodies to KDM4B, H3K36me3, and IgG. (c) The p21 promoter was used as positive enrichment control and GAPDH promoter was used as a negative enrichment control. **Statistically significant change (P < 0.01), ***Statistically significant change (P < 0.001) determined using two-way ANOVA between treatment groups versus untreated control based on two separate experiments. IL: Interleukin, TIMP: Tissue inhibitor of metalloproteinase, ChIP: Chromatin immunoprecipitation, IgG: Immunoglobulin G.

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KDM4B knockdown induces H3K36me3 and tissue inhibitor of metalloproteinase-2 expression and decreases matrix metalloproteinase-9 activation

In order to demonstrate the specific role of KDM4B in inducing fibrosis in asthma, KDM4B siRNA was used to knock down its expression. The efficacy of KDM4B knockdown was confirmed by Western blotting, which shows a 50% reduction in total protein levels [Figure 5]a, [Figure 5]b, [Figure 5]c. KDM4B knockdown resulted in upregulation of H3K36 trimethylation. IL-13 stimulation did not show a similar reduction of H3K36 trimethylation in KDM4B-depleted cells when compared to cells upon IL-13 addition alone. Furthermore, TIMP-2 levels were significantly increased even after IL-13 stimulation in KDM4B-depleted cells. This effect was accompanied with reduction in formation of active MMP-9 protein which indicates inhibition of its activation [Figure 5]a, [Figure 5]b, [Figure 5]c.

Figure 5: Relative protein expression of (a and b) KDM4B, H3K36me3, TIMP-2, and MMP-9 by Western blot on asthmatic fibroblasts, in the presence of IL-13 and JIB-04 treatment after KDM4B and scrambled siRNA knockdown. (c) Graphical data are represented as mean ± SEM, **Statistically significant change (P < 0.01), ***Statistically significant change (P < 0.001) determined using two-way ANOVA between treatment groups versus untreated control based on two separate experiments. Representative immunoblots depicting protein levels in asthma fibroblasts where tubulin was used as a loading control. IL: Interleukin, SEM: Standard error of the mean, MMP: Matrix metalloproteinase, TIMP: Tissue inhibitor of metalloproteinase.

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Discussion

The dysregulation of the histone demethylase KDM4B, a well-known epigenetic modulator, was recently reported in bronchial fibroblasts as well as bronchial biopsies from asthma patients.[19] IL-13 stimulation further induced the expression and activity of KDM4B, rendering it as a potential therapeutic target to ameliorate subepithelial fibrosis in asthma. Therefore, in this study, we aimed to target KDM4B using the pan-selective small molecule inhibitor, JIB-04, that inhibits the demethylase activity of the Jumonji family of histone demethylases.[22] As such, JIB-04 regulates the histone methylation levels on target genes with potential to block growth signaling in cultured cells. In addition to inhibiting the activity of KDM4B and its downstream demethylation of H3K36, JIB-04 was associated with the reduced expression of multiple ECM markers, such as MMP-2, MMP-9, collagen-1, and fibronectin, and increased expression of TIMP-2 in asthmatic fibroblasts. Inhibition of KDM4B also prevented IL-13-induced fibrotic response in these cells.

JIB-04 significantly reduced the viability of asthmatic fibroblasts, as early as 24 h and as low a concentration as 0.5 μM [Figure 1a]. However, the optimal reduction was demonstrated with 0.5 μM of JIB-04 at 48 h. Treatment with 0.5 μM of JIB-04 for 48 h was capable of inhibiting cell viability despite IL-13 induction. Therefore, this dosing regimen was chosen as optimal for further experiments. JIB-04 treatment was reported to affect multiple cellular signaling pathways, including the Wnt/β-catenin signaling that is involved in the proliferation and maintenance of cells. [30,31] This may explain the reduced cell viability observed in our study. However, the JIB-04-induced reduction in cell viability was relinquished within 72 h, which may indicate either the reduced half-life of JIB-04 or activation of compensatory signaling pathways that enabled cell recovery from drug effect.

Being a pan-selective inhibitor of Jumonji enzymes, KDM4B showed moderate sensitivity to JIB-04.[22] This was observed in our study as well, where KDM4B protein expression was moderately altered with JIB-04 treatment [Figure 1b]. However, JIB-04 potently inhibited the H3K36 demethylation activity of KDM4B, particularly in the presence of IL-13.

IL-13 is central to the pathogenesis of asthma and has been associated with increased bronchial hyperresponsiveness, activation of fibroblasts, ECM production, goblet cell differentiation, and antibody switching from IgM to IgE.[32] Since TIMP-1 and TIMP-2 inhibit the activities of all known MMPs,[33] they, together with their main targets MMP-9 and MMP-2, were investigated in our study. IL-13 being a key mediator of airway remodeling, it boosted the expression of MMP-2 and MMP-9, while suppressing the expression of TIMP-2 [Figure 2]. The balance between MMPs and TIMPs is skewed in asthma with increased MMP/TIMP ratio favoring tissue injury. The suppression of elastin expression in asthmatic airway fibroblasts by IL-13 was mediated by MMPs, in particular MMP-1 and MMP-2.[23] In addition, IL-13 stimulated increased production of collagen type 1 and an invasive phenotype in asthmatic airway fibroblasts in an MMP-dependent manner. [34,35] As such, MMP-2 inhibition blocked transforming growth factor-beta signaling and IL-13-induced collagen type-1 production in airway fibroblasts. Since TIMP-2 is known to block MMP-2 activity, the IL-13-induced downregulation of TIMP-2 observed in our study may boost MMP-2 signaling. MMPs are thus, important mediators of IL-13-induced extracellular remodeling in asthma.

While MMP-2 and MMP-9 are both gelatinases with similarity in their structure, domain organizations, and substrate profiles, they differ in their tissue expression and the chemokine gradient profile that they control (PMID: 11686860). As opposed to the constitutive expression of MMP-2 in the airways of naïve and allergen-challenged mice (PMID: 11887181), MMP-9 expression was induced in mouse lung during allergic inflammation (PMID: 15059974). This spatiotemporal regulation of MMP-2 and MMP-9 in lung tissue may be responsible for their differential induction by IL-13 as observed in our study.

Our results demonstrated the potent inhibition of IL-13-induced fibrotic response in asthmatic fibroblasts by JIB-04, both at the gene and protein levels [Figure 3], [Figure 4], [Figure 5]. The increased expression of total MMP, MMP-2, MMP-9, collagen-1, and fibronectin induced by IL-13 was effectively suppressed by JIB-04, while IL-13-induced downregulation of TIMP-2 was reversed in the presence of JIB-04. These results suggest the ability of JIB-04 to restore the MMP-TIMP balance to some extent in asthmatic fibroblasts. JIB-04 may thus contribute to ameliorating IL-13-induced airway remodeling in asthma.

Methylation of H3K36 is usually associated with gene activation.[36] Since KDM4B is responsible for the demethylation of H3K36, we also investigated the DNA interaction of KDM4B and H3K36me3 using the ChIP assay. We observed increased binding of KDM4B to the promoter region of TIMP-2 with IL-13 stimulation and this was inhibited in the presence of JIB-04 that was accompanied with increased H3K36me3 association [Figure 4a]. Thus, our results suggest a plausible mechanism by which KDM4B suppresses TIMP-2 expression. The effect of JIB-04 in boosting KDM4B detachment resulting in increased trimethylation of H3K36 at the TIMP-2 promoter site may be responsible for enhancing TIMP-2 expression and as a result suppressing collagen and fibronectin expression. Finally, our results demonstrated that KDM4B depletion resulted in inducing H3K36me3 that resulted in upregulating TIMP-2 expression and inhibiting MMP-9 activation [Figure 5].

Despite the widespread effects of IL-13 on almost all aspects of asthma pathobiology, the disappointing results in clinical trials of biologicals targeting IL-13 urge the search for alternative approaches to target IL-13 signaling in asthma. Epigenetic regulation of gene expression in asthma in gaining attention in recent times and its regulation of asthma pathobiology makes it an attractive therapeutic approach. JIB-04 thus serves as an epigenome-modifying tool, and it would be interesting to explore further the effects of this drug against the various hallmark features of asthma, including airway remodeling and airway inflammation.

[TAG:2]Conclusion[/TAG:2]

As shown in [Figure 6], the pro-fibrotic phenotype induced by the increased KDM4B activity in asthmatic fibroblasts was effectively suppressed by JIB-04. Therefore, early inhibition of KDM4B activity may reduce the progression or even prevent the development of subepithelial fibrosis in the airways of asthmatic individuals.

Figure 6: Schematic illustration of the epigenetic role of KDM4B in subepithelial fibrosis in asthma. (a) IL-13 stimulation of asthmatic fibroblasts promotes a pro-fibrotic microenvironment by increasing the activity of the epigenetic modifier, KDM4B, which is responsible for demethylating H3K36 at the TIMP-2 promoter site resulting in reduced TIMP-2 expression. (b) Inhibition of KDM4B using JIB-04 has the potential to reduce fibrotic activity by increasing the trimethylation of H3K36 and subsequent DNA binding, thereby promoting TIMP-2 expression. Created with BioRender.com. IL: Interleukin, TIMP: Tissue inhibitor of metalloproteinase.

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Study limitations

Some of the limitations of our study include the sample size of fibroblasts used to demonstrate the role of KDM4B in IL-13-mediated fibrotic expression in asthma, as variability between patients is often observed in primary cultured cells. However, we included fibroblasts from nonsmokers alone to reduce the confounding variables. In addition, the underlying anti-proliferative effect of JIB-04 on asthmatic fibroblasts is yet to be explored. Furthermore, the lack of in vivo data is another shortcoming of our study and we aim to further validate our ex vivo data using a KDM4B-deficient asthmatic mouse model.

Ethical policy and institutional review board statement

The samples used in this study were collected as part of the original study that was conducted in accordance with the Declaration of Helsinki, and approved by the MUHC Research Ethics Board with reference number 2003-1879 and BMB-02-039-t.

Acknowledgments

We would like to thank Mr. Abdalla Eltayeb for staining lung tissues.

Authors' contributions

KB and QH conceived the research concept; KB developed the experimental design. KB, HD, LS, JS, and MH performed the experiments. KB, QH, and RO acquired resources. KB and RKR prepared the first draft and all authors reviewed and approved final draft of the manuscript. All authors are responsible for the contents and integrity of this manuscript.

Data availability statement

No dataset was utilized in this study.

Financial support and sponsorship

This work was supported by the University of Sharjah Competitive Grant, Ref. number: 1901090263.

Conflict of interests

QH is an editor of the Advances in Biomedical and Health Sciences Journal. No conflict of interests declared.

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