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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 17  |  Issue : 1  |  Page : 47-56

Histone deacetylase-2 inducer like theophylline has a potential to improve glucocorticoid responsiveness in severe uncontrolled asthma by reducing P-glycoprotein/MRP-1


1 Department of Biotechnology, Amity Institute of Biotechnology, Amity University, Lucknow Campus; Department of Pulmonary Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Pulmonary Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Biotechnology, Amity Institute of Biotechnology, Amity University, Lucknow Campus, Lucknow, Uttar Pradesh, India
4 Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission03-May-2021
Date of Decision01-Sep-2021
Date of Acceptance12-Oct-2021
Date of Web Publication15-Mar-2022

Correspondence Address:
Zia Hashim
Department of Pulmonary Medicine, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow - 226 014, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/injr.injr_85_21

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  Abstract 


Background: Epigenetic factors play an essential role in regulating the action of steroids in asthma. Steroids suppress inflammation by recruiting histone deacetylase-2 (HDAC-2), which removes the acetyl group of the nuclear factor kappa B-enabled inflammatory gene complex and disables its action. P-glycoprotein (P-gp)/Multidrug resistance-associated protein 1 (MRP-1), the energy-dependent efflux pumps, reduces the bioavailability of steroids in the cell by pumping it out. The expression of P-gp/MRP-1 and HDAC-2 has been linked to regulating the action of steroids in asthma. Therefore, the use of HDAC-2 inducer like theophylline might improve asthma control by improving steroid responsiveness.
Objective: HDAC-2 inducer, theophylline, can improve glucocorticoid responsiveness in severe uncontrolled asthma (UA).
Materials and Methods: We classified subjects into severe UA (n = 7, age: 52.36 ± 11.94 years), controlled asthma (CA) (n = 23, age: 50.65 ± 16.11 years), and treatment-naïve patients (n = 10, age: 50.60 ± 14.38 years). Peripheral blood mononuclear cells (PBMCs) were isolated and treated with theophylline (1 μM) and Trichostatin-A (TSA) (0.8 μM) for 48 h, for induction/suppression of HDAC-2, respectively. The messenger RNA (mRNA) expression extracted from PBMCs was studied for HDAC-2and P-gp/MRP-1 by quantitative real-time polymerase chain reaction.
Results: The mRNA expression of P-gp/MRP-1 was higher, whereas HDAC-2 mRNA levels were significantly lower in UA at baseline. Exposure to theophylline reduced mRNA expression of P-gp/MRP-1 (fold change: 2.57/2.15 in UA; 1.27/1.26 in CA, respectively, P < 0.0001), but the HDAC-2 mRNA expression increased (fold change: 5.56 in UA; 6.85 in CA, respectively, P < 0.0001). However, TSA treatment resulted in higher mRNA levels for P-gp/MRP-1 (fold change: 7.45/7.26 in UA; 3.34/3.29 in CA, respectively, P < 0.0001), whereas it significantly lowered expression for HDAC-2(fold change: 1.39 in UA; 2.46 in CA, respectively, P < 0.0001).
Conclusion: HDAC-2 inducer theophylline has a potential to induce response against steroid resistance in severe UA.

Keywords: Histone deacetylase-2, P-glycoprotein/MRP-1, severe uncontrolled asthma, steroid responsiveness, theophylline, trichostatin A


How to cite this article:
Mishra R, Hashim Z, Gupta M, Chaturvedi R, Singh H, Nath A, Misra DP, Khan A, Rai MK, Srivastava S, Chaturvedi S, Agarwal V. Histone deacetylase-2 inducer like theophylline has a potential to improve glucocorticoid responsiveness in severe uncontrolled asthma by reducing P-glycoprotein/MRP-1. Indian J Rheumatol 2022;17:47-56

How to cite this URL:
Mishra R, Hashim Z, Gupta M, Chaturvedi R, Singh H, Nath A, Misra DP, Khan A, Rai MK, Srivastava S, Chaturvedi S, Agarwal V. Histone deacetylase-2 inducer like theophylline has a potential to improve glucocorticoid responsiveness in severe uncontrolled asthma by reducing P-glycoprotein/MRP-1. Indian J Rheumatol [serial online] 2022 [cited 2022 Oct 1];17:47-56. Available from: https://www.indianjrheumatol.com/text.asp?2022/17/1/47/338614




  Introduction Top


Asthma is a chronic respiratory disease that occurs all over the globe, affecting millions of people irrespective of age and gender. In 2019, asthma affected around 262 million people and caused 461,000 deaths.[1] The prevalence of asthma in India is lower than in the West. The largest study from India INSEARCH, done in 2012, reports a prevalence of 2.05%, approximating 17.23 million adults >15 years of age.[2],[3] It causes substantial morbidity, and despite the continued efforts to develop a standardized approach to management and availability of effective drugs, it remains a significant cause of mortality.[4] Asthma is now recognized to have different phenotypes based on differences in airway inflammation and response to various therapeutic agents. The inflammation is centered around eosinophils in most asthma patients, but a subset of patients also has neutrophilic inflammation. The management of asthma is a combination of long-acting beta-2 agonists (LABA) and inhaled corticosteroids (ICSs). However, one-fifth of asthma remains uncontrolled even on the highest dose of ICSs is termed “difficult to treat,” which may be because of inadequate compliance or comorbidities.[5] However, about 5%–10% of cases continue to have severe symptoms despite the maximum ICS and LABA combination dose, even with proper adherence to maximum optimized therapy and control of comorbidities.[5],[6],[7] This group is referred to as “Severe Asthma.” A study from the Netherlands estimated that 3.6% (95% confidence interval, 3.0% to 4.1%) of asthmatics had severe asthma, representing 10.4 patients per 10,000 inhabitants.[8] Such cases need a detailed evaluation of the role of epigenetic factors and asthma phenotyping. They are managed with second-line treatments including short bursts of oral steroids, monoclonal antibodies against immunoglobulin E, interleukin-5 (IL-5), IL-5 receptor, IL-4 receptor, IL-13, and if significant remodeling has already occurred, bronchial thermoplasty.[6],[7] However, frequent oral glucocorticoids, even in low dose bursts, lead to considerable side effects. The role of biologics and bronchial thermoplasty is limited by the high cost and availability, especially in developing countries [Figure 1].
Figure 1: Severe asthma versus difficult-to-treat asthma. Broad classification of asthma and basic phenotypes: Atopic asthma, nonatopic eosinophilic asthma, and neutrophilic asthma. Allergens are presented to dendritic cells, which lead to Th-2 cell activation. Th-2 cells are the principal driver of eosinophilic inflammation by interleukin-4, interleukin-5, and interleukin-13. TH2 cells also modulate goblet cell overexpression, increased mucus secretion, and airway hyper-responsiveness, fibrogenic function, airway remodeling. Interleukin-4 drives Th-2 cell differentiation and production of downstream cytokines, including interleukin-5 and interleukin-13. IL 5 causes the differentiation and maturation of eosinophils, basophils, and mast cells. interleukin-8 affects the differentiation of neutrophils. Neutrophilic asthma phenotype is usually glucocorticoid resistant

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The role of pharmacogenomic factors in severe uncontrolled asthma (UA) is being investigated mainly in cases depicting features of steroid-resistant asthma.[9] Some mechanisms for glucocorticoid resistance are decreased number of Glucocorticoid Receptors (GRs), defect in nuclear translocation of GRs, dysfunction of GRs, increased expression of pro-inflammatory transcription factors, altered expression of molecules such as histone deacetylase-2 (HDAC-2), P-glycoprotein (P-gp), and multidrug resistance-associated protein 1 (MRP-1), smoking and oxidative stress, and recurrent viral infections.[9],[10],[11],[12] The increased expression of inflammatory genes in inflammatory lung diseases is regulated by the acetylation of core histones. Corticosteroids suppress inflammatory genes in asthma by inhibiting histone acetyltransferase and recruiting HDAC-2 to NF-kB-activated inflammatory gene complex to deacetylate the acetylated glucocorticoid receptor.[10] Reduced expression of HDAC-2 has been observed as one of the plausible mechanisms for steroid resistance in various diseases including chronic obstructive pulmonary disease (COPD) and asthma.[9] The upregulation of P-gp and Multidrug resistance-associated protein-1 (MRP-1) modulates steroid's pharmacokinetics.[11] P-gp is an efflux protein whose primary function is to efflux out the xenobiotic, including steroids, to the outside of cells. Overexpression of P-gp has been associated with steroid nonresponsiveness. Furthermore, its increased functionality has been linked with reduced action of steroids in steroid-resistant nephrotic syndrome (SRNS).[11],[13] Similarly, P-gp has been observed to be overexpressed in COPD patients, unresponsive to corticosteroids.[14] Our previous study observed that P-gp and HDAC-2 were in a reciprocal relationship in patients with asthma, and HDAC-2 was upregulated in steroid-responsive patients compared to steroid nonresponsive patients[15] [Figure 2].
Figure 2: Relationship of glucocorticoids to histone deacetylase-2. Glucocorticoids (GCs), upon entering the cell, connect with the Glucocorticoid Receptor (GCR) found in the cytosol. Subsequently, the GC-GCR complex comes within the nucleus and attaches to the Glucocorticoid Receptor Elements (GREs) found in the promoter regions of different genes. Based on its binding to either positive GREs or negative GREs, gene transactivation or transrepression has occurred. In another transrepression pathway, the GC-GCR complex causes the recruitment of histone deacetylase-2, which binds specific genes to GRE and results in the deacetylation of the MDR-1 gene, other pro-inflammatory genes. Thus, histone deacetylase-2inhibitors (Trichostatin A) may suppress steroid response, whereas histone deacetylase-2 stimulators (Theophylline). Other mechanisms of glucocorticoid resistance are also shown

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MRP-1 is another protein from the same family as P-gp, which has also been reported to be overexpressed in many diseases with steroid-resistant phenotypes including asthma.[16] In one of our recent studies, we had observed a significant role of MRP-1 overexpression in steroid-resistant asthma.[17] It has recently been reported that MRP-1 homozygous mutant G2677T/A promotes steroid resistance by inducing P-gp expression.[11] The overexpression of HDAC-2 significantly reduces the expression of P-gp, and MRP-1 causing a reversal of multidrug resistance including steroid resistance.[18] The previously reported reciprocal relationship between P-gp and HDAC-2 in Asthma and interdependence of P-gp and MRP-1 has stimulated the evaluation of the interaction between P-gp/MRP-1 and HDAC-2 in patients with UA. The present study aimed to compare the expression of HDAC-2 and P-gp/MRP-1 in patients with UA and CA. We also evaluated the effect of the use of HDAC-2 stimulator and inhibitor on the expression of these biomolecules.


  Materials and Methods Top


Patient recruitment

We jointly conducted a prospective observational study over 2 years in the Department of Clinical Immunology and Rheumatology and the Department of Pulmonary Medicine at a tertiary care hospital in North India. The Institutional Ethics Committee, Human Research (Ethics Cell No: 2017/2/IMP/95) approved the study. Patients with a diagnosis of asthma were prospectively screened as per the GINA guidelines, 2016.[19] Patients were included if their symptoms were uncontrolled on high-dose ICS/LABA, even after proper control of comorbidities and adequate compliance. Patients on another controller medication such as tiotropium or short courses of low-dose oral glucocorticoids were also included. The level of asthma control was defined by the absence of (i) daytime symptoms more than twice a week, (ii) any nighttime awakening, (iii) a requirement of reliever medication more than twice a week, or (iv) any activity limitation because of asthma in the past 4 weeks. Asthma was labeled as Severe UA if either three or four criteria were present despite being on 800 mcg of budesonide or 1000 mcg of fluticasone, with adequate compliance and proper inhalational technique and control of other comorbidities such as COPD overlap or obstructive sleep apnea (GINA score 3 or 4).[6],[19] Patients who were candidates for biologic therapy such as omalizumab were also included. The patients who had none of these symptoms on their usual medications were labeled as CA. The patients with (i) partially CA (GINA score 1 or 2), (ii) difficult-to-treat asthma because of poor compliance or other comorbidities such as COPD overlap or obstructive sleep apnea, (iii) history of smoking, (iv) presence of comorbidities such as cardiac dysfunction, chronic renal and hepatic diseases, neurological disorders, and other chronic lung diseases other than asthma were excluded. Forty patients were included as the study participants after obtaining informed written consent. The enrolled patients were subgrouped into severe UA, CA, and treatment-naïve patients (Baseline).

Treatment-naïve patients were considered as the baseline group who had received no treatment and were followed up after 4 weeks of ICS therapy. Patients had an initial detailed evaluation of their symptoms and a documented physical examination. The spirometry with bronchodilator reversibility testing was performed for all the patients at their baseline visit to establish the diagnosis of severe asthma and was also repeated after 4 weeks of treatment with ICSs.

Peripheral blood mononuclear cells isolation and processing

Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood and diluted 1:1 with 1XPBS. PBMCs were isolated over Histopaque-1077 (Sigma, St. Louis, MO 63103, USA) and washed three times. Cells were resuspended at 1 × 106 cells/ml in RPMI 1640 (Sigma-Aldrich, 3050 Spruce Street, Saint Louis, MO 63103, USA). Briefly, PBMCs were isolated by density gradient centrifugation on Ficoll-Histopaque.[20]

Chemicals

The chemicals and reagents for the processing of PBMCs were acquired from reputed legal firms. RNA-iso Plus was acquired from Takara Bio Inc., Nojihigashi, Kusatsu, Japan. cDNA Synthesis kit (Cat No-K1632) had been purchased from Thermo Fisher Scientific Inc., Bartlesville, OK, USA. Light-Cycler® 480 SYBR Green I Master was purchased from Roche Diagnostics, Indianapolis, IN, USA.

Reagents

Roswell Park Memorial Institute (RPMI) Media (Cat No-R6504), Sodium bicarbonate, and Sodium Pyruvate were purchased from Sigma, St Louis, MO, USA 100X Antibiotic-Antimycotic (Cat No-15240062) and Fetal Bovine Serum (FBS, Cat No-10270106) were purchased from Gibco-Grand Island, NY, USA. Dimethyl sulfoxide (DMSO, Cat No-D2650) and [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT, Cat No-M5655) were purchased from Sigma, St Louis, MO, USA.

Cytotoxicity assay

The cytotoxicity was executed using the standard 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl 18 tetrazolium bromide (MTT) test. The effect of theophylline and TSA on the proliferative ability of PBMCs was quantified by mitochondrial-dependent reduction of tetrazolium dye, MTT to insoluble purple formazan. Briefly, PBMCs were seeded in 96-well plates at 20 × 103 cells per well in RPMI-1640 growth medium with 10% FBS and incubated overnight at 37°C in a humidified atmosphere containing 5% CO2. The culture medium of each well was scrapped and replaced by the modern media on the 3rd day. On the 4th day, the cells were synchronized with a low serum medium for 24 h. After synchronization, the cells were incubated with TSA for 24 h. After incubation at 37°C at 5% CO2 for 24 h, the medium was discarded, and the MTT solution was applied to each well (final concentration, 500 μg/ml) after 3 h of incubation at 37°C. The supernatant was withdrawn, and 150 μl of fresh DMSO was added to each well. The solution was read at 570 nm with a microplate reader (Bio-Rad 550; Bio-Rad, Japan).

Cell culture conditions and stimulation of cells

PBMCs were isolated and cultured for 24 h to monitor the expression levels of P-gp and MRP-1. PBMCs were maintained at a concentration of 2.5 × 106 cells/ml in the RPMI-1640 medium with 10% FBS and 1% penicillin/streptomycin/amphotericin-B at 37°C in a humidified atmosphere with 5% CO2 for a duration of 24 h for stable development. The viability percentage for theophylline at 1 μM and TSA at 0.8 μM was 80%, and the use of higher concentrations resulted in reduced viability. Therefore, we used a concentration of 1 μM for theophylline and 0.8 μM for TSA. Cells were subsequently treated with 1 μM theophylline and 0.8 μM TSA for a time of 48 h. After 48 h of incubation, cultures were moved to 1.5 ml of centrifugal tubes and centrifuged onto pellet cells. Cell pellets were used as a source for the extraction of RNA messenger RNA (mRNA) levels of P-gp/MRP-1 were quantified in real-time polymerase chain reaction (PCR), normalized to housekeeping glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Real-time study of quantitative chain polymerase

RNA was isolated from PBMCs using RNA-iso Plus (Trizol Method). A total of 1 μg RNA was processed for cDNA synthesis using the cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Bartlesville, OK, USA) as per the manufacturer's protocol. Real-time PCR reactions were conducted for each triplicate cDNA sample using Light-Cycler® 480 SYBR Green I Master and gene-specific primer pairs for P-gp, HDAC-2, and GAPDH. The PCR cycle was as follows: 50°C for 2 min for one process, 95°C for 10 min for one cycle, 95°C for 15 s, and 60°C for 1 min for 40 cycles for the LC480 light cycle (Roche, U. S. A.). Semi-quantitative real-time PCR data for each target gene were expressed as 2–ΔCt relative quantitation versus endogenous GAPDH, with error bars reflecting the mean, standard error for triplicate reactions, and data, defined as fold transition.

Statistical analysis

The continuous data were represented in mean ± standard deviation, analyzed by independent t-test and ANOVA. The constant data were considered as normally distributed if SD ≤ 1 / 2 of the mean value. We analyzed the using SPSS 23.0 statistical package (SPSS Inc., Chicago, IL, USA). The association was determined by binary logistic regression analysis. The statistical significance was considered as P < 0.05.


  Results Top


After the screening of forty patients who presented to the OPD with a diagnosis of asthma, out of these, 23 had CA (age: 50.65 ± 16.11 years, male = 14) had CA. In contrast, seven (age: 52.36 ± 11.94 years, male = 4) had severe UA, whereas 10 (age: 50.60 ± 14.38 years, male = 6) were treatment naïve (baseline). The demographic and biochemical characteristics of these groups of patients were compared and are presented in [Table 1] and [Table 2]. The difference in the gender distribution among the study groups was found to be statistically significant (P = 0.045). Similarly, as expected, the differences in the daytime symptoms (>twice a week, and ≤ twice a week) (P < 0.001), need for rescue medication (< twice or no, ≥ twice a week) (P < 0.001), presence of nighttime symptom on awakening (yes/no) (P < 0.001), and any limitation in activity (yes/no) (P < 0.001) were found to be statistically significant between UA as compared to the CA and baseline groups (P < 0.001).
Table 1: Demographic and clinical detail of controlled asthma and uncontrolled asthma patient

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Table 2: Demographic and clinical detail of baseline and uncontrolled asthma patient

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P-glycoprotein/MRP-1 and histone deacetylase-2 expression in response to the steroid treatment

P-gp/MRP-1 mRNA expression was significantly lower in patients with CA than patients UA [Figure 3] and [Figure 4]. The HDAC-2 mRNA expression was significantly higher in the CA group than in the UA [Figure 5].
Figure 3: P-glycoprotein messenger RNA gene expression. The first bar depicts the baseline P-glycoprotein distribution in both patients. P-glycoprotein expression in PBMCs of Controlled asthma is represented by the second bar. P-glycoprotein expression in PBMCs of uncontrolled asthma patients is represented by the third bar. The messenger RNA levels of P-glycoprotein were measured using a real-time polymerase chain reaction technique in peripheral blood mononuclear cells at baseline, during controlled, and in uncontrolled patients. Three different tests were used to create the results. The mean, standard error of the mean is used to reflect the combined results from all of the experiments P < 0.05 significant deviations from the test group

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Figure 4: MRP-1 messenger RNA gene expression. The first bar depicts the baseline MRP-1distribution in both patients. MRP-1 expression in PBMCs of controlled asthma is represented by the second bar. MRP-1 expression in PBMCs of uncontrolled asthma is represented by the third bar. The messenger RNA levels of MRP-1 were measured using a real-time polymerase chain reaction technique in peripheral blood mononuclear cells at baseline, during controlled, and in uncontrolled patients. Three different tests were used to create the results. The mean, standard error of the mean is used to reflect the combined results from all of the experiments. P < 0.05 showed significant deviations from the test group

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Figure 5: Histone deacetylase-2 messenger RNA gene expression. The first bar depicts the baseline histone deacetylase-2 distribution in both patients. Histone deacetylase-2 expression in PBMCs of controlled asthma is represented by the second bar. Histone deacetylase-2expression in PBMCs of uncontrolled asthma is represented by the third bar. The messenger RNA levels of histone deacetylase-2were measured using a real-time polymerase chain reaction technique in peripheral blood mononuclear cells at baseline, during controlled, and in uncontrolled patients. We used three different tests to create the results. The mean, standard error of the mean is used to reflect the combined results from all of the experiments. P < 0.05 showed significant deviations from the test group

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P-glycoprotein/MRP-1 and histone deacetylase-2 expression in response to exposure with histone deacetylase stimulator (theophylline) and inhibitor (trichostatin A)

The PBMCs from the patients in UA and CA groups were cultured with theophylline (HDAC stimulator) to assess the effect on P-gp/MRP-1 mRNA expression. Compared to the baseline values, theophylline exposure decreased the mRNA levels of P-gp/MRP-1 (fold change 2.57/2.15, *P < 0.0001 in UA) (fold change 1.27/1.26, *<0.0001 in CA) [Figure 6]. The expression of HDAC-2 mRNA increased at a maximum concentration of 1 μM of theophylline (fold change 5.56, *P < 0.0001 in UA) (fold change 6.85, *P < 0.0001 in CA) as compared to the baseline values [Figure 6]. Further, we determined whether HDAC inhibition would modulate P-gp/MRP-1 levels. Compared to the baseline values, TSA (HDAC inhibitor) upregulated the mRNA expression of P-gp/MRP-1 (fold change 7.45/7.26, *P < 0.0001 in UA) (fold change 3.34/3.29, *P < 0.0001 in CA) [Figure 7]. The HDAC-2 mRNA expression was downregulated with exposure to TSA [Figure 7] (fold change 1.39, *P < 0.0001 in UA) (fold change 2.46, *P < 0.0001 in CA) relative to the baseline values. Our results show that inhibition of HDAC contributes to enhanced expression of P-gp/MRP-1 in UA and CA patients. Therefore, these results suggest that HDAC-2 might have a reciprocal relationship with P-gp/MRP-1.
Figure 6: Effect of the histone deacetylase-2stimulator theophylline on P-glycoprotein, MRP-1, and histone deacetylase-2expression levels in CA and severe UA patients (a and b). Quantitative real-time polymerase chain reaction of P-glycoprotein, MRP-1 (a), and histone deacetylase-2 (b) in peripheral blood mononuclear cells of severe UA and CA in patients treated with different concentrations of theophylline (0, 0.01, 0.1, and 1.0 μM) for 48 h. The experiments represent three separate series. The pooled data for all experiments are represented as mean ± SEM. Significant differences in control are indicated by *P < 0.05

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Figure 7: Effect of the histone deacetylase-2 inhibitor Trichostatin A on P-glycoprotein, MRP-1, and histone deacetylase-2 expression levels in CA and severe UA patients (a and b). P-glycoprotein, MRP-1, and histone deacetylase-2quantitative real-time polymerase chain reaction in severe UA and CA patient's peripheral blood mononuclear cells treated with varying concentrations of Trichostatin A (0, 0.2, 0.4, and 0.8 M) for 48 h. Three different tests were used to create the results. The mean SEM is used to describe the combined results from all of the experiments. *P = 0.05 significant deviations from the control group

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  Discussion Top


Although severe asthma is about 5%–10% of total asthma cases, it still consumes about half of the financial burden because of asthma.[12] Asthma is an inflammatory disease with multiple phenotypes. Major clinically relevant phenotypes are atopic asthma, nonatopic eosinophilic asthma, and neutrophilic asthma. Steroid resistance is a subset of severe asthma, usually neutrophilic, because of several factors. If the resistance is reversed, a significant number of severe asthma patients can be managed cost effectively. The management of severe asthma because of steroid resistance is quite challenging.[4],[6],[19] Hence, the role of inhibitors and inducers of epigenetic factors: P-gp/MRP1 and HDAC-2, in severe, UA was explored in this study.[9],[10],[11] In this study, we have reported an increased expression of P-gp/MRP-1 and reduced expression of HDAC 2 in PBMCs of patients with UA, compared to CA, same as for the mRNA expression levels of HDAC2 was lower and P-gp/MRP-1 was higher in patients with asthma.[15] The exposure to the inducer of HDAC-2 had a downregulating impact on P-gp/MRP-1. In contrast, the use of an inhibitor of HDAC-2 had an upregulating effect on P-gp/MRP-1, implying a reciprocal interaction between HDAC-2 and P-gp/MRP-1. Steroids are one of the primary substrates for P-gp/MRP1, and their overexpression may reduce the bioavailability of steroids to cells, which may lead to a lack of response to steroid therapy.[21]

P-gp is present in many tissues including the intestine, blood–brain barrier, liver, thyroid, and PBMCs, macrophages, dendritic cells, and T and B lymphocytes in differing amounts.[22],[23] In one study, Wasilewska et al. reported that P-gp overexpression in CD3 lymphocytes was associated with insufficient steroid response.[23] The overexpression of P-gp in cases with poorly CA has been reported in earlier studies. We have reported similar findings in our previous work.[15],[23] MRP-1 is also expressed by most tissues, with high levels found in lungs, testis, kidneys, skeletal muscle, and PBMCs.[24],[25],[26] MRP-1 plays a vital role in delivering medication into pulmonary cells and protecting against exogenous and endogenous harmful chemicals entering the lungs.[27],[28] The previous studies on MRP-1 have substantiated its role in determining steroid responsiveness in lung diseases such as COPD and asthma.[17] In their research, Youssef et al. have reported a substantial increment in MDR-1 gene expression in peripheral blood lymphocytes and serum IL2r levels in patients with UA. relative to CA.[29]

Funaki et al. also found an increase in MDR-1 gene expression in PBMCs in patients who did not respond to steroids or relapsed after steroid therapy.[30] These findings were also supported by the evidence from one of our recent works on the subject. We had also observed that MRP-1 expression was significantly higher in UA patients than in patients with CA.[17] Furthermore, in this study, the mRNA gene expression of HDAC-2 was decreased in PBMCs of UA patients relative to CA patients and controls (treatment naïve patients). Decreased HDAC-2 gene expression has been associated with increased gene expression of the pro-inflammatory cytokine genes[31],[32] through NF-kappa B-mediated pathway, thereby affecting the response to anti-inflammatory drugs such as steroids.[10] In a study by Xu et al., it was seen that knockdown of HDAC1/HDAC-2 genes correlated with increased expression of P-gp, MRP-1, and MRP-2 in cell lines obtained from human colorectal adenocarcinomas (HCT-8 and HCT-118). It was also observed that the increased expression of HDAC1/HDAC-2 decreases gene expression of P-gp/MRP-1.[18] The findings from this study advocate the results of our study showing reduced P-gp/MRP-1 expression with HDAC stimulation and an inverse relationship between HDAC-2 expression and P-gp/MRP-1. The additional highlight of our research is the use of cultured PBMCs from patients with CA and UA rather than cell lines. To study the interaction between HDAC-2 and P-gp/MRP-1 expression, we had cultivated the PBMCs of CA and UA patients with HDAC-2 inducer (Theophylline) and inhibitor (Trichostatin A). We observed that exposure to the HDAC-2 inducer restored the expression of HDAC-2 and downregulated the expression of P-gp and MRP-1, while the use of HDAC-2 inhibitor had an opposite impact. Previous observations suggest that HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) and TSA increased the expression of P-gp and its encoding gene ABCB1.[33] On the contrary, HDAC-2 inducers such as theophylline have been confirmed to reverse glucocorticoid tolerance through modulation of HDAC-2 in macrophages of COPD and asthma patients.[34] The molecular mechanisms involved in inhibiting HDAC-2-mediated upregulation of P-gp/MRP-1 expression are not well known. There are, however, attempts to shed light on the mechanisms involved. A study on the placental cells showed that HDAC-2 was involved in transcriptional suppression of P-gp; even the dissociation of HDAC-2 from the promoter region could lead to recruitment of P300, nuclear transcription factor-Y, and P300/CBP-associated factor to the promoter region, which could lead to induction of P-gp.[35] This may be the mechanism involved even in asthma under which HDAC-2 regulates the expression of P-gp and MRP-1, leading to steroid resistance. The findings of our study have a potential for clinical implication. The current management plan for UA may consider the use of immunosuppressive drugs such as calcineurin inhibitors, azathioprine, and mycophenolate mofetil, which are potentially toxic.[25],[26] Compared to these toxic drugs, theophylline or other inducers of HDAC-2 seem to be much safer and easier to use. Thus, inducers of HDAC-2 may have the potential to contribute to the therapeutics of the UA patients and may lead to better management of steroid-resistant asthma patients. This research has a translational potential as well since the medications targeting P-gp such as calcineurin inhibitors (cyclosporine-A and tacrolimus), metformin, ketoconazole, proton pump inhibitors (omeprazole, lansoprazole, and others), and verapamil may have the potential to modulate P-gp expression and may therefore be beneficial in the treatment of severe UA patients. Tacrolimus binds explicitly and inhibits P-gp and decreases the IL-2 regulated MDR-1 gene expression.[36] This may be the explanation why steroid-resistant patients may respond to tacrolimus and cyclosporine therapy.[37] There is also a scope of using sustained-release preparation of theophylline and phosphodiesterase (PDE)-4 inhibitor, roflumilast, in steroid-resistant asthma.[38] Furthermore, to the best of our knowledge, this is a novel study, in which we have measured the expression of P-gp/MRP-1 in patients with CA and severe UA, along with the documentation of response to HDAC-2 stimulation/inhibition on these molecules. However, the present study has a few limitations. Because of the limitation of the duration of the study period, the sample size is small with inadequate gender distribution. Using cultured PBMCs is one highlight of our work. However, using a more representative cell line like the bronchoalveolar lavage fluid cells (BALF cells) would have made this work more meaningful. However, as it was unethical to perform bronchoscopy on patients with CA, BALF cell analysis could not be performed. Lack of protein expression evaluation and long-term follow-up are some of the additional limitations of our study.


  Conclusion Top


HDAC-2 inducer theophylline might improve asthma and control by improving steroid responsiveness in UA by reducing P-gp/MRP-1 and is reciprocally related to HDAC-2, P-gp/MRP-1 suppression. Research into the molecular mechanism of steroid resistance and other targets for steroid resistance in severe asthma is essential. It is crucial for developing novel, affordable drugs that may reduce the cost of treatment effect as newer methods such as monoclonal antibodies and bronchial thermoplasty are available at a high cost and selected centers only. The investigators should further explore the role of other PDE-4 inhibitors like roflumilast.

Acknowledgments

Ravi Mishra is a registered Ph. D. student from Amity Institute of Biotechnology, Amity University, Uttar Pradesh, Lucknow Campus-226028, India, with Registration number: A7130716004. We acknowledge Amity University for its support.

Financial support and sponsorship

This work received funds from the Intramural Grant (PGI/DIR/RC/1057/2017) of Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, Indian Council of Medical Research, New Delhi, India. (67/25/2019/IMM-BMS), funded Ravi Mishra with SRF for this study.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
GBD 2019 Diseases and Injuries Collaborators. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020;396:1204-22.  Back to cited text no. 1
    
2.
Jindal SK. Burden of asthma in India. Int J Tuberc Lung Dis 2013;17:145.  Back to cited text no. 2
    
3.
Jindal SK, Aggarwal AN, Gupta D, Agarwal R, Kumar R, Kaur T, et al. Indian study on epidemiology of asthma, respiratory symptoms and chronic bronchitis in adults (INSEARCH). Int J Tuberc Lung Dis 2012;16:1270-7.  Back to cited text no. 3
    
4.
Mauer Y, Taliercio RM. Managing adult asthma: The 2019 GINA guidelines. Cleve Clin J Med 2020;87:569-75.  Back to cited text no. 4
    
5.
Chung KF, Wenzel SE, Brozek JL, Bush A, Castro M, Sterk PJ, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J 2014;43:343-73.  Back to cited text no. 5
    
6.
Global Initiative for Asthma. Global Strategy for the Diagnosis and Prevention. Global Initiative for Asthma. Available form: https://ginasthma.org'gina-reports. [Last accessed on 2021 Jun 01].  Back to cited text no. 6
    
7.
Agnihotri NT, Saltoun C. Acute severe asthma (status asthmaticus). Allergy Asthma Proc 2019;40:406-9.  Back to cited text no. 7
    
8.
Hekking PW, Wener RR, Amelink M, Zwinderman AH, Bouvy ML, Bel EH. The prevalence of severe refractory asthma. J Allergy Clin Immunol 2015;135:896-902.  Back to cited text no. 8
    
9.
Barnes PJ. Corticosteroid resistance in patients with asthma and chronic obstructive pulmonary disease. J Allergy Clin Immunol 2013;131:636-45.  Back to cited text no. 9
    
10.
Barnes PJ. Histone deacetylase-2 and airway disease. Ther Adv Respir Dis 2009;3:235-43.  Back to cited text no. 10
    
11.
Prasad N, Singh H, Jaiswal A, Chaturvedi S, Agarwal V. Overexpression of P-glycoprotein and MRP-1 are pharmacogenomic biomarkers to determine steroid resistant phenotype in childhood idiopathic nephrotic syndrome. Pharmacogenomics J 2021;21:566-73.  Back to cited text no. 11
    
12.
Ito K, Mercado N. Therapeutic targets for new therapy for corticosteroid refractory asthma. Expert Opin Ther Targets 2009;13:1053-67.  Back to cited text no. 12
    
13.
Singh H, Agarwal V, Chaturvedi S, Misra DP, Jaiswal AK, Prasad N. Reciprocal relationship between HDAC2 and P-glycoprotein/MRP-1 and their role in steroid resistance in childhood nephrotic syndrome. Front Pharmacol 2019;10:558.  Back to cited text no. 13
    
14.
Berg T, Hegelund-Myrbäck T, Öckinger J, Zhou XH, Brännström M, Hagemann-Jensen M, et al. Expression of MATE1, P-gp, OCTN1 and OCTN2, in epithelial and immune cells in the lung of COPD and healthy individuals. Respir Res 2018;19:68.  Back to cited text no. 14
    
15.
Mishra R, Chaturvedi R, Hashim Z, Nath A, Khan A, Gupta M, et al. Role of P-gp and HDAC2 and their reciprocal relationship in uncontrolled asthma. Curr Pharm Biotechnol 2021;22:408-13.  Back to cited text no. 15
    
16.
Yoshioka M, Sagara H, Takahashi F, Harada N, Nishio K, Mori A, et al. Role of multidrug resistance-associated protein 1 in the pathogenesis of allergic airway inflammation. Am J Physiol Lung Cell Mol Physiol 2009;296:L30-6.  Back to cited text no. 16
    
17.
Mishra R, Hashim Z, Nath A, Singh H, Agarwal V, Chaturvedi R. Role of MRP–1 in the regulation of steroid responsiveness in uncontrolled asthma. Int J Pharm Sci & Res 2020;11:6260-66. doi: 10.13040/IJPSR.0975-8232.11(12).6260-66.  Back to cited text no. 17
    
18.
Xu Y, Jiang Z, Yin P, Li Q, Liu J. Role for class I histone deacetylases in multidrug resistance. Exp Cell Res 2012;318:177-86.  Back to cited text no. 18
    
19.
Global Initiative for Asthma.Global Strategy for the Diagnosis and Prevention. Global Initiative for Asthma. Available from: https://ginasthma.org. [Last accessed on 2021 Jun 01].  Back to cited text no. 19
    
20.
Sahaf B, Atkuri K, Heydari K, Malipatlolla M, Rappaport J, Regulier E, et al. Culturing of human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease. Proc Natl Acad Sci U S A 2008;105:5111-6.  Back to cited text no. 20
    
21.
Lama G, Luongo I, Tirino G, Borriello A, Carangio C, Salsano ME. T-lymphocyte populations and cytokines in childhood nephrotic syndrome. Am J Kidney Dis 2002;39:958-65.  Back to cited text no. 21
    
22.
Klimecki WT, Futscher BW, Grogan TM, Dalton WS. P-glycoprotein expression and function in circulating blood cells from normal volunteers. Blood 1994;83:2451-8.  Back to cited text no. 22
    
23.
Fung KL, Pan J, Ohnuma S, Lund PE, Pixley JN, Kimchi-Sarfaty C, et al. MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer. Cancer Res 2014;74:598-608.  Back to cited text no. 23
    
24.
Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992;258:1650-4.  Back to cited text no. 24
    
25.
Flens MJ, Zaman GJ, van der Valk P, Izquierdo MA, Schroeijers AB, Scheffer GL, et al. Tissue distribution of the multidrug resistance protein. Am J Pathol 1996;148:1237-47.  Back to cited text no. 25
    
26.
Stride BD, Valdimarsson G, Gerlach JH, Wilson GM, Cole SP, Deeley RG. Structure and expression of the messenger RNA encoding the murine multidrug resistance protein, an ATP-binding cassette transporter. Mol Pharmacol 1996;49:962-71.  Back to cited text no. 26
    
27.
Gustavsson L, Bosquillon C, Gumbleton M, Hegelund-Myrbäck T, Nakanishi T, Price D et al. Drug transporters in the lung: expression and potential impact on pulmonary drug disposition. In: Drug transporters: role and importance in ADME and drug development. The Royal Society of Chemistry, Cambridge, 2016;1: pp 184-228.  Back to cited text no. 27
    
28.
Kuzuya Y, Adachi T, Hara H, Anan A, Izuhara K, Nagai H. Induction of drug-metabolizing enzymes and transporters in human bronchial epithelial cells by beclomethasone dipropionate. IUBMB Life 2004;56:355-9.  Back to cited text no. 28
    
29.
Youssef DM, Elbehidy RM, Abdelhalim HS, Amr GE. Soluble interleukine-2 receptor and MDR1 gene expression levels as inflammatory biomarkers for prediction of steroid response in children with nephrotic syndrome. Iran J Kidney Dis 2011;5:154-61.  Back to cited text no. 29
    
30.
Funaki S, Takahashi S, Wada N, Murakami H, Harada K. Multiple drug-resistant gene 1 in children with steroid-sensitive nephrotic syndrome. Pediatr Int 2008;50:159-61.  Back to cited text no. 30
    
31.
Schnaper HW. The immune system in minimal change nephrotic syndrome. Pediatr Nephrol 1989;3:101-10.  Back to cited text no. 31
    
32.
Barnes PJ. Targeting the epigenome in the treatment of asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 2009;6:693-6.  Back to cited text no. 32
    
33.
Wang H, Huang C, Zhao L, Zhang H, Yang JM, Luo P, et al. Histone deacetylase inhibitors regulate P-gp expression in colorectal cancer via transcriptional activation and mRNA stabilization. Oncotarget 2016;7:49848-58.  Back to cited text no. 33
    
34.
Cosio BG, Tsaprouni L, Ito K, Jazrawi E, Adcock IM, Barnes PJ. Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. J Exp Med 2004;200:689-95.  Back to cited text no. 34
    
35.
Kim SN, Kim NH, Lee W, Seo DW, Kim YK. Histone deacetylase inhibitor induction of P-glycoprotein transcription requires both histone deacetylase 1 dissociation and recruitment of CAAT/enhancer binding protein beta and pCAF to the promoter region. Mol Cancer Res 2009;7:735-44.  Back to cited text no. 35
    
36.
Belliard AM, Tardivel S, Farinotti R, Lacour B, Leroy C. Effect of hr-IL2 treatment on intestinal P-glycoprotein expression and activity in Caco-2 cells. J Pharm Pharmacol 2002;54:1103-9.  Back to cited text no. 36
    
37.
Gulati S, Prasad N, Sharma RK, Kumar A, Gupta A, Baburaj VP. Tacrolimus: a new therapy for steroid-resistant nephrotic syndrome in children. Nephrol Dial Transplant 2008;23:910-3.  Back to cited text no. 37
    
38.
Reddy AT, Lakshmi SP, Banno A, Reddy RC. Glucocorticoid receptor α mediates roflumilast's ability to restore dexamethasone sensitivity in COPD. Int J Chron Obstruct Pulmon Dis 2020;15:125-34.  Back to cited text no. 38
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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