CC BY
6# Research Results in Pharmacology
Research Results in Pharmacology 8(1): 59-70 UDC: 616.24-036.12:615.214:615.357 DOI 10.3897/rrpharmacology. 8.75467
3 Research Article
Nortriptyline overcomes corticosteroid resistance in NK and NKT-like cells from peripheral blood of patients with chronic obstructive pulmonary disease
Aliaksei G. Kadushkin1, Anatoli D. Tahanovich1, Lyudmila V. Movchan2, Volha V. Dziadzichkina1, Olga V. Levandovskaya3, Tatsiana V. Shman2
1 Belarusian State Medical University, Dzerzhinski Ave., 83, Minsk, 220116Belarus
2 Republican Scientific and Practical Center for Pediatric Oncology, Hematology and Immunology, Frunzenskaya St., 43, Borovlyany, Minsk district, 223053 Belarus
3 Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology, Semashko St., 8, Minsk, 220045 Belarus Corresponding author: Aliaksei G. Kadushkin (kadushkyn@gmail.com)
Academic editor: Tatyana Pokrovskaia ♦ Received 18 September 2021 ♦ Accepted 12 January 2022 ♦ Published 31 March 2022
Citation: Kadushkin AG, Tahanovich AD, Movchan LV, Dziadzichkina VV, Levandovskaya OV, Shman TV (2022) Nortriptyline overcomes corticosteroid resistance in NK and NKT-like cells from peripheral blood of patients with chronic obstructive pulmonary disease. Research Results in Pharmacology 8(1): 59-70. https://doi.org/10.3897/rrpharmacology.8.75467
Abstract
Introduction: An antidepressant nortriptyline potentiates glucocorticoid (GC) action with synergistic suppression of inflammatory mediator release, but the precise molecular mechanism is unknown.
Materials and methods: Peripheral blood cells from patients with chronic obstructive pulmonary disease (COPD) (n = 21) were incubated with nortriptyline (1 ^M or 10 pM), budesonide (10 nM), or their combinations, followed by stimulation with phorbol myristate acetate (PMA) and ionomycin. Cytokine production, glucocorticoid receptor p (GRP), histone deacetylase 2 (HDAC2) and histone H4 acetylation of K8 (HAT) expression, p65 NF-kB and p38 mi-togen-activated protein kinase (p38 MAPK) phosphorylation in NK (CD3-CD56+) and NKT-like (CD3+CD56+) cells were analyzed by flow cytometry.
Results: We observed that nortriptyline (10 ^M) significantly attenuated the effects of PMA/ionomycin on the synthesis of interferon y (IFNy), interleukin 4 (IL-4), and IL-8, expression of GRp and HAT, as well as p65 NF-kB and p38 MAPK phosphorylation in NK and NKT-like cells, whereas nortriptyline (1 ^M) only inhibited IL-4 production by NK and NKT-like cells.
Discussion: The combination of nortriptyline (10 ^M) and budesonide decreased IFNy, tumor necrosis factor a, IL-4, IL-8, and GRp expression, as well as phosphorylated p38 MAPK and p65 NF-kB levels by NK and NKT-like cells above that of budesonide alone. Furthermore, the same association of drugs enhanced HDAC2 expression in NK and NKT-like cells.
Conclusion: Collectively, our results show that nortriptyline might enhance GC function through modulation of HAT, HDAC2, GRp, phospho-p38 MAPK expression. These data provide a strong rationale for combining nortriptyline with budesonide to treat COPD.
Keywords
COPD, HDAC2, nortriptyline, p38 MAPK, p65 NF-kB, NK cells, NKT-like cells, steroid resistance.
Copyright Kadushkin AG et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation that progressively rises and accompanied by inflammatory response in the airways. Globally, this disease affects 384 million people, while about 65 million patients have moderate to severe COPD (Vogelmeier et al. 2017). The burden of COPD is expected to increase over the coming decades due to the unceasing exposure to risk factors and population aging.
Cigarette smoking is recognized as the main environmental risk factor for COPD. Smoking cessation is an effective approach to slow down the disease progression and reduce COPD-related mortality (Tashkin 2015). However, resolution of inflammation is perceived not to occur even after elimination of this risk factor (Willemse et al. 2005; Ström et al. 2018). Cigarette smoking induces oxidative stress and subsequent activation of the transcription factor NF-kB. This transcription factor stimulates the expression and production of proinflammatory cytokines and chemokines by activated macrophages, airway epithelial cells, neutrophils and lymphocytes (Kadushkin and Taganovich 2016).
Glucocorticoids (GCs), drugs with anti-inflammatory mechanism of action, are widely used to treat COPD, however their therapeutic benefits are unassertive (Leung and Sin 2018). Reduced steroid responsiveness is a feature of COPD and a barrier to its effective treatment. Molecular mechanisms contributing to steroid resistance include altered expression of glucocorticoid receptor (GR) ß, macrophage migration inhibitory factor, histone deacetylase 2 (HDAC2), p38 mitogen-activated protein kinase (p38 MAPK) as a consequence of oxidative stress (Kadushkin and Taganovich 2016). In patients with COPD, a reduced steroid sensitivity of natural killer (NK) and natural killer T (NKT) cells has been reported (Hodge and Hodge 2019). The percentages of these lymphocytes are increased in the airways of COPD patients (Hodge et al. 2013).
NK cells are effector cells of the innate immune system and exhibit their effects indirectly through interaction with dendritic cells, macrophages, T-lymphocytes and en-dothelial cells. NK cells induce apoptosis of autologous lung epithelial cells via the secretion of perforin and gran-zyme B (Freeman et al. 2014; Abel et al. 2018). In patients with COPD, there is enhanced cytotoxicity of NK cells in the airways, as well as increased expression of granzyme B by NK cells in blood and bronchoalveolar lavage fluid (Hodge et al. 2013). Moreover, these lymphocytes are an important source of cytokines and chemokines such as interferon y (IFNy), tumor necrosis factor a (TNFa), interleukin 4 (IL-4), IL-8, and others (Abel et al. 2018).
Similar to NK cells, NKT cells are among the first cells, which respond to antigens entering the body. They regulate immune response by altering the activity of T helper cells and cytotoxic T lymphocytes, NK cells, regulatory T lymphocytes, dendritic and myeloid cells (Krijgsman et al. 2018). These cells synthesize T helper type 1 (Th1)-spe-cific (TNFa and IFNy) and Th2-associated (IL-4 and IL-13) cytokines, as well as other cytokines and chemokines (Krijgsman et al. 2018). In patients with COPD, an increased
natural cytotoxicity of NKT cells against autologous structural lung cells was revealed (Freeman et al. 2014), indicating their involvement in the pathogenesis of COPD.
Steroid resistance in patients with COPD prompted clinicians and scientists to search for the drugs that can enhance the anti-inflammatory effects of GCs. Experimental studies made possible to assume that nortriptyline is able to overcome steroid insensitivity (Mercado et al. 2011). Nortriptyline is a tricyclic antidepressant and the main active metabolite of amitriptyline, which is also a tricyclic antidepressant. Nortriptyline is used for the treatment of depression and anxiety as well as for smoking cessation in patients with COPD (Tselebis et al. 2016; Howes et al. 2020). Two independent studies have demonstrated a synergy between GCs and nortriptyline in reducing proinflammatory cytokine release from peripheral blood mononuclear cells (PBMCs) of healthy subjects and human monocytic U937 cells (Lehar et al. 2009; Mercado et al. 2011). However, the effects of nortriptyline have not been evaluated in cells of COPD patients, and the molecular mechanism of nortriptyline action on cells treated with corticosteroids is still not fully clear.
In this study, the aim was to examine anti-inflammatory efficacy of nortriptyline, alone and in combination with GC budesonide, on the production of cytokines by blood NK and NKT-like cells of patients with COPD and to investigate possible molecular mechanisms underlying the combine action of these drugs.
Materials and methods
Study participants
A total of 21 patients with COPD were recruited in the study (table 1). COPD was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (Vogelmeier et al. 2017). Participants were former or current smokers with at least a 10-year smoking history, a ratio of forced expiratory volume in 1 second to forced vital capacity (FEVj/FVC) < 0.70, and FEVj% predicted < 80%. We defined former smokers as those subjects who had quit smoking for more than 6 months. Patients were excluded if they had a clinical diagnosis of asthma or other active lung disease, allergy, blood coagulation disorders, or an exacerbation of COPD within 6 weeks of study entry. We also excluded subjects taking regular oral corticosteroids. Approval for this study was obtained from the ethics committee of Belarusian State Medical University, Minsk, Belarus (Protocol no. 8, January 21, 2019). All subjects gave their written informed consent.
Cell stimulation
Heparinized peripheral blood was collected from all study subjects and samples were processed less than 1 hour after venipuncture. Whole blood was mixed in sterile tubes with equal volume of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn Scientific,
Ebsdorfergrund, Germany). Cells were then treated in the presence or absence of nortriptyline (1 and 10 ^M, Sigma-Aldrich, St. Louis, MO, USA) and budesonide (10 nM, Glentham Life Sciences Ltd, Corsham, Wiltshire, UK), or with their combinations for 1 hour. After drug incubations, leukocytes were stimulated with phorbol my-ristate acetate (PMA, 50 ng/mL) (Cayman Chemical, Ann Arbor, Michigan, USA) and ionomycin (1 ^g/mL) (Cayman Chemical, Israel), as previously outlined (Kadushkin et al. 2021). Brefeldin A (10 ^g/mL) (Cayman Chemical, Israel) was added to inhibit cytokine secretion. Cells were co-incubated with the drugs and stimulants in a humidified 5% CO2/95% air atmosphere at 37 °C for 6 hours. To remove adherent cells, 100 ^L of ice-cold 20 mM EDTA was then added to the culture tubes for 10 minutes.
Table 1. Summary of patient characteristics, smoking history, and spirometry
Characteristics of participants COPD patients
Patients, n 21
Sex ratio, male/female 17/4
Age, years 65.1±1.5
Body mass index, kgm-2 26.7±1.2
Smoking status (active/former) 10/11
Smoking, pack-years 32.5±2.4
Post-bronchodilator FEVt (% predicted) 52.4±4.1
Post-bronchodilator FEV/FVC ratio (%) 57.0±2.5
Note: Data are presented as n or mean ± SEM. FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity.
Cytokine and HDAC2 expression in NK and NKT-like cells
Following stimulation as described above, cells were stained with anti-human CD3 PE-DyLight 594, CD56 PE-Cy7 and CD45 APC-Cy7 monoclonal antibodies (Exbio, Prague, Czech Republic) for 15 min in the dark at room temperature. Cells were centrifuged at 500*g for 5 min, supernatant discarded, 3 mL of wash buffer (5% FCS in CellWASH (BD, Poland)) was then added and the tubes centrifuged. After decanting supernatant, leukocytes were fixed using 100 ^L of Reagent A (Fixation Medium from FIX & PERM Cell Fixation & Cell Permeabilization Kit, Life Technologies, Bleiswijk, Netherlands). Following washing (as above), the cells were permeabilized using 100 ^L of Reagent B (Permeabilization Medium from FIX & PERM Cell Fixation & Cell Permeabilization Kit, Life Technologies). After blocking Fc receptors with human Fc block (BD Pharmingen, San Jose, CA, USA) for 10 min in the dark at room temperature, monoclonal antibodies to IL-4 PE, IFNy PE, TNFa PE (all from Beckman Coulter), IL-8 FITC (R&D systems Europe, Abingdon, UK), or HDAC2 Alexa Fluor 488 (Abcam, Cambridge, UK) were added to the cells. Appropriate isotype-mat-ched controls were used in all experiments. Cells were incubated with intracellular antibodies in the dark for 15 minutes at room temperature, followed by washing and fixation in 1% paraformaldehyde (500 ^l). Fixed preparations were stored at 4 °C in the dark and analyzed within
12 hours. Cells were examined using Navios flow cyto-meter (Beckman Coulter, Brea, CA, USA) and data were analyzed using Kaluza Analysis software.
NK cells were identified as CD3-CD56+ events. Additionally, we referred CD3+CD56+ cells as NKT-like cells. This designation of CD3+CD56+ cells is due to the feature of T lymphocytes to express CD56, marker of NK and NKT cells (Krijgsman et al. 2018). Unfortunately, we did not evaluate, whether CD3+CD56+ cells were CD1d-restricted, although it is required for assignment of these cells to the population of true NKT cells.
GR, GRp and histone acetyltransferase (HAT) expression in NK and NKT-like cells
To determine expression of GRp and acetylated lysine antibody in NK and NKT-like cells, aliquots of blood were stimulated and fixed with Reagent A (Fixation Medium from FIX & PERM Cell Fixation & Cell Permeabilizati-on Kit) as described above. After washing in wash buffer, cell cytoplasmic membrane was permeabilised using Reagent B (Permeabilization Medium from FIX & PERM Cell Fixation & Cell Permeabilization Kit), and anti-GRp polyclonal antibody (GeneTex, Irvine, CA, USA) or an-ti-Histone H4 (acetyl K8) monoclonal antibody (Abcam, Cambridge, UK) were added to the tubes. Cells were incubated with antibodies in the dark at room temperature for 15 minutes, and were further washed and stained with goat anti-rabbit IgG H&L Alexa Fluor 488 (Thermo Fisher Scientific, Eugene, Oregon, USA) for 15 minutes in the dark at room temperature. To determine expression of GR, cells were stained with appropriately diluted anti-GR mAb (Abcam) for 15 minutes, followed by washing and staining with goat anti-mouse IgG H&L Alexa Fluor 488 (Abcam) for 15 minutes in the dark at room temperature. After the final wash, cells were resuspended in 1% para-formaldehyde and data were acquired as described above.
p38 MAPK and p65 NF-kB phosphorylation in NK and NKT-like cells
After incubation ofblood cells with budesonide and nortrip-tyline for 1 hour as described above, appropriately diluted CD3 PE-DyLight 594, CD56 PE-Cy7 and CD45 APC-Cy7 monoclonal antibodies (Exbio) were added for 15 minutes in the dark at room temperature. Blood cultures were then stimulated with PMA (250 ng/mL) and ionomycin (1 ^g/ mL) for 15 minutes at 37 °C. To lyse red blood cells and fix leukocytes, 200 ^l of blood were mixed with 4 mL of pre-warmed to 37 °C BD Phosflow Lyse/Fix Buffer (BD Biosciences, San Diego, CA, USA). The tubes were then incubated in a 37 °C water bath for 10 minutes. Leukocytes were spun down at 500*g for 5 minutes and washed once with CellWASH, followed by permeabilization with pre-cooled at -20 °C 1 mL of BD Phosflow Perm Buffer III (BD Biosciences, San Jose, CA, USA) for 30 minutes on ice. After washing twice with BD Pharmingen Stain Buffer (BD Biosciences), the cells were stained with PE mouse anti-p38 MAPK (pT180/pY182) and PE mouse anti-NF-
kB p65 (pS529) (BD Biosciences) for 1 hour in the dark at room temperature. Isotype controls were run with each set of samples. Following a final wash, cells were resuspended in 500 ^l of BD Pharmingen Stain Buffer and analyzed within 2 hours on a Navios flow cytometer using Kaluza Analysis software (Beckman Coulter, Brea, CA, USA).
Statistical analysis
Data were analyzed using GraphPad Prism 7.00 (Graph-Pad Software Inc., San Diego, CA, USA) and expressed as mean ± standard error of mean (SEM). Results were assessed for a Gaussian distribution using Shapiro-Wilk normality test. Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by
Tukey post hoc test. Differences were considered significant at the 95% confidence level.
Results
Anti-inflammatory properties of nortriptyline and bu-desonide on NK and NKT-like cells
While there was negligible baseline synthesis of cytokines (data not shown), substantial percentage of NK and NKT-li-ke cells treated with PMA and ionomycin in the presence of brefeldin A produced IL-4, IL-8, IFNy, and TNFa (Figs 1, 2). Initial experiments showed that the majority of the NKT-like cells stimulated with PMA and ionomycin expressed IFNy
Figure 1. Percentage of NK (A and C) and NKT-like (B and D) cells from peripheral blood of COPD patients producing IL-4 and IL-8 after treatment with budesonide and nortriptyline. Note: Peripheral blood cells from patients with chronic obstructive pulmonary disease (COPD, n = 6) were incubated with nortriptyline (Nt, 1 ^M or 10 ^M), budesonide (Bud, 10 nM), or their combinations for 1 hour, followed by stimulation with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (Ion, 1 ^g/mL) for 6 hours. Production of interleukin 4 (IL-4, A and B) and IL-8 (C and D) in NK and NKT-like cells was examined by flow cytometry. Data are presented as mean ± SEM. One-way ANOVA followed by post hoc Tukey test: * - P<0.05 versus PMA + Ion control; ♦ - P<0.05 versus Bud 10 nM; # - P<0.05 versus Nt 10 ^M.
+ PMA + Ion
Figure 2. Percentage of NK (A and C) and NKT-like (B and D) cells from peripheral blood of COPD patients producing IFNy and TNFa after treatment with budesonide and nortriptyline. Note: Peripheral blood cells from patients with chronic obstructive pulmonary disease (COPD, n = 6) were incubated with nortriptyline (Nt, 1 pM or 10 pM), budesonide (Bud, 10 nM), or their combinations for 1 hour, followed by stimulation with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (Ion, 1 pg/mL) for 6 hours. Production of interferon y (IFNy, A and B) and tumor necrosis factor a (TNFa, C and D) in NK and NKT-like cells was examined by flow cytometry. Data are presented as mean ± SEM. One-way ANOVA followed by post hoc Tukey test: *- P<0.05 versus PMA + Ion control; ♦ - P<0.05 versus Bud 10 nM; # - P<0.05 versus Nt 10 pM.
and TNFa, while fewer cells produced IL-4 and IL-8. More than half of NK cells were also found to express TNFa.
Budesonide suppressed PMA/ionomycin-induced production of IL-4, IL-8, and TNFa in NK and NKT-like cells, as well as IFNy synthesis in NK cells. However, this drug did not affect IFNy expression by NKT-like cells. Nortriptyline 10 ^M reduced synthesis of IL-4, IL-8, and IFNy in NK and NKT-like cells from COPD patients. In contrast, nortriptyline 1 ^M only attenuated the increase of IL-4 by NK and NKT-like cells. Treating PMA/ion-omycin-stimulated whole blood cells with combination of nortriptyline 10 ^M and budesonide significantly decreased IL-4, IL-8, IFNy, and TNFa expression by NK and NKT-like cells above that of budesonide alone. The association of nortriptyline 1 ^M and budesonide showed
additive effects, reducing IL-4 and IL-8 production in NK and NKT-like cells from COPD patients.
Effects of nortriptyline and budesonide on p38 MAPK and p65 NF-kB phosphorylation
To assess the impact of nortriptyline on molecular pathways leading to steroid resistance, we examined its effect on the phosphorylation status of p38 MAPK and p65 NF-kB. Phosphorylation (and thus activation) of p38 MAPK and p65 NF-kB in NK and NKT-like cells was increased in response to PMA and ionomycin (Suppl. material 1: Table S1). Budesonide was unable to reverse phosphorylated p38 MAPK and p65 NF-kB levels induced by PMA/iono-mycin (Table 2). In contrast, nortriptyline 10 pM reduced
Table 2. Mean fluorescence intensity (MFI) of GR, GRp, phosphorylated p38 MAPK and p65 NF-kB staining in NK and NKT-like blood cells from COPD patients
Cell type
PMA + Ion
MFI GR
NK cells NKT-like cells MFI GRp NK cells NKT-like cells MFI p-p38 MAPK NK cells NKT-like cells MFI p-p65 NF-kB NK cells NKT-like cells
30.87±2.26 40.12±6.97
3.56±0.25 3.34±0.18
2.56±0.23 3.97±0.35
1.78±0.20 3.32±0.43
PMA + Ion + Bud PMA + Ion + Nt PMA + Ion + Nt PMA + Ion + Bud PMA + Ion + Bud 10 nM 1 цМ 10 цМ 10 nM + Nt 1 цМ 10 nM + Nt 10 цМ
27.73±2.30 36.00±7.50
3.43±2.24 3.13±0.18
2.50±0.26 3.82±0.37
1.73±0.19 3.20±0.41
33.43±1.14 40.42 ± 6.27
3.30±0.25 3.12±0.19
2.47±0.22 3.79±0.33
1.69±0.16 3.14±0.40
32.92±1.34 39.75±6.95
3.17±0.23*^ 2.94±0.20*^§
2.37±0.24* 3.72±0.34*
1.58±0.17* 2.93±0.40*
32.40±1.05 38.96±6.63
3.16±0.23*^ 2.91±0.20*^§
2.28±0.25*^ 3.64±0.36*^
1.60±0.21* 2.92±0.41*^
31.80±1.91 40.01±8.59
2.99±0.19*> 2.78±0.19*^
2.24±0.22*^ 3.61±0.364
1.57±0.21*^ 2.81±0.41*>
Note: The values shown are mean ± SEM of 6 experiments. * - P<0.05 versus PMA+Ion; ♦ - P<0.05 versus PMA+Ion + Bud 10 nM; § -P<0.05 versus PMA + Ion + Nt 1 ^M. Bud, budesonide; COPD, chronic obstructive pulmonary disease; GR, glucocorticoid receptor; Ion, ionomycin; NK cells, natural killer cells; NKT-like cells, natural killer T-like cells; Nt, nortriptyline; PMA, phorbol myristate acetate; p-p38 MAPK, phosphorylated p38 mitogen-activated protein kinase; p-p65 NF-kB, phosphorylated p65 nuclear factor-кВ.
phosphorylation of p38 MAPK and p65 NF-kB in NK and NKT-like cells from COPD patients. Importantly, NK and NKT-like cells of COPD patients that were pretreated with nortriptyline became significantly more responsive to the suppressive effects of GCs. As shown by flow cytometry, preincubation of whole peripheral blood from COPD patients with nortriptyline resulted in a significantly greater budesonide-mediated suppression of PMA/ ionomycin-induced levels of phosphorylated p38 MAPK and p65 NF-kB.
Impact of nortriptyline and budesonide on GR and GRp expression
The expression of GR was not modified under any experimental condition (Table 2). In particular, GR expression was not modified by PMA/ionomycin stimulation in the presence of brefeldin A (Suppl. material 1: Table S1) and following nortriptyline or budesonide exposure.
The human GR gene encodes two splicing variants, GRa and GRp. While GRa mediates most of the known GC functions, GRp may have dominant negative effect on GRa, leading to steroid resistance (Kino et al. 2009). Therefore, we next evaluated whether nortriptyline, alone and in combination with GCs, is able to modulate GRp expression by NK and NKT-like cells. Treatment of whole blood cells with budesonide or nortriptyline 1 ^M showed no effect on GRp expression. Nortriptyline 10 ^M effectively decreased GRp expression in the presence of PMA and ionomycin. Moreover, nortriptyline (1 ^M to 10 ^M) combined with budesonide markedly suppressed GRp expression by NK and NKT-like cells.
Effects of nortriptyline and budesonide on HDAC2 and histone acetyltransferase (HAT) expression
In other experiments, we assessed the expression of HDAC2, which suppresses activated inflammatory
genes. Reduced activity and expression of HDAC2 leads to the resistance of corticosteroids to the anti-inflammatory actions (Barnes 2011). At the same time, histone H4 (acetyl K8) was evaluated, as it gives evidence for the activity of HAT, which upregulates pro-inflammatory gene transcription. Budesonide was without significant effect on any of these two molecules in lymphocyte subsets (Fig. 3A, B). Nt alone was unable to reverse the downregulation of HDAC2 expression induced by PMA/ionomycin in CD3-CD56+ and CD3+CD56+ cells. In contrast, the combination of Nt (1 ^M to 10 ^M) with budesonide at 10 nM enhanced the percentage of NK and NKT-like cells expressing HDAC2 from COPD patients (Fig. 4).
We also observed that nortriptyline (10 ^M) significantly attenuated the effects of PMA/ionomycin on histone H4 (acetyl K8) expression in NK and NKT-like cells (Fig. 3C, D). Furthermore, the association of Nt (1 ^M to 10 ^M) with budesonide at 10 nM reduced the expression of histone H4 (acetyl K8) in CD3-CD56+ and CD3+CD56+ cells.
Discussion
GC insensitivity is a significant barrier to successful COPD treatment. Our current study demonstrates that a tricyclic antidepressant nortriptyline exhibits anti-inflammatory effects, alone and in combination with GCs, on cytokine production by NK and NKT-like cells from COPD patients. Combination therapy with corticosteroids and nortriptyline affords the advantage over GCs alone, as the degree of cytokine inhibition was enhanced when budesonide was used in association with nortriptyline. Moreover, we found that nortriptyline is able to overcome molecular mechanisms that can contribute to reduced responsiveness of NK and NKT-like cells to corticosteroids. The data obtained convincingly prove the feasibility of
Figure 3. Effects of nortriptyline and budesonide on histone deacetylase 2 (HDAC2) and histone H4 (acetyl K8) expression in NK (A and C) and NKT-like (B and D) blood cells from COPD patients. Note: Peripheral blood cells from patients with chronic obstructive pulmonary disease (COPD, n = 6) were incubated with nortriptyline (Nt, 1 pM or 10 pM), budesonide (Bud, 10 nM), or their combinations for 1 hour and stimulated with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (Ion, 1 pg/mL) for 6 hours. Expression of histone deacetylase 2 (HDAC2, A and B) and histone H4 acetylation of K8 (HAT, C and D) in NK and NKT-like cells was detected by flow cytometry. Data are presented as mean ± SEM. One-way ANOVA followed by post hoc Tukey test: * - P<0.05 versus PMA + Ion control; ♦ - P<0.05 versus Bud 10 nM; # - P<0.05 versus Nt 10 pM.
a combined use of nortriptyline and corticosteroids for suppressing the inflammatory process in COPD patients.
IL-4 is involved in several aspects of airway inflammation and remodeling, such as differentiation of naive CD4+ T cells into Th2 cells, alternative macrophage activation, and collagen synthesis in fibroblasts (Junttila 2018). IL-4 stimulates airway epithelial cells to produce and release chemokine CCL26/eotaxin-3 that recruit eosinophils into the inflamed lung through CCR3 receptors (Abonyo et al. 2005). It has been reported that nearly a third of patients with stable COPD have evidence of eosinophilic airway inflammation (Tashkin and Wechsler 2018), while higher blood eosinophil counts have been associated with an
increased risk of exacerbations among individuals with COPD (Vedel-Krogh et al. 2016). In this work, we observed that exposure of NK and NKT-like cells to PMA and ionomycin increased IL-4 production and this was inhibited by pretreatment with either budesonide or nor-triptyline (1 pM and 10 pM). More importantly, the combination of budesonide and nortriptyline (1 pM to 10 pM) was more potent at reducing IL-4 synthesis in NK and NKT-like cells when compared with budesonide alone, implying that nortriptyline, even at low concentrations, has the potential to enhance GC sensitivity in COPD.
IL-8 is known for its ability to attract neutrophils to the sites of inflammation in the lung tissue. There is an
00 00 Tf
iK <
H
<
EC
PMA + Ion
48.7%
i
10° 10' 102 103 CD3 PE-DyLight 594
PMA + Ion + Bud 10 nM + Nt 10 nM
10°-
31.0%
10° 10' 102 103
10^
00 00
■f 102r
to
<
cj «>'-
U <
O X
79.9%
i§;
10°
10'
102
10!
CD3 PE-DyLight 594
10'-
10°-
99.6%
Figure 4. Representative dot plots showing the combined effect of 10 nM budesonide (Bud) and 10 ^M nortriptyline (Nt) on the percentage of NKT-like cells expressing histone acetyltransferase (HAT) and histone deacetylase 2 (HDAC2) from a patient with chronic obstructive pulmonary disease.
increase in serum IL-8 level in patients with stable COPD, with a further rise during an acute exacerbation (Zhang and Bai 2018). Higher blood IL-8 level in COPD patients was associated with emphysema progression over 5 years after measuring cytokine level (Bradford et al. 2017). In this regard, reducing blood IL-8 level seems logical and may be effective in COPD. In the current study, the percentage of IL-8-producing NK and NKT-like cells was significantly reduced after budesonide and nortriptyline 10 ^M treatment. We also found that inhibitory effect of budesonide on IL-8 synthesis in NK and NKT-like cells was increased in the presence of nortriptyline (1 ^M and 10 ^M). In addition, the synergistic action of GCs and nortriptyline to decrease the release of IL-8 from stimulated human monocytic U937 cells has been reported previously (Mercado et al. 2011). Therefore, IL-8 suppression using nortriptyline combined with GCs may be a good therapeutic approach in COPD where this cytokine plays an important role.
IFNy induces the expression of MIG, IP-10 and I-TAC by bronchial epithelial cells and macrophages, which serve as ligands for the chemokine receptor CXCR3. This receptor is expressed on lymphocytes, including NK and NKT cells, and involved in their recruitment in the peripheral airways of COPD patients (Hughes and Nibbs 2018). Stimulation of macrophages with IFNy and lipo-polysaccharide leads to their polarization towards the M1 phenotype, which is characterized by the production
of pro-inflammatory cytokines, including TNFa and IL-1p (Smith et al. 2016). Moreover, IFNy contributes to the reduced sensitivity of alveolar macrophages of COPD patients to GCs through the activation of the transcription factor STAT1 (Southworth et al. 2012). In this work, budesonide decreased production of IFNy in NK cells, but did not alter IFNy synthesis in NKT-like cells. Interestingly, a previous study detected a greater inhibitory effect of other GC, methylprednisolone, on IFNy production by NK cells compared with the effect of this drug on IFNy synthesis by NKT-like cells from COPD patients (Hodge et al. 2014). Moreover, we observed that combination of nortriptyline (10 ^M) and budesonide had additive effects, inhibiting IFNy expression by both NK and NKT-like cells.
In patients with COPD, TNFa activates transcription factors NF-kB and AP-1, which promote the synthesis of proinflammatory mediators (Barnes 2016). TNFa upregulates expression of cytokines (IL-5, IL-6, IL-8, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor), chemokines (CCL5, CCL11, MCP-1), matrix metalloproteinases (MMP-9, MMP-12), adhesion molecules (ICAM-1, VCAM-1, E-selectin), and mucins (MUC1, MUC2, MUC5AC) in inflammatory and structural cells in the lung (Commins et al. 2010). TNFa also contributes to cachexia and skeletal muscle wasting in patients with COPD (Webster et al. 2020). Blood TNFa level was found to be increased in patients with COPD
compared with healthy controls (Yao et al. 2019). Here, addition of budesonide to culture tubes resulted in a decrease of TNFa production in NK and NKT-like cells of COPD patients. Furthermore, the percentage of TNFa-producing NK and NKT-like cells rose significantly upon addition of nortriptyline 10 pM to budesonide compared with cells cultured in the presence of budesonide alone. Our data agrees with another study that reported synergistic attenuation of TNFa secretion from stimulated PBMCs of healthy subjects by the combination of nortriptyline plus GCs (Lehar et al. 2009). This indicates that COPD subjects could benefit from nortriptyline/corticosteroid combination therapy.
To determine molecular basis for combined nortripty-line and budesonide action on NK and NKT-like cells, we tested their effects on known markers of steroid resistance. There is compelling evidence that inflammatory gene repression is mediated by histone deacetylases. HDAC2 has been shown to cause deacetylation of GR, enabling this receptor to bind to the p65 NF-KB-activated complex and subsequently repress inflammatory gene expression (Ito et al. 2006). In patients with COPD, reduced HDAC2 expression was first demonstrated in lung macrophages (Cosio et al. 2004) and was recently found in PBMCs (Tan et al. 2016). Impaired HDAC2 causes hyperacetylation of his-tones and enhances inflammatory gene expression. Moreover, reduced HDAC2 activity and expression prevents the association between GR and NF-kB, leading to steroid resistant cytokine production (Ito et al. 2006). We report here a rapid suppression of HDAC2 in NK and NKT-like cells following stimulation with PMA plus ionomycin. Budesonide or nortriptyline alone produced no change in HDAC2 expression by NK and NKT-like cells from that seen in control untreated samples. In addition, our study showed beneficial effects of nortriptyline (1 pM and 10 pM) combined with budesonide in enhancing HDAC2 expression by NK and NKT-like cells from COPD patients.
Of note, gene expression is affected by a balance between acetylation and deacetylation of histones. Inflammatory gene activation is associated with the histone acetylation, which is induced by coactivators having intrinsic HAT activity (Barnes 2011). In this work, there was no alteration of PMA/ionomycin-induced histone H4 acetylation of K8 in NK and NKT-like cells following either budesonide or nortriptyline 1 pM treatment. However, higher concentration of nortriptyline (10 pM), as well as the association of nortriptyline (1 pM to 10 pM) and budesonide inhibited histone H4 acetylation status. These results suggest that suppression of HAT activity and induction of HDAC2 expression may be possible mechanism by which nortriptyline regains steroid sensitivity of NK and NKT-like cells from COPD patients.
Previous studies have shown that dominant negative isoform of GR, GRp, can inhibit hormone binding to GRa, leading to steroid resistance (Kino et al. 2009). Extending those findings, we revealed that budesonide alone had no effect on PMA/ionomycin-stimulated GRp expression in NK and NKT-like cells from COPD patients. Intriguingly, nortriptyline 10 pM alone reduced percentage of GRp-ex-pressing NK and NKT-like cells. Similarly, nortriptyline
(1 pM to 10 pM) and budesonide combined caused greater GRp inhibition in NK and NKT-like cells compared with either cultures with no drugs or budesonide alone. Our data imply that nortriptyline is able to repress GRp, and thus may afford GRa to implement its functions.
Proinflammatory transcription factor NF-KB mediates induction of various proinflammatory genes in innate immune cells and regulates the maturation and effector functions of NK and NKT cells (Lougaris et al. 2017; Stankovic et al. 2011). Heightened NF-kB activation is a hallmark of COPD (Schuliga 2015). Moreover, targeting NF-KB signaling is supposed to increase effectiveness of COPD treatment (Schuliga 2015). In the present study, budesonide had no effect on p65 NF-kB activation in NK and NKT-like cells after stimulation with PMA/ionomy-cin. In contrast, nortriptyline (10 pM) efficiently reduced p65 NF-kB protein phosphorylation in these cells, suggesting that its anti-inflammatory properties are superior to those of corticosteroids in COPD.
p38 MAPK signaling is increased in COPD and can cause the inefficiency of GCs to suppress inflammation (Renda et al. 2008). Activated p38 MAPK causes GR phosphoryla-tion leading to inhibition of GR nuclear translocation and modification of GR transcriptional activity (Galliher-Beck-ley and Cidlowski 2009). Our data show that budesonide did not change PMA/ionomycin-induced expression of phospho-p38 MAPK in NK and NKT-like cells of patients with COPD. Additionally, there are other data showing that corticosteroid prednisolone prescribed orally to patients with COPD did not inhibit p38 MAPK activation in whole blood (Singh et al. 2010). We observed that nortriptyline 10 pM decreased budesonide-insensitive expression of phos-pho-p38 MAPK in NK and NKT-like cells of patients with COPD. Moreover, combination of nortriptyline (1 pM to 10 pM) with budesonide inhibited the p38 MAPK pathway to a greater degree than budesonide alone did. Thus, suppression of p38 MAPK signaling and subsequent inhibition of inflammatory mediators may be achieved by combination treatment with a corticosteroid and nortriptyline.
Collectively, our results show that nortriptyline augments budesonide's anti-inflammatory effects on IL-4, IL-8, TNFa and IFNy production by NK and NKT-like cells of patients with COPD. Nortriptyline might enhance the function of GCs through modulation of HAT, HDAC2, GRp, phospho-p38 MAPK expression. These data provide a strong rationale for combining nortriptyline with budesonide to treat COPD. Importantly, nortriptyline may also be beneficial in other steroid-refractory diseases, such as asthma, where GCs are the first-line therapy for controlling airway inflammation, but 5-10% of asthma patients respond unsatisfactorily to steroid-based therapies (Henderson et al. 2020). Future research should demonstrate if this assumption is correct.
Conflict of interest
The authors have declared that no competing interests exist.
References
■ Abel AM, Yang C, Thakar MS, Malarkannan S (2018) Natural killer cells: Development, maturation, and clinical utilization. Frontiers in Immunology 9: 1869. https://doi.org/10.3389/fimmu.2018.01869 [PubMed] [PMC]
■ Abonyo BO, Alexander MS, Heiman AS (2005) Autoregulation of CCL26 synthesis and secretion in A549 cells: a possible mechanism by which alveolar epithelial cells modulate airway inflammation. American Journal of Physiology - Lung Cellular and Molecular Physiology 289(3): L478-L488. https://doi.org/10.1152/ ajplung.00032.2005 [PubMed]
■ Barnes PJ (2016) Inflammatory mechanisms in patients with chronic obstructive pulmonary disease. The Journal of Allergy and Clinical Immunology 138(1): 16-27. https://doi.org/10.1016/). jaci.2016.05.011 [PubMed]
■ Barnes PJ (2011) Glucocorticosteroids: Current and future directions. British Journal of Pharmacology 163(1): 29-43. https://doi. org/10.1111/j.1476-5381.2010.01199.x
■ Bradford E, Jacobson S, Varasteh J, Comellas AP, Woodruff P, O'Neal W, DeMeo DL, Li X, Kim V, Cho M, Castaldi PJ, Hersh C, Silverman EK, Crapo JD, Kechris K, Bowler RP (2017) The value of blood cytokines and chemokines in assessing COPD. Respiratory Research 18(1): e180. https://doi.org/10.1186/s12931-017-0662-2 [PubMed] [PMC]
■ Cosio BG, Tsaprouni L, Ito K, Jazrawi E, Adcock IM, Barnes PJ (2004) Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. Journal of Experimental Medicine 200(5): 689-695. https://doi.org/10.1084/jem.20040416 [PubMed] [PMC]
■ Commins SP, Borish L, Steinke JW (2010) Immunologic messenger molecules: Cytokines, interferons, and chemokines. Journal of Allergy and Clinical Immunology 125(2 Suppl 2): S53-S72. https://doi. org/10.1016/j.jaci.2009.07.008 [PubMed]
■ Freeman CM, Stolberg VR, Crudgington S, Martinez FJ, Han MK, Chensue SW, Arenberg DA, Meldrum CA, McCloskey L, Curtis JL (2014) Human CD56+ cytotoxic lung lymphocytes kill autol-ogous lung cells in chronic obstructive pulmonary disease. PLoS ONE 9(7): e103840. https://doi.org/10.1371/journal.pone.0103840 [PubMed] [PMC]
■ Galliher-Beckley AJ, Cidlowski JA (2009) Emerging roles of glu-cocorticoid receptor phosphorylation in modulating glucocorticoid hormone action in health and disease. IUBMB Life 61(10): 979-986. https://doi.org/10.1002/iub.245 [PubMed]
■ Henderson I, Caiazzo E, McSharry C, Guzik TJ, Maffia P (2020) Why do some asthma patients respond poorly to glucocorticoid therapy? Pharmacological Research 160: 105189. https://doi. org/10.1016/j.phrs.2020.105189 [PubMed] [PMC]
■ Hodge G, Holmes M, Jersmann H, Reynolds PN, Hodge S (2014) Targeting peripheral blood pro-inflammatory cytotoxic lymphocytes by inhibiting CD137 expression: Novel potential treatment for COPD. BMC Pulmonary Medicine 14: e85. https://doi. org/10.1186/1471-2466-14-85 [PubMed] [PMC]
■ Hodge G, Mukaro V, Holmes M, Reynolds PN, Hodge S (2013) Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway. Respirology 18(2): 369-376. https:// doi.org/10.1111/j.1440-1843.2012.02287.x [PubMed]
■ Hodge G, Hodge S (2019) Therapeutic targeting steroid resistant pro-inflammatory NK and NKT-Like cells in chronic inflammatory lung disease. International Journal of Molecular Sciences 20(6): 1511. https://doi.org/10.3390/ijms20061511 [PubMed] [PMC]
■ Howes S, Hartmann-Boyce J, Livingstone-Banks J, Hong B, Lind-son N (2020) Antidepressants for smoking cessation. Cochrane Database of Systemic Reviews 4(4): CD000031. https://doi. org/10.1002/14651858.CD000031.pub5 [PubMed] [PMC]
■ Hughes CE, Nibbs RJB (2018) A guide to chemokines and their receptors. FEBS Journal 285(16): 2944-2971. https://doi.org/10.1111/ febs.14466 [PubMed] [PMC]
■ Ito K, Yamamura S, Essilfie-Quaye S, Cosio B, Ito M, Barnes PJ, Adcock IM (2006) Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression. Journal of Experimental Medicine 203(1): 7-13. https://doi.org/10.1084/ jem.20050466 [PubMed] [PMC]
■ Junttila IS (2018) Tuning the cytokine responses: an update on inter-leukin (IL)-4 and IL-13 receptor complexes. Frontiers in Immunology 9: 888. https://doi.org/10.3389/fimmu.2018.00888 [PubMed] [PMC]
■ Kadushkin AG, Tahanovich AD, Movchan LV, Kolesnikova TS, Khadasouskaya AV, Shman TV (2021) The effect of glucocorticoids in combination with azithromycin or theophylline on cytokine production by NK and NKT-like blood cells of patients with chronic obstructive pulmonary disease. Biomeditsinskaia Khimiia 67(4): 352-359. https://doi.org/10.18097/pbmc20216704352 [PubMed]
■ Kadushkin AG, Taganovich AD (2016) Molecular mechanisms of corticosteroid resistance in patients with chronic obstructive pulmonary disease. Pulmonologiya 26(6): 736-747. https://doi. org/10.18093/0869-0189-2016-26-6-736-747
■ Kino T, Su YA, Chrousos GP (2009) Human glucocorticoid receptor isoform beta: recent understanding of its potential implications in physiology and pathophysiology. Cellular and Molecular Life Sciences 66(21): 3435-3448. https://doi.org/10.1007/s00018-009-0098-z [PubMed] [PMC]
■ Krijgsman D, Hokland M, Kuppen PJK (2018) The role of natural killer T cells in cancer-a phenotypical and functional approach. Frontiers in Immunology 9: 367. https://doi.org/10.3389/fim-mu.2018.00367 [PubMed] [PMC]
■ Lehär J, Krueger AS, Avery W, Heilbut AM, Johansen LM, Price ER, Rickles RJ, Short 3rd GF, Staunton JE, Jin X, Lee MS, Zimmermann GR, Borisy AA (2009) Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nature Biotechnology 27(7): 659-666. https://doi.org/10.1038/nbt.1549 [PubMed] [PMC]
■ Leung JM, Sin DD (2018) Inhaled corticosteroids in COPD: the final verdict is.... European Respiratory Journal 52(6): 1801940. https:// doi.org/10.1183/13993003.01940-2018 [PubMed]
■ Lougaris V, Patrizi O, Baronio M, Tabellini G, Tampella G, Damiati E, Frede N, van der Meer JWM, Fliegauf M, Grimbacher B, Parolini S, Plebani A (2017) NFKB1 regulates human NK cell maturation and effector functions. Clinical Immunology 175: 99-108. https:// doi.org/10.1016/j.clim.2016.11.012 [PubMed]
■ Mercado N, To Y, Ito K, Barnes PJ (2011) Nortriptyline reverses corticosteroid insensitivity by inhibition of phosphoinositide-3-ki-nase-S. Journal of Pharmacology and Experimental Therapeutics 337(2): 465-470. https://doi.org/10.1124/jpet.110.175950
■ Renda T, Baraldo S, Pelaia G, Bazzan E, Turato G, Papi A, Maestrelli P, Maselli R, Vatrella A, Fabbri LM, Zuin R, Marsi-co SA, Saetta M (2008) Increased activation of p38 MAPK in COPD. European Respiratory Journal 31(1): 62-69. https://doi. org/10.1183/09031936.00036707 [PubMed]
■ Schuliga M (2015) NF-kappaB signaling in chronic inflammatory airway disease. Biomolecules 5(3): 1266-1283. https://doi. org/10.3390/biom5031266 [PubMed] [PMC]
■ Singh D, Smyth L, Borrill Z, Sweeney L, Tal-Singer R (2010) A randomized, placebo-controlled study of the effects of the p38 MAPK inhibitor SB-681323 on blood biomarkers of inflammation in COPD patients. The Journal of Clinical Pharmacology 50(1): 94-100. https://doi.org/10.1177/0091270009347873 [PubMed]
■ Smith TD, Tse MJ, Read EL, Liu WF (2016) Regulation of macrophage polarization and plasticity by complex activation signals. Integrative Biology (Camb) 8(9): 946-955. https://doi.org/10.1039/ c6ib00105j [PubMed] [PMC]
■ Southworth T, Metryka A, Lea S, Farrow S, Plumb J, Singh D (2012) IFN-y synergistically enhances LPS signalling in alveolar macrophages from COPD patients and controls by corticosteroid-resistant STAT1 activation. British Journal of Pharmacology 166(7): 20702083. https://doi.org/10.1111/j.1476-5381.2012.01907.x [PubMed] [PMC]
■ Stankovic S, Gugasyan R, Kyparissoudis K, Grumont R, Banerjee A, Tsichlis P, Gerondakis S, Godfrey DI (2011) Distinct roles in NKT cell maturation and function for the different transcription factors in the classical NF-kB pathway. Immunology and Cell Biology 89(2): 294-303. https://doi.org/10.1038/icb.2010.93 [PubMed]
■ Ström J, Pourazar J, Linder R, Blomberg A, Lindberg A, Bucht A, Behndig AF (2018) Cytotoxic lymphocytes in COPD airways: increased NK cells associated with disease, iNKT and NKT-like cells with current smoking. Respiratory Researh 19(1): e244. https://doi. org/10.1186/s12931-018-0940-7 [PubMed] [PMC]
■ Tan C, Xuan L, Cao S, Yu G, Hou Q, Wang H (2016) Decreased histone deacetylase 2 (HDAC2) in peripheral blood monocytes (PB-MCs) of COPD patients. PLoS ONE 11(1): e0147380. https://doi. org/10.1371/journal.pone.0147380 [PubMed] [PMC]
■ Tashkin DP, Wechsler ME (2018) Role of eosinophils in airway inflammation of chronic obstructive pulmonary disease. International Journal of Chronic Obstructive Pulmonary Disease 13: 335-349. https://doi.org/10.2147/C0PD.S152291 [PubMed] [PMC]
■ Tashkin DP (2015) Smoking cessation in chronic obstructive pulmonary disease. Seminars in Respiratory and Critical Care Medicine 36(4): 491-507. https://doi.org/10.1055/s-0035-1555610 [PubMed]
■ Tselebis A, Pachi A, Ilias I, Kosmas E, Bratis D, Moussas G, Tzanakis N (2016) Strategies to improve anxiety and depression in patients with COPD: a mental health perspective. Neuropsychiatric Disease and Treatment 12: 297-328. https://doi.org/10.2147/NDT. S79354 [PubMed] [PMC]
■ Vedel-Krogh S, Nielsen SF, Lange P, Vestbo J, Nordestgaard BG (2016) Blood eosinophils and exacerbations in chronic obstructive pulmonary disease. The Copenhagen General Population Study. American Journal of Respiratory and Critical Care Medicine 193(9): 965-974. https://doi.org/10.1164/rccm.201509-18690c [PubMed]
■ Vogelmeier CF, Criner GJ, Martinez FJ, Anzueto A, Barnes PJ, Bourbeau J, Celli BR, Chen R, Decramer M, Fabbri LM, Frith P, Halpin DM, López Varela MV, Nishimura M, Roche N, Rodri-guez-Roisin R, Sin DD, Singh D, Stockley R, Vestbo J, Wedzicha JA, Agustí A (2017) Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease 2017 report. GOLD Executive Summary. American Journal of Respiratory and Critical Care Medicine 195(5): 557-582. https://doi.org/10.1164/rc-cm.201701-0218PP [PubMed]
■ Webster JM, Kempen LJAP, Hardy RS, Langen RCJ (2020) Inflammation and skeletal muscle wasting during cachexia. Frontiers in Physiology 11: 597675. https://doi.org/10.3389/fphys.2020.597675 [PubMed] [PMC]
■ Willemse BW, ten Hacken NH, Rutgers B, Lesman-Leegte IG, Post-ma DS, Timens W (2005) Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomatic smokers. European Respiratory Journal 26(5): 835-845. https://doi.org/10.1183/090319 36.05.00108904 [PubMed]
■ Yao Y, Zhou J, Diao X, Wang S (2019) Association between tumor necrosis factor-a and chronic obstructive pulmonary disease: a systematic review and meta-analysis. Therapeutic Advances in Respiratory Diseases 13: 1-17. https://doi.org/10.1177/1753466619866096 [PubMed] [PMC]
■ Zhang J, Bai C (2018) The significance of serum interleukin-8 in acute exacerbations of chronic obstructive pulmonary disease. Tanaffos 17(1): 13-21. [PubMed] [PMC]
Supplementary material 1 Table S1
Authors: Aliaksei G. Kadushkin, Anatoli D. Tahanovich, Lyud-mila V. Movchan, Volha V. Dziadzichkina, Olga V. Levando-vskaya, Tatsiana V. Shman
Data type: Table (docx. file)
Explanation note: Basal and stimulated intracellular expression of enzymes, glucocorticoid receptor, phosphorylated p38 MAPK and p65 NF-kB staining in NK and NKT-like cells from patients with chronic obstructive pulmonary disease.
Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/ odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Link: https://doi.org/10.3897/rrpharmacology.8.75467.suppl1
Author contributions
■ Aliaksei G. Kadushkin, PhD in Medical Sciences, Associate Professor of the Department of Biological Chemistry, e-mail: kadushkyn@gmail.com, ORCID ID https://orcid.org/0000-0002-1620-8477. The author conceived the idea of the study and played a leading role in conducting the experiments, interpreting the data, and writing the article.
■ Anatoli D. Tahanovich, Doctor Habil. of Medical Sciences, Professor, Head of the Department of Biological Chemistry, e-mail: taganovich@bsmu.by, ORCID ID https://orcid.org/0000-0002-0668-2888. The author consulted on the research idea, concept and design, and participated in writing the final version of the article.
■ Lyudmila V. Movchan, PhD in Biological Sciences, Doctor of Laboratory Diagnostics of Clinical Diagnostic Laboratory, e-mail: movchan-l@mail.ru, ORCID ID https://orcid.org/0000-0002-0441-0109. The author performed flow cytometry analysis and interpreted the data obtained.
■ Volha V. Dziadzichkina, PhD in Medical Sciences, Associate Professor of the Department of Obstetrics and Gynecology, e-mail: dyadichkinaov@gmail.com, ORCID ID https://orcid.org/0000-0003-1198-7755. The author took part in the design of the experiment and statistical data processing.
■ Olga V. Levandovskaya, Head of the Transfusiology Center, e-mail: o_lev77@mail.ru, ORCID ID https://orcid. org/0000-0003-0595-1262. The author conducted the experiments and participated in the data interpreting.
■ Tatsiana V. Shman, PhD in Biological Sciences, Head of the Immunological Research Laboratory, e-mail: shman@ oncology.by, ORCID ID https://orcid.org/0000-0001-8669-8005. The author provided an advice on planning, designing and conducting the experiments, as well as editing the article.
