P38 MAPK and glucocorticoid receptor crosstalk in bronchial epithelial cells

Abstract p38 MAPK inhibition may have additive and synergistic anti-inflammatory effects when used with corticosteroids. We investigated crosstalk between p38 MAPK inhibitors and corticosteroids in bronchial epithelial cells to investigate synergistic effects on cytokine production and the molecular mechanisms involved. Effects of the p38 MAPK inhibitor BIRB-796 and dexamethasone alone and in combination on LPS, polyI:C or TNFα -induced IL-6, CXCL8 and RANTES were assessed in 16HBEs (human epithelial cell line) and on TNFα-induced IL-6 and CXCL8 in primary human epithelial cells from asthma patients and healthy controls. 16HBEs were used to assess effects of BIRB-796 alone and in combination with dexamethasone on glucocorticoid receptor (GR) activity by reporter gene assay, expression of GR target genes and nuclear localisation using Western blot. The effects of BIRB-796 on TNFα stimulated phosphorylation of p38 MAPK and GR at serine (S) 226 by Western blot. Epithelial levels of phosphorylated p38 MAPK and GR S226 were determined by immunohistochemistry in bronchial biopsies from asthma patients and healthy controls. BIRB-796 in combination with dexamethasone increased inhibition of cytokine production in a synergistic manner. Combination treatment significantly increased GR nuclear localisation compared to dexamethasone alone. BIRB-796 inhibited TNFα-induced p38 MAPK and GR S226 phosphorylation. Phosphorylated GR S226 and p38 MAPK levels were increased in bronchial epithelium of more severe asthma patients. Molecular crosstalk exists between p38 MAPK activation and GR function in human bronchial epithelial cells, which alters GR activity. Combining a p38 MAPK inhibitor and a corticosteroid may demonstrate therapeutic potential in severe asthma. Key messages • Combination of corticosteroid and p38 inhibitor in human bronchial epithelial cells • Combination increased cytokine inhibition synergistically and nuclear GR • p38 MAPK inhibition reduced TNFα-induced phosphorylation of GR at S226 but not S211 • Phosphorylated GRS226 and p38 is increased in bronchial epithelium in severe asthma • Combining a p38 inhibitor and a corticosteroid may be effective in asthma treatment Electronic supplementary material The online version of this article (10.1007/s00109-020-01873-3) contains supplementary material, which is available to authorized users.

Culture supernatants were removed and cells were lysed in RLT buffer. Total RNA was purified from cell lysates using RNeasy kits (Qiagen, Crawley, UK) according to manufacturer's instructions. DNA contamination was prevented by on-column addition of DNase (Qiagen, Crawley, UK) according to manufacturer's instructions. Reverse transcription was performed on 50 ng of RNA using the Verso cDNA kit (Thermo Scientific). The resulting cDNA was reacted with ABsolute blue qPCR mix (Thermo Scientific) in 25 µl reactions containing premade ABI Taqman gene expression assays for GILZ (Hs00608272_m1), FKBP5 (Hs00188025_m1) and the endogenous control was glyceraldehyde-3phosphate dehydrogenase (GAPDH) (Catalogue no: 4352934E) (Applied Biosystems, Warrington, UK). Controls without RT-enzyme showed there was no genomic DNA amplification. Thermal cycling was carried out on a Stratagene MX3005P (Agilent Technologies, West Lothian, UK). Relative expression levels were determined using the 2 -ΔΔCt (untreated vs treatment).

IHC methods
Bronchial biopsies were fixed in 10% neutral buffered formalin for 8 hours and were processed using an automated tissue processing machine on a routine overnight schedule. Biopsies were embedded in histological grade paraffin wax and 3µm sections were cut with a Leica RM2235 rotary microtome. For immunohistochemical staining, slides were dewaxed using xylene and dehydrated through a series of industrial denatured alcohols. Heat-induced epitope retrieval was achieved using citrate buffer (pH 6.0) and a microwave. Staining was carried out with rabbit anti-human glucocorticoid receptor Ser226 (Abcam, ab93104; 1:150 overnight at 4ᵒC) and rabbit anti-human phosphorylated p38 MAPK (Cell Signaling Technology, #9211; 1:400 overnight at 4ᵒC) coupled with an ImmPRESS™ Excel Amplified HRP Polymer Staining Kit (Anti-Rabbit IgG) with 3,3' diaminobenzidine as a substrate (Vector, MP-7601). Sections were counterstained in Gills haematoxylin. A rabbit igG isotype, diluted to the same concentration as the relevant primary antibody was used as a negative control (Cell Signaling Technology, #3900).
Images of the stained slides were captured using a Nikon Eclipse 80i microscope (Nikon UK Ltd) with an attached QImaging digital camera (Media Cybernetics). The percentages of glucocorticoid receptor Ser226 and phosphorylated p38 MAPK positive cells were calculated using the cell counting tool in ImageJ (version 1.49, NIH). Numbers of positively-and negatively-stained cells were counted from the epithelium of each biopsy and the number of positively-stained cells was expressed as a percentage of the total number of epithelial cells.

Data analysis
Two analyses were performed to assess whether a combination of dexamethasone and BIRB-796 exhibited synergy: a dose-sparing analysis to assess whether equivalent responses can be achieved at lower doses of compound than expected given the monotherapy response of the two compounds, and an efficacy-enhancing analysis to assess whether the combination results in a significantly greater maximal effect than either compound alone as monotherapies. The dose-sparing analysis calculates a combination index with confidence intervals using the method described by (Harbron, 2010). The efficacy-enhancing analysis fits Hill dose-response curves to the monotherapy and combination results using both common and separate parameters for maximal response and tests for the improvement in fit from allowing the parameter to vary by using an F test. Both analyses were performed assuming a slope parameter in the Hill dose-response equation equal to one. Robustness analyses were also performed estimating the slope parameters and found to give the same conclusions.
The maximal inhibition of poly I:C stimulated IL-6, CXCL8 and RANTES secretion was similar (p>0.05); maximal inhibition of LPS stimulated cytokine production was also similar between cytokines. BIRB-796 caused significantly greater maximal inhibition of TNFα induced IL-6 compared to CXCL8 and RANTES (p=0.02 for both comparisons) and greater inhibition of TNFα induced CXCL8 compared to RANTES (p=0.02) (Supplementary Table 1).
There were no significant differences in levels of inhibition between dexamethasone and BIRB-796 at any concentration for cytokines induced by poly I:C ( Figure 1). The inhibition of TNFα induced CXCL8 or RANTES were similar for dexamethasone and BIRB-796 at all concentrations, while the effect of dexamethasone on TNFα induced IL-6 was significantly greater compared to BIRB-796 at 10-1000 nM (p<0.05 for all comparisons). The inhibition of LPS induced CXCL8 or RANTES was similar at all concentrations, while the effect of dexamethasone on IL-6 was significantly greater compared to BIRB-796 at 1-1000 nM (p<0.05 for all comparisons).

S226 and S211
In additional experiments, TNFα alone and in combination with dexamethasone showed increased p38 MAPK phosphorylation which was reduced by BIRB-796 (Supplementary   Primary bronchial epithelial cells from healthy subjects (HNS) (n=5) or patients with asthma (n=5) were pre-treated with dexamethasone (100 nM) and BIRB-796 (100 nM) alone or in combination or with vehicle (DMSO 0.05%) for 1 hour before 24-hour stimulation with TNFα (10 ng/ml) or media (Basal). Supernatants were collected and assayed for IL-6 (A) or CXCL8 (B) release by ELISA. Data presented as Mean±SEM percentage inhibition of LPS induced cytokine levels. 2way ANOVA followed by Bonfferoni's post analysis used to compare between HNS and Asthma for all conditions.