The glucocorticoid dexamethasone alleviates allergic inflammation through a mitogen‐activated protein kinase phosphatase‐1‐dependent mechanism in mice

Glucocorticoids are widely used in the treatment of allergic and inflammatory diseases. Glucocorticoids have a widespread action on gene expression resulting in their pharmacological actions and also an array of adverse effects which limit their clinical use. It remains, however, to be studied which target gene effects are essential for the anti‐allergic activity of glucocorticoids. Mitogen‐activated protein kinase phosphatase‐1 (MKP‐1) inhibits proinflammatory signalling by suppressing the activity of mitogen activated protein kinase (MAP kinase) pathways. MKP‐1 is one of the anti‐inflammatory genes whose expression is enhanced by glucocorticoids. In the present study, we aimed to investigate the role of MKP‐1 in the therapeutic effects of the glucocorticoid dexamethasone in acute allergic reaction. The effects of dexamethasone were studied in wild‐type and MKP‐1 deficient mice. The mice were first sensitized to ovalbumin, and the allergic reaction was then induced by a subcutaneous ovalbumin injection in the hind paw. Inflammatory edema was quantified with plethysmometer and expression of inflammatory factors was measured by quantitative reverse transcription polymerase chain reaction (RT‐PCR). Dexamethasone reduced the ovalbumin‐induced paw edema at 1.5, 3 and 6 h time points in wild‐type mice by 70%, 95% and 89%, respectively. The effect was largely abolished in MKP‐1 deficient mice. Furthermore, dexamethasone significantly attenuated the expression of ovalbumin‐induced inflammatory factors cyclooxygenase‐2 (COX‐2); inducible nitric oxide synthase (iNOS); interleukins (IL) 1β, 6 and 13; C‐C motif chemokine 11 (CCL‐11); tumour necrosis factor (TNF) and thymic stromal lymphopoietin (TSLP) in wild‐type mice by more than 40%. In contrast, in MKP‐1 deficient mice dexamethasone had no effect or even enhanced the expression of these inflammatory factors. The results suggest that dexamethasone alleviates allergic inflammation through an MKP‐1‐dependent mechanism. The results also demonstrate MKP‐1 as an important conveyor of the favourable glucocorticoid effects in ovalbumin‐induced type I allergic reaction. Together with previous findings, the present study supports the concept of MKP‐1 enhancing compounds as potential novel anti‐inflammatory and anti‐allergic drugs.

MKP-1 as an important conveyor of the favourable glucocorticoid effects in ovalbumin-induced type I allergic reaction.Together with previous findings, the present study supports the concept of MKP-1 enhancing compounds as potential novel anti-inflammatory and anti-allergic drugs.
K E Y W O R D S allergy, edema, glucocorticoids, inflammation, MKP-1

| INTRODUCTION AND BACKGROUND
Allergic diseases such as atopic dermatitis, allergic rhinitis and food allergies affect approximately 20% of the global population. 1 Topical glucocorticoids, together with non-sedating antihistamines, are widely used in the treatment of IgE-mediated allergies to environmental allergens.Generally, glucocorticoids have a widespread effect on gene expression, but essential target genes to alleviate allergic inflammation remain poorly known.Identification of more specific pharmaceutical targets in the treatment of allergic diseases is warranted since longterm use of topical glucocorticoids is associated with risk of adverse effects.
Mitogen-activated protein kinase phosphatase-1 (MAP kinase phosphatase-1, MKP-1) is an enzyme which reduces the activity of pro-inflammatory MAP kinases through dephosphorylation. 2 The anti-inflammatory and anti-allergic activity of glucocorticoids is based on their effects on gene expression. 3Glucocorticoids suppress the expression of many pro-inflammatory genes and enhance that of several anti-inflammatory factors.MKP-1 is one of the genes upregulated by glucocorticoids, and it has been involved in the anti-inflammatory feedforward mechanisms of glucocorticoids. 4For instance, we have recently shown that the glucocorticoid dexamethasone suppresses catabolic enzyme expression in chondrocytes by enhancing MKP-1. 5We hypothesized that MKP-1 could also be a factor involved in the mechanisms of action of glucocorticoids in allergic inflammation.In the present study, MKP-1 dependency of the anti-allergic effects of dexamethasone was studied for the first time in vivo using an ovalbumin-induced acute allergic reaction in mice as a model.

| Mice
Wild-type and MKP-1 deficient C57BL/6 mice 6 originating from the R. Bravo laboratory at Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ, USA) were used in the experimental model of allergy.Mice were cared under standard conditions and anaesthetized as previously described. 7The study was performed in accordance with the EU directive on the protection of animals used for scientific purposes (Directive 2010/63/ EU) and upon approval of National Animal Experiment Board.The animal experiments were conducted in the preclinical facility of Tampere University, Tampere, Finland, and the laboratory analyses were performed in the laboratory of the Immunopharmacology Research Group, Tampere University, Tampere, Finland, in 2021.

| Ovalbumin-induced acute allergic paw edema
Ovalbumin-induced allergic paw edema model was carried out as previously described. 7In short, mice were sensitized to ovalbumin with an i.p. injection on day 1, boosted with another i.p. injection of ovalbumin on day 6 and challenged with a s.c.injection into the hind paw of anaesthetized mice on day 14 (Figure 1).On day 14, the mice were treated with an i.p. injection of dexamethasone (2 mg/kg, in phosphate-buffered saline (PBS), Orion Corporation, Espoo, Finland) or vehicle (PBS) 1 h prior to the ovalbumin challenge.The paw volumes were measured with plethysmometer (Ugo Basile, Comerio, Italy) before and 1.5, 3 and 6 h after the ovalbumin challenge.The ovalbumin-induced paw edema volumes at each time point were calculated by subtracting from the volume of the ovalbumin-challenged paw the volume of the same paw before the ovalbumin-challenged.The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies. 8he number of mice per group was based on sample size calculations to detect the effect of dexamethasone on ovalbumin-induced paw edema expecting at least 50% decrease with 30% standard deviation (SD) and with Type I significance level (α) of 0.05 and Type II significance level (β) of 0.2.Based on these study parameters, the minimum number of mice required per group was six.

| RNA extraction
After the 6 h paw volume measurements, mice were sacrificed.The paw tissues from both paws (ovalbuminchallenged paw and contralateral control paw) were stored in RNA later (Sigma-Aldrich, St. Louis, MO, USA).Tissue pieces were homogenized using the Precellys tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), and GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) was used to extract total RNA according to the manufacturer's guidelines.

| Quantitative RT-PCR
cDNA was reverse-transcribed from the total RNA with Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), subjected to quantitative polymerase chain reaction (PCR) using TaqMan Universal PCR Master Mix and detected with the Applied Biosystems™ 7500 Real-Time PCR System (Foster City, CA, USA).To detect mouse COX-2, iNOS, IL-6, TNF and GAPDH mRNA, primer and probe sequences and concentrations were optimized as instructed in TaqMan ® Universal PCR Master Mix Protocol Part Number 4304449 Rev. C (Applied Biosystems) and purchased from Metabion (Martinsried, Germany, sequences shown in Table 1).To detect mouse IL-1β (Mm00434228_m1), IL-13 (Mm00434204_m1), CCL-11 (Mm00441238_m1) and TSLP (Mm00498739_m1) TaqMan ® Gene Expression assays obtained from Thermo Fisher Scientific were used.PCR reaction was performed as previously described. 5The mRNA expression levels were normalized against GAPDH mRNA levels and were quantified using the standard curve method (genes detected with the optimized inhouse primers and probes) or the ΔΔCt method (genes detected with the TaqMan ® Gene Expression assays).

| Statistical analysis
Data are expressed as mean + standard error of the mean (SEM).Results were analysed by ANOVA with Bonferroni posttest using GraphPad Prism Version 9 (GraphPad Software, San Diego, California, USA).Normality was confirmed using Kolmogorov-Smirnov test.Most of the data was normally distributed, but some of the data were lognormal, and for those, the ANOVA results were calculated from the LN values.* = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001.
To examine inflammatory gene expression, the tissue from ovalbumin-challenged and vehicle-injected (control) paw was collected for RT-PCR analysis.Ovalbumin challenge clearly enhanced the expression of inflammatory genes in sensitized wild-type (Figure 3) and MKP-1 deficient (Figure 4) mice.The percentage increase (supporting information Figure S1) as well as the expression level of many of the inflammatory genes (supporting information Figure S2) was even higher in MKP-1 deficient mice.
Notably, dexamethasone had no effect on the expression of these genes in the control paw in either genotype (Figures 3 and 4).

| DISCUSSION
The present study showed that the glucocorticoid dexamethasone alleviated allergic inflammation in the ovalbumin-induced acute allergic reaction by reducing inflammatory edema and the expression of inflammatory factors in wild-type but not in MKP-1 deficient mice.The results suggest that the anti-allergic effects of glucocorticoids are largely dependent on MKP-1.
Glucocorticoids are widely used in the treatment of inflammatory diseases and also in allergic conditions preferentially as topical preparations. 1Glucocorticoids exert their effects in the target cells by binding to the glucocorticoid receptor (GR), which is activated and migrates to the nucleus. 3The glucocorticoid-GR complex binds to the glucocorticoid-responsive element (GRE) and upregulates the transcription of target genes.Additionally, glucocorticoids inhibit the activation and/or activity of proinflammatory transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1). 3By these mechanisms, glucocorticoids suppress the expression of a large number of pro-inflammatory genes and enhance the expression of many anti-inflammatory factors.1][12][13][14] An excessive proinflammatory response is a general appearance of MKP-1 deficiency in those models.However, these previous studies have mainly been focusing on type I inflammatory reactions and macrophage responses.In mouse macrophages, glucocorticoids enhance the expression of MKP-1 which results in the dephosphorylation and inhibition of p38 and JNK MAP kinases. 15Moreover, in mast cells which play a central role in allergic reactions, glucocorticoids have also been reported to increase MKP-1 expression. 16The results of the present study suggest that MKP-1 has also a meaningful role in acute allergic reaction because allergen challenge induced an increased edema formation and inflammatory gene expression in sensitized MKP-1 deficient mice as compared with wildtype mice.In addition, the anti-allergic effects of dexamethasone were absent or significantly reduced in MKP-1 deficient mice.
The present study showed that the glucocorticoid dexamethasone reduced allergen-induced paw edema in sensitized wild-type mice.The anti-allergic effect of dexamethasone has also been found in ovalbumininduced paw edema model in the rat, but the mechanism of dexamethasone action was not studied in detail. 17Interestingly, we found in the present study that the effect of dexamethasone on the edema formation was significantly reduced in MKP-1 deficient mice.To our knowledge, the effects of glucocorticoids on acute allergic inflammation in MKP-1 deficient mice have not been previously studied.In accordance with the previously mentioned study, 17 our results demonstrate the resolving effects of the glucocorticoid dexamethasone in ovalbumin-induced acute allergic reaction, and we show here for the first time that this effect is, at least in part, MKP-1-dependent.
Dexamethasone was also found to attenuate the expression of the inflammatory factors COX-2, iNOS, IL-1β, IL-6, IL-13, CCL-11, TNF and TSLP in ovalbuminchallenged paw tissue from wild-type mice.Reduced expression of those factors is likely to result in an alleviation of the acute allergic paw edema and inflammation: COX-2 and iNOS synthesize vasodilatating and proinflammatory prostaglandins and NO. 18Interleukins 1β, 6 and 13 as well as TNF, in addition to their other proinflammatory effects, upregulate the expression of chemokines and leukocyte adhesion molecules in endothelium resulting in the recruitment of blood leukocytes. 1 Chemokines CCL-11 and TSLP recruit particularly granulocytes. 1otably, in MKP-1 deficient mice, dexamethasone did not inhibit the expression of any of the measured inflammatory factors, and COX-2, IL-1β, IL-6 and CCL-11 expression was even enhanced.The lack of the inhibitory effect of dexamethasone on inflammatory gene expression in MKP-1 deficient mice strongly supports the mediator role of enhanced MKP-1 expression and subsequently reduced MAP kinase activity in the glucocorticoid effect, 19 whereas the enhancing effect of dexamethasone on COX-2, IL-1β, IL-6 and CCL-11 expression in MKP-1 deficient mice is less obvious to explain.Glucocorticoids have, however, been reported to enhance the expression of the toll-like receptor 2 (TLR2), 20,21 the purinergic receptor P2Y2R 22 and the inflammasome component NLRP3 23 possibly through transcriptional synergy between activated GR and the inflammatory transcription factor NF-κB or AP-1. 19All of those effects are likely to enhance inflammatory factor expression, particularly in inflammatory conditions and under reduced activity or in the absence of the MKP-1 mediated anti-inflammatory mechanisms of glucocorticoids.
The present study was conducted in an experimental ovalbumin-induced allergic inflammation in mice displaying MKP-1 as a potential future drug target mediating the anti-allergic effects of glucocorticoids.The model mimics acute type I hypersensitivity reaction; to apply the findings to other types of allergies and to chronic allergic diseases, additional studies in animal models and further in clinical settings are needed.In this preliminary study, the effects on gene expression were assessed at mRNA level and additional research is needed to confirm their translation to protein level.Also, the use of pharmacological inhibition of MKP-1 in addition to its genetic deletion would have strengthen the results, but unfortunately, specific MKP-1 inhibitors are not currently available.
Specific medications that act by enhancing MKP-1 expression are not yet available, but their potential adverse effects should also be considered.We assume that targeted enhancement of MKP-1 (leading to the dephosphorylation and inhibition of p38 and JNK MAP kinases) would have significantly less adverse effects than glucocorticoids in general.Inhibitors of MAP kinases are under investigation, and their adverse effect profile is likely to be rather similar to that of MKP-1 enhancing compound would have.Most common adverse effects regarding MAP kinase inhibitors include fatigue, diarrhoea, rash and nausea. 24However, these adverse effects (as adverse drug effects in general) may in part result from unspecific effects and the adverse effect profile of therapies aiming to specifically enhance MKP-1 expression remains to be revealed in further studies.
In conclusion, the present results show that the glucocorticoid dexamethasone alleviates experimental ovalbumin-induced acute allergic reaction in mice through an MKP-1-mediated mechanism.The results also support the role of MKP-1 as a regulatory/anti-inflammatory factor in allergic inflammation.Together with previous results, the present findings endorse the concept of MKP-1 enhancing compounds as potential novel drugs to treat allergic inflammation.

F
I G U R E 2 Dexamethasone reduced ovalbumin-induced paw edema in wild-type (WT) mice but not in MKP-1 deficient (knockout, KO) mice.The mice were first sensitized to ovalbumin, and after 14 days, they were challenged with ovalbumin injection into the hind paw.Dexamethasone (2 mg/kg) was injected i.p. 1 h prior to the ovalbumin challenge.Paw edema was measured before and 1.5, 3 and 6 h after the ovalbumin challenge.The ovalbumin-induced paw edema volumes at each time point were calculated by subtracting from the volume of the ovalbumin-challenged paw at the indicated time point the volume of the same paw before the ovalbumin challenge.Data are expressed as the mean+SEM, n = 8-9.Results were analysed with ANOVA followed by Bonferroni post-test in each time point, * = p < 0.05, *** = p < 0.001, **** = p < 0.0001, ns = not significant.TA B L E 1 Primer and probe sequences.

F I G U R E 3
Dexamethasone reduced ovalbumin-induced expressions of inflammatory factors in sensitized wild-type (WT) mice.The mice were first sensitized to ovalbumin, and after 14 days, they were challenged with ovalbumin injection into the hind paw.Dexamethasone (2 mg/kg) was injected i.p. 1 h prior to the ovalbumin challenge.The mice were sacrificed 6 h after the ovalbumin challenge, and COX-2, iNOS, IL-1β, IL-6, IL-13, CCL-11, TNF and TSLP mRNA levels in the paw tissues were measured with RT-PCR and normalized against GAPDH mRNA.The contralateral hind paws injected with vehicle (PBS) served as a control (the first two bars).The ovalbumin-induced expression levels were set as 100%, and other values are given in relation to that.Data are expressed as the mean+SEM, n = 6-9.Results were analysed with ANOVA followed by Bonferroni post-test, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.F I G U R E 4 Dexamethasone had no effect on ovalbumin-induced expression of inflammatory factors iNOS, IL-13, TNF and TSLP in MKP-1 deficient mice hind paws, and the expression of COX-2, IL-1β, IL-6 and CCL-11 was even enhanced.The mice were first sensitized to ovalbumin, and after 14 days, they were challenged with ovalbumin injection into the hind paw.Dexamethasone (2 mg/kg) was injected i.p. 1 h prior to the ovalbumin challenge.The mice were sacrificed 6 h after the ovalbumin-challenge, and COX-2, iNOS, IL-1β, IL-6, IL-13, CCL-11, TNF and TSLP mRNA levels in the paw tissues were measured with RT-PCR and normalized against GAPDH mRNA.The contralateral hind paws injected with vehicle (PBS) served as a control (the first two bars).The ovalbumin-induced expression levels were set as 100%, and other values are given in relation to that.Data are expressed as the mean+SEM, n = 6-9.Results were analysed with ANOVA followed by Bonferroni post-test, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.