Superior effect of MP‐AzeFlu compared to monotherapy with fluticasone propionate or azelastine on GILZ, MKP‐1 and TTP anti‐inflammatory gene expression in healthy and inflamed upper airway mucosa

Allergic rhinitis (AR) is a Th2 IgE- mediated disease with elevated levels of pro- inflammatory mediators, eosinophil infiltration of the nasal mucosa, and mucus hypersecretion. Antihistamines and intranasal corticosteroids, including MP- AzeFlu (intranasal fluticasone and azelastine), are recommended as the first- line therapy for AR. 1 Intranasal corticosteroids are also the first- line treatment for chronic rhinosinusitis with nasal polyps (CRSwNP) or without (CRSsNP), while antihistamines are only indicated in chronic rhinosinusitis (CRS) with concomitant AR. 2 MP- AzeFlu has demonstrated efficacy in AR, and a superior effect compared to these drugs administered individually, on nasal and ocular symptoms and quality of life; moreover, MP- AzeFlu has shown earlier and faster AR control in moderate- to- severe AR. 3 While MP-AzeFlu is guideline- recommended and its clinical efficacy has been widely studied, the mechanisms by which this therapy exerts its effect in AR are largely unknown. vitro models have limitations. In this artificial setting, interactions between cells are lost. Furthermore, the concentration of drugs used do not correlate with clinical doses used in the patient care setting. Thus, these findings may not translate to clinical outcomes and further studies are needed to confirm the findings. In conclusion, the superior clinical effect of MP- AzeFlu on both NP and NM compared with monotherapy may be partly related to the greater up- regulation of the anti- inflammatory gene expression of GILZ and, to a lesser extent, MKP- 1 and TTP expression.

MP-AzeFlu has demonstrated efficacy in AR, and a superior effect compared to these drugs administered individually, on nasal and ocular symptoms and quality of life; moreover, MP-AzeFlu has shown earlier and faster AR control in moderate-to-severe AR. 3 While MP-AzeFlu is guideline-recommended and its clinical efficacy has been widely studied, the mechanisms by which this therapy exerts its effect in AR are largely unknown.
Our laboratory has used an in vitro model of eosinophilic inflammation based on the interaction between cultured primary isolated nasal mucosa epithelial cells and isolated peripheral blood eosinophils to study the effect and potency of anti-inflammatory drugs. 4,5 Using this model, we have previously reported greater anti-inflammatory effects of the combination of an intranasal corticosteroid and an antihistamine, including MP-AzeFlu, in both nasal mucosa and nasal polyp epithelial cells, compared to the effect of these drugs administered alone. 5 We have also demonstrated that glucocorticoids induce the transcription of anti-inflammatory genes such as glucocorticoid-induced leucine zipper (GILZ), mitogenactivated protein kinase (MAPK) phosphatase 1 (MKP-1) and tristetraprolin (TTP) in nasal mucosa fibroblasts. 6 While detailed insights are lacking regarding the mechanisms of pathogenesis in CRSwNP, it is now clear that IgE-mediated Th2 inflammatory pathways play a critical role in this disease. 7 H 1 receptor antagonists decrease eosinophil survival 4 and inhibit the release of pro-inflammatory mediators by mast cells 8 and the production of proinflammatory cytokines by nasal epithelial cells. 4,9 In addition, azelastine (AZE) has been shown to enhance the anti-inflammatory effect of budesonide and improve nasal symptoms in AR patients through the induction of MKP-1 expression. 9 As such, it is reasonable to hypothesize that an increased induction of anti-inflammatory genes could partially explain the superior anti-inflammatory effect of MP-AzeFlu when compared to fluticasone propionate (FP) or AZE alone.
In the present study, we propose to analyse the anti-inflammatory effect of MP-AzeFlu, compared to monotherapy with FP or AZE, on GILZ, MKP-1 and TTP gene expression in healthy and inflamed upper airway mucosa.

| Study population
Nasal mucosa (NM) tissues were obtained from patients undergo-

| Experimental design
Fibroblasts were isolated using a specific and selective growth culture medium. The purity of fibroblast cultures was confirmed by positive immunostaining to vimentin (fibroblast marker) and negative to cytokeratin 1 (epithelial cell marker). 6 Cells were incubated with serum-free medium and treated for 2-24 h with MP-AzeFlu

| Ethics issues
Prior to study initiation, the protocol was approved by the HCB Ethics Committee. Written informed consent was obtained from all patients prior to their participation.

| Statistical methods
All results are expressed as mean ± standard error of the mean  Table 1).

MKP-1 protein expression was not detected when cells were incu-
bated with MP-AzeFlu and FP.

| TTP
MP-AzeFlu and FP at dilution 1:10 2 increased TTP mRNA expression in NM (at 2 h and 6 h) and NP (at 6 h).
AZE had no effect on TTP mRNA expression. MP-AzeFlu at dilution 1:10 2 showed a superior effect in the up-regulation of TTP mRNA expression compared to monotherapy with FP and AZE in NM (at 2 h) and with AZE in NM and NP (at 6 h; Table 1). The effect of drugs on TTP protein expression was not studied due to very low gene expression.

| DISCUSS ION
In

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. Sònia