The mixture of siRNAs targeted to IL-4 and IL-13 genes effectively reduces the airway hyperreactivity and allergic inflammation in a mouse model of asthma

https://doi.org/10.1016/j.intimp.2021.108432Get rights and content

Highlights

  • Th2 cytokines IL-4 and IL-13 play a key role in the pathogenesis of bronchial asthma.

  • Intranasal delivery of siRNAs targeting IL-4 and IL-13 inhibited their expression.

  • Inhibition of IL-4 and IL-13 expression leads to weakening of inflammation in Lungs.

Abstract

Bronchial asthma (BA) is one of the most common chronic inflammatory disease of airways. There are huge experimental data indicating that Th2-cytokines IL-4 and IL-13 play a key role in BA pathogenesis. They are implicated in the IgE synthesis, eosinophil infiltration to the lungs and in the development of airway hyperreactivity (AHR), that makes these cytokines the promising targets. Neutralization of IL-4 and IL-13 or its common receptor chain (IL-4Rα) by monoclonal antibodies substantially reduce asthma symptoms. RNA interference provides a novel method for regulation of gene expression by siRNA molecules. In this study we evaluated whether the siRNA targeted to IL-4 and IL-13 reduce BA symptoms in mice model.

Experimental BA was induced in BALB/c mice by sensitization to ovalbumin allergen followed by intranasal challenge. The intranasal delivery of siRNAs targeted to IL-4 and IL-13 inhibited the lung expression of these cytokines by more than 50% that led to the attenuation of AHR and pulmonary inflammation; the quantity of eosinophils in lungs which are one of the major inflammatory cells involved in allergic asthma pathogenesis decreased by more than 50% after siRNA treatment. These data support the possibility of a dual IL-4 and IL-13 inhibition by locally delivered siRNAs which in turn leads to the suppression of allergen-induced pulmonary inflammation and AHR.

Introduction

Bronchial asthma (BA) is a chronic inflammatory disease of the airways, which affects more than 300 million people worldwide [1]. The global mortality rate of the disease is 13 and 9 per 100 000 for men and women, respectively [2]. BA provides significant economic issues; Europe Union spends about €17.7 billion annually to treat the disease, annual cost of BA in the United States is about $18 billion [3]. In Russian Federation expenses for BA treatment reach 14 billion rubles a year [4]. The majority of BA cases around the world (up to 80%) are allergic and associated with the sensitization to the allergens [1], [5]. Allergic asthma is characterized by common clinical symptoms (mucus hypersecretion, pulmonary inflammation, AHR) and by the presence of allergen-specific IgE-antibodies in the serum, increase in the proportion of eosinophils in the blood and BAL [5].

According to clinical and animal studies, it has been found that allergic asthma is developed through Th2-dependant mechanisms [6], [7]. Activated Th2-lymphocytes induce, via the production of cytokines (IL-4, IL-5 and IL-13), an inflammatory cascade comprised of eosinophil action, IgE production, and mast-cell activation, all of which are necessary mediators causing AHR [6], [7]. Cytokines IL-4 and IL-13 are the most important in the development of Th2-mediated allergic inflammation. IL-4 and IL-13 have a common receptor (chains IL-4Rα and IL-13Rα1) because of this, they have similar biological functions: induce IgE isotype switching, stimulate VCAM-1 expression, which is necessary to recruit eosinophils to the lung. This heterodimeric receptor (IL-4Rα/IL-13Rα1) is expressed on many immune and non-immune cells. Binding of IL-4 and IL-13 to the receptor leads to activation of tyrosine kinases of the Janus family (Jak1–3 and Tyk2), which phosphorylate transcription factor STAT6. STAT6 dimers translocate to the nucleus, where upregulate the transcription of IL-4/IL-13-responsive genes involved in Th2 differentiation, isotype class switching of B cells to IgE synthesis, AHR development, and mucus production [8], [9].

Allergen-specific immunotherapy and the use of glucocorticoids and bronchodilators are the most common ways of BA treatment [10]. However, some patients, especially those with severe asthma are often resistant to conventional therapies [11], [12], [13]. To treat these severe forms of the disease, inhibitors of the IL-4Rα/STAT6 pathway have been developed [14], [15]. Dupilumab is a novel drug, based on monoclonal antibodies targeted to IL-4Rα (common receptor chain for both IL-4 and IL-13), which was recently approved to treat BA [16], [17], however its broad use is restricted by high cost.

At the same time, the new ways to regulate the gene expression are developing. RNA interference is a technology of negative post-transcriptional regulation of gene expression by small interfering RNA molecules (siRNA) [18], [19]. In this study we used intranasally delivered siRNAs targeted to both IL-4 and IL-13 to reduce allergic inflammation in a mouse model of BA. Our results indicate that suppression of these cytokines decreases the allergen-induced inflammatory response and suggests that inhaled anticytokine siRNAs could be an effective anti-inflammatory approach for respiratory diseases.

Section snippets

Construction of plasmids carrying mouse IL-13 gene

To clone mouse IL-13, the coding sequence of corresponding gene was amplified by PCR from concanavalin A stimulated mouse splenocyte cDNA, verified by sequencing, and cloned into the expression vector pUCHR IRES GFP [20]. Expression of the gfp gene, located downstream of the internal ribosome entry site (IRES), was used as an indicator of the level of IL-13 expression.

Design of siRNAs

To select the optimal sequences of siRNA molecules that can effectively suppress the target gene, we used the specialized

Design siRNAs and evaluation of it’s activity in vitro

siRNAs targeted to mouse IL-4 was developed in our previous study [22]. The most potent gene silencing provided variant siIL4-408 [22]. To design siRNAs against mouse IL-13 gene OligoWalk software was used [21]. In total 47 different variants of siRNAs were predicted. Five of them (named as siIL13-95, siIL13-195, siIL13-230, siIL13-280 and siIL13-371) were chosen for synthesis and evaluation of silencing activity in in vitro experiments. Selected variants matched following criteria: 1) the

Discussion

As mentioned above, asthma - is a common chronic inflammatory disease of the airways. Despite the availability of a broad-spectrum anti-inflammatory drugs the question of uncontrolled or poorly controlled BA cases still exists, that drives the development of new therapeutic approaches. Recent advances in studies of molecular mechanisms of asthma pathogenesis revealed that Th2-cytokines (especially IL-4 and IL-13) take important part in the initiation and maintenance of this pathology. For

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The study was supported by RFBR grant No 20-34-90151 and RSF grant No 19-15-00272.

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