MG149 inhibits histone acetyltransferase KAT8-mediated IL-33 acetylation to alleviate allergic airway inflammation and airway hyperresponsiveness

Abstract

Asthma is one of the most common heterogeneous airway diseases. Worldwide, the prevalence of doctor-diagnosed asthma in adults was reported to be 4.3% in 2003. In China, the overall prevalence of asthma during 2012 and 2015 was 4.2%, representing 45.7 million Chinese adults. Although comprehensive approaches have been used in clinical practice, a considerable number of people are still hospitalized for acute exacerbation of asthma every year. In China, 15.5% of asthmatics reported at least one emergency room visit and 7.2% reported at least one hospital admission due to exacerbation of respiratory symptoms. A need exists to clarify the mechanism of asthma’s pathogenesis and to identify more appropriate and precise therapies for asthma. Post-translational modifications (PTMs) are involved in many physiological and pathological processes by regulating the stability, localization, and activity of proteins. The lysine acetylation modification has emerged as one of the major PTMs, which regulates gene transcription and various cellular functions. Previous studies have found an increase in lysine acetyltransferase (KAT) activity and some reduction in lysine deacetylase (KDAC) activity in asthma, which implies that these enzymes may be potential drug targets in asthma. Histone acetyltransferase KAT8 is a member of the MYST family of proteins, which is highly conserved across species and involved in a wide range of cellular functions, including gene expression, DNA damage repair, cell death, stem cell development, and oncogenesis. However, little is known about whether KAT8 plays a role in asthma and how it works. There is only one report on this topic, in which Bosch et al. found that pro-inflammatory gene expression was decreased in lipopolysaccharide and interferongamma stimulated murine precision-cut lung slices after MG149 (a KAT8 inhibitor) treatment, which indicated that MG149 had the potential to develop applications for the treatment of inflammatory lung diseases. Whether MG149 can play role in relieving inflammation in vivo and how it exerts an anti-inflammatory effect remain unclear. In order to explore the effects of MG149 on airway inflammation, we developed a house dust mite (HDM)-challenged mouse model of allergic asthma (Supplementary Fig. S1a). For the MG149 intervention group, the anaesthetized C57BL/6 mice were pretreated with MG149 60minutes prior to HDM administration. We found that mice exposed to HDM developed airway hyperresponsiveness (AHR), and administration of MG149 significantly reduced the airway resistance (Supplementary Fig. S1b). Additionally, administration of MG149 also significantly decreased total protein levels and total cell numbers in bronchoalveolar lavage fluid (BALF) compared with HDM-challenged mice (Supplementary Fig. S1d, e). Hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining showed that MG149 could relieve the inflammatory cell infiltration and mucus secretion (Supplementary Fig. S1f, g). Additionally, we also found that MG149 treatment significantly reduced collagen deposition around the airways (Supplementary Fig. S1h). Collectively, these results demonstrated that MG149 could relieve HDMinduced AHR and airway inflammation in a murine allergic asthma model. Next, we sought to study the mechanism underlying KAT8’s function in this process. Western blotting of mouse lung homogenate demonstrated that HDM treatment increased the expression of IL-33, an effect that was reduced by administration of MG149 (Supplementary Fig. S2a). The changes of IL-33 protein levels were consistent with the results of immunohistochemistry (IHC) of lung sections and enzyme-linked immunosorbent assay (ELISA) of mouse lung homogenate (Supplementary Fig. S2b–d). Given that IL-33 was a key cytokine participating in the pathogenesis of asthma, we hypothesized that IL-33 was related to this process by which KAT8 inhibition alleviated allergic asthma. To verify this hypothesis, we developed allergic asthma model in both wild-type (WT) and Il33 mice. The observed series of changes in airway resistance, total IgE, total BALF cell numbers, and total BALF protein levels in WT mice after HDM stimulation and MG149 treatment were the same as before, but it was intriguing that MG149 did not play a role in the absence of IL-33 (Fig. 1a, b, Supplementary Fig. S3). Next, we measured the type 2 cytokines in mediastinal lymph nodes (mLNs) and lung tissue to further assess type 2 immune response and the efficacy of MG149. Similar results were found in that there was no difference between HDM-treated Il33 mice and HDM+MG149 treated Il33 mice (Fig. 1b–f, Supplementary Figs. S4–8). Taken together, these data indicated that KAT8 played a role in asthma, and this process was dependent on IL-33. We next explored how KAT8 functioned in asthma. We performed confocal microscopy to localize KAT8, and found that the airway epithelial cells of asthmatics highly expressed IL-33, while the control group only expressed IL-33 in the basal cells of the airways (Fig. 1g, Supplementary Fig. S9). More importantly, KAT8 was also expressed highly in the lungs of asthma patients and co-localized with IL-33 (Fig. 1g, Supplementary Fig. S9). We also localized KAT8 in mouse lung sections and found that IL-33 and KAT8 were also co-localized in mouse alveolar epithelial cells (Supplementary Fig. S10). These results suggested that KAT8 was spatially co-localized with IL-33, which further confirmed the possible interaction between the two proteins. In view of the above, we suspected that KAT8 may interact with IL-33. First, we constructed overexpression vectors of mouse IL-33 and KAT8, and found that mouse IL-33 interacted with KAT8 via co-immunoprecipitation (coIP) (Supplementary Fig. S11a). Because IL-33 and KAT8 were both highly conserved proteins, we speculated that human IL-33 and KAT8 may also interact. We then performed coIP to confirm the interaction between human