Indirubin induces tolerogenic dendritic cells via aryl hydrocarbon receptor activation and ameliorates allergic asthma in a murine model by expanding Foxp3-expressing regulatory T cells

Background: Allergic asthma is a chronic bronchial inflammatory disease closely associated with abnormal immune responses of dendritic cells (DCs) and allergen-specific type 2 T helper (Th2) cells. Indirubin (IR), a natural aryl hydrocarbon receptor (AhR) ligand, exerts anti-inflammatory and immunomodulatory properties. Purpose: In this study, we aimed to clarify whether IR exhibits immunomodulatory action on DCs via AhR activation and investigated the antiallergic effects of IR in a mouse model of allergic asthma. Methods: Lipopolysaccharide (LPS)-activated bone marrow-derived DCs were treated with IR. Their mRNA expressions, cytokine production, and phenotype patterns were determined by a quantitative real-time PCR, ELISA, flow cytometry, and RNA sequencing. The mixed lymphocyte reaction was utilized to evaluate the regulatory function of IR-treated DCs on T-cell differentiation. Moreover, mice with ovalbumin (OVA)-induced allergic asthma were treated with IR. Thereafter, the airway hyperresponsiveness (AHR), allergen-specific IgE production, cytokine levels, airway inflammation, and T-cell responses were evaluated. Results: Treatment of LPS-stimulated DCs with 20 mu M IR significantly reduced IL-12 and TNF-alpha production while increasing IL-10 secretion. Meanwhile, these DCs expressed decreased levels of CD80 but increased levels of Jagged 1 surface molecules. However, the effects of IR on DCs were reversed by pretreatment with the AhR antagonist, CH223191. Additionally, the coculture of these tolerogenic-like DCs with allogeneic CD4(+) T cells promoted the generation of Foxp3(+) regulatory T (Treg) cells. A transcriptomic analysis identified several downregulated genes that are involved in regulating cell migration, cytokine secretion, and inflammatory responses in DCs after IR treatment. In an asthmatic murine model, oral administration of 25 mg kg(-1) body weight of IR efficiently alleviated the development of AHR, OVA-specific IgE production, and levels of Th2-type cytokines (IL-4, IL-5, and IL-13) and the CCL11 chemokine. IR treatment also attenuated inflammatory cell recruitment and mucus production in the lungs. Notably, an enhanced frequency of Foxp3(+) Treg cells and reduced effector T-cell proliferation associated with increased levels of IL-10 and TGF-beta were observed in IR-treated mice. Conclusion: IR can induce tolerogenic-like BMDCs which promote the differentiation of Treg cells. Importantly, the expansion of Foxp3(+) Treg cells contributed to the therapeutic efficacy of IR against allergic asthma.