Indirubin, a bisindole alkaloid from Isatis indigotica, reduces H1N1 susceptibility in stressed mice by regulating MAVS signaling

Abstract

Isatis indigotica has a long history in treating virus infection and related symptoms in China. Nevertheless, its antivirus evidence in animal studies is not satisfactory, which might be due to the lack of appropriate animal model. Previously, we had utilized restraint stress to establish mouse H1N1 susceptibility model which was helpful in evaluating the anti-virus effect of medicines targeting host factors, such as type I interferon production. In this study, this model was employed to investigate the effect and mechanism of indirubin, a natural bisindole alkaloid from Isatis indigotica, on influenza A virus susceptibility. In the in vitro study, the stress hormone corticosterone was used to simulate restraint stress. Our results demonstrated that indirubin decreased the susceptibility to influenza virus with lowered mortality and alleviated lung damage in restraint-stressed mice model. Moreover, indirubin promoted the expression of interferon-β and interferon inducible transmembrane 3. In addition, indirubin maintained the morphology and function of mitochondria following influenza A virus infection. Further study revealed that indirubin promoted interferon-β production through promoting mitochondrial antiviral signaling pathway. Our study indicated that indirubin could be a candidate for the therapy of influenza.

Keywords: MAVS; STING; indirubin; influenza virus; virus susceptibility.

Figure 1. Indirubin attenuates the morbidity and mortality caused by influenza infection in stressed mice

Chemical structure of indirubin (A). Effects of indirubin on body weight changes (B), morbidity (C) and survival rate (D) of mice after influenza infection. “-” indicates no treatment. Indirubin-H and Indirubin-L respectively represent the higher dose of indirubin (5 mg·kg⁻¹·d⁻¹) and the lower dose of indirubin (2.5 mg·kg⁻¹·d⁻¹). The difference was considered statistically significant at ^†P < 0.05 vs. Virus group; ^*P < 0.05 and ^**P < 0.01 vs. “Stress+Virus” group. Data were obtained from 10 - 14 animals in each group.

Figure 2. Indirubin protects against pneumonia caused by influenza infection in stressed mice

(A) Histologic sections of lung tissues on the 5^th day after influenza virus challenge, stained by H&E to exam histopathologic changes (Black scale bar = 100 μm, Blue scale bar = 25 μm). Black arrows represent congesting vessels and yellow arrows stand for inflammatory cells infiltration. (B) Effects of indirubin administrations on the lung index in H1N1-infected restraint-stressed mice. The protein expressions of IL-1β, IL-6 (C), TNF-α and IL-10 (D) in the lung tissues. “-” indicates no treatment. Indirubin-H and Indirubin-L respectively represent the higher dose of indirubin (5 mg·kg⁻¹·d⁻¹) and the lower dose of indirubin (2.5 mg·kg⁻¹·d⁻¹). The difference was considered statistically significant at ^†P < 0.05, ^††P < 0.01 vs. Normal group; ^#P < 0.05, ^##P < 0.01 vs. Virus group; ^*P < 0.05, ^**P < 0.01 vs. “Stress+Virus” group. Data are expressed as mean ± SEM from 3 independent determinations.

Figure 3. Indirubin promotes IFN-β generation through MAVS antiviral signaling after influenza infection in stressed mice

The protein expressions of NP (A), IFN-β, IFITM3 (B), MAVS (C) and p-IRF3, NF-κB (D) in the lung tissues on the 5^th day post infection. “-” indicates no treatment. The difference was considered statistically significant at ^††P < 0.01 vs. Normal group; ^#P < 0.05, ^##P < 0.01 vs. Virus group; ^*P < 0.05, ^**P < 0.01 vs. “Stress+Virus” group. Data are expressed as mean ± SEM from 3 independent determinations.

Figure 4. Indirubin promotes IFN-β generation through MAVS antiviral signaling after influenza infection in CORT-loaded A549 cells

NP, IFN-β and IFITM3 protein expressions in CORT-loaded A549 cells treated with different concentrations of indirubin (A). The protein expressions of MAVS, p-IRF3 (B), NF-κB, IFN-β and IFITM3 (C) in A549 cells. “-” indicates no treatment. The difference was considered statistically significant at ^†P < 0.05, ^††P < 0.01 vs. Normal group; ^#P < 0.05, ^##P < 0.01 vs. Virus group; ^*P < 0.05, ^**P < 0.01 vs. “CORT+Virus” group. Data are expressed as mean ± SEM from 3 independent determinations. H1N1-infected CORT-loaded A549 cells were fixed and stained for NP (D) and MAVS (E) expression (representative images from n = 3 per group, NP and MAVS: green, DAPI nuclear stain: blue, scale = 50 μm).

Figure 5. STING is involved in the regulation of IFN-β production by indirubin

The structure of indirubin bound to STING (A). An expanded view of the indirubin-binding pocket in the STING (B). Blue spheres represented THR266 residue and THR266 residue of STING protein, respectively. Binding mode between indirubin and STING (C). The protein expressions of STING (D, E) in A549 cells. The difference was considered statistically significant at ^††P < 0.01 vs. Normal group; ^##P < 0.01 vs. Virus group; ^*P < 0.05 vs. “CORT+Virus” group. Data are expressed as mean ± SEM from 3 independent determinations. The protein expressions of STING (F), IFN-β and IFITM (G) in A549 cells with or without siRNA transfection of STING. ^*P < 0.05, ^**P < 0.01. “-” indicates no treatment. Data are expressed as mean ± SEM from 3 independent determinations.

Figure 6. The maintenance of mitochondrial morphology contributes to the effect of indirubin on MAVS signaling

Expression of Mfn2 in restraint-stressed mice (A) and CORT-loaded A549 cells (B). The difference was considered statistically significant at ^††P < 0.01 vs. Normal group; ^#P < 0.05, ^##P < 0.01 vs. Virus group; ^**P < 0.01 vs. “Stress+Virus” or “CORT+Virus” group. “-” indicates no treatment. Data are expressed as mean ± SEM from 3 independent determinations. The morphology of mitochondria and the location of MAVS in A549 cells were observed by the transfection of pAcGFP1-Mito Vector and the immunofluorescence of MAVS (C) (MAVS: red, Mitochondria: green, DAPI nuclear stain: blue, scale = 5 μm). Mitochondrial membrane potential in A549 cells was determined by flow cytometry using JC-1 staining (D).

Figure 7. Schematic diagram of the mechanism of indirubin-induced attenuation of H1N1 pathogenesis in the susceptible model

MAVS localizes to mitochondria to exert an anti-viral effect. H1N1 infection triggers host cell activation of the NF-κB and IRF3 signaling pathway following MAVS sensing. STING acts as a mitochondria cofactor for MAVS signaling to promote NF-κB and IRF3 production. CORT impacts the function of mitochondria, which has adverse effects on MAVS signaling. Indirubin promotes the production of IFN-β and IFITM3 by recovering MAVS signaling and maintaining mitochondrial function affected by CORT in the susceptible model. Moreover, indirubin is found to promote MAVS signaling by regulating STING following influenza A virus infection. In addition, indirubin attenuates NF-κB dependent pro-inflammatory cytokine production to alleviate pneumonia in restraint-stressed mice.