Endosomal NOX2 oxidase exacerbates virus pathogenicity and is a target for antiviral therapy

Abstract

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.Production of reactive oxygen species is an ancient antimicrobial mechanism, but its role in antiviral defense in mammals is unclear. Here, To et al. show that virus infection activates endosomal NOX2 oxidase and restricts TLR7 signaling, and that an endosomal NOX2 inhibitor decreases viral pathogenicity.

Fig. 1

Seasonal and pandemic influenza A viruses induce endosomal ROS production via activation of NOX2 oxidase. a, b Confocal microscopy of wild-type (WT) mouse primary alveolar macrophages that were infected with influenza A virus strain HKx31 (MOI of 10) for 1 h and labeled with antibody to the early endosome antigen 1 (EEA1) and antibodies to either a influenza A virus nucleoprotein (NP) or b NOX2, and then with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Also shown is the quantification of results (n = 5). c, d Time-dependent elevation in endosomal ROS levels in mouse primary alveolar macrophages as assessed by OxyBURST (100 μM) confocal fluorescence microscropy and labeled with DAPI (n = 5). e, f Endosomal ROS production in WT, NOX2^−/y and superoxide dismutase (SOD; 300 U/ml)-treated WT mouse primary alveolar macrophages as assessed by OxyBURST confocal fluorescence microscopy in the absence or presence of HKx31 virus and labeled with DAPI (n = 5). g Uninfected and HKx31 virus-infected mouse primary alveolar macrophages were labeled with OxyBURST and the acidified endosome marker Lysotracker (50 nM). Some cells were treated with bafilomycin A (Baf-A; 100 nM) to suppress acidification of endosomes (n = 4). h Human alveolar macrophages infected with seasonal H3N2 (A/New York/55/2004, A/Brisbane/9/2007), seasonal H1N1 (A/New Caledonia/20/1999, A/Solomon Islands/3/2006) and pandemic A(H1N1) pdm09 strains (A/California/7/2009, A/Auckland/1/2009) and labeled with OxyBURST for endosomal ROS (n = 4). i, j Endosomal ROS production in WT mouse primary alveolar macrophages as assessed by OxyBURST fluorescence microscopy exposed to either heat (56 ^ºC)-inactivated HKx-31 virus (to block virus fusion) or UV-inactivated HKx-31 virus (to block replication) and labeled with DAPI (n = 4). a–i Images are representative of >150 cells analyzed over each experiment. Original magnification ×100. a, b, d, f and j Data are represented as mean ± S.E.M. a and b Students’ unpaired t-test *P < 0.05. d, f and j One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. *P < 0.05 and **P < 0.01. Scale bars: 10 µm

Fig. 2

Co-localization of TLR7 with influenza A virus, NOX2 and EEA1 is a signaling platform for endosomal ROS generation to influenza A virus via a TLR7 and PKC-dependent mechanism. a–c Confocal microscopy of mouse primary alveolar macrophages that were untreated or infected with influenza A virus HKx31 (MOI of 10) and labeled with antibodies to TLR7 and either a influenza A virus NP, b NOX2 or c EEA1, and then with 4′,6′-diamidino-2-phenylindole (DAPI). Quantification data from multiple experiments are also shown (n = 5). d Endosomal ROS production in WT and TLR7^−/− mouse primary alveolar macrophages as assessed by Oxyburst (100 μM) fluorescence microscopy in the absence or presence of HKx31 virus and labeled with DAPI (n = 6). e Immunofluorescence microscopy for assessment of NOX2 and p47phox association. WT and TLR7^−/− immortalized bone marrow-derived macrophages (BMDMs) were untreated or infected with HKx31 virus, (MOI of 10) in the absence or presence of bafilomycin A (Baf-A; 100 nM) or dynasore (Dyna; 100 μM), and then labeled with antibodies to NOX2 and p47phox. Also shown is the quantification of the results (n = 5). f, g Endosomal ROS production in WT and NOX2^−/y mouse primary alveolar macrophages as assessed by Oxyburst fluorescence microscopy in the absence or presence of f imiquimod (Imiq; 10 μg/ml) and g ssRNA (100 μg/ml) and co-labeled with DAPI. (n = 5). h, i Cytosolic PKC activity as assessed by FRET analysis in WT and TLR7^−/− BMDMs. Cells were either treated with vehicle controls or with bafilomycin A (100 nM) or dynasore (10 μM) and then exposed for 25 min to influenza A virus (HKx31, MOI of 10) or imiquimod (10 μg/ml) (n = 3). a–g Images are representative of >150 cells analyzed over each experiment. Original magnification ×100. All data are represented as mean ± S.E.M. a, b, c, f and g Student’s unpaired t-test *P < 0.05. d, e, h and i One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. *P < 0.05. Scale bars: 10 µm

Fig. 3

Endosomal ROS production to ssRNA and DNA viruses are via TLR7 and TLR9-dependent mechanisms, respectively. a Endosomal ROS production in WT and TLR7^−/− bone marrow-derived macrophages as assessed by OxyBURST (100 μM) fluorescence microscopy in the absence or presence of influenza A virus (HKx31 virus), rhinovirus (rhino), respiratory synctitial virus (RSV), human parainfluenza virus (PIV), human metapneumovirus (HMPV), sendai virus, dengue virus, human immunodeficiency virus (HIV), mumps virus (MuV), Newcastle disease virus (NDV), rotavirus (UK and bovine strains), herpes simplex virus 2 (HSV-2), and vaccinia virus and labeled with 4′,6′-diamidino-2-phenylindole (DAPI). Also shown is the quantification of the results (n = 5). b Endosomal ROS production in WT and TLR9^−/− mouse primary alveolar macrophages as assessed by OxyBURST fluorescence microscopy in the absence or presence of HKx31 virus, rhinovirus, sendai virus, dengue virus, and herpes simplex virus 2 (HSV-2) and labeled with DAPI (n = 5). a and b Images are representative of >150 cells analyzed over each experiment. Original magnification ×100. All data are represented as mean ± S.E.M. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. ^# P < 0.05 compared to WT control. *P < 0.05 comparisons indicated by horizontal bars. Scale bars: 10 µm

Fig. 4

Bacteria-induced ROS production is distinct from virus-dependent ROS mechanisms. a Phagosomal superoxide production to Haemophilus influenzae and Streptococcus pneumoniae as assessed by OxyBURST (100 μM) fluorescence microscopy in WT and TLR7^−/− immortalized bone marrow derived macrophages. Images are representative of >150 cells analyzed over each experiment. Original magnification ×100. b Is the quantification of the results (n = 5). All data are represented as mean ± S.E.M. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. *P < 0.05 compared to WT control. Scale bar: 10 µm

Fig. 5

Endosomal NOX2 oxidase suppresses cytokine expression in response to TLR7 activation in vitro and in vivo. a, b WT and TLR7^−/− immortalized bone marrow-derived macrophages (BMDMs) were untreated or treated with imiquimod (Imiq; 10 μg/ml) in the absence or presence of a apocynin (Apo; 300 μM) or b bafilomycin A (Baf-A; 100 nM), and IFN-β, IL-1β, TNF-α and IL-6 mRNA expression determined by QPCR after 24 h (n = 6). c, d WT and NOX2^−/y mice were administered with imiquimod (50 μg per mouse, intranasal) and c total airway inflammation quantified by bronchoalveolar lavage fluid analysis and d cytokine expression assessed 24 h later (n = 5). a, b, d Responses are relative to GAPDH and then expressed as a fold-change above WT controls. a–d Data are represented as mean ± S.E.M. a, b and d Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons. c One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. Statistical significance was accepted when P < 0.05. *P < 0.05; **P < 0.01

Fig. 6

Endosomal NOX2 oxidase-derived hydrogen peroxide (H₂O₂) inhibits cytokine expression in response to TLR7 activation in vitro and in vivo. a WT mouse primary alveolar macrophages were either left untreated or treated with FITC-labeled catalase for 5 min prior to infection with HKx31 virus (MOI of 10). Cells were labeled for Lysotracker (50 nM) and colocalization of Lysotracker and FITC catalase assessed by confocal microscopy. Images are representative of >100 cells analyzed over each experiment. Original magnification ×100 (n = 3). b WT and TLR7^−/− immortalized bone marrow-derived macrophages (BMDMs) were left untreated or treated for 1 h with catalase (1000 U/ml) and IFN-β and IL-1β, mRNA expression determined by QPCR after 24 h (n = 7). c WT BMDMs were left untreated or treated for 1 h with imiquimod (Imiq) in the absence or presence of catalase (1000 U/ml), IFN-β and IL-1β, mRNA expression assessed 24 h later by QPCR (n = 6). d WT BMDMs were treated for 30 min with either DMSO (0.1%) or dynasore (Dyna; 100 μM) and then with catalase (1000 U/ml) for 1 h. Cytokine mRNA expression determined by QPCR after 24 h (n = 6). e WT and TLR2^−/− immortalized BMDMs were treated with catalase (1000 U/ml) for 1 h and cytokine mRNA expression determined by QPCR after 24 h (n = 6). f WT and UNCB93^−/− immortalized BMDMs were treated with catalase (1000 U/ml) for 1 h and cytokine mRNA expression determined by QPCR after 24 h (n = 6). g–i WT BMDMs were treated for 1 h with either catalase or imiquimod (10 μg/ml) and g TLR7, h NLRP3 or i TREML4 mRNA expression determined by QPCR after 24 h (n = 6). j Mice were intranasally treated with catalase (1000 U per mouse) and then lung expression of TREML4 was determined by QPCR (n = 5). k and l Catalase (1000 U per mouse, intranasal) was administered to WT mice and k total BALF airway inflammation and l lung cytokine expression assessed 24 h later (n = 5). b–j and l Responses are relative to GAPDH and then expressed as a fold-change above WT controls. b–h and l Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons. i and j Mann–Whitney Wilcoxon test. All data are represented as mean ± S.E.M. Statistical significance was taken when the P < 0.05. *P < 0.05. Scale bar: 10 µm

Fig. 7

C98 on TLR7 regulates activity of the receptor and is a target for endosomal H₂O₂. a TLR7^−/− BMDMs were transfected with empty vector, WT TLR7 or with either TLR7 with cysteines 98, 260, 263, 270, 273, and 445 mutated to alanine (TLR7 6 mut), TLR7 with cysteines 98 and 445 mutated to alanine (TLR7C98A/445 A) or with TLR7 with cysteines 445 (TLR7C445A) or 98 (TLR7C98A) mutated to alanine. After 48 h, cells were left untreated or treated for 1 h with either catalase (1000 U/ml) or imiquimod (Imiq, 10 μg/ml) and cytokine expression assessed 24 h later (n = 6). Responses are relative to GAPDH and then expressed as a fold-change above TLR7^−/− controls. Data are represented as mean ± S.E.M. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. Statistical significance was accepted when P < 0.05. *P < 0.05. (NS) Denotes not significant. b Multiple sequence alignment with CLUSTAL OMEGA showing across species conservation of Cys 98 on TLR7

Fig. 8

Inhibition of NOX2 oxidase increases expression of Type I IFN and IL-1β, and antibody production to influenza A virus infection. a Alveolar macrophages from WT and Nox2^−/y mice were either left untreated (naïve) or infected with HKx31 influenza A virus (MOI of 10) for analysis of IFN-β, IL-1β, TNF-α, and IL-6 mRNA expression by QPCR after 24 h (n = 8). b, c WT and Nox2^−/y mice were infected with live HKx31 influenza A virus (1 × 10⁵ PFU per mouse) and b cytokine mRNA expression and IFN-β protein expression in c BALF or d serum were assessed 3 days later (n = 5). e–i WT and Nox2^−/y mice were infected with inactivated HKx31 influenza A virus (equivalent to 1 × 10⁴ PFU per mouse) for measurements at day 7 of: e body weight; f airway inflammation and differential cell counts (i.e., macrophages, neutrophils, and lymphocytes); g cytokine expression in whole lung (responses are shown as fold change relative to GAPDH) and h serum and i BALF antibody levels (n = 6). Data are shown as mean ± SE. a Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons. b–i Unpaired t-test; statistical significance taken when the P < 0.05. *P < 0.05. **P < 0.01

Fig. 9

Endosome targeted delivery of a NOX2 oxidase inhibitor protects mice following influenza A virus infection in vivo. a–e Alveolar macrophages from WT mice were treated with the Cy5 cholestanol-PEG linker fluorophore (Cy5-chol; 100 nM) for 30 min and infected with HKx31 influenza A virus (MOI of 10). Cells were then counter labeled with antibodies to either: a and b EEA1, c NOX2 or d influenza A nucleoprotein (NP). All cells were then stained with 4′,6′-diamidino-2-phenylindole (DAPI) and imaged with confocal microscopy. b Cells were pretreated with dynasore (100 μM) for 30 min prior to exposure to Cy5-cholestanol. e Quantification of data from (a–d, n = 5). f RAW 264.7 macrophages were either untreated or treated with various concentrations of cholestanol-conjugated gp91ds-TAT (Cgp91), ethyl conjugated gp91ds-TAT (Egp91) or unconjugated gp91ds-TAT (Ugp91) for 30 min prior to quantifying ROS production by L-O12 (100 μM)-enhanced chemiluminescence (n = 7). g Superoxide production via the xanthine/xanthine oxidase cell-free assay in the absence or presence of Ugp91ds-TAT, (1 μM) or Cgp91ds-TAT (1 μM) (n = 6). h, i Ugp91ds-TAT (0.02 mg/kg/day) or Cgp91ds-TAT (0.02 mg/kg/day) were delivered intranasally to WT mice once daily for 4 days. At 24 h after the first dose of inhibitor, mice were either treated with saline or infected with HKx31 influenza A virus (1 × 10⁵ PFU per mouse). Mice were culled at day 3 post-infection and h airway inflammation was assessed by BALF cell counts and i lung IFN-β mRNA was determined by QPCR (n = 7). (NS) denotes not significant. j–m Mice were subjected to the NOX2 inhibitor treatment regime and virus infection protocol as in h except NOX2 inhibitors were delivered at a dose of 0.2 mg/kg/day (n = 7). Analysis of j airway inflammation by BALF counts, k body weight (% weight change from the value measured at Day -1), l ROS production by BALF inflammatory cells with L-O12 enhanced chemiluminescence and m viral mRNA by QPCR. Data are represented as mean ± S.E.M. e Unpaired t-test; statistical significance taken when the P < 0.05. f, g, h, j, k, l One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. i and m Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons. Statistical significance was accepted when P < 0.05. *P < 0.05; **P < 0.01. Scale bars: 10 µm