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