Fig 2: USP19 removes K27-linked polyubiquitin moieties from TRIF. a USP19 but not its C607S mutant deubiquitinates TRIF. HEK293 cells were transfected with HA-TRIF and Myc-Ub together with empty vector, Flag-USP19 or Flag-USP19(CS). Twenty hours after transfection, ubiquitination assays were performed with the indicated antibodies. The detailed information of ubiquitination assays is described in the Methods. b USP19-deficiency potentiates polyubiquitination of TRIF after poly(I:C) and LPS stimulation. Usp19+/+ and Usp19−/− BMDMs were left untreated or treated with poly(I:C) (100 μg/ml) or LPS (100 ng/ml) respectively for the indicated times. Cell lysates were immunoprecipitated with anti-TRIF. The immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies. c Overexpression of USP19 removes K27-linked polyubiquitin moieties from TRIF. HEK293 cells were transfected with Flag-TRIF, Myc-USP19, HA-ubiquitin or its mutants (KO, K-only; KR, K is mutated to R) together with a control and USP19 expression plasmid for 24 h before ubiquitination assays with the indicated antibodies. d USP19-deficiency potentiates poly(I:C)- and LPS-induced K27-linked polyubiquitination of TRIF. The 293-TLR3 cells or 293-TLR4 cells were left untreated or treated with poly(I:C) (100 μg/ml) or LPS (100 ng/ml) respectively for the indicated times before ubiquitination assays with the indicated antibodies. Data are representative of two or three experiments with similar results. Source data are provided as a Source Data file

Fig 3: High but not low concentration of LL-37 stimulates poly I:C-induced upregulation of TLR3 in BEAS-2B cells. (A) TLR3 mRNA expression was evaluated using quantitative real-time RT-qPCR in cells treated with poly I:C (0.2 µg/mL), LL-37 (4 µM) or LL-37 (1 and 4 µM) and poly I:C (0.2 µg/mL) in combination for 24 h. (B) TLR3 protein expression was evaluated using Western blot in cells treated with poly I:C (0.2 µg/mL) alone or poly I:C in combination with LL-37 (4 µM) for 24 h. The TLR3 immunoreactive band was observed at the expected molecular weight of 115-130 kDa and normalized to GAPDH (37 kDa) serving as internal control. (C) Cell viability was assessed using the MTT assay in cells treated with LL-37 (0.1, 1, 4 and 10 µM) for 4 h. (A-C) Data are presented as mean ± SEM, n = 8 (A,B) and n = 6–8 (C) in each group. Statistical significance was calculated using a one-way ANOVA, followed by Tukey’s post hoc test. *** represents p < 0.001 vs. control. For comparisons indicated by the bars, ** and *** represent p < 0.01 and p < 0.001, respectively.

Fig 4: TLR3 is required for NK cell response to reovirus. NK cells were stimulated with 1, 10, or 100 MOI reovirus for 12 h. Untreated NK cells were used as a control. a Fold changes in TLR3 gene expression of NK cells relative to control cells, measured by qPCR. b Western blot analysis of TLR3 expression of NK cells. c NK cells were transfected with siTLR3, and knockdown NK cells were then stimulated with 10 MOI reovirus or 1 μg/mL Poly(I:C) for 12 h. Cytotoxicity of control and TLR3 knockdown NK cells against DLD-1 cells was measured by CCK-8 assay. d NK cells were activated with 10 MOI reovirus or 1 μg/mL Poly(I:C) in the presence or absence of 10 μM TLR3/dsRNA complex inhibitor for 12 h. Cytotoxicity of chemically inhibited and control NK cells against DLD-1 cells was measured by CCK-8 assay. e TNF-α and IFN-γ levels in culture supernatants were quantified by ELISA. Error bars represent means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: The expression level of TLR3 (A, B), RIG-I (C, D), and MDA5 mRNA (E, F) and protein expression in cultured normal and inflammatory sinonasal epithelial cells obtained from healthy control and patients with CRSwNP, respectively. Their levels were evaluated 24, 48, and 72 h after RV 16 inoculation and poly(I: C) treatment, which were evaluated by RT – qPCR (A, C, E) and western blot (B, D, F). Right panels located in the figure show representative protein bands evaluated with western blot. Data are mean ± SEM from 7 different epithelial donors. Normal ep + rhinovirus indicates that cultured normal epithelial cells were infected with RV 16. Inflam ep + rhinovirus indicates that cultured inflammatory epithelial cells were infected with RV 16. Normal ep + poly (I: C) means poly(I: C)-treated cultured normal epithelial cells. Inflam + poly (I:C) means poly (I:C)-treated cultured inflammatory epithelial cells. * indicates a significant difference between each time-points.