Fig 1: The specific RIG-I agonist-induced type I IFN production is upregulated by NLRC5 or NLRX1 silencing while the NF-?B signaling pathway is not affected in GEN2.2 cells. (A–D) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h then pre-treated with 0.25 µM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were stimulated with the specific RIG-I agonist 5'ppp-dsRNA (RIGL, 1 µg/ml). The IFNA1 and IFNB mRNA expression levels were assessed by real-time PCR after 3 h (A) and IFN-a, IFN-ß (B), TNF, IL-6, and IL-8 (C) protein levels were measured by ELISA after 6 (B) or 24 h (C). (D) Kinetics of I?Ba degradation was determined by western blotting. (D) A representative blot is shown. (A–C) Data are represented as means ± SD of 3-5 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. pre-CpG-A-treated samples; #p < 0.05, ####p < 0.0001, n.d., not determined.
Fig 2: Type I IFN signature genes are upregulated in ENL skin lesions. Skin lesions of non-reactional multibacillary patients (NR, black) or ENL patients (ENL, red) were processed in TRIzol® reagent for RNA extraction and gene expression was determined by RT-qPCR for the following ISGs: (A) EIF2AK2 (NR = 12, ENL = 16); (B) MX1 (NR = 11, ENL = 17); (C) IFNB (NR = 7, ENL = 10); (D) IFNAR1 (NR = 13, ENL = 12); (E) TBK1 (NR = 18, ENL = 12); and (F) IFI16 (NR = 18, ENL = 11). RPL13 was used as an endogenous control. Box plots show median, interquartile range, sample minimum and maximum. Each dot represents a donor. *p < 0.05; ***p < 0.001.
Fig 3: Thalidomide treatment decreases IFN-I pathway activity in skin lesions of ENL patients. (A) Follow-up of ENL patients before (ENL) and after 7 days of thalidomide treatment (ENLThal). Skin lesions were processed in TRIzol® for RNA extraction. Gene expression was determined by RT-qPCR for the following genes: EIF2AK2 (n = 6), MX1 (n = 7), IFNAR1 (n = 5), IFNB (n = 7), TBK1 (n = 7), and IFI16 (n = 7). RPL13 was used as an endogenous control. (B) Top– Longitudinal follow-up of MX1 protein levels by Western blot in skin lesions before (ENL = 3) and on Day 7 of thalidomide treatment (ENLThal = 3). GAPDH was used as an endogenous constitutive gene. Bottom – Densitometry analysis displayed in arbitrary units. (C) Immunofluorescence for IFN-a (green) of skin lesions of ENL patients (n = 3) and ENLThal (n = 3). Cell nuclei are in blue by DAPI staining. White arrows (?) indicate IFN-a labeling in the merge images. Scale bar = 50 µm. (D) Representative immunohistochemical analysis of IFI16 and IRF3 proteins in skin lesions of ENL and ENLThal patients. Inflammatory infiltrate is indicated by the asterisk symbol. The photomicrographs are representative of four patients from each group. Scale bar = 100 µm. *p < 0.05.
Fig 4: NLRX1 but not NLRC5 affects the specific RIG-I agonist-induced type I IFN and pro-inflammatory responses in human moDCs. (A–E) moDCs transfected with the indicated siRNAs were stimulated with the RIG-I ligand 5'ppp-dsRNA (RIGL, 1 µg/ml). The mRNA expression levels of IFNA1 and IFNB were assessed by real-time PCR after 12 h (A) and IFN-a, IFN-ß (B), TNF, IL-6, and IL-8 (C) protein levels were measured by ELISA after 24 h. (D,E) Kinetics of I?Ba degradation was determined by western blotting. (D) A representative blot is shown. (E) Bar graphs show the relative density of I?Ba measured at 60 min of stimulation. (A-C, E) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.01 ****p < 0.0001 vs. untreated; #p < 0.05, ###p < 0.001, ####p < 0.0001, n.d., not determined.
Fig 5: Identification of SARS-CoV-2 proteins that induce IRF3 deamidation to inhibit IFN induction(A) NHBE cells were infected with Sendai virus (SeV) (100 HAU/ml) or SARS-CoV-2 (MOI = 1). Total RNA was extracted, reverse transcribed and analyzed by real-time PCR with primers specific for IFNB1, ISG15, ISG56, CCL5 and Mx1.(B) NHBE cells were transfected with poly(I:C) and RNA isolated from mock- or SARS-CoV-2-infected NHBE cells (MOI = 1, 72 hpi). RNA extraction and real-time PCR were performed as in (A).(C) Modulation of IFN-ß induction was determined by a promoter activity in 293T cells expressing indicated SARS-CoV-2 proteins, with SeV infection.(D) Inhibition of antiviral gene expression by SARS-CoV-2 proteins in 293T cells infected with SeV was examined by real-time PCR with primers specific for indicated genes.(E) Inhibition of IFN-ß induction by selected SARS-CoV-2 proteins was determined by reporter assay of 293T cells expressing TBK-1 and IRF3.(F) Effect of selected SARS-CoV-2 proteins on IRF3 charge status was determined by two-dimensional gel electrophoresis and immunoblotting analyses using lysates of 293T cells transfected with plasmids containing indicated genes.(G) Interactions between exogenous IRF3 and SARS-CoV-2 proteins were analyzed by co-immunoprecipitation in transfected 293T cells.For (F) and (G), Strep indicates SARS-CoV-2 proteins. Error bars indicate standard deviation (SD) of technical triplicates. Statistical significance was calculated using unpaired, two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.See related Figure S1.
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