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: 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 4: 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-α (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-α 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 5: Deamidation impedes IRF3 to activate antiviral immune responses by blocking its DNA binding activity(A) IRF3 expression was analyzed by immunoblotting in Irf3−/−Irf7−/− MEFs reconstituted with wild-type IRF3 and its mutants.(B and C) The mRNA abundance of antiviral genes in reconstituted MEF as described in (A), with Sendai virus infection, was analyzed by real-time PCR.(D) A heatmap of the expression of IFN-related genes analyzed by RNA sequencing using total RNA extracted from Sendai virus-infected Irf3−/−Irf7−/− MEFs reconstituted with IRF3-WT and IRF3-N85D.(E) IFN-β and CXCL10 in the medium of reconstituted Irf3−/−Irf7−/− MEF (as described in A) infected with Sendai virus for 12 hours were assessed by ELISA.(F) Structure of the IRF3-containing Ifnb enhanceosome (PDB: 206G). Deamidated residue (N85) was highlighted in red.(G) Quantification by ChIP-qPCR of mouse Ifnb and Ifna4 promoter sequences that were precipitated in Sendai virus-infected Irf3−/−Irf7−/− MEFs reconstituted with IRF3-WT and IRF3-N85D.(H) IRF3-WT and IRF3-N85D proteins were purified from 293T cells by affinity chromatography and analyzed by Coomassie blue staining (left panel). BSA, bovine serum albumin. In vitro IRF3-DNA binding was performed and analyzed by EMSA (right panel).(I and J) Human ACE2-expressing Irf3−/−Irf7−/− MEFs were reconstituted with IRF3-WT, IRF3-N85D, IRF3-N85A and Vector. Effect of IRF3 and its mutants on SARS-CoV-2 RNA abundance (I) was assessed by real-time PCR with total RNA extracted at 24 h after SARS-CoV-2 infection (MOI =0.01). Medium of the cells infected with SARS-CoV-2 was used for plaque assay to determine infectious viral progeny (J).Error bars indicate 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 S4.
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