Fig 1: ROS-deficient pDCs with hyperactivated STING/IFN-α/JAK1/STAT1 cascade exacerbate PIL.(A and B) IFN-α production by BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice upon 24-hour stimulation by (A) TLR7/9 agonists and (B) cGAMP. (C) IFN-α production by BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice upon 20-hour stimulation by IFN-α. (D) P-STAT1 in BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice upon 30-minute stimulation by IFN-α. Representative histograms are presented. (E) Expression of ISGs in BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice upon 4-hour stimulation by IFN-α. (F) P-STAT1 in BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice with or without H2O2 upon 30-minute stimulation by IFN-α. (G) P-JAK1 in BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice upon stimulation by IFN-α for indicated times. (H) Serum levels of autoantibodies against dsDNA, nucleosomes, Sm/RNP, and cardiolipin and (I) urinary protein concentrations in Balb/c mice receiving Balb/c pDCs and Balb/c mice receiving Balb/c.Ncf1m1j/m1j pDCs (n = 7) at different time points after pristane injection. (J) Frequency and numbers of CD45+ cells within kidneys from Balb/c mice receiving Balb/c pDCs and Balb/c mice receiving Balb/c.Ncf1m1j/m1j pDCs (n = 7) at 5 MPI. Representative plots are presented. Two-way ANOVA with Holm-Šídák multiple-comparison test (A–E and G–I), 1-way ANOVA with Tukey’s multiple-comparison test (F), and 2-tailed Mann-Whitney U test (J). *P < 0.05, **P < 0.01, ****P < 0.0001.
Fig 2: ROS regulate accumulation of pDCs and type I IFN responses at the initial stage of PIL.(A) Frequency and numbers of pDCs within peritoneal exudates (PECs) from naive and pristane-treated Balb/c and Balb/c.Ncf1m1j/m1j mice (n = 4–5) at day 3 after pristane injection. Representative flow cytometry plots from pristane-treated Balb/c and Balb/c.Ncf1m1j/m1j are presented. (B) Expression of ISGs within PECs from Balb/c and Balb/c.Ncf1m1j/m1j (n = 4–5) at day 3 after pristane injection. (C) Frequency and numbers of pDCs within PECs from naive and pristane-treated B10.Q, B10.Q.Ncf1m1j/m1j, and Ncf1m1j/m1j.MNTg (n = 3–4) at day 3 after pristane injection. Representative flow cytometry plots from pristane-treated B10.Q, B10.Q.Ncf1m1j/m1j, and Ncf1m1j/m1j.MNTg mice are presented. (D) Expression of Stat1 within PECs from CD11c-Cre+.TN3 (n = 14) and CD11c-Cre–.TN3 mice (n = 9) at day 3 after pristane injection. (E) Frequency and numbers of pDCs within PECs from B10.Q (n = 7) and B10.Q.Ncf1R90/90H (n = 7) at day 3 after pristane injection. Representative flow cytometry plots are presented. (F) Level of IFN-α in peritoneal fluids from B10.Q (n = 4) and B10.Q.Ncf1R90/90H (n = 5) at day 3 after pristane injection. (G) Protein level of STAT1 within PECs from B10.Q (n = 7) and B10.Q.Ncf1R90/90H (n = 7) at day 3 after pristane injection. Statistical significance is determined by 2-way ANOVA with Holm-Šídák multiple-comparison test (A), 2-tailed Mann-Whitney U test (B and D–G), and 1-way ANOVA with Tukey’s multiple comparison test (C). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Generation of pDCs is enhanced via AKT/mTOR pathway in ROS-deficient mice.(A) Frequency and numbers of pre-pDCs within BM from B10.Q and B10.Q.Ncf1R90/90H (n = 6) at day 3 after pristane injection. (B and C) Monarch mammalian phenotype enrichment analysis of (B) proteomic expression profile and (C) PISA of BM-derived pDCs from B10.Q and B10.Q.Ncf1m1j/m1j mice upon 20-hour stimulation by IFN-α. (D) Protein-protein interaction networks (STRING) of the matching proteins in the PISA data set that are relevant to “abnormal hematopoietic system morphology/development” in Mammalian Phenotype Ontology. The proteins related to “PI3K-Akt-mTOR signaling pathway” in WikiPathways are illustrated in red. (E) Expression of p-AKTThr308 and p-mTORSer2448 in BM SiglecH+ cells from B10.Q and B10.Q.Ncf1m1j/m1j mice upon 15-minute stimulation by imiquimod. The densitometric ratio of the phosphorylated protein to the total protein is calculated. (F) Expression of p-AKTThr308 and p-mTORSer2448 in splenocytes from B10.Q and B10.Q.Ncf1R90/90H with or without GSK2795039 or H2O2 upon 15-minute stimulation by imiquimod. Quantification of bands normalized to cyclophilin A is presented. (G) Expression of CCR2 in BM pDCs from naive B10.Q (n = 5), naive B10.Q.Ncf1R90/90H (n = 6), pristane-treated B10.Q (n = 4), and pristane-treated B10.Q.Ncf1R90/90H (n = 5) mice at day 3 after pristane injection. Statistical analysis is done by 2-tailed Mann-Whitney U test (A, E, and G) and 2-tailed Student’s t test (B and C). *P < 0.05.
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