Fig 1: IL-10 deficiency alleviates virus replication and PCV2-induced pathological changes through increasing T cell infiltration. (A) Wild-type mice and il10-/- mice were infected with PCV2, the lungs were collected, and then paraffin sections were stained with H&E for observation at 7, 14, and 28 d.p.i. Bar = 25 µm. (B) Wild-type mice and il10-/- mice were infected with PCV2, the PCV2 Rep expression in the lungs was measured by western blot at 7, 14, and 28 d.p.i. (C) Mixed feeding wild-type mice and il10-/- mice were infected with different doses of PCV2. At 28 d.p.i., the numbers of lymphocytes in lung of PCV2-infected mice were counted, and the correlation of the pulmonary lymphocyte numbers and the viral number (upper panel) or lung lesion scores (lower panel) analyzed. *p < 0.05, **p < 0.01 demonstrated that cell number is inversely correlated with PCV2 copies or lung lesion degree.
Fig 2: NELF controls IL-10 expression by constraining the transcription of AP-1.a RT-qPCR analysis of Il10 mRNA in WT and Nelfb KO BMDM stimulated with LPS for indicated time. Representative (left) and cumulative (LPS 0.5 h, n = 7, P = 0.0002) (right) data are shown. b IL-10 protein ELISA in WT and Nelfb KO BMDM stimulated with LPS for indicated time. Representative (left) and cumulative (LPS 6 h, n = 8, P = 0.0066) (right) data are shown. c NELF-E ChIP-seq tracks Jun and Fos in resting (-) and LPS-activated (+) (0.5 h) BMDM. d PRO-seq (sense strand) tracks for Jun and Fos in WT and Nelfb KO BMDM cultured with (+) or without (-) LPS for 0.5 h. e Cumulative RT-qPCR data showing expression level of Jun (n = 4, P = 0.0301) and Fos (n = 6, P = 0.0039) in WT and Nelfb KO BMDM treated with LPS for 0.5 h. f Immunoblot analysis of c-Fos, c-Jun, and p38 in whole cell lysates of WT and Nelfb KO BMDM treated with LPS for indicated time (left: a representative result of five replicates). For each replicate, c-Fos and c-Jun bands were quantified by densitometry at 60 min time point, normalized to internal control (p38) and expressed relative to WT ( = 1) (c-Jun P = 0.0041; c-Fos P = 0.0144). Mean + SD. g WT and Nelfb KO BMDM were pretreated with SP600125 (50 µM) or SB203580 (20 µM) for 0.5 h, where indicated, and Il10 expression following 0.5 h of LPS co-incubation was assessed by RT-qPCR (n = 5). Relative Il10 expression was normalized to the levels in WT cells that were set as 1. h Out of 29, 20 NELF- genes super-induced in Nelfb KO are potential (P < 0.001) AP-1 targets. *P < 0.05, **P < 0.01, ***P < 0.001, N.S. P > 0.05 by two-sided paired Student’s t test. n represents biologically independent experiments. Source data are provided as a Source Data file.
Fig 3: miR-17~92 family miRNAs promote the expression of Fos in macrophages.(A) RNA-seq analysis showing RNA expression in TKO BMDMs versus those in WT cells. RNAs upregulated in TKO BMDMs were colored red, whereas RNAs downregulated were colored blue, gene Fos was pointed out and colored bright blue, and genes Phlpp2, E2f1, Pten, and Bim were pointed out and colored green. Top 10 upregulated genes (Atp6v0c-ps2, Hmga1-rs1, H2-Q6, H2-Ea-ps, Gm8580, Gm8909, Sap25, LOC547349, H2–l, and H2–Q10) were colored yellow and top 10 downregulated genes (Mir8114, 0610010B08Rik, Gm14430, Gm8615, Asb4, Gm14305, 6230416C02Rik, Gm38431, Pira7, and Cpt1b) were colored purple. (B and C) qPCR analysis of Fos mRNA in WT and TKO BMDMs stimulated for the indicated periods with LPS. (D) Immunoblotting analysis of c-Fos ad p38 (loading control) in whole-cell lysates of WT and TKO BMDMs treated for the indicated periods with LPS. (E) Quantifications of c-Fos protein abundance in unstimulated condition in (D) by densitometry from four independent experiments. (F) qPCR analysis of Il10 mRNA in WT and TKO BMDMs transfected with control or Fos overexpression vector and stimulated for the indicated periods with LPS. (G) Cumulative results from three independent experiments of Il10 levels in LPS-stimulated 6 hr results as in (F), normalized to mRNA expression in control vector transfected WT cells. (H) ELISA of IL-10 in supernatants from WT and TKO BMDMs transfected with control or Fos overexpression vector and stimulated with LPS for 12 hr. *p<0.05 and **p<0.01 (paired Student’s t-test). Data are representative of six (B), four (D), or three (F) independent experiments or are pooled from six (C), four (E), or three (G and H) independent experiments (mean + s.d.).
Fig 4: (A) Prior to FNA, FMN constitutively produces IL-10. Microglia constitutively transcribe Il10 mRNA, but protein translation is unknown. (B) Astrocytic IL-10 production is axotomy-induced. (C) The appearance of CD4+ T cells in the FMNuc promotes upregulation of IL-10R expression on resident cells [1,9], which may provide trophic support to injured FMN. (D) CD4+ T cells must express the IL-10R to modulate microglial activation and confer neuroprotection [9].
Fig 5: Three clusters of miR-17~92 family miRNAs regulate the expression of TNF and IL-10 collectively in macrophages.(A–D) qPCR analysis of Tnf mRNA in WT and miR-17~92flox/flox Lyz2-Cre (17/92 KO) (A), miR-106a~363-/- (106a KO) (B), miR-106b~25-/- (106b KO) (C), or miR-106a~363-/- miR-106b~25-/- (106a 106b DKO) (D) BMDMs stimulated for the indicated periods with LPS. (E–H) qPCR analysis of Il10 mRNA in WT and 17/92 KO (E), 106a KO (F), 106b KO (G), or 106a 106b DKO (H) BMDMs stimulated for the indicated periods with LPS. (I and J) qPCR analysis of Tnf (I) and Il10 (J) mRNA in WT and various knockout (horizontal axes) BMDMs stimulated for 1 hr with LPS; results are presented relative to those of LPS-stimulated WT BMDMs. **p<0.01 (paired Student’s t-test). Data are representative of two (A–C and E–G) or three (D and H) independent experiments or are pooled from two to three (I and J) independent experiments (mean + s.d.).
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