Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Upregulation of IRF3 in Mϕ-LPS/IFNγ cells and HCFs under inflammatory stimulation. ( A ) Alteration of the relative mRNA expression of IRF3 in human LPS- and IFNγ-stimulated macrophages (Mϕ-LPS/IFNγ cells). The mRNA expression was normalized to GAPDH ( n = 8 samples per group). ( B ) Schematic illustration of the experimental procedure for primary culture of HCFs from donor corneas and stimulation with LPS for IRF3 analysis. ( C ) Comparison of relative mRNA expression of IRF3 between LPS-stimulated and untreated control HCFs ( n = 6 per group). ( D, E ) Schematic illustration of the in vitro coculture model using HCFs and Mϕ-LPS/IFNγ cells D and relative mRNA expression of IRF3 in HCFs cocultured with or without Mϕ-LPS/IFNγ cells E ( n = 3 samples per group). Statistical analyses were performed using the t -test. *** P < 0.001 and **** P < 0.0001.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Comparison, Control, In Vitro

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: IRF3 expression in Mϕ-LPS/IFNγ cells and the alteration of inflammatory and myeloid markers by IRF3 knockdown. ( A, B ) Relative mRNA A and protein B expression of IRF3 in Mϕ-LPS/IFNγ cells transfected with small interfering RNA targeting IRF3 (si IRF3 ) or negative control siRNA (si Con ). The mRNA levels were normalized to GAPDH ( n = 6 samples per group). ( C ) Schematic illustration of the preparation of si IRF3 -Mϕ-LPS/IFNγ cells and si Con -Mϕ-LPS/IFNγ cells used for subsequent analyses. ( D–L ) Relative mRNA expression (normalized to GAPDH ) of IL1B D , IL6 E , TNF F , PTGS2 G , ITGAM H , CD68 I , CD80 J , CD86 K , and MRC1 L in si Con -transfected Mϕs as control and Mϕ-LPS/IFNγ cells transfected with si Con or si IRF3 ( n = 3 samples per group). Statistical analyses were performed using the t -test A and 1-way ANOVA followed by Tukey's post hoc test D to L . * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Knockdown, Transfection, Small Interfering RNA, Negative Control, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: IRF3 expression in the LPS-stimulated inflammatory HCFs and the alteration of downstream signals elicited by TLR4/LPS complex by IRF3 knockdown. ( A ) Relative mRNA expression of IRF3 in HCFs transfected with small interfering RNA targeting IRF3 (si IRF3 ) or negative control siRNA (si Con ; n = 8 samples per group). ( B ) Schematic illustration of the preparation of LPS-stimulated cells in untreated HCFs and si IRF3 -transfected HCFs (si IRF3 -HCF) used for subsequent analyses. ( C–I ) Relative mRNA expression (normalized to GAPDH ) of IL1B D , IL6 E , TNF F , CXCL10 G , TLR4 H , and MYD88 I in untreated control HCFs, LPS-HCFs and LPS-stimulated si IRF3 -HCFs ( n = 3 samples per group). ( J ) Illustration showing the intracellular signal pathway elicited by TLR4 binding of LPS to activate MyD88-NF-κB pathway and IRF3 pathway to produce IL6 , IL1B , TNF , and CXCL10 transcripts in HCFs. Statistical analyses were performed using the t -test A and 1-way ANOVA followed by Tukey's post hoc test C to I . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Knockdown, Transfection, Small Interfering RNA, Negative Control, Control, Binding Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TLR4-associated inflammatory activation in HCFs by IRF3 -silenced macrophages in the coculture model. ( A ) Schematic illustration of the in vitro coculture system using human corneal stromal fibroblasts (HCFs) and Mϕ-LPS/IFNγ cells transfected with small interfering RNA targeting IRF3 (si IRF3 ) or negative control siRNA (si Con ) for subsequent analyses. ( B–H ) Relative mRNA expression (normalized to GAPDH ) of IRF3 B , IL1B C , IL6 D , TNF E , CXCL10 F , TLR4 G , and MYD88 H in HCFs without coculture or cocultured with si Con -Mϕ-LPS/IFNγ cells or si IRF3 -Mϕ-LPS/IFNγ cells ( n = 3 samples per group). Statistical analyses were performed using 1-way ANOVA followed by Tukey's post hoc test. ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Activation Assay, In Vitro, Transfection, Small Interfering RNA, Negative Control, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TLR4-IRF3 signaling-mediated corneal inflammation and opacity in Irf3 -deficient mice. ( A ) Schematic illustration of the experimental design using wild-type (WT) and homozygous Irf3 -deficient ( Irf3 ⁻/⁻; B6.Cg- Irf3 < tm2.2Ttg >) mice. After mechanical corneal epithelial removal, phosphate-buffered saline (PBS; vehicle control) or lipopolysaccharide (LPS) eye drops were topically instilled 6 times, and corneas were harvested 48 hours post-injury for analysis. ( B ) Relative mRNA expression of Irf3 (normalized to Gapdh ) in excised corneas from WT mice treated with PBS (vehicle) or LPS and from Irf3⁻/⁻ mice treated with LPS ( n = 10 corneas from 5 mice per group). ( C ) Representative photographs of the corneas before (0 hours) and after (48 hours) six topical LPS instillations in WT and Irf3 ⁻/⁻ mice. ( D–F ) Corneal opacity grades D and relative expression of Vim E (epithelial–mesenchymal transition marker) and Fap F (fibroblast activation marker) in each group ( n = 10 corneas from 5 mice per group). ( G–J ) Relative mRNA expression (normalized to Gapdh ) of Il1b G , Il6 H , Tnf I , and Cxcl10 J in the corneas ( n = 10 corneas from 5 mice per group). Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test B, D–H, and J or Kruskal-Wallis test followed by Dunn's post hoc test I . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Saline, Control, Eye Drops, Expressing, Marker, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Attenuation of monocyte recruitment and activation of inflammatory macrophages by Irf3 deficiency in the LPS-induced mouse corneal inflammation model. ( A ) Relative mRNA expression of Ccl2 (MCP-1) (normalized to Gapdh ) in excised corneas from WT mice treated with PBS (vehicle) or LPS and from Irf3 ⁻/⁻ mice treated with LPS ( n = 10 corneas from 5 mice per group). ( B ) Flow cytometric gating strategy for identifying myeloid cells, including neutrophils and monocytes, in the mouse cornea. Four corneas were pooled into one sample for each analysis. ( C–E ) Comparison of monocyte (CD45⁺CD11b⁺Ly6C⁺Ly6G⁻) and neutrophil (CD45⁺CD11b⁺Ly6C int Ly6G⁺) populations in WT and Irf3 ⁻/⁻ corneas after LPS instillation ( n = 4 pooled samples, prepared from 16 corneas from 8 mice, per group). ( F–I ) Relative mRNA expression (normalized to Gapdh ) of Itgam (CD11b) F , Adgre1 (F4/80) G , Cd80 H , and Mrc1 (CD206) I in excised corneas from WT mice treated with PBS (vehicle) or LPS and from Irf3 ⁻/⁻ mice treated with LPS ( n = 10 corneas from 5 mice per group). Statistical analyses were performed using t -test D and E 1-way ANOVA followed by Tukey's post hoc test F to I or Kruskal-Wallis test followed by Dunn's post hoc test A . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Activation Assay, Expressing, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TBK1-independent IRF3 signaling and downstream inflammation by piceatannol in LPS-stimulated HCFs. ( A–D ) Relative mRNA expression of IRF3 A , IL1B B , IL6 C , and CXCL10 D in untreated HCFs and LPS-stimulated HCFs treated with or without piceatannol (PIC; 10 µM, 24 hours,( n = 3 samples per group). ( E–G ) Relative mRNA expression of IL1B E , IL6 F , and CXCL10 G in HCFs transfected with si IRF3 (si IRF3 -HCFs) with or without LPS stimulation and/or PIC treatment ( n = 3 samples per group). ( H–K ) Phosphorylated (p-) and total TBK1 and IRF3 protein expression in LPS-stimulated HCFs treated with or without PIC as shown by Western blot analysis ( n = 3 samples per group). Statistical analyses were performed using one-way ANOVA followed by Tukey's post-hoc test ( A–G ) or t -test ( I, J ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Transfection, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TLR4-IRF3 signaling-mediated corneal inflammation by topical PIC treatment in the LPS-induced mouse model. ( A ) Schematic illustration of the experimental protocol using PIC eye drops in the LPS-induced inflammatory corneal model. After mechanical corneal epithelial removal in naïve mouse corneas, LPS was instilled twice as pretreatment, followed by four co-instillations of LPS and PIC, and finally two additional PIC-only treatments. Corneas were harvested 58 hours post-injury for subsequent analyses. ( B ) Relative mRNA expression of Irf3 (normalized to Gapdh ) in excised corneas treated with PBS (vehicle) or LPS and with or without topical PIC at different concentrations (2, 20, or 200 µg/µL, n = 5 to 10 corneas from 4 to 5 mice per group). ( C–L ) Relative mRNA expression (normalized to Gapdh ) of TLR4-IRF3 signaling–related factors, including Il1b C , Il6 D , Tnf E , Cxcl10 F , and Myd88 G , and myeloid/macrophage markers, including Itgam (CD11b) H , Cd68 I , Adgre1 (F4/80) J , Cd86 K , and Mrc1 (CD206) L , in excised corneas ( n = 7 to 8 corneas from 4 mice per group). Statistical analyses were performed using 1-way ANOVA followed by Tukey's post hoc test B to K or Kruskal-Wallis test followed by Dunn's post hoc test L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Eye Drops, Expressing