Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Differentially expressed genes (DEGs) in full-thickness bladder tissues from HIC patients and controls identified via RNA sequencing (|log₂FC|≥2; adjusted p < 0.05, two-sided Wald test using DESeq2). Gene Ontology (GO) ( B ) and KEGG ( C ) enrichment analyses of DEGs (two-sided hypergeometric test with FDR correction). D UMAP visualization of single-cell transcriptomic landscape of urothelium, with urothelial cells (UCs) identified by KRT19 and UPK1A expression. GO ( E ) and KEGG ( F ) analyses of upregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). GO ( G ) and KEGG ( H ) analyses of downregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). I , J Gene Set Enrichment Analysis (GSEA) of altered pathways in UCs (two-sided permutation test with 1000 permutations, with multiple testing corrected using the FDR). The exact p value for TLR signaling of UCs in panel I is 0.0009. K Expression of Toll-like receptor (TLR) subtypes in UCs from HIC bladders. mRNA ( L ) and protein ( M ) expression levels of TLR3 in isolated urothelium from control and HIC patients ( n = 7 control vs. 10 HIC; data show median (IQR); two-sided Mann–Whitney U test; ns not significant; Bar for panel M: 1 cm). N Immunostaining showing the distribution of TLR3 protein in the urothelium of patients with HIC ( n = 7 control vs. 10 HIC; one section and field per patient; Bar: 100 μm). IHC immunohistochemistry, HL Hunner lesions, U urothelium, LP lamina propria (blue line), M muscularis. Source data are provided as a Source Data file.

Article Snippet: To explore the role of Toll-like receptor 3 (TLR3), TCDCA-pretreated HUCs were treated with either a TLR3 agonist (Poly(I:C) Sodium Salt, CST, Cat. 61401; 5 μg/mL) or TLR3 siRNA (Toll-like Receptor 3 siRNA I, CST, Cat. 6236; 100 nM).

Techniques: RNA Sequencing, Expressing, Isolation, Control, MANN-WHITNEY, Immunostaining, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Experimental workflow of E. avium colonization in antibiotic-pretreated mice (Abx-mice, n = 6 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . B Increased abundance of E. avium confirmed by metagenomic sequencing ( n = 6 per group). C Evaluation of voiding behavior following E. avium transplantation ( n = 6 per group; one measurement per mouse). D Measurement of mechanical pain threshold post-transplantation ( n = 6 per group). E Representative histological analysis of bladder tissues after E. avium transplantation ( n = 6 per group; one section and field per mouse). F , G Quantification of blood and urinary bile acids using comprehensive targeted bile acid profiling ( n = 6 per group). H Cell viability of human urothelial cells (HUCs) following TCDCA or TUDCA treatment ( n = 3 independent experiments). I Expression of ZO-1, TNF-α, and TLR3 following TCDCA (200 µM) exposure ( n = 3 independent experiments). J Experimental workflow of intravesical instillation of TCDCA in rats ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . K Evaluation of voiding function after TCDCA instillation ( n = 5 per group; each rate measured once). L Assessment of mechanical pain threshold following TCDCA exposure ( n = 5 per group). M Representative histology of bladder tissues post-instillation ( n = 5 per group; one section per rat, one field quantified per section). For panels ( B – H , K , L ): data are presented as median (IQR). Statistical analysis was performed using the two-sided Mann–Whitney U test. IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: To explore the role of Toll-like receptor 3 (TLR3), TCDCA-pretreated HUCs were treated with either a TLR3 agonist (Poly(I:C) Sodium Salt, CST, Cat. 61401; 5 μg/mL) or TLR3 siRNA (Toll-like Receptor 3 siRNA I, CST, Cat. 6236; 100 nM).

Techniques: Sequencing, Transplantation Assay, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A , B Expression of tight junction protein ZO-1 and inflammatory marker TNF-α after TLR3 intervention in TCDCA-pretreated human urothelial cells (HUCs) ( n = 3 independent experiments). C Transepithelial resistance (TER) changes following TLR3 intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). D , E Expression of ZO-1 and TNF-α after pentosan polysulfate sodium (PPS) administration in TCDCA-pretreated HUCs ( n = 3 independent experiments). F TER changes following PPS intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). G Experimental workflow showing TLR3 inhibitor and PPS administration to rats following intravesical TCDCA instillation ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . H Assessment of voiding function (one measurement per rat) and pain threshold after TLR3 inhibition ( n = 5 per group). I Representative histological images of bladder tissues post-TLR3 inhibition ( n = 5 per group; one section and field per rat). For panels ( B , C , E , F , H ): data are presented as median (IQR). Statistical analysis was performed using the Kruskal–Wallis test. ns not significant, IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: To explore the role of Toll-like receptor 3 (TLR3), TCDCA-pretreated HUCs were treated with either a TLR3 agonist (Poly(I:C) Sodium Salt, CST, Cat. 61401; 5 μg/mL) or TLR3 siRNA (Toll-like Receptor 3 siRNA I, CST, Cat. 6236; 100 nM).

Techniques: Expressing, Marker, Inhibition, Immunohistochemistry

Journal: iScience

Article Title: Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses

doi: 10.1016/j.isci.2021.102477

Figure Lengend Snippet: SARS-CoV-2 infection does not inhibit type I IFN expression To determine if SARS-CoV-2 can modulate IFNβ gene expression and downstream stimulation of ISGs, Calu-3 cells were infected with SARS-CoV-2 for varying times, following which cells were mock transfected or transfected with poly(I:C). Mock-infected and mock-transfected cells served as controls. Transcript levels were quantified using qPCR. Protein expression was observed and quantified using immunoblot analysis. (A) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) for 0, 24, 48, and 72 h. Cells were fixed and stained to visualize the nucleus and SARS-CoV-2 nucleocapsid (N) protein. Scale bar indicates 300 μm. (B) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h and transfected with 100 ng of poly(I:C) or mock transfected for 6 h (n = 6). Primers for the UpE region were designed to quantify SARS-CoV-2 genome levels (see methods). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels at 18 hpi with Ct values observed at 0 hpi (immediately after removal of virus inoculum). Gel (below): UpE qPCR amplicons were visualized on an agarose gel. (C) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 6). (D) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 6). (E) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 6). (F) IFIT1, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for 24 h (n = 3). (G) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (H) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (I) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (J) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (K) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). (L) pTBK1-S172, TBK1, pIRF3-S396, IRF3, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for an additional 24 h (n = 3). (M) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h, followed by transfection with 1,000 ng of rhodamine-labeled poly(I:C) or mock transfection for 3 h. Cells were fixed and stained to visualize SARS-CoV-2 nucleocapsid (N) protein and rhodamine-labeled poly(I:C). SARS-CoV-2 N and poly(I:C)-rhodamine containing cells are indicated by arrows. Cells that only contained detectable levels of poly(I:C)-rhodamine are indicated by arrow heads. Scale bar indicates 150 μm. Data are represented as mean ± SD, n = 3 or 6, p∗∗<0.01, ∗∗∗<0.001, and ∗∗∗∗<0.0001 (Student's t test and Tukey's multiple comparisons test). pTBK1-S172 and pIRF3-S396 protein expression levels are expressed as ratios of pTBK1-S172/TBK1 and pIRF3-S396/IRF3 levels, respectively. Blots were quantified using Image Studio (Li-COR) (n = 3). Ct, cycle threshold. See also Figure S3 .

Article Snippet: Poly(I:C)-Rhodamine , InvivoGen , Cat#tlrl-piwr.

Techniques: Infection, Expressing, Transfection, Western Blot, Staining, Virus, Agarose Gel Electrophoresis, Labeling