Journal: Nature Communications

Article Title: Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

doi: 10.1038/s41467-023-40094-3

Figure Lengend Snippet: a Subclustering analysis of fibroblasts. Populations shown at the upper right are cholesteatoma fibroblasts. Populations shown at the lower left are skin fibroblasts. Cholesteatoma fibroblasts are labeled blue, and skin fibroblasts are labeled red at the lower left. Cholesteatoma fibroblasts were associated with five subclusters labeled 1, 7, 8, 10, and 11. b Trajectory mapping performed using Monocle 3. Undifferentiated and differentiated cells are labeled blue and red, respectively. The most differentiated cells were observed in cholesteatoma (labeled red). The differentiated cells were identical to cholesteatoma fibroblasts in subcluster 8. c INHBA expression in fibroblasts in UMAP. Cholesteatoma fibroblasts showed high levels of INHBA expression. The area of high INHBA expression was identical to the area in cholesteatoma fibroblasts in subcluster 8. d Violin plots of INHBA expression in each cluster. Subcluster 8 shows a high INHBA normalized value. e Immunofluorescence staining of cholesteatoma perimatrix. Colocalization of vimentin and INHBA immunofluorescence showed that fibroblasts in the perimatrix expressed INHBA. CD45 was not colocalized with activin A. Leukocytes exhibited no activin A expression. INHBA was labeled with Alexa Fluor 568, vimentin was labeled with Alexa Fluor 488, and CD45 was labeled with Alexa Fluor 647. Scale bars: 10 µm. f INHBA mRNA expression level, corrected for GAPDH mRNA expression, was significantly higher in cholesteatoma fibroblasts than in control skin fibroblasts ( n = 6). Data represent means ± SDs. g IL-1β, PGE 2 , and TNF-α promoted INHBA expression in human primary skin fibroblasts. IL-6 did not promote INHBA expression in ear pinna-derived fibroblasts ( n = 4). The exact p -value between the control and IL-1β is 0.0000003. Data represent means ± SDs. Statistical significance was determined using the ratio paired two-tailed t -test ( f ) and two-tailed unpaired t -test ( g ). Source data are provided as a Source Data file.

Article Snippet: Human primary skin fibroblasts (C-12302; PromoCell, Heidelberg, Germany) were cultured for 3 days in DMEM supplemented with 10% FBS containing recombinant murine IL-1β (1 ng/mL; 201-LB-005; R&D Systems), PGE 2 (10 µM; 165–10813; Wako), TNF-α (50 ng/mL; 210-TA-005; R&D Systems), or IL-6 (50 ng/mL; 201-IL-010; R&D Systems).

Techniques: Labeling, Expressing, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

doi: 10.1038/s41467-023-40094-3

Figure Lengend Snippet: IL-1β, PGE 2 , and TNF-α secreted from infiltrating CD45 + cells, particularly macrophages, induced activin A-expressing pathogenic fibroblasts; the activin A acted in conjunction with RANKL to promote ectopic osteoclastogenesis.

Article Snippet: Human primary skin fibroblasts (C-12302; PromoCell, Heidelberg, Germany) were cultured for 3 days in DMEM supplemented with 10% FBS containing recombinant murine IL-1β (1 ng/mL; 201-LB-005; R&D Systems), PGE 2 (10 µM; 165–10813; Wako), TNF-α (50 ng/mL; 210-TA-005; R&D Systems), or IL-6 (50 ng/mL; 201-IL-010; R&D Systems).

Techniques: Expressing