Identification of a Transcription Factor That Drives Polarization Toward Tissue-destructive Fibroblasts in Arthritis

MINGLU YAN¹, Noriko Komatsu¹, Ryunosuke Muro¹, Hiroyuki Takaba¹, Takeshi Nitta¹, Kazuo Okamoto¹, Masayuki Tsukasaki² and Hiroshi Takayanagi¹, ¹University of Tokyo, Tokyo, Japan, ²The Univerisity of Tokyo, Tokyo, Japan

Meeting: ACR Convergence 2022

Keywords: Fibroblasts, Synovial, rheumatoid arthritis

Session Information

Date: Saturday, November 12, 2022

Title: RA – Animal Models Poster

Session Type: Poster Session A

Session Time: 1:00PM-3:00PM

Background/Purpose: Fibroblasts exert important homeostatic functions but can also drive disease pathogenesis. In rheumatoid arthritis (RA), synovial fibroblasts (SFs) contribute to the joint destruction by producing the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand RANKL as well as matrix metalloproteinases (MMPs) to induce bone erosion and cartilage damage, respectively. However, the mechanisms underlying the tissue-destructive fibroblast phenotype remain unclear.

Methods: We performed single-cell RNA-sequencing (scRNA-seq) and epigenomic analyses of arthritic SFs to study the transcriptional mechanism underlying the tissue-destructive gene expression in SFs. Luciferase assay was performed to examine the functions of candidate enhancer elements and transcription factor (TFX) in vitro. We further generated the enhancer knockout (KO) mice and fibroblast-specificTFXconditional knockout mice and analyzed their phenotypes under steady state and arthritic conditions.

Results: Epigenomic analysis of arthritic SFs identified five distal enhancer elements (E1 to E5) upstream of the RANKL gene locus. Among the homologous regions of the five elements in mice, E3 was found to be capable of efficiently inducing RANKL transcriptional activity in an in vitro reporter assay. We further identified transcription factor TFX binds to E3 region and further enhanced RANKL gene transcription. Mice lacking E3 region displayed a reduced joint damage with a decreased RANKL expression in SFs under arthritic conditions, while retaining normal bone metabolism at steady state. Fibroblast-specific TFX deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation.

Conclusion: Therefore, TFX drives the polarization toward tissue-destructive fibroblasts in arthritis. These findings provide a mechanistic basis for pathogenic fibroblast polarization in arthritis and have important clinical implications.

Fig. 1: A distal enhancer element regulates RANKL gene expression in arthritic SFs. A, Epigenomic analyses of SFs from RA patients at the TNFSF11 locus (hg19) by ATAC–seq (GSE128644), H3K27ac ChIP–seq (GSE128642), BRD4 ChIP–seq and Pol II ChIP–seq (GSE148399). B, The TNFSF11 (left) and AKAP11 (right) expression levels in RA patient-derived SFs treated with or without IL_1 and JQ1 (GSE148395). P values were determined by one-way ANOVA analysis followed by Turkey’s post hoc test. C, Reporter assay for the measurement of mouse E1-E5 enhancer activity for RANKL gene transcription. P values were determined by one-way ANOVA analysis followed by Turkey’s post hoc test. D, Tnfsf11 mRNA expression in the arthritic SFs stimulated with or without TNF/PGE2. P values were determined by two-way ANOVA analysis followed by Turkey’s post hoc test.

Fig.2: E3 deletion ameliorates arthritis-induced bone damage. A, Schematic showing murine serum transfer-induced arthritis (STIA) model. B, Arthritis scores of WT and E3-KO mice under STIA condition. P values were determined by two-tailed Mann-Whitney U-test. C, Eroded volume per bone volume of the ankle joints in WT and E3-KO mice under untreated and STIA (day 10) conditions analyzed by micro-CT. P values were determined by two-way ANOVA analysis followed by Turkey’s post hoc test. D, Representative micro-CT images of the ankles of WT and E3-KO mice under untreated and STIA conditions. The red area indicated the erosive cavities evaluated by micro-CT analysis. E, Micro-CT analysis of bone volume per tissue volume, trabecular spacing and number of WT and E3-KO mice at the age of 10 weeks (n=5_13) under physiological conditions. P values were determined by two-way ANOVA analysis followed by Turkey’s post hoc test. F, Representative micro-CT images of the femurs of WT and E3-KO male mice at the age of 10 weeks, micro-CT scale bars: 1 mm.

Fig. 3: TFX deletion in SFs attenuates arthritic joint damage. A, Schematic depicting the E3 region upstream of the TNFSF11 transcription start site and ChIP of TFX binding in the E3 region analyzed by q-PCR in RA patient SFs (with or without TNF/PGE2 stimulation). Data were expressed as mean ± s.e.m. B, Reporter assay for the measurement of the mouse E3 enhancer activity for RANKL gene transcription. P values were determined by two-way ANOVA analysis followed by Turkey’s post hoc test. C, Spearman’s correlation analysis between TFX expression and Tnfsf11, Mmp13 and Mmp3 expression in arthritic SFs isolated from STIA mice (GSE129451). D, Arthritis scores of TFX-flox mice and TFX-ΔFib mice under STIA condition. E, Eroded volume per bone volume of the ankle joints of TFX-flox and TFX-ΔFib mice under untreated and STIA conditions as analyzed by micro-CT. P values were determined by two-way ANOVA analysis followed by Turkey’s post hoc test. F, Representative TRAP and Safranin O staining images of the histological sections of ankle joints and the histopathological scores for bone destruction and cartilage damage under STIA condition. P values were determined by two-tailed t-test.

Disclosures: M. YAN, None; N. Komatsu, None; R. Muro, None; H. Takaba, None; T. Nitta, None; K. Okamoto, None; M. Tsukasaki, None; H. Takayanagi, None.

To cite this abstract in AMA style:

YAN M, Komatsu N, Muro R, Takaba H, Nitta T, Okamoto K, Tsukasaki M, Takayanagi H. Identification of a Transcription Factor That Drives Polarization Toward Tissue-destructive Fibroblasts in Arthritis [abstract]. Arthritis Rheumatol. 2022; 74 (suppl 9). https://acrabstracts.org/abstract/identification-of-a-transcription-factor-that-drives-polarization-toward-tissue-destructive-fibroblasts-in-arthritis/. Accessed .

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