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Background <p>Rheumatoid arthritis (RA) involves multiple pro-inflammatory cytokines; fibroblast-like synoviocytes (FLS) are central effector cells that integrate these signals to produce pathogenic mediators. Tumor necrosis factor (TNF) is a major upstream driver of coordinated inflammatory mediator induction in FLS; however, the underlying mechanisms remain incompletely defined, particularly intracellular feed-forward amplification sustaining and enhancing TNF-driven transcriptional outputs. Complement factor H (FH; gene <i>CFH</i>), a Sushi domain-only complement regulator, is elevated in RA serum. FH is regarded as an extracellular inhibitor of complement activation; however, its potential intracellular expression and functions in FLS have not been explored. We aimed to investigate intracellular FH expression and function using an in vitro transcribed messenger RNA (IVT mRNA)-based approach.</p> Methods <p>FH expression in sera, synovial tissues, and fibroblast-like synoviocytes (FLS) were assessed by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, RT-qPCR, and immunoblotting. To examine intracellular FH function, FLS were transfected with <i>CFH</i>-encoding IVT mRNA to express FH intracellularly, followed by next-generation sequencing (RNA-seq), RT-qPCR, ELISA, phosphorylation assays, and pathway inhibition. Interactions between TNF-driven signaling and intracellular FH were evaluated using TNF- and/or FH-neutralizing antibodies, with or without <i>CFH</i>-targeting small interfering RNA (siRNA).</p> Results <p>FH was highly expressed in RA synovium and predominantly localized intracellularly in FLS. To assess intracellular FH function, FLS were transfected with <i>CFH</i>-encoding IVT mRNA, which induced the coordinated upregulation of RA-relevant mediators, including granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, IL-6, and multiple chemokines. Mechanistically, intracellular FH activated nuclear factor-κB (NF-κB) signaling, promoting receptor-interacting serine/threonine-protein kinase 2 (RIPK2) phosphorylation and p65 nuclear translocation, and the competitive nucleotide-binding oligomerization domain 2 (NOD2) antagonist suppressed FH-driven gene induction. TNF and intracellular FH additively enhanced pathogenic mediator production. <i>CFH</i>-targeting siRNA attenuated TNF-induced cytokine and chemokine expression; blockade of extracellular FH had no detectable effect. Combined TNF neutralization and <i>CFH</i> knockdown produced more effective suppression of cytokine induction than either intervention alone.</p> Conclusions <p>Intracellular FH is a previously unrecognized amplifier of TNF-driven inflammation in RA-FLS via a NOD2/RIPK2/NF-κB axis, establishing a feed-forward loop boosting pathogenic mediator production. Dual targeting of TNF and intracellular FH may represent a therapeutic strategy to suppress synovial inflammation more effectively.</p>

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TNF-driven intracellular complement factor H–NF-κB feed-forward loop amplifies inflammatory mediator production in rheumatoid arthritis synovial fibroblasts

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月4日

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  • Hiroki Kobayashi,
  • Sho Mokuda,
  • Michinori Ishitoku,
  • Hirofumi Watanabe,
  • Tomohiro Sugimoto,
  • Yusuke Yoshida,
  • Junya Masumoto,
  • Shintaro Hirata

摘要

Background

Rheumatoid arthritis (RA) involves multiple pro-inflammatory cytokines; fibroblast-like synoviocytes (FLS) are central effector cells that integrate these signals to produce pathogenic mediators. Tumor necrosis factor (TNF) is a major upstream driver of coordinated inflammatory mediator induction in FLS; however, the underlying mechanisms remain incompletely defined, particularly intracellular feed-forward amplification sustaining and enhancing TNF-driven transcriptional outputs. Complement factor H (FH; gene CFH), a Sushi domain-only complement regulator, is elevated in RA serum. FH is regarded as an extracellular inhibitor of complement activation; however, its potential intracellular expression and functions in FLS have not been explored. We aimed to investigate intracellular FH expression and function using an in vitro transcribed messenger RNA (IVT mRNA)-based approach.

Methods

FH expression in sera, synovial tissues, and fibroblast-like synoviocytes (FLS) were assessed by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, RT-qPCR, and immunoblotting. To examine intracellular FH function, FLS were transfected with CFH-encoding IVT mRNA to express FH intracellularly, followed by next-generation sequencing (RNA-seq), RT-qPCR, ELISA, phosphorylation assays, and pathway inhibition. Interactions between TNF-driven signaling and intracellular FH were evaluated using TNF- and/or FH-neutralizing antibodies, with or without CFH-targeting small interfering RNA (siRNA).

Results

FH was highly expressed in RA synovium and predominantly localized intracellularly in FLS. To assess intracellular FH function, FLS were transfected with CFH-encoding IVT mRNA, which induced the coordinated upregulation of RA-relevant mediators, including granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, IL-6, and multiple chemokines. Mechanistically, intracellular FH activated nuclear factor-κB (NF-κB) signaling, promoting receptor-interacting serine/threonine-protein kinase 2 (RIPK2) phosphorylation and p65 nuclear translocation, and the competitive nucleotide-binding oligomerization domain 2 (NOD2) antagonist suppressed FH-driven gene induction. TNF and intracellular FH additively enhanced pathogenic mediator production. CFH-targeting siRNA attenuated TNF-induced cytokine and chemokine expression; blockade of extracellular FH had no detectable effect. Combined TNF neutralization and CFH knockdown produced more effective suppression of cytokine induction than either intervention alone.

Conclusions

Intracellular FH is a previously unrecognized amplifier of TNF-driven inflammation in RA-FLS via a NOD2/RIPK2/NF-κB axis, establishing a feed-forward loop boosting pathogenic mediator production. Dual targeting of TNF and intracellular FH may represent a therapeutic strategy to suppress synovial inflammation more effectively.