IL6/sIL6R regulates TNFα-inflammatory response in synovial fibroblasts through modulation of transcriptional and post-transcriptional mechanisms

BMC Mol Cell Biol. 2020 Oct 30;21(1):74. doi: 10.1186/s12860-020-00317-7.

Abstract

Introduction: The clinical efficacy of specific interleukin-6 inhibitors has confirmed the central role of IL6 in rheumatoid arthritis (RA). However the local role of IL6, in particular in synovial fibroblasts (SF) as a direct cellular target to IL6/sIL6R signal is not well characterized. The purpose of the study was to characterize the crosstalk between TNFα and IL6/sIL6R signaling to the effector pro-inflammatory response of SF.

Methods: SF lines were stimulated with either TNFα, IL6/sIL6R, or both together, for the time and dose indicated for each experiment, and where indicated, cells were treated with inhibitors actinomycin D, adalimumab, ruxolitinib and cycloheximide. mRNA expression of cytokines, chemokines and matrix metalloproteases (MMPs) were analyzed by quantitative RT-PCR. Level of IL8/CXCL8 and CCL8 in culture supernatants was measured by ELISA. Mononuclear and polymorphonuclear cells migration assays were assessed by transwell using conditioned medium from SF cultures. Statistical analyses were performed as indicated in the corresponding figure legends and a p-value < 0.05 was considered statistically significant.

Results: The stimulation of SF with IL6/sIL6R and TNFα, cooperatively promotes the expression of mono- and lymphocytic chemokines such as IL6, CCL8 and CCL2, as well as matrix degrading enzymes such as MMP1, while inhibiting the induction of central neutrophil chemokines such as IL8/CXCL8. These changes in the pattern of chemokines expression resulted in reduced polymorphonuclear (PMN) and increased mononuclear cells (MNC) chemoattraction by SF. Mechanistic analyses of the temporal expression of genes demonstrated that the cooperative regulation mediated by these two factors is mostly induced through de novo transcriptional mechanisms activated by IL6/sIL6R. Furthermore, we also demonstrate that TNFα and IL6/sIL6R cooperation is partially mediated by the expression of secondary factors signaling through JAK/STAT pathways.

Conclusions: These results point out to a highly orchestrated response to IL6 in TNFα-induced SF and provide additional insights into the role of IL6/sIL6R in the context of RA, highlighting the contribution of IL6/sIL6R to the interplay of SF with other inflammatory cells.

Keywords: Cross-talk; IL6; Inflammatory response; JAK/STAT; Post-transcriptional mechanisms; Rheumatoid arthritis; Soluble receptor; Synovial fibroblast; TNFα; Transcriptional mechanisms.

MeSH terms

  • Adalimumab / pharmacology
  • Arthritis, Rheumatoid / enzymology
  • Arthritis, Rheumatoid / genetics
  • Arthritis, Rheumatoid / metabolism*
  • Cell Line
  • Cell Movement / drug effects
  • Chemokine CCL8 / metabolism
  • Chemokines / genetics
  • Chemokines / metabolism
  • Cycloheximide / pharmacology
  • Cytokines / genetics
  • Cytokines / metabolism
  • Dactinomycin / pharmacology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Gene Expression Regulation / drug effects*
  • Gene Expression Regulation / genetics
  • Humans
  • Inflammation
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism
  • Interleukin-6 / pharmacology*
  • Interleukin-8 / metabolism
  • Janus Kinases / metabolism
  • Kinetics
  • Matrix Metalloproteinases / genetics
  • Matrix Metalloproteinases / metabolism
  • Nitriles
  • Pyrazoles / pharmacology
  • Pyrimidines
  • Receptors, Interleukin-6 / metabolism*
  • STAT Transcription Factors / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Synovial Membrane / cytology
  • Synovial Membrane / drug effects*
  • Synovial Membrane / metabolism*
  • Tumor Necrosis Factor-alpha / pharmacology*

Substances

  • CXCL8 protein, human
  • Chemokine CCL8
  • Chemokines
  • Cytokines
  • IL6 protein, human
  • IL6R protein, human
  • Interleukin-6
  • Interleukin-8
  • Nitriles
  • Pyrazoles
  • Pyrimidines
  • Receptors, Interleukin-6
  • STAT Transcription Factors
  • TNF protein, human
  • Tumor Necrosis Factor-alpha
  • Dactinomycin
  • ruxolitinib
  • Cycloheximide
  • Janus Kinases
  • Matrix Metalloproteinases
  • Adalimumab