RhoA/ROCK-dependent pathway is required for TLR2-mediated IL-23 production in human synovial macrophages: Suppression by cilostazol
- DOI: 10.1016/j.bcp.2013.08.017
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Abstract
IL-23 is produced by antigen presenting cells and plays critical roles in immune response in rheumatoid arthritis (LA).
In this study, we investigated whether the RhoA/Rho-kinase pathway is required to elevate TLR2-mediated IL-23 production in synovial macrophages from patients with rheumatoid arthritis (RA),
and then examined the suppressive effect of cilostazol on these pathways.
IL-23 production was elevated by lipoteichoic acid (LTA), a TLR2 ligand, and this elevation was more prominent in RA macrophages than in those from peripheral blood of normal control.
LTA increased the activation of RhoA in association with increased the nuclear translocation of NF-κB and its DNA-binding activity.
Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA), Y27632 (Rho-kinase) or BAY11-7082 (NF-κB) inhibited IL-23 production by LTA.
Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-κB activation.
Cilostazol suppressed the TLR2-mediated activation of RhoA, decreased NF-κB activity with down-regulated IL-23 production, and these effects were reversed by Rp-cAMPS, as an inhibitor of cAMP-dependent protein kinase.
The expression of IL-23, which colocalized with CD68(+) cells in knee joint of CIA mice, was significantly attenuated by cilostazol along with the decreased severity of arthritis.
Taken together, the RhoA/Rho-kinase pathway signals TLR2-stimulated IL-23 production in synovial fluid macrophages via activation of NF-κB. Thus it is summarized that cilostazol suppresses TLR2-mediated IL-23 production by suppressing RhoA pathway via cAMP-dependent protein kinase activation.
Graphical abstract
Abbreviations
- CIA, collagen induced arthritis;
- IL-23, interleukin-23;
- LTA, lipoteichoic acid;
- RA, rheumatoid arthritis;
- TLR2, toll like receptor 2
Keywords
- RA synovial macrophages;
- RhoA;
- TLR2;
- IL-23;
- Cilostazol
1. Introduction
Rheumatoid arthritis (RA) is a common autoimmune and chronic inflammatory joint disease characterized by increased infiltration of macrophages, proliferation of synovial fibroblast with joint destruction [1]. When abundant monocytes/macrophages in the synovial fluid of RA patients are activated, they produce high levels of cytokines and chemokines, such as interleukin-1β (IL-β), tumor necrosis factor α (TNFα), IL-6, and MCP-1, which contribute to chronic inflammation and joint destruction [2] and [3].
Toll-like receptors (TLRs) are conserved receptors that recognize pathogen-associated molecular patterns, and play important roles in innate and adaptive immunity [4]. TLR2 are mainly expressed on cells, such as macrophages and dendritic cells; and act as primary sensors by recognizing diverse ranges of stimuli [5]. The lipoteichoic acid (LTA) and peptidoglycan are recognized mainly by TLR-2 [6]. It has been reported that TLR2 stimulation causes the preferential induction of IL-8 and IL-23 p19 [7]. IL-23 is involved in autoimmune diseases like RA and psoriasis, in which the cellular function of IL-23 is associated with the self-reactive productions of IL-17, IL-6, and TNF-α, and thus IL-23 plays a critical role in development of autoimmune inflammation [8]. Furthermore, they reported that mice deficient in IL-23 (p19−/−) were relatively resistant to the development of joint and bone inflammation in a collagen-induced arthritis (CIA) model.
The Rho-GTPase family of monomeric RhoA, Rac1 and Cdc42 is known to cycle between the inactivated GDP-bound state and the activated GTP-bound state [9]. In the active state, Rho is implicated in various cellular processes, such as the cell cycle, cytoskeletal regulation, cellular growth and apoptosis [10] and [11]. Furthermore, it has been reported that RhoA is a key regulator of transcription factors, NF-κB [12], and that inhibition of Rho-kinase reduces the severity of synovial inflammation in rats with CIA [13].
Although it has been demonstrated that IL-23 production plays a crucial role in inflammatory reactions associated with rheumatoid arthritis [14], the signal pathway by which TLR2 induces IL-23 production in RA synovial macrophages has not been defined. On the other hand, cilostazol, a type-III phosphodiesterase inhibitor, has been reported to have anti-inflammatory effects due to the cAMP-dependent protein kinase activation-coupled suppression of NF-κB gene transcription [15].
Therefore, in the present study, we undertook to investigate the signal transduction pathways responsible for TLR2-mediated IL-23 production in synovial fluid macrophages from RA patients: in particular, the present study highlighted implication of the RhoA/ROCK signal pathway in the regulation of TLR2-mediated IL-23 production in RA macrophages. We found that increased IL-23 production by TLR2 involves the activation of NF-κB via a RhoA/ROCK pathway. Further, cilostazol was found to inhibit TLR2-mediated IL-23 production by suppressing RhoA activity via the activation of cAMP-dependent protein kinase, and to suppress the expression of IL-23 in the knee joints of CIA mice.
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