Elsevier

Cytokine

Volume 142, June 2021, 155502
Cytokine

Cyanidin attenuates IL-17A cytokine signaling mediated monocyte migration and differentiation into mature osteoclasts in rheumatoid arthritis

https://doi.org/10.1016/j.cyto.2021.155502Get rights and content

Highlights

  • Cyanidin, a potent inhibitor of IL-17 signaling associated pathogenesis in RA.

  • Cyanidin modulates IL-17A/IL-17RA interaction.

  • Cyanidin blocks IL-17A/p38 MAPK signaling mediated monocyte migration.

  • Cyanidin suppresses osteoclastogenesis via regulating RANKL and OPG expression.

Abstract

Interleukin (IL)-17A signaling pathway plays a critical role in the initiation and progression of rheumatoid arthritis (RA) and represents a viable target for RA therapy. Cyanidin, a flavonoid compound, is a novel inhibitor of IL-17A/IL-17RA (receptor subunit A) interaction in several inflammatory diseases. However, the therapeutic efficacy of cyanidin on IL-17A cytokine signaling induced monocyte migration and fibroblast-like synoviocytes (FLS) released RANKL mediated osteoclastogenesis in RA has not yet been deciphered. In the present study, cyanidin impeded IL-17A induced migration of monocytes isolated from adjuvant-induced arthritic (AA) rats. At the molecular level, cyanidin blocked the activation of p38MAPK signaling in response to IL-17A. Importantly, cyanidin downregulated IL-17A induced expression of HSP27, CXCR4, and CCR7 in AA monocytes via modulating IL-17/p38 MAPK signaling axis. Alternatively, cyanidin significantly suppressed the formation of matured osteoclasts and bone resorption in a coculture system consisting of IL‐17 treated AA‐FLS and rat bone marrow-derived monocytes/macrophages. Further, cyanidin significantly inhibited the expression of RANKL and increased the expression of OPG in AA-FLS via blunted activation of IL-17A/STAT-3 signaling cascade. Interestingly, cyanidin impaired IL-17A induced overexpression of IL-17RA. Taken together, our study proposes a novel therapeutic function of cyanidin towards targeted inhibition of IL-17A/IL-17RA signaling mediated disease severity and bone erosion in RA.

Introduction

Rheumatoid arthritis (RA) is the most common autoimmune arthropathy. Pathological hallmarks of the disease involve a multifarious progressive inflammation leading to cartilage destruction and bone erosion. This eventually results in unremitted pain, loss of joint function with reduced ambulation, and irreversible joint deformity. Monocytes, the key effector cells of the innate immune response, play an influential role in the early inflammation and joint dysfunction of RA. Indeed, massive emigration of monocytes is detected in the synovial tissues that secrets several pro-inflammatory mediators and chemokines which, in turn, recruits more immune cells into the inflamed synovium entailing increased inflammatory profile in patients with RA. Previous studies have revealed that monocytes possess significant effects on the polarization and expansion of lymphocytes thus promoting pathogenic T cell responses in RA [1]. The primacy of monocytes as precursor cells of the bone-resorbing osteoclasts has also been highlighted [2]. Therefore, migration of monocytes can be considered as an important pathological event with a deleterious effect on inflammation and osteoclast-mediated bone erosion causing joint dysfunction in RA.

Osteoclasts are giant polykaryons derived from monocyte/macrophage-lineage hematopoietic cells. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) is the main protagonist involved in monocyte differentiation and its maturation into osteoclasts. Alternatively, osteoprotegrin (OPG) a soluble decoy receptor for RANKL impedes osteoclast differentiation through its competitive binding against the RANK receptor. In RA pathological settings, enhanced RANKL levels promote increased osteoclast differentiation and excessive bone resorption [3]. Of note, RANKL is majorly secreted by fibroblast-like synoviocytes (FLS), the resident synovial cells of the inflamed joints in RA [4]. RANKL, upon release, interacts with its receptor RANK expressed on osteoclast precursor cells and activates a broad range of downstream signaling cascades resulting in robust expression of nuclear factor of activated T cells (NFATC) 1, a key transcription factor for osteoclast differentiation. As a result, several osteoclast-related marker genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (Ctsk), and matrix metalloproteinases (MMPs) are upregulated in matured osteoclasts that degrade cartilage and actively resorbs bone [5]. Denosumab, a humanized anti-RANKL monoclonal antibody, effectively prevents bone loss in RA but shows no effect on inflammation. Moreover, profound inhibition of RANKL by denosumab leads to osteonecrosis [6] and thus, opens a critical window of opportunity for the introduction of a novel therapeutic target for inhibiting both inflammation and bone erosion process in RA.

Interleukin (IL)-17 is the predominant pro-inflammatory cytokine critically implicated in the pathogenesis of RA. T 17 helper T cells (Th) are a distinct subset of CD4 + helper T cells that majorly secretes IL-17; though minimal levels of IL-17 are also secreted from Yδ T cells, natural killer (NK) cells and neutrophils. The IL-17 cytokine family comprises of six members (IL-17A-F) that play a significant protective role in providing host immune response against invasive pathogens and in controlling bacterial burden. Conversely, unperturbed activation of these cytokines contributes to the pathologies of several inflammatory and autoimmune diseases. While, IL-17A and IL-17F exhibits pro-inflammatory effects in RA, IL-17A is considered to possess more profound catabolic effects thus leading to tissue destruction and autoimmunity. Besides IL-17F physiological functions outweighs its pro-inflammatory effects and hence targeting IL-17F can disturb cellular homeostatic processes. Consequently, IL-17A has been an attractive therapeutic target in RA. [7]. Highly elevated levels of IL-17A are also found in the serum and synovial fluid of RA patients. In RA cohort study in patients with knee effusions, IL-17A levels in the synovial fluid significantly co-related with C-reactive protein (CRP) levels, and disease duration [8]. Notably, IL-17A overexpression enhanced the severity of synovial inflammation and accelerated bone erosion in a collagen-induced arthritis model in mice. Moreover, IL-17A accelerates FLS aggressive phenotype and production of pathogenic mediators leading to disease perpetuation of RA [9]. In a similar study, IL-17 receptor A knockout significantly reduced cartilage destruction and joint damage in a streptococcal cell wall induced model of chronic relapsing arthritis; wherein, TNF blockers showed no protective effects [10]. Collectively, these data exemplify the irrefutable role of IL-17A in the pathogenesis of RA.

IL-17A signals via its corresponding cell surface receptor IL-17 receptor subunit A (IL-17RA). After binding to its receptor, it activates several inflammatory signaling cascades including, nuclear factor kappa B (NF -κB), CCAAT/enhancer-binding protein (C/EBP), mitogen-activated protein kinases (MAPK) and janus kinase (JAK)/ signal transducers and activators of transcription (STAT) [11]. NF-κB activator 1 (Act1) is the key adaptor protein that necessitates the expression of IL-17A signaling related genes involved in the initiation of joint pathology in collagen induced model of arthritis. Reportedly, Th17/IL-17/NF-κB axis has been shown to promote inflammation and joint destruction in patients with RA [12]. Previous studies have shown that IL-17 is an important cytokine involved in enhanced monocyte cell migration via p38MAPK signaling with no effects on ERK, JNK, and PI3K pathways activation in RA [13]. It has also been reported that IL-17 is chemotactic for monocytes similar to migration induced by chemokine (C-C motif) ligand (CCL)-2, CCL-5, and CCL-3 [12]. IL-17 may mediate monocyte migration directly through p38 signaling activated expression of HSP27 associated with regulation of actin filament dynamics [13]. The strongest evidence has postulated CCR7 receptors on monocytes to promote its adhesion and trans-endothelial migration [14]. Moreover, an enhanced expression of monocyte chemokine receptor CXC motif ligand (CXCR)-4 in RA has also been noted [15]. However, the role of IL-17A on the expression of HSP27, CXCR-4, and CCR-7 that mediates monocyte migration has not yet been investigated. On the other hand, IL-17 promotes osteoclast differentiation from human monocytes through the upregulation of RANKL expression in FLS. Importantly, a previous study has demonstrated that inhibiting JAK/STAT signaling downstream of IL-17 stimulation significantly reduced RANKL expression in AA-FLS [9]. Consequently, IL-17 signaling blocking might be beneficial in RA pathological settings. Several anti-IL-17 monoclonal antibodies including, secukinumab and ixekizumab, and the anti-IL-17R antibodies are currently been investigated in the treatment of RA. However, poor cellular penetration, short half-life, high manufacturing costs, and loss of responsiveness in the majority of the patients remains a major challenge. Therefore, natural small molecular inhibitors that mimic anti-IL17 monoclonal antibody effects, yet evade the associated side effects can be developed in the treatment against RA.

Cyanidin (Fig. 1A) is a naturally occurring flavonoid compound that has been found specifically in berries and other fruits. Cyanidin has been considered to be a powerful therapeutic candidate in several diseases because of its pharmacological properties such as antioxidant, anti-inflammatory, anti-thrombogenic, anti-viral, chemopreventive, and anti-osteoporotic properties. Importantly, a recent study has shown that cyanidin acts as a small molecule inhibitor of IL-17A interaction with IL-17RA in preclinical mice models of psoriasis and asthma [16]. Yet, in our previous study, cyanidin has been considered as a potential therapeutic candidate in inhibiting IL-17A dependent FLS aggressive phenotype and production of pathogenic mediators in RA via attenuation of JAK-STAT signaling cascade in RA [17]. Intriguingly, a recent study has demonstrated that cyanidin protects against osteoclast-mediated bone loss in an OVX-induced osteoporosis mouse model [18]. However, the therapeutic effect of cyanidin on IL-17A induced monocyte migration and RANKL production from RA FLS has not yet been studied. Therefore, as per our knowledge, the current study for the first time will unravel the therapeutic effect of cyanidin and its underlying molecular mechanism in inhibiting IL-17A induced expression of HSP27, CXCR4, CCR7 that critically impacts monocyte migration. Moreover, it will evaluate cyanidin as a likely therapeutic candidate in inhibiting IL-17A dependent RANKL release from AA-FLS mediated osteoclast formation and bone erosion in RA.

Section snippets

Chemicals and antibodies

Cyanidin chloride (purity ≥ 95%) was acquired from Sigma-Aldrich (St. Louis, MO, USA), and dissolved in dimethyl sulfoxide (DMSO). Recombinant murine IL-17A was purchased from PeproTech (Rocky Hill, NJ). Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotic–antimycotic solution, type II collagenase, bovine serum albumin (BSA), and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were procured from Sigma‐Aldrich (St. Louis, USA). The STAT-3 inhibitor

Effect of cyanidin on monocyte cell viability

MTT assay was performed to determine the cytotoxicity of cyanidin on freshly isolated AA monocytes. As shown in Fig. 1C, AA monocytes were incubated with increasing concentration (µM) (0, 2.5, 5, 7.5, 10, 15, 20, 30) of cyanidin for 3 h. Based on the results, cyanidin concentration from 0 to 10 µM showed no significant reduction in cell viability of monocytes (Fig. 1C). While it was noted that cyanidin dramatically reduced the cell viability of monocyte cells at its highest concentrations

Discussion

Targeted inhibition of the IL-17A signaling axis has emerged as an attractive therapeutic approach implicated in the treatment of RA. More recently, combined blockade of TNF-a and IL-17A is more effective in patients who do not respond or exhibit loss of response to anti-TNF-a treatment alone, thus highlighting the potential therapeutic value of blocking IL-17A especially in RA refractory settings [20]. Accordingly, several biopharmaceuticals that can block IL-17A or IL-17RA and downstream

Conclusions

In conclusion, our study suggests that cyanidin is a potent inhibitor of IL-17 signaling associated pathogenesis in RA. Not only it effectively blocks IL-17A/p38 MAPK signaling mediated monocyte migration, but also suppresses osteoclastogenesis via regulating IL-17A/STAT-3 dependent RANKL and OPG expression in AA-FLS. The precise therapeutic mechanism of action of cyanidin was based on its ability to modulate IL-17A/IL-17RA interaction. This study further suggests that cyanidin has a great

CRediT authorship contribution statement

Snigdha Samarpita: Conceptualization, Investigation, Methodology, Writing - original draft. Mahaboobkhan Rasool: Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, New Delhi (File no. EMR/2017/000523).

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