Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease characterized by synovial hyperplasia, joint destruction, and extraarticular manifestations, and it has a significant impact on both morbidity and mortality (). Although the number of effective medications for RA has expanded rapidly, the pathogenesis of RA, in particular the role of microRNA (miRNA), remains to be determined.
MicroRNAs are endogenous small (∼22 nucleotides), single-stranded, noncoding RNAs that mediate messenger RNA (mRNA) cleavage, translational repression, and mRNA destabilization (). Currently, >2,200 human miRNAs are registered in the miRBase database (release 19) (). As fine-tuning regulators of gene expression, miRNAs have been implicated in important cellular processes such as apoptosis and differentiation (), and it has been estimated that one-third of all mRNAs may be regulated by miRNAs ().
In the past several years, research has shown that patients with RA have alterations in cellular miRNA. Dysregulation of miRNA in peripheral blood mononuclear cells (PBMCs), T lymphocytes, synovial fibroblasts, and osteoclasts, each considered a key effector of joint destruction, has been shown to contribute to inflammation, degradation of the extracellular matrix, and invasive behavior of resident cells ([6-9]). MicroRNAs are also present in human plasma (circulating miRNAs) (). Altered expression of circulating miRNAs in patients with RA has been reported by our group and by others ([11-13]).
MicroRNA-451 (miR-451) is one of the miRNAs that is conserved perfectly among vertebrates and expressed abundantly in plasma ([14, 15]). It plays an important role as a tumor suppressor by regulating cell proliferation, invasion, and apoptosis ([16, 17]). MicroRNA-451 also regulates cytokine production by dendritic cells (). However, the role of miR-451 in autoimmune arthritis is unknown.
In the present study, we showed that cellular miR-451 levels in neutrophils were lower in patients with RA than in healthy controls. We also demonstrated that enhancement of miR-451 suppressed neutrophil chemotaxis via down-regulation of p38 MAPK phosphorylation and that systemic administration of miR-451 with atelocollagen significantly suppressed neutrophil migration in an air-pouch model of local inflammation and severity of arthritis in SKG mice. These findings suggest that miR-451 has potential as a therapeutic target in RA.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
MicroRNAs are of interest because of their critical role as fine regulators of gene expression at the posttranscriptional level within cells in many diseases, and because they are potential targets in the treatment of disease. The abnormal pattern of miRNA expression has been reported in various diseases, and the suppression of overexpressed miRNAs or reconstitution of the expression by restoration of silenced miRNAs is a therapeutic target in many fields ().
MicroRNA-451 has been shown to play an important role in erythropoiesis () and in suppressing tumor growth by regulating cell proliferation and apoptosis ([16, 17]). It has also been suggested that miR-451 contributes to CD4+ T cell function (). Thus, we assumed that miR-451 was relevant to autoimmune arthritis and that reduced expression of miR-451 in neutrophils implied that miR-451 was possibly crucial to neutrophil function.
Transcriptional regulation of miR-451 is largely unknown; however, miR-451 was shown to be transcriptionally activated by transcription factor 3 in T cells (). E2A activity is inhibited by a Ras/ERK/MAPK pathway (), which is activated by GM-CSF stimulation (). Early growth response 1, an inhibitory factor of transcription factor 3, is stimulated by IFNγ independently of STAT-1 (). Stimulation with GM-CSF or IFNγ might down-regulate the expression of miR-451 in neutrophils via this pathway.
Neutrophil chemotaxis driven by the complement pathway has an important function in animal models of arthritis (), and the possible contribution of neutrophils to RA pathology, even in the early phases, has been reported (). Neutrophils are the first cell type to arrive at sites of inflammation. They secrete immune mediators that can activate neutrophils and other immune cells, triggering positive regulatory feedback that may lead to acute and persistent inflammation. Neutrophils live longer in inflammatory sites, where they augment the release of powerful destructive enzymes. These cells are also thought to be involved in bone remodeling and bone resorption through membrane-bound RANKL, which activates monocyte fusion into fully functional osteoclasts (). Neutrophils can interact with other cells, such as antigen-presenting cells and osteoclast-like cells, and can regulate their function. Thus, neutrophils are considered to be more than simple final effectors.
Rab5a and CPNE3 are newly identified direct targets of miR-451. Rab5a is a small GTPase, and it is essential for endosome formation and trafficking in neutrophils and macrophages ([46, 47]). CPNE3 exists in the cytosol of neutrophils and is expressed in immature neutrophil precursors (). However, the true function of CPNE3 in neutrophils is unknown because the short lifespan of neutrophils limits the ability to investigate this using transfection or in vivo microinjection studies.
Systemic administration of miR-451 suppressed arthritis in SKG mice. Although the reduced chemotaxis of neutrophils by miR-451 explains this suppression at least in part, the contribution of other cells may also be assumed. Via Rab5a and CPNE3, miR-451 down-regulated the production of IL-6 from FLS, which play a fundamental role in driving the inflammation associated with RA at both the local and systemic levels. We also confirmed that miR-451 down-regulated the proliferation of FLS independent of Rab5a and CPNE3 (data not shown). A recent study demonstrated that inhibition of miR-451 in dendritic cells increased the production of IL-6, TNF, CCL3, CCL5, and IFNβ via 14-3-3ζ when cells were transfected with influenza (). Proinflammatory mediators, including TNFα, IL-1β, and cyclooxygenase 2, are downstream of p38 MAPK (). Systemic administration of miR-451 might relieve inflammation in autoimmune arthritis via these mechanisms.
The present study has a few limitations. It is difficult to deliver precursor miR-451 specifically to neutrophils. We confirmed that the expression of miR-451 in neutrophils from mice transfected with double-stranded miR-451 was 1.8 times as high as in neutrophils from control mice. However, surrounding cells, which were also transfected with miR-451, might prime the condition of neutrophils in vivo prior to isolation. More efficient direct methods to deliver miRNA are needed for the clinical use of small RNAs. Although we showed that miR-451 inhibits the phosphorylation of p38 MAPK via 14-3-3ζ and Rab5a, it remains to be determined how crucial these genes are for the regulation of p38 MAPK by miR-451.
Another limitation of this study is that we could not identify the function of CPNE3 in neutrophils, and we could not elucidate fully the mechanism by which miR-451 suppresses autoimmune arthritis. The function of CPNE3 in neutrophils and the role of miR-451 in other cells including T cells, B cells, and synovial cells should be investigated in the future. Nevertheless, this study shows for the first time the obvious suppressive function of miR-451 in neutrophil chemotaxis and in autoimmune arthritis.
In conclusion, we demonstrated that miR-451 suppressed neutrophil chemotaxis via p38 MAPK in vitro and in vivo and ameliorated autoimmune arthritis, and our analysis identified new direct targets of miR-451. These findings suggest that miR-451 is a potential target in the treatment of RA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Yoshitomi had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Murata, Yoshitomi.
Acquisition of data. Murata, Yoshitomi, Furu, Ishikawa, Shibuya, Ito.
Analysis and interpretation of data. Murata, Yoshitomi, Matsuda.