miR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes
Introduction
Inflammation is a major factor for the progression of several chronic diseases/disorders, including rheumatoid arthritis and osteoarthritis [1,2]. The inflammatory response includes a very complex network of mechanisms and messengers that interact each other in a finely regulated way [1,2]. Therefore the inflammation machinery must be very tightly regulated, and in this last twenty years, a new class of regulators has emerged in the form of microRNAs (miRNAs). A number of authors have focused their attention on this field and the large investigation in this area has led to a plenty of information about these key immune modulators and their mechanism of action [3,4].
miRNAs, are small non coding RNA molecules (approximately 22 nucleotides in length). They regulate gene expression binding the 3′-UTRs of their target mRNAs, degrading mRNAs for cleavage or repressing their translation [5]. The importance of miRNAs in modulation of normal and pathologic immune function has been shown in multiple studies in which deregulation of miRNAs was demonstrated to accompany diseases associated with excessive or uncontrolled inflammation [[6], [7], [7], [8]].
miRNAs are commonly transcribed by type II RNA polymerase [9]. This characteristic subjects miRNA expression to positive and negative regulation at the transcriptional level, like to protein coding genes. The canonical miRNA biogenesis pathway starts with transcription of the miRNA gene, leading to formation of primary miRNA (pri-miRNA) [9]. Then pri-miRNA undergoes processing via the two well-known RNAse III enzyme complexes Drosha and Dicer. In the final stage the mature miRNA binds to RNA-induced silencing complexes (RISC) and leads it to complementary regions in messenger RNAs (mRNAs) [10]. The discovery of miRNAs as important regulators of development in model organisms suggested the potential role of miRNAs in the immune system.
The miRNA miR146a was one of the first small RNA that has been shown to be involved in the regulation of the immune system, mainly, in the development of different pathologies diseases in which the tissue degeneration is involved, such as osteoarthritis (OA) rheumatoid arthritis (RA) [35–14], cancer and sepsis [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]], and in different cell types [[17], [18], [19]], including chondrocytes [20]. It has been extensively studied for its role in innate immunity. This miRNA plays an essential role in the negative regulation of the production of pro-inflammatory cytokines, thus modulating the severity of the inflammation response [21]. The miR-146a and 146b, are located on chromosomes 5 and 10 in humans, respectively. Both of them, have been shown, to be are up-regulated following stimulation of cell surface toll-like receptors (TLRs), tumor necrosis factor-α receptors (TNFRs), Interleukin-1 receptors (IL1Rs) and advanced glycation end products receptors (AGERs) [[22], [23], [24], [25]]. Several reports have demonstrated that miR-146a expression is directly controlled by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to its promoter [22,26,27]. In particular, NFκB activation induces the transcription of pro-inflammatory cytokines that in turn are responsible of the downstream innate immunity signalling [28]. The NFκB is subject to refined modulation and miRNAs are a special molecules involved to the fine regulation of NFκB activity [29].
A number of evidences have suggested the role of TLRs in many chronic inflammatory diseases such as OA and RA. These investigations enlighten a specific experimental model of miRNAs that shed new light on the possible implication of miRNAs in chronic diseases in which the degradation ad the destruction of the cartilage is especially involved. It has been reported that miR-146a reduced the expression of tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF-6) and interleukin-1-receptor-associated kinase-1 (IRAK-1) and was also able to inhibit the synthesis of adaptor molecules which are fundamental to prime the inflammatory response after the TLRs activation [22]. An increasing number of studies have demonstrated the involvement of miR-146a, which exhibits a marked negative effects on the inflammatory responses, in degradative diseases. Recent reports have shown that microRNAs may serve as physiological ligands for TLRs and other specific activators/mediators of the inflammatory pathways. It has been demonstrated that the miR-146a reduces the release of deleterious inflammation cytokines by regulating negatively the activation of the TLRs inflammation pathway, as well as it has also shown an inhibiting action on the NF-κB activation [28,[31], [32], [33]]. Inflammatory cytokines also play a fundamental role in cartilage degeneration. Cartilage chondrocytes respond to pro-inflammatory cytokines, e.g. interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-16, IL-17, and mechanical loading, by secreting degradative enzymes, such as metalloproteinases, and other catabolic mediators [34,35].
Increasing evidence, emerging from several investigations, has demonstrated that the inflammatory response, primed by different agents, produces an increment in the expression of growth factors and cytokines which in turn promote the degradation of the extracellular matrix (ECM) [36], specially the high molecular weight native hyaluronan (HA) [[37], [38], [39], [40], [41], [42]]. HA depolymerization generates small HA fragments that have been shown to prime a marked inflammation response in many cell types [42–444]. The small HA oligosaccharides derived from native HA degradation resulted to exert angiogenic, inflammatory and immunogenic action depending on their size [[37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48]].
A number of studies have confirmed that HA oligosaccharides may interact with different receptors such as TLR-2, TLR-4 and the cluster determinant 44 (CD44) thus directly triggering the inflammatory response or amplifying the inflammatory process previously induced by other agents [46,47]. Therefore, the generation HA fragments in pathologies may act as an endogenous danger signal able to activate both innate and acquired immunity. The activated NF-kB in turn translocates into the nucleus with the consequent transcription of different inflammatory mediators, such as tumor necrosis-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-17 (IL-17), matrix metalloproteases (MMPs) etc.[17, 38–39, 48].
Starting by these previous data, the scope of the present investigation was to study the involvement of the miR146a in the inflammatory response in human cultured chondrocytes stimulated with the small oligosaccharide 6-mer HA.
Section snippets
Materials
The 6-mer HA oligosaccharides as sodium salt was obtained from, PG Research (Tokyo, Japan). The product was endotoxin free (endotoxin content <0.1 ng/mg). Human TLR-4 (cat. ABIN6730906), CD44 (cat. ABIN366268) and IL-6 (cat. ABIN625026) commercial ELISA kits were provided from Antibodies-online, (Limerick, PA, USA). Human MMP-13 (cat. RAB0364) commercial ELISA kit and microRNA 146a mimic (cat. HMI0229), microRNA negative control (cat. HMC0002), synthetic microRNA 146a inhibitor (cat. HSTUD0228)
miR146a expression after treatment with 6-mer HA, miR146a mimic, or miR146 inhibitor
Fig. 1, panel A, shows the expression of miR146a in chondrocytes at 24 h after cell treatment with 10–80 μg/mL of 6-mer HA. An approximately threefold increase in miR146a levels was observed in response to treatment with 40 and 80 μg/mL 6-mer HA for 24 h. The exposition of chondrocytes to 6-mer HA at 10 mg/mL dose produced a significant increase of about double, while the dose of 20 μg/mL augmented significantly miR146a expression more than one and half times compared to the control. Therefore,
Discussion
In the present study, we found that miR146a was significantly expressed in the chondrocytes treated with 6-mer HA. Starting by this evidence we investigated the involvement of miR146a on TLR-4, CD44, NF-kB and pro-inflammatory/damaging intermediates. It is tough a consensus has emerged that, regardless of the TLR-stimulus utilized, the induction of different miRNAs, and in particular of the miR146a, suggest their role in the pathways downstream of TLR stimulation. Several evidences, regarding
Declaration of Competing Interest
None.
Acknowledgements
This study was supported by a grant (FFABR 2019) Funding for University Research Basic Activities for Teachers, University of Messina.
Author contributions
AA, AD and MS designed and performed the experiments, analyzed the data and wrote the manuscript. GM performed RT-PCR analysis, performed the image analysis and assayed cytokine ELISA. GMC and SC directed the study.
Declaration of Competing Interest
The authors declare no conflict of interest.
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