Introduction
Chronic disease is a multi-factorial and polygenic disease, therefore, its pathogenesis is influenced by several genetic and environmental factors under diverse molecular pathways. In RA, the progressive destruction of articular cartilages and bones are observed [1]. Typically, the level of pro-inflammatory cytokines such as IL-1β and TNF-α becomes highly elevated in synovium at the initial stage of the RA pathology [2,3]. Although the level of pro-inflammatory cytokines in OA are generally lower than that in RA, joint inflammation still plays a role and thereby contributes to pain in OA, the main subjective symptom in the patients [4,5]. The homeostatic balance of joint tissues such as cartilage and synovium is regulated by extracellular stimuli including cytokines and mechanical stress [6,7]. The impaired balance causes the growth or the death of joint cells, correspondingly resulting in hyperproliferation of Fibroblast-Like Synoviocytes (FLSs) or apoptotic cell death of chondrocytes in RA and OA. In addition, up-regulated proteolytic enzymes degrade the extracellular matrix of cartilage and bone [8].
A mitochondrion, an indispensable organelle in eukaryote cells, not only supplies chemical energy in the form of ATP but also buffers calcium gradients and regulates programmed cell death [9]. In addition, Reactive Oxygen Species (ROS) is produced mainly during the oxidative phosphorylation in mitochondria. Excessive ROS generation damages cellular functions by oxidizing lipids, proteins and nucleic acids, along with enhancing inflammatory response as a second messenger in the pathogenesis of chronic diseases. In response to cellular demands and/or environmental stresses, mitochondria undergo morphological transitions that is regulated by dynamic collaboration of membrane fusion/fission proteins. The dysfunction of the organelle has been shown to be a mechanism underlying various inflammatory, autoimmune and age-related diseases [6,10].
In fact, mitochondrial dysfunction promotes and aggravates inflammatory response in human FLSs and chondrocytes [11,12]: IL-1β, in conjunction with the inhibitors of mitochondrial respiratory chain, synergetically induces the inflammatory cytokines/mediators such as IL-8, COX-2 and PGE2, though the ROS-mediated NF-κB pathway. In addition, the stimulative cytokine reduces the mitochondrial biomass and membrane potential, resulting in the reduction of cellular ATP level and the induction of apoptotic cell death [13]. According to these reports, an antioxidant resveratrol attenuates the inhibitory effects of the inflammatory stimuli in both chondrocytes and FLSs [14,15]. These antiinflammatory substances may represent a future strategy in controlling the inflammatory response of joint tissues. The relationship between mitochondrial dysfunction and articular inflammation has been increasingly built up. For instance, the mitochondrial mutations are highly associated with the inflammation level of synovial tissue in inflammatory arthritis [16]. In addition, the excessive oxidative stress induced by defective mitochondria is closely correlated with synovial inflammatory progressions [17-19]. Interestingly, RA patients show higher incidence of mitochondrial mutations in FLSs compared with OA patients [20].
While energy metabolism dominantly depends on glycolysis in chondrocytes, mitochondrial oxidative phosphorylation plays a significant role in the cells [21,22]. Indeed, when human chondrocytes were exposed to either IL-1β or TNF-α, the energy producing organelle was fragmented and the respiratory system became inefficient, concomitantly with a decrease in the activity of ATP production and the elevation of superoxide level [23]. However, the relationship between mitochondrial morphology and function has not been quantitatively described in line with inflammatory response in joint cells. In the current work, we firstly investigated mitochondrial morphology and function in IL-1β-stimulated FLSs.
A better understanding of inflammation-related mitochondrial regulation shall be beneficial in developing a novel therapeutic approach for the prevention of arthropathies. In fact, TNF blocking therapy effectively suppresses oxidative stress and hypoxia-induced mitochondrial mutagenesis in inflammatory arthritis [17]. We have recently discovered a novel strain of microalga, Mucidosphaerium sp. RG92, and its extract inhibited the over expression of the pro-inflammatory cytokines in different types of human primary cells [24]. In addition, the algal extract suppressed the excess ROS production in FLSs, inhibited the abnormal proliferation of the cells and attenuated the gene expression of Matrix Metalloproteinases (MMPs), when the cells were stimulated by IL-1β. While those results imply that the microalgal extract could prevent the progresses of arthritis, mitochondrial involvement in the effects has yet to be revealed. Here, we describe that the algal RG92 dramatically attenuates the influence of IL-1β upon mitochondrial morphology and function in FLSs.
Materials and Methods
Materials
Primary FLSs were isolated from synovial autopsy of donors. Studies were approved human subjects/ethics protocols by Scripps Research Institute Human Subjects Institutional Review Boards. IL-1β was purchased from PeproTech (Rocky Hill, NJ). Unless otherwise stated, all other chemicals of analytical grade were obtained from either Sigma-Aldrich (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The microalgal extract was prepared as described elsewhere [24].
Cell culture
FLSs (passage 5) were sub-cultured as described elsewhere [24]. In brief, the cells were grown in DMEM supplemented with 10% foetal calf serum and 50 Uml-1 penicillin/50 μgmL-1 streptomycin (Invitrogen, Carlsbad, CA, USA) in a 5% CO₂ atmosphere at 37℃.
Functional assays
FLSs were stimulated by 10 ng/ml recombinant human IL-1β for 24 hours. In order to test the effect of the microalge-derived ingredient, the cells were incubated with the algal extract for 24 hours in advance of the IL-1β stimulation. For the investigation of mitochondrial morphology, the fluorescent images were observed after the organelle were specifically stained with 100 nM Mito Tracker Red CMXRos (Invitrogen, Carlsbad, CA) [25]. For the detection of the level of cellular ATP and ROS, "Cell" ATP Assay reagent (TOYO INK CO., LTD., Tokyo) and DCFDA Cellular ROS Detection Assay Kit (Abcam, Cambridge) were used, respectively. Data are represented as the mean +/- SD of three to six independent experiments. Statistical analysis was performed using Student's t-test.
Results and Discussion
Inflammatory stimulus uncouples ATP synthetic reaction through mitochondrial morphological change The mitochondrial morphology of primary synovial cells were observed under a fluorescent microscope. Similar to the previous report with a cutaneous dermal papilla cells [25], the energy producing organelle showed mainly two different configurations, i.e., filamentous and rounded types (Figure 1). 37% of the cellular population showed only filamentous mitochondria while 6% population showed only rounded mitochondria. The rest of the cells showed mixture of the two types (Table 1).
As shown in Figure 2 and Table 1, when the cells were treated with IL-1β, the balance between the two morphologies was considerably affected, with the decreased level of filamentous mitochondria (9%) and the increased level of rounded mitochondria (26%). We previously demonstrated that the filamentous mitochondria produce more chemical energy ATP than the rounded mitochondria in other fibroblast-like cells, which is beneficial in cellular migration [25,26]. Therefore, we investigated mitochondrial functions in the presence or the absence of IL-1β. As shown in Figure 3, the level of cellular ATP and ROS were significantly decreased and increased, respectively, in the presence of the pro-inflammatory cytokine. Therefore, it is likely that IL-1β induces the uncoupling of the chemical process of ATP synthesis in the articular cells, in parallel with the morphological transition of mitochondria. In fact, as shown in Figure 4a, a linear relationships were observed in both ATP and ROS levels over the population of filamentous mitochondria. The opposite relationship was shown over the population of rounded mitochondria (Figure 4b). These results confirm that the mitochondrial configuration plays an important role in the coupled functions of ATP synthesis of the organelle [25,26]. It was previously reported that the surface of cristae increases through the collaboration of fusion proteins when energy is highly required [27,28]. It is intriguing to investigate fine structure of IL-1β-stimulated mitochondria in the future.
Microalgal RG92 prevents mitochondria from undergoing inflammatory damageWe have recently discovered a novel strain of microalga, Mucidosphaerium sp. (RG92), from a famous hot spring spa in Beppu in Japan. The algal extract shows anti-inflammatory effects in different types of human primary cells including FLSs: when the cells were stimulated by IL-1β, the gene expression of TNF-α, IL-1β, IL-6 and MMP-1, 3, 9 were upregulated. The over expression of the risk factors of RA were effectively suppressed by the algal extract, which suggests that the novel microalga is an useful tool for the preventative application of the articular disease [24]. Here we examined the effect of the algal extract on mitochondrial activities in FLSs. As shown in Figure 2 and Table 1, the percentage of the cells that show filamentous and rounded mitochondria became back to the normal levels by the algal extract in the presence of IL-1β, while the numbers were unaffected by the bio-product in the absence of the pro-inflammatory cytokine. Importantly, the algal extract suppressed both the inhibition of ATP production and the enhancement of ROS generation that were induced by IL-1β (Figure 3). The effects of the extract were similar to a typical ROS scavenger NAC. Therefore, the algal extract restores the inhibitory effect of IL-1β in the process of the mitochondrial oxidative phosphorylation.
ConclusionsIn the current work, we investigated mitochondrial configuration and functions in FLSs and found that IL-1β reduces the number of filamentous form of the organelle, concomitantly with the reduction of ATP level and the excess generation of ROS in the cells. The microalgal RG92 attenuated the effect of IL-1β on mitochondrial morphology and functions (Figure 5). The effects of the extract might be closely correlated to its antiinflammatory functions and the detailed molecular mechanism is to be examined [24]. Even so, the discovery of the mitochondrial involvement in the cellular process of RA and OA along with that of the novel function of the algal extract will provide new methodology in the therapeutic approach of the diseases.
Abbreviations
IL: Interleukin; MMP: Matrix Metalloproteinase; NAC: N-Acetyl-L-Cysteine; ROS: Reactive Oxygen Species; TNF: Tumor Necrosis Factor
Conflicts on Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Acknowledgement
We would like to thank Dr. Martin Lotz for providing the FLSs.
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