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TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells
Experimental Cell Research 384 (2019) 111594
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TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells
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Dakang Suna,∗, Xinye Anb, Bing Jib a b
Clinical Medicine Laboratory, Binzhou, 256603, China Laboratory of Clinical Medicine, Hospital Affiliated to Binzhou Medical University, Binzhou, 256603, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Tripartite motif 34 (TRIM34) Multinucleated giant cells Cell fusion Phagocytosis Aggregates
Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.
1. Introduction MGCs are mainly formed by the fusion of monocyte-derived macrophages and considered as characteristic feature at chronic inflammatory sites [1,2]. Macrophages may fuse with somatic cells to rehabilitate damaged tissues or with tumor cells to enhance metastatic process [3,4]. Recent evidences indicate that MGCs contribute to remodel granuloma-associated extracellular matrix, purge apoptotic debris and restrict spread of infection [5]. However they may also result in inflammatory tissue damage through facilitating matrix metalloproteinase production [6,7]. MGCs are mainly classified into Langhans' cells, foreign body giant cells (FBGCs), osteoclasts and so forth [8]. These cells perform highly specific biological functions in different tissues. Langhans’ cells are often found in infective granulomatous diseases with a circular arrangement of nuclei. They normally contain about 10–20 nuclei and the diameters are highly variable but seldom exceed 50 μm [5]. FBGCs are ordinarily found around foreign materials such as surgical sutures that are difficultly digestible. FBGCs contain about 10–200 nuclei diffusely
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distributed throughout cytoplasm and are endowed with enhanced capacity to degrade and absorb implanted biomedical materials [5]. Osteoclasts can be derived from monocytes of healthy individuals and play essential roles in efficient extracellular resorption of mineral and organic bone matrix components [9]. TRIM34 belongs to the tripartite motif family and is comprised of RING, B-Box, coiled-coil and B30.2 domains [10]. Like many other TRIM family members, TRIM34 can be stimulated dramatically by type I interferon (IFN) in macrophages, lymphocytes and HeLa cells. In addition, Influenza virus or phosphorothioate CpG DNA can induce the expression of TRIM34 in mouse macrophages and dendritic cells in a type I IFN-dependent pathway [11]. In this study, when investigating the subcellular localization of TRIM34 in HEK293T cells, we accidently discovered that TRIM34 proteins induced HEK293T cells to fuse together and promoted the formation of MGCs. Moreover, we surveyed the influence factor on cell fusion, such as cell density, fixation and etc. We also observed nuclei shapes of fusion cells induced by TRIM34. Moreover, we took advantage of cell fusion assay to observe the function of TRIM34 on MGCs
Corresponding author. Hospital Affiliated to Binzhou Medical University, Binzhou, 256603, China. E-mail address: [email protected] (D. Sun).
https://doi.org/10.1016/j.yexcr.2019.111594 Received 26 March 2019; Received in revised form 27 August 2019; Accepted 31 August 2019 Available online 02 September 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. TRIM34 facilitated cell fusion in HEK293T cells. (A–E) pEGFP-N3-TRIM34 or pEYFP-N1-TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) The localization of TRIM34-EGFP in HEK293T cells was examined. Scale bar, 10 μm. (B) TRIM34-EGFP induced cell fusion in HEK293T cells. Scale bar, 10 μm. (C–D) The formation of MGCs, induced by TRIM34-EGFP, was quantitatively evaluated. HEK293T cells were transfected with pEGFP-N3 or pEGFP-N3-TRIM34 vectors for 24 h, and the mock-transfected HEK293T cells were used as control in the assay. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. Scale bar, 10 μm. (E) TRIM34-EYFP induced cell fusion in HEK293T cells. Scale bar, 10 μm. (F) The fusion of HEK293T cells, induced by TRIM34-EGFP, was examined through DiD staining. HEK293T cells were transfected with pEGFP-N3-TRIM34 vectors for 24 h, followed by staining with cell membrane probes DiD for 20 min. Scale bar, 5 μm. Representative results were presented, two additional experiments yielded similar results.
room temperature for 15 min, permeabilized (0.1% Triton X-100), stained with DAPI for 10 min. Images were acquired by Leica TCS SP5 confocal microscope equipped with 63 × oil immersion objectives. Results were obtained in sequential scan mode and samples were selectively excited at 405 nm, 488 nm, 514 nm, 543 nm or 633 nm laser wavelength, according to the test requirement. Z-stacks were obtained by sequential scanning. 3D Viewer, plugin of ImageJ 1.50i software, was used to display 3D distribution of TRIM34-EGFP or TRIM34DsRed1 fluorescent proteins in MGCs.
formation. Furthermore, we respectively observed the membrane changes in primary multinucleated cells stage and the phagocytosis function in secondary multinucleated cells stage. 2. Materials and methods 2.1. Plasmid construction and DNA transfection Human TRIM34 cDNAs were obtained by RT-PCR from HeLa cells induced by INFα and cloned into pEGFP-N3 vector (Clontech) using XhoⅠ/HindⅢ restriction enzymes to generate pEGFP-N3-TRIM34. TRIM34 coding sequence was also transferred into XhoⅠ/HindⅢ sites of pEYFP-N1 (Clontech) or pDsRed1-N1 vector (Clontech) to generate pEYFP-N1-TRIM34 or pDsRed1-N1-TRIM34 vectors.
2.6. Statistical analysis Statistical analyses were performed by one-way analysis of variance (ANOVA, Bonfferoni-Dunn test) with Graphpad prism 7.0 (San Diego, USA). Each experiment was repeated at least three times and the data was presented as means ± SD. p < 0.05 was considered as statistically significant difference.
2.2. Cell culture and transfection HEK293T cells were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum, 4.5 g/liter glucose, 4.0 mM L-glutamine and sodium pyruvate. Cells were maintained in a humidified 5% CO2 incubator at 37 °C. HEK293T cells were cultured on confocal dishes (NEST, diameter: 20 mm) or 6 cm cell culture dish for 24 h prior to transfection. Cells were transiently transfected with corresponding plasmids using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.
3. Results 3.1. TRIM34 facilitated the cell fusion in HEK293T cells To examine the localization of TRIM34, pEGFP-N3-TRIM34 vector was transfected into HEK293T cells for 24 h. Confocal images showed that TRIM34-EGFP was diffusely distributed throughout cytoplasm and formed variably sized aggregates in HEK293T cells (Fig. 1A). Interestingly, we found that TRIM34 could induce cell fusion, resulting in the formation of MGCs (Fig. 1B). As shown in Fig. 1C–D, TRIM34-EGFP drastically boosted the formation of MGCs compared with the mock group and EGFP-vector group. Similarly, TRIM34-EYFP also induced the formation of MGCs, which hinted that the effect of TRIM34 on cell fusion was not an occasional event (Fig. 1E). By DiD staining to label cell membrane, it also showed that TRIM34 could induce the formation of MGC, which size was significantly larger than that of peripheral cells (Fig. 1F).
2.3. DiD, LysoTracker and MitoTracker staining After transfection for 24 h, part of HEK293T cells were stained by cell membrane probes DiD (5 μM), DiIC18(5) (US EVERBRIGHT® INC, D4019) for 20 min. Then the live cells were rinsed with prewarmed DMEM two times and resuspended in DMEM, followed by confocal microscope assay. After transfection for 24 h, part of HEK293T cells were stained by LysoTracker Deep Red (50 nM) (Molecular Probes: L12492) for 30 min. LysoTracker probes can label the acidic organelles, mainly refer to lysosome, simultaneously nuclei contour are displayed. Then the live cells were rinsed with PBS two times and resuspended in PBS, followed by confocal microscope assay. After transfection for 24 h, part of HEK293T cells were stained by MitoTracker Deep Red (100 nM) (Molecular Probes: M22426) for 30 min. Then the live cells were rinsed with PBS two times and resuspended in PBS, followed by confocal microscope assay.
3.2. Multidimensional observing the cell fusion induced by TRIM34 Confocal images showed that MGCs could even be formed in the low cell density areas, which excluded the effect of excessive squeeze on cell fusion (Fig. 2A). We also found that TRIM34-mediated cell fusion was a gradual process. As shown in Fig. 2B, two adjacent HEK293T cells with TRIM34-EGFP expressing were fused into a single cell at the upper level (arrow 2); however they were still two separate cells at the lower level (arrow 1). HEK293T cells were also stained by LysoTracker Deep Red, which mainly exhibited lysosome specific location and showed nuclei contour simultaneously. The red fluorescence signal of LysoTracker disappeared at the junction of two adjacent HEK293T cells (Fig. 2C, arrow), suggesting that the two cells with high levels of TRIM34 expression fused into one cell. To observe the formation of MGCs in living cells, HEK293T cells were stained by MitoTracker Deep Red which was specifically located to mitochondria. As shown in Fig. 2D, TRIM34 evidently promoted the formation of MGCs in live HEK293T cells (arrow 1). The mitochondria accumulated in the MGC and its long and short diameter were about 18.7 μm and 16.9 μm respectively. The area of accumulated mitochondria (arrow 2, R1: 2.46) was about twice as
2.4. Cell fusion assay pEGFP-N3-TRIM34 and pDsRed1-N1-TRIM34 vectors were respectively transfected into HEK293T cells for 24 h. Then transfected cells were digested by trypsin and resuspended in DMEM complete medium after centrifugation. TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells were co-cultured within the ratio of 1:1 or cultured solely as control for 24 h, followed by confocal microscope assay. 2.5. Confocal microscope assay Cells were washed with PBS, fixed with 4% paraformaldehyde at 3
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Fig. 2. Multidimensional observation of the influence factors on cell fusion induced by TRIM34. TRIM34-EGFP vectors were transfected into HEK293T cells for 24 h (A–D). Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope (A–B). (A) In the low density areas of HEK293T cells, TRIM34 induced the formation of MGCs. Scale bar, 10 μm. (B) MGCs were observed at different depth by confocal microscope. Scale bar, 10 μm. (C) After transfection, HEK293T cells were stained by LysoTracker Deep Red (50 nM) for 30 min. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. Scale bar, 10 μm. (D) After transfection, HEK293T cells were stained by MitoTracker Deep Red (100 nM) for 30 min. Then the live cells were directly observed by confocal microscope. Scale bar, 10 μm. R1: the area of accumulated mitochondria in MGCs, R2: the cross-sectional area of normal cell.
elucidate the features of primary multinucleated cells stage, we observed the membrane changes of HEK293T cells transfected with pEGFP-N3-TRIM34 via DiD staining. Confocal images showed that the TRIM34-EGFP+ cell was in contact with the TRIM34-EGFP- cell (Fig. 7A, arrow 1). Then, the membrane at the junction became blurred with the fluorescence intensity of DiD weakened (Fig. 7B, arrow 2). Afterwards the membrane between two cells disappeared and the fusion cell gradually formed (Fig. 7C, arrow 3). With the integration of other cells, the volume of fusion cell increased continuously leading to the formation of primary multinucleated cell (Fig. 7D, arrow 4). When capable of swallow adjacent cells, the primary multinucleated cell turned into the secondary multinucleated cell (Fig. 7D, arrow 5).
big as the nearby normal cell (R2:1.15) analyzed by ImageJ software. 3.3. Nuclei polymorphy of the fusion cells induced by TRIM34 Nuclei shapes of MGCs were examined in HEK293T cells with high levels of TRIM34 expression by confocal microscope. As shown in Fig. 3A–F, the nuclei of MGCs presented various shapes, such as oval shape, heart shape, pear shape, horseshoe shape, ring like shape and irregular shape etc. 3.4. TRIM34 enhanced the formation of MGCs Confocal microscope showed that TRIM34 resulted in MGCs formation. As shown in Fig. 4A, the diameter of MGC reached to 55–65 μm. Nuclei in the MGC were arranged in ring like shape and distributed around the plasma membrane. Substantial TRIM34-EGFP proteins accumulated to the central areas of MGC (Fig. 4A). At the same time, some TRIM34-EGFP proteins located to the vicinity of MGCs cytomembrane. 3D reconstruction of confocal z-stacks showed that TRIM34-EGFP proteins formed macroaggregates, which major diameter reached to over 3 μm (Fig. 4B, left panel, arrow). Interactive 3D Surface Plot analyzing tool showed that the intensities of TRIM34-EGFP aggregates were very high in the central region of MGC, compared with the peripheral areas (Fig. 4B, right panel).
3.8. Phagocytosis function of secondary multinucleated cells In this study we discovered that some fusion cells, induced by TRIM34, had the quality of phagocytosis and thus we named them as secondary multinucleated cells. As shown in Fig. 8A1 (arrow 1), the membrane of multinucleated cell showed obvious invagination when the phagocytosis was in the early phase. During the metaphase of phagocytosis, about half of the target cell was engulfed by the multinucleated cell (Fig. 8A2, arrow 2). Finally, the target cell was totally swallowed at the last phase of phagocytosis (Fig. 8A3, arrow 3). From the above results, we discovered that TRIM34-EGFP- cells (Fig. 8A1, arrow 1) or TRIM34-EGFP+ cells (Fig. 8A2, arrow 2) could be engulfed by secondary multinucleated cells. Namely, the phagocytosis of secondary multinucleated cell had no obvious selectivity for cells expressing TRIM34-EGFP or not. Moreover, we found that secondary multinucleated cells could initiate multiple-process phagocytosis at the same time (Fig. 8B). Confocal images showed that the secondary multinucleated cell could concurrently engulf several target cells in different phagocytosis period, such as the early stage (arrow 1 and arrow 2), middle stage (arrow 3) or completion stage (arrow 4).
3.5. TRIM34 triggered the formation of MGCs by means of cell fusion To elucidate the cellular mechanism of MGCs formation, cell fusion assay was performed in HEK293T cells transfected with pEGFP-N3TRIM34 or pDsRed1-N1-TRIM34 vectors separately. Then, TRIM34EGFP expressing cells and TRIM34-DsRed1 expressing cells were cocultured for another 24 h. Confocal images showed that TRIM34-EGFP and TRIM34-DsRed1 fluorescent proteins from two or more different cells could be detected in the same fusion cell (Fig. 5A, arrow 1). Zstack images were also acquired to verify whether TRIM34-EGFP and TRIM34-DsRed1 co-existed in the same fusion cell. 3D reconstruction of confocal z-stacks showed that some TRIM34-DsRed1 aggregates (red) co-existed in the TRIM34-EGFP (green) preferential expressing cell (Fig. 5B3-B5, arrow 3). Moreover, the volume of accumulated nuclei in MGC was about two times larger than that of control cells.
4. Discussion In general, persistent existence of pathogens or foreign material leads to the formation of MGCs [12]. MGCs are primarily formed by the fusion of macrophages and play important roles in many physiological and pathological processes [13–16]. Langhans’ cells exist in many infectious granulomatous disorders such as tuberculosis and sarcoidosis, while foreign body giant cells are featured by foreign body granulomas [2,17]. In addition, spontaneous formation of MGCs can arise in physiological conditions. For example, osteoclasts are commonly multinucleates in adult mammals due to cell fusion [18]. Osteoclasts attach firmly to bones and form extracellular acidic compartment, which facilitate the dissolution and resorbing bone matrix. On the contrary, they are also related to bone diseases such as rheumatoid arthritis, osteoporosis and bone lesions [19,20]. In addition, Hart et al. described that MGCs, derived from microglias, accumulated in brain tissues with age. MGCs in aged mice were commonly found in the white matter and contained as many as 6 or 7 nuclei with approximately 20–30 μm in diameter [21]. Several reports describe that MGCs can be formed from other cell types, such as endothelial cells and fibroblast etc. HEK293 cells are a kind of human embryonic kidney epithelial cell. Purinergic P2X7 receptor overexpression in HEK293 cells promoted the fusion process leading to the MGCs formation [22]. Endothelial cells, derived from
3.6. Process of cell fusion mediated by TRIM34 It was probably a multistage process that TRIM34 mediated cell-cell membrane fusion, leading to MGCs formation. We suppose that the process might contain four major stages. First, the formation of MGCs occurred among adjacent HEK293T cells. The gap between two nuclei abounded with cytoplasm, which displayed as yellow for TRIM34-EYFP expression (Fig. 6A). Second, the gap between two nuclei became narrow gradually (Fig. 6B, arrow 2). Third, the gap between two nuclei was partly disrupted and the two adjacent cells fused into a single-cell (Fig. 6C, arrow 3). Finally, the intervals between multiple cells disappeared and MGCs came into being (Fig Fig. 6D). 3.7. Membrane changes during primary multinucleated cells stage The process of MGCs formation may be divided into primary multinucleated cells stage and secondary multinucleated cells stage. To 5
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Fig. 3. The nuclei shapes of TRIM34-induced fusion cells were polymorphic. (A–F) pEGFP-N3- TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) Oval shape, (B) Heart shape, (C) Pear shape, (D) Horseshoe shape, (E) Ring like shape, (F) Irregular shape. Scale bar, 10 μm.
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Fig. 4. TRIM34 promoted the formation of MGCs. pEGFP-N3-TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) TRIM34 expression induced the formation of MGCs in HEK293T cells. Scale bar, 20 μm. (B) 3D reconstruction of z series was analyzed by ImageJ software. The left panel showed image from the top of cells, and the middle panel showed the side view from the top of cells. The right panel showed the intensities of TRIM34-EGFP aggregates by Interactive 3D Surface Plot tool of ImageJ in Thermal LUT mode. Z-axis represented the intensities of TRIM34-EGFP. Representative results were presented, two additional experiments yielded similar results.
about 3–4 times larger than that of nearby normal cells (Fig. 1B). As the doubling time of HEK293T cell was around 24 h, the failure of cytokinesis was not essential for the formation of MGCs mediated by TRIM34. Accumulated evidences have shown that in vitro generation of MGCs often occurs as a result of cell fusion [26]. Cytokines, such as IFNγ and IL-4, adhesion proteins and receptors have been demonstrated to enhance the formation of MGCs [22,27,28]. IFN-γ plays a key role in MGCs formation for it enhances the cell clustering and cell-to-cell adhesion [29,30]. Antibodies against IFN-γ restrained MGCs generation both in vitro and in vivo [31]. Keegan AD demonstrated that IL-4 accelerated the formation of MGCs via regulating the expression of Ecadherin, which resulted in homotypic cell fusion and incorporation of large foreign bodies [32]. IFN-γ or IL-4 might not perform the direct role in cell fusion but through it mediated signaling pathways. In this study, we discovered that TRIM34, as a single factor, could directly induce MGCs formation even in the areas with low cell density, excluding the influence of excessive squeeze on cell fusion (Fig. 2A). In newly formed MGCs, we also found that TRIM34 could induce adjacent cells to fuse together at the upper level, while they were still two separate cells at the lower level (Fig. 2B). By DiD (Fig. 1F) or MitoTracker Deep Red (Fig. 2D) staining, confocal images evidently showed that TRIM34 promoted the formation of MGCs in live cells, which eliminated the potential influence of paraformaldehyde fixation and the detergent of Triton X-100. 3D image reconstruction showed that TRIM34-EGFP proteins could form macroaggregates in MGCs, while nuclei were arranged in ring like shape around the plasma membrane (Fig. 4A–B). Cell fusion assay further demonstrated that TRIM34EGFP+ cells and TRIM34- DsRed1- cells could fuse into a MGC (Fig. 5A–B). In the present study, we suppose the progress of MGCs
brain capillary, could also be induced into MGCs by cell fusion [23]. Holt DJ demonstrated that MGCs could originate from fibroblast in vitro and in vivo in pathologies such as fibrosis and senescence tissue [24]. Furthermore, there is evidence to suggest that somatic cells might fuse with macrophages. HIV infected T cells can contact and fuse with macrophage targets. Then, the newly formed lymphocyte-macrophage fusion cells can fuse with neighboring uninfected macrophages for HIV1 spreading [25]. In this study, we found that TRIM34 could lead to a marked increase in the efficiency of cell fusion compared with mock group and EGFPvector group in HEK293T cells (Fig. 1C–D). Moreover the nuclei shapes of MGCs, induced by TRIM34, were diversiform such as oval shape, heart shape, pear shape, horseshoe shape, ring like shape and irregular shape etc (Fig. 3A–F). Furthermore, confocal microscope showed that TRIM34-EGFP proteins appeared as aggregates and dispersed in the cytoplasm of HEK293T cells (Fig. 1A), suggesting TRIM34 proteins could bind to each other and form macromolecular complexes. The intracellular aggregates might be sensed by an unknown mechanism and trigger the cell fusion process, just like the formation of FBGCs initiated by extracellular biomedical materials. Currently, the cellular and molecular mechanisms of MGCs formation are still poorly understood. It has been suggested that MGCs may arise from the failure of cytokinesis. Kim YS reported that cyclin D1 and p16 were overexpressed in foreign body giant cells, osteoclast giant cells and Langhans giant cells, leading to cell-cycle arrested at the G1/S transition [8]. Brown GC described that inflammatory stimulus could induce the multinucleation of cultured microglia as a result of failure of cytokinesis. In this study, when HEK293T cells were transfected with pEGFP-N3-TRIM34 for only 24 h, the formation of MGCs could be detect by confocal microscope (Fig. 1B). The volume of MGCs nuclei was
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Fig. 5. The function of TRIM34 on MGCs formation was verified by cell fusion assay. (A) pEGFP-N3-TRIM34 or pDsRed1-N1-TRIM34 vectors were respectively transfected into HEK293T cells for 24 h. Cells transfected with pEGFP-N3-TRIM34 or pDsRed1-N1-TRIM34 vectors were co-cultured or cultured solely for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by the confocal microscope. The scale bar represented 10 μm. Representative results were presented, two additional experiments yielded similar results. (B) Z-stack confocal images of co-cultured HEK293T cells expressing TRIM34-EGFP and TRIM34DsRed1 were collected and analyzed by ImageJ. Scale bar, 10 μm. (B1) Single stack slice showed the coexistence of TRIM34-EGFP and TRIM34-DsRed1 in a fusion cell. The arrow 2 indicated TRIM34-DsRed1 red fluorescence protein. (B2) 3D Reconstruction image was viewed from the bottom of cells. (B3) Image was viewed from the top of cells. (B4) Images showed the side view from the top of cells. (B5) Image was viewed in y-z plane. Representative results were presented, two additional experiments yielded similar results.
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Fig. 6. The progress of cell fusion induced by TRIM34. (A–D) pEYFP-N1-TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) TRIM34-EYFP was expressed in multiple adjacent HEK293T cells. Scale bar, 10 μm. (B) The interval between two nuclei was narrowed down gradually. Scale bar, 10 μm. (C) The interval between two cells was partly disrupted. Scale bar, 10 μm. (D) The interval between multi-cells disappeared and MGCs came into being. Scale bar, 10 μm.
formation can be divided into two phases. Adjacent cells firstly formed primary multinucleated cell through cell fusion mediated by TRIM34 (Fig. 7A–D, arrow 1–4). Afterwards, some primary multinucleated cells acquired the ability to swallow adjacent cells and then turned into the secondary multinucleated cells (Fig. 7D, arrow 5). More importantly, as Fig. 8B shown, the secondary multinucleated cell could simultaneously devour several adjacent cells in different phagocytosis phase, which better explained the reason why TRIM34 could induce MGCs formation in short period. Meanwhile virus or germs are able to induce cell fusion [33]. Syncytia, namely MGCs, can be found in autopsy samples from human respiratory syncytial virus infections [34]. Fusion protein of RSV can be expressed in infected airway epithelial cells. They accumulated to
plasma membrane and promoted the fusion of infected cells with adjacent cells, leading to the formation of MGCs [34]. Moreover, FAST (fusion-associated small transmembrane) proteins of reovirus could accumulate to cell membrane, resulting in the fusion of virus-infected cells with neighboring normal cells [35]. Furthermore, burkholderia pseudomallei could escape into the cytosol of host cells and enhance the formation of MGCs [36]. In murine macrophages and dendritic cells, the expression of TRIM34 was highly up regulated upon virus infection in an IFN-I-dependent manner. In addition, TRIM34 expression was also stimulated by LPS and interferon-γ in human macrophages. It was possible that TRIM34 might exert direct actions in MGCs formation induced by interferon or virus. In the future work, HEK293T cells, transfected with TRIM34-EGFP or TRIM34-EYFP vectors, may be used 9
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Fig. 7. TRIM34 induced cytomembrane changes in primary multinucleated cells stage. HEK293T cells were transfected with pEGFP-N3-TRIM34 vectors for 24 h. Then the cells were stained by DiD (5 μM) for 20 min and observed by confocal microscope. (A). A TRIM34-EGFP+ cell was in contact with a TRIM34-EGFPcell. Scale bar, 5 μm. (B). The contact sites of two adjacent cells became thinning compared with peripheral cytomembrane. Scale bar, 5 μm. (C). The contact sites of two adjacent cells were disintegrated. Scale bar, 5 μm. (D). With cell fusion of many adjacent cells, the primary multinucleated cell came into being. The arrow 4 indicated the primary multinucleated cell. Scale bar, 10 μm. Representative results were presented, two additional experiments yielded similar results.
have similar functions.
as a cell model to imitate MGCs formation. By means of live cell imaging system, it can easily and dynamically display the process of cell fusion and phagocytosis during the formation of MGCs. At the same time, it is still not clear whether TRIM34 proteins are able to induce other cell types to form MGCs and whether other TRIM family members
5. Conclusions In short, our data demonstrates that TRIM34 proteins are able to 10
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Fig. 8. The phagocytosis function of secondary multinucleated cell. HEK293T cells were transfected with pEGFP-N3-TRIM34 vectors for 24 h. Then cells were stained by DiD (5 μM) for 20 min and observed by confocal microscope. Scale bar, 10 μm. Representative results were presented, two additional experiments yielded similar results. (A1). In the early phase of phagocytosis, the multinucleated cell began swallowing adjacent cell with obvious invagination of membrane. (A2). The multinucleated cell engulfed half of the target cell in the metaphase of phagocytosis. (A3). The target cell was totally engulfed in the last phase of phagocytosis. (B). Secondary multinucleated cell could simultaneously swallow several target cells in a multi-process manner.
Conflicts of interest
boost the formation of MGCs by means of cell fusion and phagocytosis in epithelial cells. Our study provides new insight into the function of TRIM family protein on MGCs formation.
The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Competing interests None.
[18]
Funding
[19]
This study was supported by Projects of medical and health technology development program in Shandong province (grant numbers 2018WSB30002); the Natural Science Foundation of Shandong Province, China, (grant numbers ZR2012CM009); Science and Technology Development Plan Project of Shandong Province (grant number 2011YD18015).
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells
T
Dakang Suna,∗, Xinye Anb, Bing Jib a b
Clinical Medicine Laboratory, Binzhou, 256603, China Laboratory of Clinical Medicine, Hospital Affiliated to Binzhou Medical University, Binzhou, 256603, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Tripartite motif 34 (TRIM34) Multinucleated giant cells Cell fusion Phagocytosis Aggregates
Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.
1. Introduction MGCs are mainly formed by the fusion of monocyte-derived macrophages and considered as characteristic feature at chronic inflammatory sites [1,2]. Macrophages may fuse with somatic cells to rehabilitate damaged tissues or with tumor cells to enhance metastatic process [3,4]. Recent evidences indicate that MGCs contribute to remodel granuloma-associated extracellular matrix, purge apoptotic debris and restrict spread of infection [5]. However they may also result in inflammatory tissue damage through facilitating matrix metalloproteinase production [6,7]. MGCs are mainly classified into Langhans' cells, foreign body giant cells (FBGCs), osteoclasts and so forth [8]. These cells perform highly specific biological functions in different tissues. Langhans’ cells are often found in infective granulomatous diseases with a circular arrangement of nuclei. They normally contain about 10–20 nuclei and the diameters are highly variable but seldom exceed 50 μm [5]. FBGCs are ordinarily found around foreign materials such as surgical sutures that are difficultly digestible. FBGCs contain about 10–200 nuclei diffusely
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distributed throughout cytoplasm and are endowed with enhanced capacity to degrade and absorb implanted biomedical materials [5]. Osteoclasts can be derived from monocytes of healthy individuals and play essential roles in efficient extracellular resorption of mineral and organic bone matrix components [9]. TRIM34 belongs to the tripartite motif family and is comprised of RING, B-Box, coiled-coil and B30.2 domains [10]. Like many other TRIM family members, TRIM34 can be stimulated dramatically by type I interferon (IFN) in macrophages, lymphocytes and HeLa cells. In addition, Influenza virus or phosphorothioate CpG DNA can induce the expression of TRIM34 in mouse macrophages and dendritic cells in a type I IFN-dependent pathway [11]. In this study, when investigating the subcellular localization of TRIM34 in HEK293T cells, we accidently discovered that TRIM34 proteins induced HEK293T cells to fuse together and promoted the formation of MGCs. Moreover, we surveyed the influence factor on cell fusion, such as cell density, fixation and etc. We also observed nuclei shapes of fusion cells induced by TRIM34. Moreover, we took advantage of cell fusion assay to observe the function of TRIM34 on MGCs
Corresponding author. Hospital Affiliated to Binzhou Medical University, Binzhou, 256603, China. E-mail address: [email protected] (D. Sun).
https://doi.org/10.1016/j.yexcr.2019.111594 Received 26 March 2019; Received in revised form 27 August 2019; Accepted 31 August 2019 Available online 02 September 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. TRIM34 facilitated cell fusion in HEK293T cells. (A–E) pEGFP-N3-TRIM34 or pEYFP-N1-TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) The localization of TRIM34-EGFP in HEK293T cells was examined. Scale bar, 10 μm. (B) TRIM34-EGFP induced cell fusion in HEK293T cells. Scale bar, 10 μm. (C–D) The formation of MGCs, induced by TRIM34-EGFP, was quantitatively evaluated. HEK293T cells were transfected with pEGFP-N3 or pEGFP-N3-TRIM34 vectors for 24 h, and the mock-transfected HEK293T cells were used as control in the assay. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. Scale bar, 10 μm. (E) TRIM34-EYFP induced cell fusion in HEK293T cells. Scale bar, 10 μm. (F) The fusion of HEK293T cells, induced by TRIM34-EGFP, was examined through DiD staining. HEK293T cells were transfected with pEGFP-N3-TRIM34 vectors for 24 h, followed by staining with cell membrane probes DiD for 20 min. Scale bar, 5 μm. Representative results were presented, two additional experiments yielded similar results.
room temperature for 15 min, permeabilized (0.1% Triton X-100), stained with DAPI for 10 min. Images were acquired by Leica TCS SP5 confocal microscope equipped with 63 × oil immersion objectives. Results were obtained in sequential scan mode and samples were selectively excited at 405 nm, 488 nm, 514 nm, 543 nm or 633 nm laser wavelength, according to the test requirement. Z-stacks were obtained by sequential scanning. 3D Viewer, plugin of ImageJ 1.50i software, was used to display 3D distribution of TRIM34-EGFP or TRIM34DsRed1 fluorescent proteins in MGCs.
formation. Furthermore, we respectively observed the membrane changes in primary multinucleated cells stage and the phagocytosis function in secondary multinucleated cells stage. 2. Materials and methods 2.1. Plasmid construction and DNA transfection Human TRIM34 cDNAs were obtained by RT-PCR from HeLa cells induced by INFα and cloned into pEGFP-N3 vector (Clontech) using XhoⅠ/HindⅢ restriction enzymes to generate pEGFP-N3-TRIM34. TRIM34 coding sequence was also transferred into XhoⅠ/HindⅢ sites of pEYFP-N1 (Clontech) or pDsRed1-N1 vector (Clontech) to generate pEYFP-N1-TRIM34 or pDsRed1-N1-TRIM34 vectors.
2.6. Statistical analysis Statistical analyses were performed by one-way analysis of variance (ANOVA, Bonfferoni-Dunn test) with Graphpad prism 7.0 (San Diego, USA). Each experiment was repeated at least three times and the data was presented as means ± SD. p < 0.05 was considered as statistically significant difference.
2.2. Cell culture and transfection HEK293T cells were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum, 4.5 g/liter glucose, 4.0 mM L-glutamine and sodium pyruvate. Cells were maintained in a humidified 5% CO2 incubator at 37 °C. HEK293T cells were cultured on confocal dishes (NEST, diameter: 20 mm) or 6 cm cell culture dish for 24 h prior to transfection. Cells were transiently transfected with corresponding plasmids using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.
3. Results 3.1. TRIM34 facilitated the cell fusion in HEK293T cells To examine the localization of TRIM34, pEGFP-N3-TRIM34 vector was transfected into HEK293T cells for 24 h. Confocal images showed that TRIM34-EGFP was diffusely distributed throughout cytoplasm and formed variably sized aggregates in HEK293T cells (Fig. 1A). Interestingly, we found that TRIM34 could induce cell fusion, resulting in the formation of MGCs (Fig. 1B). As shown in Fig. 1C–D, TRIM34-EGFP drastically boosted the formation of MGCs compared with the mock group and EGFP-vector group. Similarly, TRIM34-EYFP also induced the formation of MGCs, which hinted that the effect of TRIM34 on cell fusion was not an occasional event (Fig. 1E). By DiD staining to label cell membrane, it also showed that TRIM34 could induce the formation of MGC, which size was significantly larger than that of peripheral cells (Fig. 1F).
2.3. DiD, LysoTracker and MitoTracker staining After transfection for 24 h, part of HEK293T cells were stained by cell membrane probes DiD (5 μM), DiIC18(5) (US EVERBRIGHT® INC, D4019) for 20 min. Then the live cells were rinsed with prewarmed DMEM two times and resuspended in DMEM, followed by confocal microscope assay. After transfection for 24 h, part of HEK293T cells were stained by LysoTracker Deep Red (50 nM) (Molecular Probes: L12492) for 30 min. LysoTracker probes can label the acidic organelles, mainly refer to lysosome, simultaneously nuclei contour are displayed. Then the live cells were rinsed with PBS two times and resuspended in PBS, followed by confocal microscope assay. After transfection for 24 h, part of HEK293T cells were stained by MitoTracker Deep Red (100 nM) (Molecular Probes: M22426) for 30 min. Then the live cells were rinsed with PBS two times and resuspended in PBS, followed by confocal microscope assay.
3.2. Multidimensional observing the cell fusion induced by TRIM34 Confocal images showed that MGCs could even be formed in the low cell density areas, which excluded the effect of excessive squeeze on cell fusion (Fig. 2A). We also found that TRIM34-mediated cell fusion was a gradual process. As shown in Fig. 2B, two adjacent HEK293T cells with TRIM34-EGFP expressing were fused into a single cell at the upper level (arrow 2); however they were still two separate cells at the lower level (arrow 1). HEK293T cells were also stained by LysoTracker Deep Red, which mainly exhibited lysosome specific location and showed nuclei contour simultaneously. The red fluorescence signal of LysoTracker disappeared at the junction of two adjacent HEK293T cells (Fig. 2C, arrow), suggesting that the two cells with high levels of TRIM34 expression fused into one cell. To observe the formation of MGCs in living cells, HEK293T cells were stained by MitoTracker Deep Red which was specifically located to mitochondria. As shown in Fig. 2D, TRIM34 evidently promoted the formation of MGCs in live HEK293T cells (arrow 1). The mitochondria accumulated in the MGC and its long and short diameter were about 18.7 μm and 16.9 μm respectively. The area of accumulated mitochondria (arrow 2, R1: 2.46) was about twice as
2.4. Cell fusion assay pEGFP-N3-TRIM34 and pDsRed1-N1-TRIM34 vectors were respectively transfected into HEK293T cells for 24 h. Then transfected cells were digested by trypsin and resuspended in DMEM complete medium after centrifugation. TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells were co-cultured within the ratio of 1:1 or cultured solely as control for 24 h, followed by confocal microscope assay. 2.5. Confocal microscope assay Cells were washed with PBS, fixed with 4% paraformaldehyde at 3
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Fig. 2. Multidimensional observation of the influence factors on cell fusion induced by TRIM34. TRIM34-EGFP vectors were transfected into HEK293T cells for 24 h (A–D). Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope (A–B). (A) In the low density areas of HEK293T cells, TRIM34 induced the formation of MGCs. Scale bar, 10 μm. (B) MGCs were observed at different depth by confocal microscope. Scale bar, 10 μm. (C) After transfection, HEK293T cells were stained by LysoTracker Deep Red (50 nM) for 30 min. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. Scale bar, 10 μm. (D) After transfection, HEK293T cells were stained by MitoTracker Deep Red (100 nM) for 30 min. Then the live cells were directly observed by confocal microscope. Scale bar, 10 μm. R1: the area of accumulated mitochondria in MGCs, R2: the cross-sectional area of normal cell.
elucidate the features of primary multinucleated cells stage, we observed the membrane changes of HEK293T cells transfected with pEGFP-N3-TRIM34 via DiD staining. Confocal images showed that the TRIM34-EGFP+ cell was in contact with the TRIM34-EGFP- cell (Fig. 7A, arrow 1). Then, the membrane at the junction became blurred with the fluorescence intensity of DiD weakened (Fig. 7B, arrow 2). Afterwards the membrane between two cells disappeared and the fusion cell gradually formed (Fig. 7C, arrow 3). With the integration of other cells, the volume of fusion cell increased continuously leading to the formation of primary multinucleated cell (Fig. 7D, arrow 4). When capable of swallow adjacent cells, the primary multinucleated cell turned into the secondary multinucleated cell (Fig. 7D, arrow 5).
big as the nearby normal cell (R2:1.15) analyzed by ImageJ software. 3.3. Nuclei polymorphy of the fusion cells induced by TRIM34 Nuclei shapes of MGCs were examined in HEK293T cells with high levels of TRIM34 expression by confocal microscope. As shown in Fig. 3A–F, the nuclei of MGCs presented various shapes, such as oval shape, heart shape, pear shape, horseshoe shape, ring like shape and irregular shape etc. 3.4. TRIM34 enhanced the formation of MGCs Confocal microscope showed that TRIM34 resulted in MGCs formation. As shown in Fig. 4A, the diameter of MGC reached to 55–65 μm. Nuclei in the MGC were arranged in ring like shape and distributed around the plasma membrane. Substantial TRIM34-EGFP proteins accumulated to the central areas of MGC (Fig. 4A). At the same time, some TRIM34-EGFP proteins located to the vicinity of MGCs cytomembrane. 3D reconstruction of confocal z-stacks showed that TRIM34-EGFP proteins formed macroaggregates, which major diameter reached to over 3 μm (Fig. 4B, left panel, arrow). Interactive 3D Surface Plot analyzing tool showed that the intensities of TRIM34-EGFP aggregates were very high in the central region of MGC, compared with the peripheral areas (Fig. 4B, right panel).
3.8. Phagocytosis function of secondary multinucleated cells In this study we discovered that some fusion cells, induced by TRIM34, had the quality of phagocytosis and thus we named them as secondary multinucleated cells. As shown in Fig. 8A1 (arrow 1), the membrane of multinucleated cell showed obvious invagination when the phagocytosis was in the early phase. During the metaphase of phagocytosis, about half of the target cell was engulfed by the multinucleated cell (Fig. 8A2, arrow 2). Finally, the target cell was totally swallowed at the last phase of phagocytosis (Fig. 8A3, arrow 3). From the above results, we discovered that TRIM34-EGFP- cells (Fig. 8A1, arrow 1) or TRIM34-EGFP+ cells (Fig. 8A2, arrow 2) could be engulfed by secondary multinucleated cells. Namely, the phagocytosis of secondary multinucleated cell had no obvious selectivity for cells expressing TRIM34-EGFP or not. Moreover, we found that secondary multinucleated cells could initiate multiple-process phagocytosis at the same time (Fig. 8B). Confocal images showed that the secondary multinucleated cell could concurrently engulf several target cells in different phagocytosis period, such as the early stage (arrow 1 and arrow 2), middle stage (arrow 3) or completion stage (arrow 4).
3.5. TRIM34 triggered the formation of MGCs by means of cell fusion To elucidate the cellular mechanism of MGCs formation, cell fusion assay was performed in HEK293T cells transfected with pEGFP-N3TRIM34 or pDsRed1-N1-TRIM34 vectors separately. Then, TRIM34EGFP expressing cells and TRIM34-DsRed1 expressing cells were cocultured for another 24 h. Confocal images showed that TRIM34-EGFP and TRIM34-DsRed1 fluorescent proteins from two or more different cells could be detected in the same fusion cell (Fig. 5A, arrow 1). Zstack images were also acquired to verify whether TRIM34-EGFP and TRIM34-DsRed1 co-existed in the same fusion cell. 3D reconstruction of confocal z-stacks showed that some TRIM34-DsRed1 aggregates (red) co-existed in the TRIM34-EGFP (green) preferential expressing cell (Fig. 5B3-B5, arrow 3). Moreover, the volume of accumulated nuclei in MGC was about two times larger than that of control cells.
4. Discussion In general, persistent existence of pathogens or foreign material leads to the formation of MGCs [12]. MGCs are primarily formed by the fusion of macrophages and play important roles in many physiological and pathological processes [13–16]. Langhans’ cells exist in many infectious granulomatous disorders such as tuberculosis and sarcoidosis, while foreign body giant cells are featured by foreign body granulomas [2,17]. In addition, spontaneous formation of MGCs can arise in physiological conditions. For example, osteoclasts are commonly multinucleates in adult mammals due to cell fusion [18]. Osteoclasts attach firmly to bones and form extracellular acidic compartment, which facilitate the dissolution and resorbing bone matrix. On the contrary, they are also related to bone diseases such as rheumatoid arthritis, osteoporosis and bone lesions [19,20]. In addition, Hart et al. described that MGCs, derived from microglias, accumulated in brain tissues with age. MGCs in aged mice were commonly found in the white matter and contained as many as 6 or 7 nuclei with approximately 20–30 μm in diameter [21]. Several reports describe that MGCs can be formed from other cell types, such as endothelial cells and fibroblast etc. HEK293 cells are a kind of human embryonic kidney epithelial cell. Purinergic P2X7 receptor overexpression in HEK293 cells promoted the fusion process leading to the MGCs formation [22]. Endothelial cells, derived from
3.6. Process of cell fusion mediated by TRIM34 It was probably a multistage process that TRIM34 mediated cell-cell membrane fusion, leading to MGCs formation. We suppose that the process might contain four major stages. First, the formation of MGCs occurred among adjacent HEK293T cells. The gap between two nuclei abounded with cytoplasm, which displayed as yellow for TRIM34-EYFP expression (Fig. 6A). Second, the gap between two nuclei became narrow gradually (Fig. 6B, arrow 2). Third, the gap between two nuclei was partly disrupted and the two adjacent cells fused into a single-cell (Fig. 6C, arrow 3). Finally, the intervals between multiple cells disappeared and MGCs came into being (Fig Fig. 6D). 3.7. Membrane changes during primary multinucleated cells stage The process of MGCs formation may be divided into primary multinucleated cells stage and secondary multinucleated cells stage. To 5
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Fig. 3. The nuclei shapes of TRIM34-induced fusion cells were polymorphic. (A–F) pEGFP-N3- TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) Oval shape, (B) Heart shape, (C) Pear shape, (D) Horseshoe shape, (E) Ring like shape, (F) Irregular shape. Scale bar, 10 μm.
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Fig. 4. TRIM34 promoted the formation of MGCs. pEGFP-N3-TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) TRIM34 expression induced the formation of MGCs in HEK293T cells. Scale bar, 20 μm. (B) 3D reconstruction of z series was analyzed by ImageJ software. The left panel showed image from the top of cells, and the middle panel showed the side view from the top of cells. The right panel showed the intensities of TRIM34-EGFP aggregates by Interactive 3D Surface Plot tool of ImageJ in Thermal LUT mode. Z-axis represented the intensities of TRIM34-EGFP. Representative results were presented, two additional experiments yielded similar results.
about 3–4 times larger than that of nearby normal cells (Fig. 1B). As the doubling time of HEK293T cell was around 24 h, the failure of cytokinesis was not essential for the formation of MGCs mediated by TRIM34. Accumulated evidences have shown that in vitro generation of MGCs often occurs as a result of cell fusion [26]. Cytokines, such as IFNγ and IL-4, adhesion proteins and receptors have been demonstrated to enhance the formation of MGCs [22,27,28]. IFN-γ plays a key role in MGCs formation for it enhances the cell clustering and cell-to-cell adhesion [29,30]. Antibodies against IFN-γ restrained MGCs generation both in vitro and in vivo [31]. Keegan AD demonstrated that IL-4 accelerated the formation of MGCs via regulating the expression of Ecadherin, which resulted in homotypic cell fusion and incorporation of large foreign bodies [32]. IFN-γ or IL-4 might not perform the direct role in cell fusion but through it mediated signaling pathways. In this study, we discovered that TRIM34, as a single factor, could directly induce MGCs formation even in the areas with low cell density, excluding the influence of excessive squeeze on cell fusion (Fig. 2A). In newly formed MGCs, we also found that TRIM34 could induce adjacent cells to fuse together at the upper level, while they were still two separate cells at the lower level (Fig. 2B). By DiD (Fig. 1F) or MitoTracker Deep Red (Fig. 2D) staining, confocal images evidently showed that TRIM34 promoted the formation of MGCs in live cells, which eliminated the potential influence of paraformaldehyde fixation and the detergent of Triton X-100. 3D image reconstruction showed that TRIM34-EGFP proteins could form macroaggregates in MGCs, while nuclei were arranged in ring like shape around the plasma membrane (Fig. 4A–B). Cell fusion assay further demonstrated that TRIM34EGFP+ cells and TRIM34- DsRed1- cells could fuse into a MGC (Fig. 5A–B). In the present study, we suppose the progress of MGCs
brain capillary, could also be induced into MGCs by cell fusion [23]. Holt DJ demonstrated that MGCs could originate from fibroblast in vitro and in vivo in pathologies such as fibrosis and senescence tissue [24]. Furthermore, there is evidence to suggest that somatic cells might fuse with macrophages. HIV infected T cells can contact and fuse with macrophage targets. Then, the newly formed lymphocyte-macrophage fusion cells can fuse with neighboring uninfected macrophages for HIV1 spreading [25]. In this study, we found that TRIM34 could lead to a marked increase in the efficiency of cell fusion compared with mock group and EGFPvector group in HEK293T cells (Fig. 1C–D). Moreover the nuclei shapes of MGCs, induced by TRIM34, were diversiform such as oval shape, heart shape, pear shape, horseshoe shape, ring like shape and irregular shape etc (Fig. 3A–F). Furthermore, confocal microscope showed that TRIM34-EGFP proteins appeared as aggregates and dispersed in the cytoplasm of HEK293T cells (Fig. 1A), suggesting TRIM34 proteins could bind to each other and form macromolecular complexes. The intracellular aggregates might be sensed by an unknown mechanism and trigger the cell fusion process, just like the formation of FBGCs initiated by extracellular biomedical materials. Currently, the cellular and molecular mechanisms of MGCs formation are still poorly understood. It has been suggested that MGCs may arise from the failure of cytokinesis. Kim YS reported that cyclin D1 and p16 were overexpressed in foreign body giant cells, osteoclast giant cells and Langhans giant cells, leading to cell-cycle arrested at the G1/S transition [8]. Brown GC described that inflammatory stimulus could induce the multinucleation of cultured microglia as a result of failure of cytokinesis. In this study, when HEK293T cells were transfected with pEGFP-N3-TRIM34 for only 24 h, the formation of MGCs could be detect by confocal microscope (Fig. 1B). The volume of MGCs nuclei was
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Fig. 5. The function of TRIM34 on MGCs formation was verified by cell fusion assay. (A) pEGFP-N3-TRIM34 or pDsRed1-N1-TRIM34 vectors were respectively transfected into HEK293T cells for 24 h. Cells transfected with pEGFP-N3-TRIM34 or pDsRed1-N1-TRIM34 vectors were co-cultured or cultured solely for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by the confocal microscope. The scale bar represented 10 μm. Representative results were presented, two additional experiments yielded similar results. (B) Z-stack confocal images of co-cultured HEK293T cells expressing TRIM34-EGFP and TRIM34DsRed1 were collected and analyzed by ImageJ. Scale bar, 10 μm. (B1) Single stack slice showed the coexistence of TRIM34-EGFP and TRIM34-DsRed1 in a fusion cell. The arrow 2 indicated TRIM34-DsRed1 red fluorescence protein. (B2) 3D Reconstruction image was viewed from the bottom of cells. (B3) Image was viewed from the top of cells. (B4) Images showed the side view from the top of cells. (B5) Image was viewed in y-z plane. Representative results were presented, two additional experiments yielded similar results.
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Fig. 6. The progress of cell fusion induced by TRIM34. (A–D) pEYFP-N1-TRIM34 vectors were transfected into HEK293T cells for 24 h. Then the cells were fixed, stained for nuclei using DAPI (blue) and observed by confocal microscope. (A) TRIM34-EYFP was expressed in multiple adjacent HEK293T cells. Scale bar, 10 μm. (B) The interval between two nuclei was narrowed down gradually. Scale bar, 10 μm. (C) The interval between two cells was partly disrupted. Scale bar, 10 μm. (D) The interval between multi-cells disappeared and MGCs came into being. Scale bar, 10 μm.
formation can be divided into two phases. Adjacent cells firstly formed primary multinucleated cell through cell fusion mediated by TRIM34 (Fig. 7A–D, arrow 1–4). Afterwards, some primary multinucleated cells acquired the ability to swallow adjacent cells and then turned into the secondary multinucleated cells (Fig. 7D, arrow 5). More importantly, as Fig. 8B shown, the secondary multinucleated cell could simultaneously devour several adjacent cells in different phagocytosis phase, which better explained the reason why TRIM34 could induce MGCs formation in short period. Meanwhile virus or germs are able to induce cell fusion [33]. Syncytia, namely MGCs, can be found in autopsy samples from human respiratory syncytial virus infections [34]. Fusion protein of RSV can be expressed in infected airway epithelial cells. They accumulated to
plasma membrane and promoted the fusion of infected cells with adjacent cells, leading to the formation of MGCs [34]. Moreover, FAST (fusion-associated small transmembrane) proteins of reovirus could accumulate to cell membrane, resulting in the fusion of virus-infected cells with neighboring normal cells [35]. Furthermore, burkholderia pseudomallei could escape into the cytosol of host cells and enhance the formation of MGCs [36]. In murine macrophages and dendritic cells, the expression of TRIM34 was highly up regulated upon virus infection in an IFN-I-dependent manner. In addition, TRIM34 expression was also stimulated by LPS and interferon-γ in human macrophages. It was possible that TRIM34 might exert direct actions in MGCs formation induced by interferon or virus. In the future work, HEK293T cells, transfected with TRIM34-EGFP or TRIM34-EYFP vectors, may be used 9
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Fig. 7. TRIM34 induced cytomembrane changes in primary multinucleated cells stage. HEK293T cells were transfected with pEGFP-N3-TRIM34 vectors for 24 h. Then the cells were stained by DiD (5 μM) for 20 min and observed by confocal microscope. (A). A TRIM34-EGFP+ cell was in contact with a TRIM34-EGFPcell. Scale bar, 5 μm. (B). The contact sites of two adjacent cells became thinning compared with peripheral cytomembrane. Scale bar, 5 μm. (C). The contact sites of two adjacent cells were disintegrated. Scale bar, 5 μm. (D). With cell fusion of many adjacent cells, the primary multinucleated cell came into being. The arrow 4 indicated the primary multinucleated cell. Scale bar, 10 μm. Representative results were presented, two additional experiments yielded similar results.
have similar functions.
as a cell model to imitate MGCs formation. By means of live cell imaging system, it can easily and dynamically display the process of cell fusion and phagocytosis during the formation of MGCs. At the same time, it is still not clear whether TRIM34 proteins are able to induce other cell types to form MGCs and whether other TRIM family members
5. Conclusions In short, our data demonstrates that TRIM34 proteins are able to 10
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Fig. 8. The phagocytosis function of secondary multinucleated cell. HEK293T cells were transfected with pEGFP-N3-TRIM34 vectors for 24 h. Then cells were stained by DiD (5 μM) for 20 min and observed by confocal microscope. Scale bar, 10 μm. Representative results were presented, two additional experiments yielded similar results. (A1). In the early phase of phagocytosis, the multinucleated cell began swallowing adjacent cell with obvious invagination of membrane. (A2). The multinucleated cell engulfed half of the target cell in the metaphase of phagocytosis. (A3). The target cell was totally engulfed in the last phase of phagocytosis. (B). Secondary multinucleated cell could simultaneously swallow several target cells in a multi-process manner.
Conflicts of interest
boost the formation of MGCs by means of cell fusion and phagocytosis in epithelial cells. Our study provides new insight into the function of TRIM family protein on MGCs formation.
The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Competing interests None.
[18]
Funding
[19]
This study was supported by Projects of medical and health technology development program in Shandong province (grant numbers 2018WSB30002); the Natural Science Foundation of Shandong Province, China, (grant numbers ZR2012CM009); Science and Technology Development Plan Project of Shandong Province (grant number 2011YD18015).
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