Supervillin contributes to LPS-induced inammatory response in THP-1 cell-derived macrophages

Supervillin (SVIL), the largest member of villin/gelsolin family, is an actin-binding and membrane-associated protein, that can also be localized to the nucleus. It has been reported that the mRNA expression of SVIL in neutrophils could be increased by lipopolysaccharide (LPS), but the underlying mechanisms remain unknown. Moreover, SVIL was also observed to be involved in the regulation of macrophages’ movement. However, it is not clear whether SVIL is involved in the LPS-induced inammatory response in macrophages. This work was to investigate the underlying molecular mechanisms of LPS regulating SVIL expression in macrophages and hence the possible role of SVIL in LPS-induced inammation. Our data showed that in THP-1-derived macrophages, LPS stimulation signicantly increased SVIL mRNA and protein expression. Inhibition of TLR4 by Resatorvid (Res) completely reversed the expression of SVIL and inammatory cytokines (IL-6, IL-1β and TNF-α) induced by LPS. Additionally, ERK1/2 and NF-κB inhibitors (U0126 and BAY) signicantly reduced SVIL and IL-6, IL-1β & TNF-α expression. Furthermore, down-regulation of SVIL by SVIL-specic shRNA signicantly attenuated the expression of IL-6, IL-1β & TNF-α induced by LPS. Taken together, as a downstream molecule of TLR4/NF-κB and ERK1/2, SVIL was involved in the inammatory response of LPS-induced elevated IL-6, IL-1β and TNF-α in macrophages. SVIL be related to TLR4-MAPK-NF-κB pathway in macrophage inammation response. In this study, we identied that LPS stimulation markedly upregulated the expression of SVIL in macrophage via activating TLR4/NF-κB and ERK1/2 MAPK signaling pathway. The deciency of SVIL signicantly attenuated the expression of IL-6, IL-1β and TNF-α in macrophages induced by LPS. This novel nding helps elucidate the involvement of SVIL in LPS-induced inammation.


Introduction
Supervillin (SVIL), a member of villin/gelsolin family, is an actin-binding and membrane-associated protein participating in cell proliferation, motility, and signal transduction etc [1,2]. Current research on SVIL mostly focuses on cancers and muscular tissue. It was reported that SVIL were involved in many cellular processes of tumor development and muscular function [3,4]. SVIL was found to localize with the nucleus to participate in the intracellular signal transduction and early myogenesis of myoblasts [5,6]. Recent evidence indicated that SVIL was a component of podosome and could bind to actin isoforms and myosin regulators contributing to regulation of podosome turnover, migration and invasion in primary human macrophages [7,8]. However, the knowledge about the functions of SVIL in macrophage is still limited. It is far from clear whether and how SVIL participates in macrophage in ammatory response.
Lipopolysaccharide (LPS) is a key component in the outer membrane of Gram-negative bacteria. LPS was observed to stimulate macrophages to produce pro-in ammatory cytokines, IL-1β, IL-6 and TNF-α [9]. It was once found that the mRNA expression of SVIL was elevated by LPS stimulation in human neutrophils [10]. However, there is no more evidence of how LPS regulates SVIL, and what role SVIL plays after LPS stimulation. It might be speculated that SVIL could also response to this LPS stimulation and participate in the subsequent pro-in ammatory process in macrophage.
Toll-like receptor 4 (TLR4) is a de nite receptor of LPS, which belongs to pattern recognition receptor (PRR) and triggers all the responses to LPS stimulation [11,12]. The mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK, P38) are activated as the downstream of TLR4 after LPS stimulation [13][14][15], leading to activation of NF-κB and hence the expression of in ammatory factors [16,17]. In smooth muscle cells, SVIL has been identi ed as a ERK scaffolding protein and contributed to the regulation of ERK binding, activation, and release from the signaling complex [18,19]. SVIL also activated EGFR-ERK1/2 and raised the motility in Hela cells [20]. In natural killer (NK) cells, SVIL was necessary for KIR2DL1-mediated ERK activation [21]. SVIL promoted liver cancer cell migration and invasion via the activation of the RhoA/ROCK-ERK/p38 pathway under hypoxia [22]. These reports highly suggest that SVIL may be closely related to TLR4-MAPK-NF-κB pathway and hence participate in macrophage in ammation response.
In this study, we identi ed that LPS stimulation markedly upregulated the expression of SVIL in macrophage via activating TLR4/NF-κB and ERK1/2 MAPK signaling pathway. The de ciency of SVIL signi cantly attenuated the expression of IL-6, IL-1β and TNF-α in macrophages induced by LPS. This novel nding helps elucidate the involvement of SVIL in LPSinduced in ammation.

Real-time PCR (RT-PCR)
Total RNA was extracted from treated THP-1-derived macrophages by using TRIzol reagent. For cDNA synthesis, HiScript II Q-RT superMix was applied to reverse transcription reaction. The cDNA was used for qRT-PCR with SYBR green under standard reaction condition by Bio-Rad CFX Connect System (Bio-Rad, Hercules, CA, USA). The primers for SVIL, IL-1β, IL-6, TNF-α, GAPDH were provided by GENERAY (Shanghai, China) and the sequence were shown in Table 1.

Protein isolation and western blots analysis
The proteins were obtained from treated cells, which were lysed by RIPA buffer, which contained PMSF (1%), protease (10%) inhibitor and phosphatase (10%) inhibitor (Roche). and the concentration of protein was assessed by BCA

Lentiviral encapsulation and infection
The sequences of shRNAs targeting SVIL (shSVIL) and a scrambled shRNA (shNC) were designed and constructed by Gene Pharma (shanghai, China). ShRNAs (shNC or shSVIL) and Lentiviral plasmids were packaged in HEK293FT cells with Lipofectamine 2000 and then lentiviral particles containing shRNA (shNC or shSVIL) were produced. THP-1 cells were infected with Lentiviruses containing shNC or shSVIL by polybrene (8 μg/ml), then the infected cells were selected by puromycin (1 μg/ml) for about 2 weeks. Cells expressing green uorescence were also differentiated by PMA and were used in subsequent experiments.

Flow cytometry analysis
The treated THP-1 Cells were harvested and stained with the Annexin V-FITC and PI to detect cell apoptosis. Brie y, the harvested cells were resuspended in 500 μl binding buffer (1x) and incubation with 5 μl Annexin V-FITC and 10 μl PI for 15 min in the dark, then cell apoptosis was detected by ow cytometry (BD FACS Calibur, NY) according to the manufacturer's instructions.
Cell cycle distribution changes were also measured by ow cytometry (BD FACS Calibur, NY). The cells were collected and xed with 75% ethanol and stored at 4℃.Then, cell cycle was detected by ow cytometry according to the manufacturer's instructions.

Cell proliferation assay
Cell proliferation was measured by CCK-8. The cells were seeded into 96-well plates at a density of 1x10 4 cells/well and incubated at 37℃. Then, cells were treated with/without indicated drugs and indicated times. Next, 10 μl CCK-8 solution was added into each well and incubated for 2.5 h. The cells viability was measured by microplate reader at the absorbance of 450nm.

Statistical analysis
The data was analyzed with GraphPad Prism 5.0 by two-tailed t-test (comparison of two groups) or one-way analysis followed by Turkey tests (comparison of multiple groups). *p < 0.05 was considered to be statistically signi cant. In our study, each experiment was repeated more than three times respectively.

LPS induced SVIL expression in THP-1 macrophages
Here, we detected the mRNA and protein expression levels of SVIL in THP-1-derived macrophages after LPS stimulation. LPS treatment signi cantly induced the mRNA expression ( Fig. 1A) and protein expression ( Fig. 1B) of SVIL in a dosedependent manner and the concentration (100 ng/ml) of LPS was selected for following experiments. As shown in Fig. 1C and D, LPS treatment induced the highest mRNA expression level of SVIL at 3 h and the protein expression reached the peak at 9 h. These results indicated that LPS stimulation could signi cantly enhance the expression of SVIL in macrophages.

TLR4/NF-κB mediated LPS-induced SVIL expression in macrophages
TLR4 has been assumed to trigger all responses to LPS since it was identi ed as the LPS receptor [23][24][25]. However, whether TLR4 participates in LPS-induced expression of SVIL is not known yet. Here, Resatorvid (Res), a speci c inhibitor of TLR4, to detect whether it could inhibit the expression of SVIL induced by LPS in macrophages. As shown in Fig. 2A and B, LPS led to a signi cant increase of SVIL mRNA expression level, and the protein expression level was also increased remarkably. LPS-induced SVIL expression was signi cantly suppressed by pretreatment of Res at both mRNA ( Fig. 2A) and protein (Fig. 2B) levels.
As the above data show, LPS caused a signi cant up-regulation in the mRNA level of SVIL via TLR4 ( Fig. 1A and C, Fig.   2A). As known, the nuclear transcription factor NF-κB is a classical downstream signal molecule of TLR4. Hence, we speculated that NF-κB was also involved in the regulation of SVIL by LPS. As shown in Fig. 2C, LPS stimulation increased the phosphorylation level of NF-κB, which could be signi cantly reversed by Res. To investigate whether NF-κB participated in LPS-induced SVIL expression, NF-κB inhibitor BAY were used and signi cant inhibition of LPS-induced phosphorylation of NF-κB was observed (Fig. 2D). As expected, pretreatment of the cells with BAY reversed LPS-induced SVIL mRNA (Fig. 2E) and protein expression (Fig. 2F). Therefore, these results suggested that TLR4/NF-κB mediated LPSinduced SVIL expression in macrophages.

ERK1/2 MAPK mediated LPS-induced SVIL expression in macrophages
Although inhibition of NF-κB could reverse the up-regulation of SVIL induced by LPS, the reversion was not complete (Fig.  2E and F). Thus, we wondered if there were other signaling pathways involved in this process. Many studies demonstrated that TLR4 activation can activate MAPK signal pathway and initiate intracellular response [26][27][28]. In order to verify whether MAPK family was involved in the regulation of SVIL by LPS, we pretreated cells with ERK1/2 inhibitor U0126, JNK inhibitor SP or P38 inhibitor SB, respectively, before LPS stimulation. Interestingly, only U0126 inhibited LPS-induced mRNA expression of SVIL, while SP and SB did not reverse this e ciency of LPS (Fig. 3A). Therefore, the following research mainly focused on the ERK1/2 MAPK signaling pathway.
As shown in Fig. 3B, LPS stimulation increased the phosphorylation of ERK1/2 and this effect could be reversed by Res.

SVIL Knockdown e ciency in THP-1 cells
To further investigate the role of SVIL in LPS-induced macrophage in ammatory response, we used SVIL-targeted shRNA to interfere with its genetic expression. Expression of LV(H1/GFP&Puro)-shNC and LV(H1/GFP&Puro)-shSVIL was con rmed by detection of green uorescent protein (GFP)-uorescence in cells (Fig. 4A). Then, we detected the mRNA expression (Fig. 4B), protein expression (Fig. 4C) of SVIL in the transfected cells. As shown, the data indicated that SVIL was signi cantly knocked down by shRNA. The shSVIL THP-1 cells with the highest downregulation e ciency of SVIL expression was selected for the present study.

The in uence of SVIL knockdown on cell cycle, cell proliferation & apoptosis
The effects of SVIL depletion on the THP-1 viability was detected by cell ow cytometry and cell proliferation assay. Cell cycle was evaluated by measuring DNA content, as shown in Fig. 5A and B, PMA signi cantly reduced the proportion of cells in G 0-1 and S phases, while the proportion of cells in G 2 /M phases was increased remarkably in shNC THP-1 cells.
After PMA stimulation, the number of cells were signi cantly inhibited compared with the control group (Fig. 5C). These results demonstrated that PMA signi cantly inhibited the proliferation of THP-1 cells in the process of inducing THP-1 to differentiate into macrophages (Fig. 4A).
As shown in Fig. 5A and B, without PMA stimulation, SVIL ablation increased the proportion of cells in G 0-1 phase and decreased the proportion of cells in G 2 /M phase, while the proportion of cells in S phase with no signi cant difference.
After THP-1 cells were induced to differentiate into macrophages by PMA, the knockdown of SVIL caused similar changes in G 0-1 and G 2 /M phases, and signi cantly increased the proportion of cells in S phase. However, CCK8 data showed that SVIL knockdown did not affect the nal change in cell numbers compared with control group with or without PMA incubation (Fig. 5C). As shown in Fig. 5D, SVIL depletion also could not induce apoptosis of THP-1 cells.
3.6 SVIL depletion inhibited LPS-induced expression of in ammatory cytokines LPS stimulation induces pro-in ammatory responses of macrophages and promotes the production of in ammatory cytokines, such as IL-1β, IL-6 and TNF-α [9,15]. Moreover, SVIL has been identi ed as an interacting protein of Sphingosine kinase 1(sphk1) [29], and sphk1 is reported to involve in macrophage in ammation induced by LPS [30,31]. Therefore, it was of interest to examine if SVIL depletion could change the regulation of LPS-induced pro-in ammatory responses in THP-1 macrophages. Indeed, LPS stimulation signi cantly induced the expression of in ammatory cytokines, TNF-α, IL-6 and IL-1β (Fig. 6A-I). The mRNA expression of all the three in ammatory cytokines induced by LPS could be inhibited by TLR4 inhibitor Res (Fig. 6A-C), NF-κB inhibitor BAY (Fig. 6D-F) and ERK1/2 inhibitor U0126 (Fig. 6G-I).
Importantly, knockdown of SVIL produced opposite effects of LPS. As shown in Fig. 7A, LPS signi cantly increased the expression of SVIL in shNC macrophages, while the same stimulation condition could not induce the SVIL expression in shSVIL macrophages. Interestingly, we found that the depletion of SVIL in macrophages signi cantly reversed the mRNA expression of in ammatory factors IL-1β, IL-6 and TNF-α induced by LPS stimulation (Fig. 7B-D).  [10], here we demonstrate that LPS could enhance the mRNA and protein expression of SVIL in THP-1-derived macrophages. SVIL has been reported to be involved in the regulation of podosome function in macrophages [7,8]. As far as we know, the speci c mechanism of LPS's SVIL-increasing effect has not been reported yet. In this paper, we found the signaling pathway of LPS regulating SVIL in macrophages. Our results may provide some useful basis for discussing the relationship between LPS and SVIL, and hence gaining a new insight into some in ammatory conditions like atherosclerosis.

Discussion
Since TLR4 was rstly identi ed as the receptor of LPS in 1998, various studies have shown that it is the most important cell surface receptor of LPS, in almost all responses caused by LPS [25]. The activation of TLR4 by LPS cause various intracellular changes, i.e. the production of in ammatory factors in macrophages [38]. And our data showed that LPS increased SVIL expression in macrophages. Therefore, in the process of regulating SVIL expression, TLR4 was the rst receptor that we considered in response to LPS stimulation. As expected, LPS activated the TLR4 receptor and markedly enhanced the expression of SVIL in macrophages. The inhibition of TLR4 with Res could signi cantly reverse this SVILinducing effect of LPS. Thus, TLR4 played a critical role in the process of LPS regulating the expression of SVIL in macrophages.
Our results showed that LPS could signi cantly increase the mRNA expression of SVIL in macrophages, which was consistent with Morozumin T's report in neutruphils [10]. In this study, we further investigated the transcription factors mediating the transcription of SVIL induced by LPS. The nuclear transcription factor-κB (NF-κB), is known to be a key downstream nuclear transcription signaling effector of the TLR4 pathway [39,40]. Therefore, it is believable that NF-κB would mediate the up-regulation of SVIL mRNA expression by LPS. As expected, LPS stimulation induced the phosphorylation of NF-κB and this effect could be inhibited by TLR4 inhibitor Res pre-treatment. BAY, the NF-κB inhibitor also signi cantly attenuated LPS-induced mRNA and protein expression of SVIL in macrophages. These results indicated that TLR4-NF-κB contributed to LPS-induced SVIL expression in macrophages. To our knowledge, it is the rst time to demonstrate that LPS-induced SVIL expression is mediated by TLR4-NF-κB pathway. Moreover, we found that inhibition of TLR4 almost completely reversed the expression of SVIL protein induced by LPS, but inhibition of NF-κB could not achieve the same e cacy. Therefore, these results suggest that in addition to NF-κB, there may exist other signaling molecules in the downstream of TLR4 that participated in the regulation of SVIL by LPS.
MAPKs are another TLR4-mediated activated protein kinases, which are also responsible for cellular responses induced by LPS, such as the production of proin ammatory cytokines [39,41]. Therefore, we used ERK, JNK and P38 inhibitors to investigate the effect of MAPK on LPS-induced SVIL expression. Although TLR4 mediated LPS-induced phosphorylation of ERK, P38 and JNK, it was found that only ERK inhibitor U0126 could partially reverse LPS-induced SVIL mRNA and protein expression, while P38 inhibitor SB and JNK inhibitor SP had no signi cant effect. This result is consistent with previous studies that SVIL participated in ERK signal transduction and promoted epithelial-mesenchymal transformation [20][21][22]. Furthermore, U0126 was observed to inhibit LPS-induced NF-κB phosphorylation, indicating that U0126 inhibited LPS-induced SVIL expression perhaps partly through NF-κB pathway. Taken together, our research provided a certain basis for exploring the relationship between SVIL and ERK. SVIL contributed to cytokinesis and mainly played a regulatory role in earlier cytokinesis by interacting with central spindle proteins and myosin II [42,43]. Similarly, SVIL could also enhance cell survival and maintain the cell proliferative activity [3,44]. Our results showed that SVIL depletion increased the proportion of cells in S phase and decreased the proportion of cells in G 2 /M phase. However, CCK8 data showed that cell proliferation was not affected. This result is partially consistent with the report that SVIL had some effect on cell division in M phase [42,43]. The S-phase-related effect remains to be elucidated.
SVIL was reported to be an interacting protein of sphk1 [29], and involved in cellular in ammation together with sphk1.
However, there were no more reports on the relationship between in ammation and SVIL, which was a macromolecular protein closely related to cytoskeleton. Due to the lack of literature about the role of SVIL in in ammation, it was challenging but interesting to demonstrate whether SVIL was involved in the regulation of LPS-induced macrophage in ammation. In our study, LPS induced the expressions of in ammatory cytokines and SVIL in macrophages.
Interestingly, we found that after interfering with the expression of SVIL, the expressions of in ammatory cytokines (IL-6, IL-1β and TNF-α) in macrophages induced by LPS were signi cantly inhibited. These data suggested that SVIL played an important role in LPS-induced in ammation.
In conclusion, this is the rst study investigating the involvement of SVIL in LPS-induced macrophages in ammatory response. As depicted in Fig. 8, our current study clearly showed that LPS induced the mRNA and protein expressions of SVIL in macrophages via activating TLR4/NF-κB and ERK1/2 MAPK signaling pathway. Additionally, depletion of SVIL could signi cantly reverse the expression of in ammatory factors in macrophages induced by LPS. The present study provides a basis for exploring the relationship between SVIL and in ammation, and SVIL may be a potential target for the therapy of vascular in ammatory diseases such as atherosclerosis.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The materials and analyzed data sets generated during the current study are available from the corresponding author on reasonable request.
The authors have no competing interests to declare that are relevant to the content of this article. Tables Table 1:         Schematic diagram of SVIL involved in LPS-induced in ammatory response in macrophages. LPS induced SVIL expression by activating TLR4/NF-κB signaling pathway. ERK1/2 MAPK pathway also participated in this process as a downstream of TLR4. Elevated SVIL was involved in the production and release of in ammatory cytokines.