GM1 Induced the inflammatory response related to the Raf-1/MEK1/2/ERK1/2 pathway in co-culture of pig mesenchymal stem cells with RAW264.7

ABSTRACT Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.


Introduction
Gangliosides are complex glycosphingolipids containing one or more sialic acids, and are a main component of cell membranes (Hakomori 1990). Some studies have reported that gangliosides are developmentally controlled in a cell type-specific manner (Yu 1994;Yamamoto et al. 1996;Yu et al. 1988). Additionally, expression of gangliosides is related to the biological processes of stem cells in vitro (Kwak et al. 2006).
Although xenotransplantation has vast clinical potential, it is limited by the problem of immune responses against xenogeneic tissue (Wright et al. 2016). Additionally, xenotransplanted cells, including vascularized organ xenografts, show loss of function within a short time of transplantation in dissonant species combinations. Previous studies reported that gangliosides are related to the inflammatory responses induced in co-culture of xenogeneic cells, such as pig endothelial cells (PAECs) and human leukocytes (Cho et al. 2012). The inflammatory responses were associated with the mitogen-activated protein kinase (MAPK) family (Yin et al. 2016).
However, the expression and role of gangliosides in inflammatory responses is unclear, and has not been investigated using xenogeneic co-culture of pig MSCs with cells from other species. In this study, we investigated the role of gangliosides in inflammatory activation using co-culture of pig adipose-derived mesenchymal stem cells (pADMSCs) with RAW 264.7 macrophages.

Materials and methods
Culture of pADMSCs and RAW264.7 cells pADMSCs were provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB). The cells were cultured in pre-warmed Dulbecco's Eagle Medium (DMEM) containing 10 ng/ml basic fibroblast growth factor (bGFGF; R&D Systems, Minneapolis, USA), 10% fetal bovine serum (FBS), and 1% (v/v) penicillin/streptomycin (P/S) solution and incubated in a humidified 5% CO 2 atmosphere at 37°C. RAW 264.7 cells were maintained in DMEM supplemented with 10% FBS and 1% P/S at 37°C in a humidified 5% CO 2 incubator.

Cell viability
Cell proliferation was determined by MTT assay 24 h after initiating culture of pADMSCs and RAW 264.7 cells. pADMSCs co-cultured with RAW 264.7 cells were transferred into 96-well plates at 1 × 10 4 cells/well and treated with LPS at 10 µM and GM1 synthase siRNA (10 nM), respectively. MTT solution (Sigma) was added to each well and incubated for 4 h and the absorbance was measured at 590 nm using a spectrophotometer.

Ganglioside extraction and purification
Lee et al. have described the methods used to extract and purify gangliosides. Briefly, cells were homogenized in distilled water at 48°C to extract total lipids, which were re-suspended in chloroform/methanol (1:1, v/v), lyophilized using N 2 gas, and subsequently dissolved in chloroform/methanol/H 2 O (15:30:4, v/v/v). The column was washed with H 2 O to remove non-hydrophobic lipids. Finally, the gangliosides were eluted with methanol, dried at 30°C under N 2 for 3 h, and stored at −80°C until analysis.

Western blot analysis
pADMSCs and RAW 264.7 cells were homogenized in RIPA buffer (Sigma), and then centrifuged at 13,000 rpm for 20 min at 4°C. Proteins (30 µg/lane) were separated on a 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ). The blots were blocked for 2 h with 5% bovine serum albumin (BSA) in Tris-buffered saline, and the membrane was incubated for 16 h with the following primary antibodies: BCl-2, Caspase-8, Caspase-9, Caspase-3, and βactin (1:500; Santa Cruz Biotechnology, Santa Cruz, USA). The blot was then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies, such as anti-mouse and anti-rabbit (Santa Cruz Biotechnology), and proteins were visualized using the ECL system (Pierce, Rockford, USA).

Statistical analysis
All data are presented as mean (SD). Multi-group associations were analyzed using one-way ANOVA and two-way ANOVA, followed by Tukey's and Bonferroni post-hoc pairwise comparisons. A p-value < 0.05 was considered statistically significant. All statistical analyses were executed using GraphPad Prism (Ver. 5.00; Graph-Pad Software Inc., La Jolla, USA).

Results
Cell viability and ganglioside expression patterns in pADMSCs, RAW 264.7 cells, and pADMSCs cocultured with RAW264.7 cells The co-culture was designed to emulate the conditions of xenograft. The cell culture groups consisted of pADMSCs only, RAW264.7 cells plus LPS, and co-culture of pADMSCs with RAW264.7 cells. Figure 1 shows cell viability as determined by MTT assays. Cell viability was significantly decreased when pADMSCs were co-cultured with RAW264.7 cells (Figure 1(A)). However, cell viability was similar to control in LPS only group (Figure 1(A)). In addition, we examined the ganglioside expression profile in pADMSCs only, RAW264.7 cells only, and pADMSCs co-cultured with RAW264.7 cells. GM2 and GD3 were weakly expressed in pADMSCs only and RAW264.7 cells only (Figure 1(B)). However, GM1 and GM3 were highly expressed in co-culture of pADMSCs with RAW264.7 cells (Figure 1(B)).
Cell viability in pADMSCs co-cultured with RAW 264.7 cells with knockdown of GM1 and GM3 synthase using siRNA We investigated the effects of GM1 and GM3 knockdown in pADMSCs using GM1 and GM3 synthase siRNA. Figure 3(A) shows the results of GM1 and GM3 knockdown in co-culture of pADMSCs with RAW264.7 cells (Figure 3(A)). Cell viability significantly decreased in pADMSC co-cultured with RAW264.7 cells as a positive control (Figure 3(B)). However, cell viability was significantly higher in pADMSCs (GM1 synthase knockdown) co-cultured with RAW264.7 cells than in pADMSCs cocultured with RAW264.7 cells (Figure 3(B)).

Involvement of the MAPK pathway with GM1 in inflammation of co-cultured pADMSCs and RAW264.7 cells
We attempted to determine the role of MAPK and to elucidate its mechanism of action in co-culture of pADMSCs with RAW264.7 cells with or without knockdown of GM1.

Discussion
Inflammation results from an excessive immune response, as part of the mechanism for protection against damaged tissue and foreign substances (Fitzpatrick 2001). Inflammatory mediators such as NO are produced by macrophages (Lee et al. 2016). Inducible iNOS and COX-2 are expressed following increased production of NO in LPS-stimulated macrophages (Zhao et al. 2013). iNOS is an important factor for the development of inflammation and subsequent maintenance of the inflammatory response. COX-2 is another important factor in inflammation ). In addition, the activities of iNOS and COX-2 related to production of TNF-α play important roles in inflammatory responses (Shin et al. 2015). NF-κB is an important transcription factor associated with inflammatory responses, which induces the expression of various inflammatory factors, including iNOS, COX-2, and TNF-α Yamada et al. 2014). In LPS-stimulated RAW264.7 macrophage cells, NF-κB is activated by the protein I-κBα (Ramaswami et al. 2012). Some reports have shown that gangliosides can induce production of cyclooxygenase-2. In this study, we observed that pro-inflammatory factors including COX-2, iNOS, p65, p50, p-IκBα, and TNF-α were significantly increased in pADMSCs co-cultured with RAW264.7 cells (Figure 2). Moreover, the increases in pro-inflammatory factors in co-culture of pADMSCs with RAW 264.7 cells were similar to the proinflammatory factor expression observed in LPS-stimulated RAW 264.7 cells, as a positive control ( Figure 2). However, pro-inflammatory factors were significantly suppressed in GM1-knockdown pADMSCs co-cultured with macrophages ( Figure 2). These results indicated that in co-culture of xenogeneic cells (pig MSCs with RAW264.7 mouse macrophages) inflammation related to the rejection of xenografts was mediated by GM1.
Several studies have reported that phosphorylation of three MAPKs (ERK, JNK, and p38) occurs by NF-κB activation (Hwang et al. 2011;Li et al. 2011). Szelenyi and Uros reported that the ERK1/2 pathway is a dominant and highly responsive pathway in inflammation (Szelenyi and Urso 2012). MEK-mediated ERK activation is the most important regulatory step in inflammation (Parthasarathy and Philipp 2014). A previous study indicated that the anti-inflammatory mechanism of flavonoids was related to inhibition of ERK phosphorylation by down-regulation of the expression of iNOS and COX-2 (Han et al. 2013), but was independent of the JNK and P38 pathway (Mazier et al. 2001). In addition, another report indicated that GM1 can activate ERKs in young rats and that GM1 induces activation of ERK1/2 by the Raf-1/MEK1/2 pathway in the VSMCs pathway (Duchemin et al. 2002). Ceramide, an important component of gangliosides, is known to be related to the ERK1/2 and the JNK pathways (Maziere et al. 2001). In particular, ceramide regulates the ERK1/2 pathway via activated RAf-1 and MEKs in various cell types (Willaime et al. 2001). In this study, we investigated how the MAPK pathway is involved with inflammation in pADMSCs co-cultured with RAW264.7 macrophage cells, and we found that expression of ERK1/2 and JNK1/2 were meanly activated in pADMSCs co-cultured with RAW264.7 cells ( Figure 5 (A)). Furthermore, activation of MEK1/2 and Raf-1, the up-stream pathway of ERK1/2, was strongly increased in pADMSCs co-cultured with RAW 264.7 cells ( Figure 5 (B)). However, other components of the MAPK pathway, including ERK1/2, JNK1/2, MEK1/2, and Raf-1, were significantly suppressed in co-culture of pADMSCs with GM1 knockdown ( Figure 5). These results indicated that ERK1/2 phosphorylation by upregulation of MEK1/2/ Raf-1 was associated with macrophage inflammation mediated by an increase of the ganglioside GM1 in coculture of xenogeneic cells (pig MSCs with RAW264.7 mouse macrophages).

Disclosure statement
No potential conflict of interest was reported by the author(s).