RON Expression Mediates Lipopolysaccharide-Mediated Dendritic Cell Maturation via March-I

The macrophage stimulating protein (MSP)–Recepteur d’origine nantais (RON) signaling pathway regulates macrophage function. Here, we verified RON receptor expression in bone marrow-derived dendritic cells (BMDCs) by real time-PCR, Western blot, and flow cytometry. Flow cytometry was used to detect the changes in MHC II and CD86 expression following the inhibition of RON in BMDCs and splenic dendritic cells (DCs). Immunoprecipitation and Western blot were used to detect the level of MHC II and CD86 ubiquitination. An enzyme-linked immunosorbent assay was used to detect cytokine release, and a mixed lymphocyte reaction was performed to evaluate DC maturity. The results show that the inhibition of RON leads to an increase in March-1 transcription, which intensifies the ubiquitination of MHC II and CD86 and ultimately leads to a decreased level of these two molecules. The mixed lymphocyte reaction provided evidence that RON inhibition decreased the ability of DCs to promote the proliferation of T cells. The MSP-RON signaling pathway may play an important role in lipopolysaccharide (LPS)-stimulated DC maturation through March-I and may protect DC differentiation following LPS stimulation.


INTRODUCTION
Dendritic cells (DCs) represent a type of antigen-presenting cell that recognizes and eliminates invading pathogenic microorganisms through the activation of Toll-like receptors (TLRs). Following DC stimulation with TLR ligands [e.g., lipopolysaccharide (LPS)], immature DCs turn into mature DCs, exhibited by increased major histocompatibility complex (MHC) II and CD86 expression and enhanced release of inflammatory factors. Mature DCs stimulate the activation of naive T cells. MHC II and CD86 expression in DCs is regulated by ubiquitination and degradation by the E3 ligases March-I and March-8 (Furuta et al., 2013;Oh and Shin, 2015). The expression of March-I in immature DCs enables the cells to recycle MHC II molecules, thereby efficiently treating antigens intracellularly, and loading the antigens onto MHC II. Moreover, MHC II ubiquitination is decreased in LPS-stimulated DCs, which could contribute to maintaining a high level of MHC II expression on the surface of DCs (Cho et al., 2015).
Recepteur d'origine nantais (RON, also known as MST1R) is a receptor in the tyrosine kinase receptor family of the MET family (Huang et al., 2020). RON participates in epithelial cell proliferation and tissue repair (Nanney et al., 1998), antagonizing LPS-induced inflammatory factor synthesis (Laskin et al., 2010). RON-specific inhibitor BMS777607 is currently undergoing a Phase II clinical trial for cancer research (Khaibullina et al., 2018). There is also evidence that the activation of RON leads to an increase in MHC class II transactivator (CIITA) expression in peritoneal macrophages and regulates the expression of MHC II in macrophages (Wilson et al., 2008).
In this study, we found that RON was expressed in DCs. The inhibition of RON led to increased March-I expression following LPS-mediated DC maturation, enhanced MHC II and CD86 ubiquitination, and inhibited DC maturation.

Mice and Cells
Six-week-old wild-type female C57BL/6 and BALB/C mice were purchased from the Shanghai Laboratory Animal Center (Slaccas, Shanghai, China). The animals were housed in a specific pathogen-free facility at the First Affiliated Hospital of Zhejiang University (Hangzhou, China). The experimental procedures in the use and care of animals were approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University.
For cell sorting, DCs were incubated with 1 µg/ml Fc block per 10 6 cells and purified using a MACS mouse DC isolation kit (Miltenyi Biotech). CD3 + T cells were purified from spleen cells using a MACS mouse CD3ϵ + T cell isolation kit (Miltenyi Biotech, Cat# 130-094-973). Cells were isolated quickly and maintained on ice to limit spontaneous DC and CD3ϵ + T cell activation.

Mixed Lymphocyte Reaction Assay
BMDCs were treated with LPS + DMSO, LPS + MSP + DMSO, LPS + MSP + BMS777607, or LPS + BMS777607 for 24 h before harvesting, following CD11c magnetic bead sorting, respectively. The stimulation concentrations were: LPS 100 ng/mL, MSP 100 ng/ml, BMS777607 400 nM, and DMSO added at the same dose as BMS777607 to control for any effect of DMSO in BMS777607 stimulations. BMDCs were resuspended in 25 µg/ml mitomycin (Biovision, Palo Alto, USA, Cat# 2713-2) for 20 min. The cells were washed three times with Dulbecco's phosphate-buffered saline (DPBS; Gibco, Cat# 14190144) and resuspended in complete RPMI-1640 medium containing 2 ng/ml IL-2 (R&D Systems, Cat# 402-ML-020). CD3 + T cells were derived from Balb/c mouse splenocytes, sorted with CD3 magnetic beads, resuspended in DPBS, mixed with a final concentration of 2.5 µM carboxyfluorescein succinimidyl ester (CFSE; BD Bioscience, Cat# 565082) for 15 min at 37°C in a water bath, and resuspended in complete RPMI-1640 medium containing 2 ng/ ml IL-2. A density of 3 × 10 5 CD3 + T cells/well was added to a Ushaped 96-well plate. BMDCs were added at a ratio of 1:10 or 1:50 (DC:T cells). T cells stained with CFSE were used as a negative control. After co-culturing for 48 h, the cells were collected and data acquisition was performed with a BD Canto II flow cytometer. Data were analyzed with FlowJo software, version 10.4.

Statistical Analysis
SPSS software was used to perform regular statistical analysis. A one-way analysis of variance with a Games-Howell's multiple comparison test was used for multiple comparisons. The data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

RON Expression in DCs
To assess whether RON was expressed in DCs, we examined the level of RON mRNA expression by RT-PCR, which demonstrated that RON was expressed in both untreated DCs and LPS-stimulated DCs ( Figure 1A). The transcriptional levels of RON in DCs were similar to those in peritoneal macrophages and were not affected by LPS stimulation ( Figure 1A). Western blotting results showed that RON was expressed in DCs and RON expression was not influenced by LPS, MSP, or BMS777607 treatment ( Figure 1B). Flow cytometry assay results also showed RON expression in DCs using the mouse anti-RON antibody, E-9 ( Figure 1C).

RON Inhibition Downregulates MHC II and CD86 Expression in BMDCs and Splenic DCs, Which Is Inconsistent With the Transcription Results for MHC II and CD86
We then detected changes in DC surface marker expression by flow cytometry following stimulation with LPS 100 ng/ml, MSP 100 ng/ml, and BMS777607 400 nM. The level of CD86 and MHC II expression was increased in each group stimulated with LPS compared with untreated DCs. The results of the MFI assay suggested that both CD86 (Figures 2A, B) and MHC II ( Figures  2C, D) expression decreased with RON inhibition after stimulation for 12 or 24 h. We examined changes in the level of CD86 and CIITA mRNA expression. CD86 was not significantly altered following RON inhibition ( Figure 2E). CIITA expression was increased by almost 13 times when 400 nM BMS777607 was used to inhibit RON (p < 0.05) compared with LPS-treated DCs ( Figure 2F). We found that RON inhibition leads to decreased MHC II and CD86 in LPSmediated splenic DCs after stimulation for 24 h (Figures 2G,  H). The inhibition of RON downregulated the expression of MHC II (p < 0.01) and CD86 (p < 0.001) in LPS-mediated splenic DCs, and these effects were antagonized by MSP.

The Inhibition of RON Enhances CD86 and MHC II Ubiquitination
Since CD86 and MHC II transcription and protein level data were inconsistent, we next considered whether the degradation of these two molecules had increased. MHC II and CD86 are ubiquitinated by the E3 ligases, March-I and March-8 (Oh and Shin, 2015). Thus, we sought to determine the changes in the gene expression of March-I and March-8. RON inhibition increased March-I expression following treatment with LPS (p < 0.01); this inhibitory effect was antagonized by MSP (p < 0.05) ( Figure 3A), whereas March-8 did not exhibit significant changes ( Figure 3A). In addition, this phenomenon was not observed in the non-LPS-treated group. Therefore, we performed an immunoprecipitation assay of CD86 and MHC II and examined the changes in the level of ubiquitination of these two molecules. MHCII and CD86 could be polyubiquitinated with 7 kDa ubiquitin (Lei et al., 2018), which resulted in proteins of various molecular weights appearing in the lane. Similar to the RT-PCR results, the ubiquitination of these two molecules increased with RON inhibition and MSP stimulation reduced the degree of ubiquitination induced by BMS777607 ( Figure 3B).

RON Inhibition Reduced the Release of IL-1b and IL-12p70, as Well as Increased the Release of IL-10 in LPS-Mediated DCs and an Allogeneic Mixed Lymphocyte Reaction Assay Revealed Reduced BMDC Maturity
To evaluate the function of DCs after RON inhibition, we next examined the level of cytokine release from DCs following LPS stimulation. RON inhibition significantly reduced the level of IL- 1b (p < 0.05) and IL-12p70 (p < 0.05) and increased the level of IL-10 (p < 0.05) in the cell culture supernatants of LPS-treated BMDCs after 24 h ( Figure 4A). Treatment with MSP antagonized the reduction of IL-12p70 release (p < 0.01) after RON inhibition in DCs ( Figure 4A). Finally, we performed co-culture experiments with BMDCs and splenic T cells isolated from Balb/c mice. The results showed that the ability of BMDCs to stimulate allogeneic T lymphocytes was significantly reduced following RON inhibition ( Figure 4B), and MSP was used to antagonize this process. Similar results were obtained when we used a DC:T cell ratio of 1:10 or 1:50 for co-cultivation and the experiment was repeated twice, independently.

DISCUSSION
DCs are the only immune cell that can activate naive T cells and they play an important role in various tissues and organs. Untreated DCs express low levels of CD86 and MHC II but are able to capture antigens via endocytosis and the Fc receptor (Pearce and Everts, 2015). After processing, the antigen fragment is loaded into the MHC II molecule and expressed on the cell surface. At the same time, when receiving pathogen-associated molecular pattern stimulation, DCs show increased surface expression of MHC II and CD86, providing the first and costimulatory signals required for T cell activation, resulting in the activation of naive T cells. LPS-stimulated DCs also secrete IL-12p70, Il-1b, and other cytokines to assist in the activation of other immune cells (Liu and Cao, 2016). RON is expressed on peritoneal macrophages (Yao et al., 2013b), osteoclasts (Andrade et al., 2017), and Kupffer cells (Stuart et al., 2011). Here, we determined that RON is expressed on both DCs and LPS-stimulated DCs, and was not influenced by LPS (Figures 1A, B). Interestingly, when the MHC II and CD86 phenotypes were detected by flow cytometry, we found that RON-inhibited DCs exhibited lower levels of MHC II and CD86 when treated with LPS in both BMDCs and splenic DCs (Figures 2A-D, G, H). These flow cytometry results did not match the above CIITA and CD86 transcription results ( Figures  2E, F). Based on this contradiction, we suspected that RON inhibition can lead to the ubiquitination and degradation of both MHC II and CD86 by the E3 ligases, March-I and/or March-8. Our RT-PCR results showed that the inhibition of RON resulted in increased transcription of the E3 ligase March-I, whereas March-8 transcription remained unchanged ( Figure 3A). Subsequent Western blotting results confirmed that there was an increase in the degree of MHC II and CD86 ubiquitination when RON was inhibited in DCs ( Figure 3B). These findings provide evidence that although CIITA transcription increases with RON inhibition, the ubiquitination of MHC II and CD86 increases, leading to the degradation of these two molecules and ultimately decreased levels of MHC II and CD86 ( Figure 4C).
Mitogen-activated protein kinase (MAPK) inhibition leads to increased CIITA transcription (Yao et al., 2006) and RON inhibition may lead to a decrease in extracellular signal-related kinase (Erk)1/2 phosphorylation and an increase in CIITA transcription, as described previously (Wilson et al., 2008). Interestingly, when the MHC II and CD86 phenotypes were detected by flow cytometry, we found that RON-inhibited DCs A B C FIGURE 4 | (A) IL-1b, IL-12p70, and IL-10 production by LPS-treated BMDCs in the presence of a RON inhibitor leads to downregulation of the ability of BMDCs stimulated with LPS to proliferate T cells. BMDCs were cultured in serum-free RPMI-1640 medium for 2 h and then stimulated with LPS, LPS + MSP, LPS + MSP + BMS777607, LPS + BMS777607, BMS777607 or unstimulated respectively. The supernatant was collected at 24 h and the assay was performed as described in the Methods. The data represent the mean ± SEM of three independent experiments. NS, no significant difference; *p < 0.05; **p < 0.01. (B) The allogeneic MLR protocol was performed as described in the Methods section. The left peak indicates a T cell after proliferation and division. The negative control was CFSE-stained T cells without co-culture with DCs. The experiment was repeated twice independently. (C) Scheme showing that the use of RON inhibitors leads to an increased March-I transcription and thus an increased CD86 and MHC II ubiquitination when LPS stimulated DCs. These changes led to a decrease of IL-1b and IL-12p70 release and increased IL-10 release. Moreover, the ability of LPS-stimulated DCs to stimulate T cell proliferation was decreased when the inhibition of RON. exhibited lower levels of MHC II and CD86 than the LPSstimulated DCs (Figures 2A-H). These flow cytometry results did not match the above CIITA and CD86 transcription results ( Figures 2E-F). We suspected that RON inhibition leads to the ubiquitination and degradation of both MHC II and CD86 by March-I and/or March-8, which would suggest that E3 ligases can ubiquitinate MHC II and CD86 in DCs. Our RT-PCR results show that the inhibition of RON resulted in increased transcription of the E3 ligase March-I, whereas March-8 transcription remained unchanged ( Figure 3A). Western blotting results confirmed that there was an increase in the degree of MHC II and CD86 ubiquitination on inhibiting RON in DCs ( Figure 3B). To our surprise, we only observed CD86 ubiquitination but not MHC II ubiquitination in the LPS + MSP + BMS777607 group ( Figure 3B), in which the transcription of March-I did not increase significantly ( Figure 3A).
Therefore, these findings suggest that RON maintains LPSstimulated DC activation by inhibiting March-I expression in LPS-stimulated DCs. Since the release of IL-1b and IL-12p70 is a marker of DC maturation, we measured the concentrations of IL-1b and IL-12p70 released by LPS-stimulated DCs. The ability of RON-inhibited LPS-stimulated DCs to release IL-1b and IL-12p70 was significantly reduced compared with normal LPSstimulated DCs ( Figure 4A). In addition, we evaluated IL-10 production, and the results showed increased release of IL-10 in LPS-stimulated DCs when RON was inhibited ( Figure 4A). At the same time, we performed an allogeneic mixed lymphocyte reaction experiment, and the results showed that RON-inhibited DC stimulation of T cell proliferation was significantly decreased ( Figure 4B).
Our findings may provide novel insight into the role of RON receptors in both innate and acquired immunity. The RON receptor may protect the process of DC differentiation when receiving external stimuli. As a tyrosine kinase receptor, RON has an important protective effect on HK2 cells from apoptosis caused by H 2 O 2 (Lee et al., 2013a;Lee et al., 2013b), and protected peritoneal macrophages from LPS-induced apoptosis (Chen et al., 2002). RON inhibition enhances LPS-induced apoptosis in DCs, which may contribute to the increased March-I transcription. However, we have been unable to clarify how RON promotes March-I transcription. This research has been impeded by the lack of a RON knockout mouse and we could only use BMS777607 to inhibit RON activation, which may have an impact on the reliability of the experimental results. Thus, further evidence is required.
In the present study, we provide evidence that RON is expressed in DCs. Our findings show that a blockade in RON signaling can lead to enhanced March-I transcription. This results in increased MHC II and CD86 ubiquitination and decreased DC maturity.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article and/or the supplementary material.

ETHICS STATEMENT
The experimental procedures regarding the use and care of animals were approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University.