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RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages
Molecular Immunology 87 (2017) 200–206
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages Shuyuan Liu1, Lisong Huang1, Zhusen Lin, Yuanqin Hu, Ruifeng Chen, Liqiu Wang, Yi Shan
MARK
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Emergency Department of Navy General Hospital, Beijing, 100037, China
A R T I C L E I N F O
A B S T R A C T
Keywords: RhoB MHC class II molecule NF-κB Macrophage Toll-like receptors
Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligandtriggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not β chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon β activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.
1. Introduction Macrophages are the first line of defense against invading microbial pathogens. In response to infection, activated macrophages produce cytokines to initiate the inflammatory response. This process depends largely on activation of the nuclear factor-κB (NF-κB) family of transcription factors. Macrophages express numerous pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) expressed on bacteria, viruses, and parasites. Recognition of PAMPs is mediated at the cell surface or intracellularly by a variety of PRRs, including transmembrane Toll-like receptors (TLRs) and cytoplasmic nucleotide oligomerization domain (NOD)-like receptors (Hayden and Ghosh, 2011). TLR activation is essential for initiating the innate immune response and enhancing adaptive immunity against invading pathogens (Aderem and Ulevitch, 2000; Medzhitov and Janeway, 2000). Less efficient activation of the TLR response may not evoke potent anti-infection immunity; however, excessive activation of TLR may induce immunopathological processes, such as endotoxin shock (Oda et al., 2014; Zhang et al., 2014) and autoimmune diseases (Hamerman et al., 2016; Wu et al., 2015). How to
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Corresponding author. E-mail address: [email protected] (Y. Shan). These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.molimm.2017.04.015 Received 26 January 2017; Received in revised form 28 March 2017; Accepted 23 April 2017 0161-5890/ © 2017 Elsevier Ltd. All rights reserved.
manipulate or control the TLR response for the prevention and treatment of inflammatory and immunological diseases largely depends on understanding the molecular basis for TLR responses. Currently, the molecular mechanisms for the initiation and regulation of TLR responses remain to be fully elucidated. The Rho GTPases are a family of small signaling G proteins, which are a subfamily of the Ras superfamily (Fritz and Kaina, 2006). The members of the Rho GTPase family (including RhoA, RhoB and RhoC) play important roles in organelle development, cytoskeletal dynamics, and cell movement (Ridley, 2001). Apart from roles it has in common with RhoA and RhoC, RhoB is also implicated in a variety of physiological and pathological processes (Vega and Ridley, 2016), including inflammatory responses, particularly in macrophages and endothelial cells (Wojciak-Stothard et al., 2012). It is reported that RhoB is involved in mannose receptor-mediated phagocytosis in human alveolar macrophages (Zhang et al., 2005), and regulates the secretion of tumor necrosis factor α (TNFα) and nitric oxide by macrophages in a model of lipopolysaccharide (LPS)-induced inflammation in mice, possibly through a pathway involving NF-κB (Wang et al., 2013). Furthermore, RhoB depletion in mouse macrophages impairs produc-
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Fig. 1. LPS increases RhoB expression in RAW264.7 cells. The mRNA (A) and protein (B) levels of RhoB in RAW264.7 cells stimulated with LPS for the indicated times. (C) The mRNA levels of RhoB in RAW264.7 cells stimulated with CpG or poly(I:C) for 2 h. Data are the mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01 compared with control cells.
Recombinant vector encoding RhoB was constructed by PCR-based amplification of cDNA from mouse peritoneal macrophages, and then subcloned into the pcDNA3.1-HA eukaryotic expression vector. Antibody to RhoB was from Santa Cruz. Antibodies to IRF3 phosphorylated at Ser396, IRF3, c-Jun N-terminal kinase (JNK) phosphorylated at Thr183 and Tyr185, JNK, phosphorylated Erk, Erk, IκBα phosphorylated at Ser32-36, IκBα, HA-Tag, Myc-Tag, Flag-Tag, anti-GAPDH and anti-β-actin were used as previously described (Chen et al., 2013).
tion of inflammatory cytokines both in normoxic and hypoxic conditions (Huang et al., 2017). Although several studies have investigated the effect of RhoB on the innate immune response, the molecular mechanism remains unclear. RhoB localizes to major histocompatibility complex class II (MHCII) molecules in dendritic cells (DCs) and regulates endosome transport by promoting actin assembly on endosomal membranes (Fernandez-Borja et al., 2005; Ocana-Morgner et al., 2009). Intracellular MHCII can act as an adaptor molecule, interacting with the tyrosine kinase Btk via the costimulatory molecule CD40, and promoting TLR-mediated innate immune responses (Liu et al., 2011). Therefore, we sought to determine whether RhoB is associated with promoting TLR signaling via interaction with MHCII. Here, we demonstrate that RhoB is a positive regulator of TLR-mediated signaling that allows efficient proinflammatory cytokine production in response to several inducers of macrophage activation. We further demonstrate the regulatory mechanism by which RhoB interacts with MHCII in endosomes and then interacts with Btk, promoting TLR-mediated innate immune responses.
2.3. Real-time quantitative RT PCR Total RNA was extracted with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized with qScript cDNA Synthesis kit (Quanta Biosciences). SYBRGreen Detection system (Bio-Rad Laboratories) was used for qPCR as previously described (Lee et al., 2008, 2009). Data were normalized to the expression of β-actin in each sample. 2.4. Luciferase reporter assay
2. Material and methods For luciferase assays, HEK293T cells were co-transfected with luciferase reporter plasmid, Renilla (RL) luciferase reporter vector and RhoB expressing plasmid. 24 h after transfection, cells were treated with LPS for 4 h, and then luciferase activity was measured using the Dual-luciferase reporter assay kit (Promega) according to the manufacturer’s instructions in a Lumat LB9507 luminometer (Berthold, Germany).
2.1. Cell culture and reagents HEK293T and RAW264.7 cells were maintained in Dulbecco's modified Eagle medium (DMEM; high glucose) supplemented with 10% fetal bovine serum (gibco, life technology). Thioglycollate-elicited mouse peritoneal macrophages were obtained and cultured in endotoxin-free RPMI-1640 medium with 10% FCS (Invitrogen). siRNA targeting RhoB (Santa Cruz Biotechnology) was used at a final concentration of 20 nM. Cells were transfected with siRNA using RNAiMAX reagent (Invitrogen) according to the manufacturer’s instruction. C57BL/6 mice (5–6 weeks old) were obtained from the Institute of Zoological Sciences, Chinese Academy of Medical Sciences (Beijing). LPS derived from Escherichia coli 0111:B4 was obtained from Sigma (St Louis, MO). CpG ODN and poly (I:C) were purchased from InvivoGen (San Diego, CA). The pRL-TK-Renilla-luciferase plasmid as obtained from Promega (Madison, WI).
2.5. Immunofluorescence staining and confocal microscopy For the colocalization analysis, cells were visualized after sequential immunostaining with specific antibody. The immunostaining process was performed as described. Briefly, cells were washed with phosphatebuffered saline, fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in phosphatebuffered saline, and then blocked in phosphatebuffered saline containing 5% bovine serum albumin and 5% fetal calf serum. Then samples were incubated with appropriate secondary antibodies. Slides were finally examined under a Zeiss LSM 510 confocal microscope (Carl Zeiss, Heidelberg, Germany). The co-localization coefficient was calculated as described previously (Ozgen et al., 2014). This analysis method gave rise to two
2.2. Plasmids and antibodies The Myc-tagged MHCII-α and V5-tagged MHCII-β expression plasmids were constructed as described previously (Chen et al., 2013). 201
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Fig. 2. Silencing of RhoB expression inhibits production of proinflammatory mediators by macrophages upon LPS stimulation. The effect of si-RhoB on RhoB protein in mouse peritoneal macrophages (A). The TLR4 protein level was analyzed by western blot (B) and cell viability was analyzed by CCK-8 assay (C) in RhoB silenced peritoneal macrophages after LPS treatment. The production of proinflammatory mediators, including IL-6, TNFα and IL-1β, was measured either by q-PCR or by ELISA in RhoB silenced peritoneal macrophages after LPS treatment (3 h and 6 h) (D-F), CpG (6 h) or poly(IC) (6 h) (G). Data are the mean ± SD of three independent experiments.
lysis buffer and boiled in 20 μl of 2 × Laemmli’s buffer. Samples were subjected to 8–12% SDS-PAGE analysis and electro-transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with the indicated primary antibodies, followed by secondary antibodies. Membranes were then washed and visualized with an enhanced chemiluminescence detection system (ECL; GE Healthcare). The relative protein levels were determined using the ImageJ program.
correlation coefficients: the green pixels overlapping with the red channel (M1) or vice versa (M2). In order to calculate the percentage of colocalization of two protein, we used M1 when calculates overlapping red pixels (MHCII) with green pixels (Lamp1), and used M2 when calculates overlapping green pixels (MHCII or RhoB) with red pixels (RhoB or Btk). 100% co-localization gives a value of 1. 2.6. Immunoblotting, and coimmunoprecipitation assay
2.7. ELISA assay
Cells were lysed in lysis buffer and freshly added protease inhibitor cocktail. The cell lysates were mixed with antibodies for 2 h, followed by the addition protein G-Sepharose beads (GE Healthcare) for an additional 2 h at 4 °C. Immunoprecipitates were washed four times with
Cytokines in supernatants were analyzed using ELISA kits for mouse TNF-α, IL-1β and interleukin (IL)-6 (R & D Systems). 202
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Fig. 3. Silencing RhoB expression inhibits TLR ligand-triggered downstream signaling pathways in macrophages. The NF-κB, MAPK and Btk pathways were analyzed by western blot in mouse peritoneal macrophages transfected with si-RhoB or si-ctrl upon LPS treatment (A). NF-κB luciferase activity was measured using the Dual-Luciferase Reporter Assay System in RAW264.7 cells transfected with si-RhoB or si-ctrl with or without LPS treatments (B). Colocalization of MHCII (red) with Lamp1 (green) in RAW264.7 cells with si-RhoB or si-ctrl Nuclei were stained with DAPI (blue). The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient Calculator and displayed as a bar graph. Bars represent the mean + SEM of at least 15 cells (C). Data are the mean ± SD of three independent experiments. ** P < 0.01 compared with cells transfected with si-ctrl but without LPS stimulation; # P < 0.05 compared with cells transfected with si-ctrl upon LPS stimulation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
3.2. RhoB increases the production of proinflammatory mediators in macrophages upon LPS stimulation
2.8. Statistical analysis All the experiments were performed at least three independent experiments. Data are presented as mean ± SD. Significance was determined with Student’s t-test, and P < 0.05 was considered statistically significant
To test whether RhoB was associated with the regulation of innate immune responses, we efficiently silenced RhoB expression in murine peritoneal macrophages by transfection with short interfering RNA (siRNA) duplexes against the mouse RhoB gene (si-RhoB) (Fig. 2A). RhoB silencing did not affect TLR4 expression (Fig. 2B) or cell viability (Fig. 2C). Notably, knockdown of RhoB significantly decreased the production of proinflammatory cytokines, including IL-6, TNFα and IL1β, in response to simulation with LPS at 3 and 6 h, both at the mRNA level and secreted into the cell culture supernatant (Fig. 2D-F). In addition, RhoB silencing also significantly decreased the expression of IL-6 and TNFα in macrophages upon stimulation of poly (I:C) and CpG (Fig. 2G). These data indicate that RhoB increases the production of proinflammatory cytokines in TLR-triggered macrophages.
3. Results 3.1. TLR agonists induce RhoB expression in macrophages To investigate the potential role of RhoB in TLR-mediated innate immunity, we first detected the expression of RhoB in LPS-stimulated macrophages. As shown in Fig. 1A, RhoB mRNA levels gradually increased, reaching a maximum at 1 h of approximately 3.5-fold compared with the control group, then decreased to baseline within 24 h after LPS stimulation. Furthermore, RhoB protein levels increased, mirroring the mRNA levels, after LPS treatment (Fig. 1B). Similar increases in RhoB mRNA were also obtained by stimulation of macrophages with CpG ODN or poly(I:C) (Fig. 1C). These results suggest that RhoB, which is induced by TLR agonists, may play an important role in TLR-mediated innate immunity.
3.3. RhoB induces LPS-initiated signaling pathways in macrophages We further investigated the effect of RhoB on TLR-mediated signaling pathways in macrophages. We observed that knockdown of RhoB markedly decreased the phosphorylation of IκBα (p-IκBα), JNK, Erk and p38 in peritoneal macrophages upon LPS stimulation (Fig. 3A). 203
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Fig. 4. RhoB interacts with intracellular MHCII and Btk. Immunoblot analysis of the interactions of RhoB with MHCII α or β chains in RAW264.7 cells cotransfected with vectors encoding HA-RhoB, Myc-MHCIIα or V5-MHCIIβ chains with LPS treatment (A). Immunoblot analysis of the interaction of Btk, MHCII and RhoB in peritoneal macrophages stimulated with LPS (B). Colocalization of RhoB (red) with MHCII (green) or RhoB (green) with MHCII (red) in RAW264.7 cells with or without LPS treatment. Nuclei were stained with DAPI (blue).The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient Calculator and displayed as a bar graph. Bars represent the mean + SEM of at least 15 cells (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
MHCII in DCs (Kamon et al., 2006; Ocana-Morgner et al., 2009). Moreover, MHCII can promote TLR-mediated innate immune responses by maintaining the activation of Btk (Liu et al., 2011). To determine whether RhoB promotes TLR ligand-triggered innate immune responses via interaction with MHCII, we transfected RAW264.7 cells with expression vectors for HA-RhoB and Myc-tagged MHCII α and β chains, followed by coimmunoprecipitation analysis. We found that RhoB interacted with the MHCII α chain, but not the β chain (Fig. 4A). Furthermore, endogenous RhoB also interacted with MHCII and the downstream Btk protein in macrophages (Fig. 4B). Immunofluorescence imaging demonstrated the colocalization of RhoB and MHCII or Btk, with or without LPS stimulation, in the cytoplasm of mouse peritoneal
Of note, knockdown of RhoB also significantly decreased the level of pBtk (Y222), which is necessary for full activation (Liu et al., 2011) (Fig. 3A). In addition, knockdown of RhoB also decreased NF-κB activity in HEK293T cells stimulated with LPS (Fig. 3B). Of note, RhoB silencing did not affect the colocalization of MHCII with lysosome associated membrane protein 1 (Lamp1, an abundant membrane protein of late endosomes) (Fig. 3C). Collectively, these data suggest that RhoB promotes TLR-triggered signaling pathways in macrophages.
3.4. RhoB interacts with MHCII in endosomes It has been reported that RhoB colocalizes intracellularly with 204
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pathways, upon LPS stimulation. The present study demonstrates that RhoB serves as a potent positive regulator of TLRs signaling and may provide a novel perspective for the design of preventive or therapeutic approaches to inflammatory autoimmune diseases. RhoB is normally expressed at low steady-state levels in cells, but can be rapidly and transiently upregulated in macrophages by several stimuli, including UV irradiation, growth factors, cytokines and LPS (Fritz et al., 1995; Jahner and Hunter, 1991; Kamon et al., 2006; OcanaMorgner et al., 2009). In the present study, we found that TLR agonists (LPS, CpG, poly(I:C)) can induce RhoB expression in macrophages, indicating that RhoB levels are regulated in response to TLR activation, and thus RhoB may be involved in the regulation of TLR-mediated signaling. Several studies have implicated RhoB in inflammatory responses, particularly in macrophages and endothelial cells, however, the detailed mechanisms remain uncharacterized. It was recently shown that a TRIF-mediated pathway activates RhoB, which is colocalized with MHCII in intracellular membrane vesicles and necessary for the alteration of cytoskeletal components to traffic intracellular MHCII-containing vesicles toward the surface of DCs after LPS stimulation (Kamon et al., 2006). MHCII is expressed by professional antigen presenting cells, including DCs, macrophages and B cells and has a crucial role in the development and function of the immune system (Miyake et al., 2017; Roche and Furuta, 2015). The classical function of MHCII is to present peptides processed from extracellular proteins to CD4+ helper T cells and to direct the processes of positive and negative selection, shaping the T cell repertoire during maturation and lineage commitment (Mucida et al., 2013; Weigand et al., 2012). In addition to the classical function of MHCII molecules of presenting antigen to CD4+ T cells, intracellular MHCII molecules can act as adaptors, interacting with the tyrosine kinase Btk via the costimulatory molecule CD40 to maintain Btk activation, and thereby promoting TLR signaling (Liu et al., 2011). Based on the major findings, we sought to determine whether RhoB plays an important role in regulating the function of endosomal MHCII in TLR ligand-triggered innate immune responses. We demonstrated that RhoB is a positive regulator of TLR-mediated signaling that allows efficient proinflammatory cytokine production in response to several macrophage activators. During LPS stimulation, RhoB is required for full initiation of several intracellular signaling pathways including the Btk, NF-κB pathway and three mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38. These signaling pathways induce the expression of many genes encoding proinflammatory mediators, such as IL-6, TNFα and IL-1β. Thus, our findings suggest that RhoB is a regulator of inflammatory signaling pathways in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. MHCII, which is a heterodimeric cell surface protein composed of an α chain and a β chain, is a type I integral membrane protein with short cytoplasmic domains and four large extracellular domains (Liu et al., 2011; Schafer et al., 1995). Various transgenic MHCII knockout mice have been generated including MHCIIAα−/− (I-Aα−/−), MHCIIAβ−/− (I-Aβ−/−) and the double knockout (I-Aαβ−/−) (Hou et al., 1995; Kontgen et al., 1993; Madsen et al., 1999). It follows that disruption of either α or β gene locus prevents the cell surface expression of MHCII molecules. Alsharifi et al. observed that the avirulent strain of Semliki Forest virus (aSFV) caused a very acute and lethal infection in I-Aα−/−, but not in I-Aβ−/− or I-Aαβ−/−, mice. I-Aα−/− mice exhibit a striking susceptibility to viral infections (Alsharifi et al., 2013). However, the detailed mechanism remains unclear. In the present study, we found that the MHCII α chain, but not β chain, associated with RhoB. Confocal microscopy also demonstrated that intracellular MHCII colocalized with RhoB and Btk in the endosomes of macrophages stimulated with LPS. Knockdown of MHCII expression significantly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and IFN-β activity by RhoB upon LPS treatment. These results indicated that RhoB activates TLR ligand-triggered innate immune responses through
Fig. 5. RhoB induces TLR-mediated signaling via interaction with MHCII. Immunoblot analysis of the interactions of RhoB with MHCII and Btk in LPS-treated RAW264.7 cells cotransfected with si-MHCII and HA-RhoB vectors (A). NF-κB and IFN-β luciferase activity were measured using the Dual-Luciferase Reporter Assay System in RAW264.7 cells cotransfected with si-RhoB or si-ctrl and the HA-RhoB vector, with or without LPS treatment (B). ** P < 0.01 compared with cells transfected with si-ctrl but without HARhoB vector; # P < 0.05 compared with cells cotransfected with si-ctrl and HA-RhoB vector.
macrophages (Fig. 4C). These results suggest that RhoB interacts with intracellular MHCII molecules in macrophages. 3.5. RhoB induces TLR-mediated signaling via interaction with MHCII To test whether the interaction of RhoB with MHCII is involved in the regulation of TLR ligand-triggered innate immune responses, we designed siRNA targeting MHCII (si-MHCII) and confirmed that siMHCII significantly decreased the protein level of MHCII (Fig. 5A). Knockdown of MHCII expression significantly reduced the interaction of RhoB with Btk in RAW264.7 cells (Fig. 5A), suggesting that the interaction of RhoB with Btk is dependent on MHCII. In addition, knockdown of MHCII expression in RAW264.7 cells attenuated the induction effect of RhoB on NF-κB and interferon (IFN)-β activity upon LPS stimulation (Fig. 5B). In contrast, overexpression of RhoB significantly increased both activities upon LPS but not without LPS stimulation. However, overexpression of RhoB by pretreatment with si-MHCII for 24 h followed by LPS treatment did not increase the activity of NFκB or IFN-β. And these treatments did not affect the viability or TLR4 expression in cells (Fig. 5B). These results indicate that RhoB activates TLR-triggered innate immune response through interaction with MHCII. 4. Discussion We are interested in whether the interaction of RhoB with endosomal MHCII is involved in TLR-mediated innate immune responses. Here, we demonstrated that RhoB, whose expression is induced by TLR agonists (LPS, CpG, poly(I:C)), can promote TLR-initiated signaling pathways and the production of proinflammatory cytokines by macrophages. Specifically, RhoB interacts with the MHCII α chain in endosomes and then with the downstream protein Btk, thereby inducing intracellular signaling pathways, including the NF-κB and MAPK 205
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binding to MHCII. However, the detailed molecular mechanism remained unclearly. RhoB as a Rho family members controls tubulin and F-actin cytoskeletal rearrangements. However, previous studies demonstrated that in unstimulated DCs, efficient surface localization of MHCII is largely independent of RhoB activity (Ocana-Morgner et al., 2009; Kamon et al., 2006). Upon LPS activation, expression and activation of RhoB in DCs and in B cells may account for the cytoskeletal actin rearrangement necessary for the surface expression of MHCII (Kamon et al., 2006), but they did not demonstrated whether RohB affected MHCII expression at endosomal membranes. Here, we did not find that knockdown of RhoB affected the localization of MHCII molecules on the endosome of macrophages upon LPS treatment. Thus, the mechanism by which the interaction of RhoB with intracellular MHCII induces the activation of Btk may not rely on Rho GTPase activity. The discovery of which region of MHC class II interacts with CD40 and Btk in the endosomes of TLR-activated macrophages, and why Btk can be activated after stimulation of human B lymphocytes with CD40L, could give new insights into thel underlying mechanism of RhoB in promoting MHC activation. In conclusion, our study demonstrated that RhoB serves as a potent positive regulator of TLR signaling pathways by interacting with MHCII in endosomes of macrophages and enhancing the production of proinflammatory cytokines. Therefore, RhoB is involved in the activation of TLR-mediated signaling, and may be a therapeutic target for the treatment of inflammatory and autoimmune diseases. References Aderem, A., Ulevitch, R.J., 2000. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787. Alsharifi, M., Koskinen, A., Wijesundara, D.K., Bettadapura, J., Mullbacher, A., 2013. MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies. PLoS One 8, e68458. Chen, W., Han, C., Xie, B., Hu, X., Yu, Q., Shi, L., Wang, Q., Li, D., Wang, J., Zheng, P., et al., 2013. Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation. Cell 152, 467–478. Fernandez-Borja, M., Janssen, L., Verwoerd, D., Hordijk, P., Neefjes, J., 2005. RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1. J. Cell Sci. 118, 2661–2670. Fritz, G., Kaina, B., 2006. Rho GTPases: promising cellular targets for novel anticancer drugs. Curr. Cancer Drug Targets 6, 1–14. Fritz, G., Kaina, B., Aktories, K., 1995. The ras-related small GTP-binding protein RhoB is immediate-early inducible by DNA damaging treatments. J. Biol. Chem. 270, 25172–25177. Hamerman, J.A., Pottle, J., Ni, M., He, Y., Zhang Bucknere, J.H., 2016. Negative regulation of TLR signaling in myeloid cells–implications for autoimmune diseases. Immunol. Rev. 269, 212–227. Hayden, M.S., Ghosh, S., 2011. NF-kappaB in immunobiology. Cell Res. 21, 223–244. Hou, S., Mo, X.Y., Hyland, L., Doherty, P.C., 1995. Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins. J. Virol. 69, 1429–1434. Huang, G., Su, J., Zhang, M., Jin, Y., Wang, Y., Zhou, P., Lu, J., 2017. RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response. Cell Mol.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages Shuyuan Liu1, Lisong Huang1, Zhusen Lin, Yuanqin Hu, Ruifeng Chen, Liqiu Wang, Yi Shan
MARK
⁎
Emergency Department of Navy General Hospital, Beijing, 100037, China
A R T I C L E I N F O
A B S T R A C T
Keywords: RhoB MHC class II molecule NF-κB Macrophage Toll-like receptors
Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligandtriggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not β chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon β activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.
1. Introduction Macrophages are the first line of defense against invading microbial pathogens. In response to infection, activated macrophages produce cytokines to initiate the inflammatory response. This process depends largely on activation of the nuclear factor-κB (NF-κB) family of transcription factors. Macrophages express numerous pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) expressed on bacteria, viruses, and parasites. Recognition of PAMPs is mediated at the cell surface or intracellularly by a variety of PRRs, including transmembrane Toll-like receptors (TLRs) and cytoplasmic nucleotide oligomerization domain (NOD)-like receptors (Hayden and Ghosh, 2011). TLR activation is essential for initiating the innate immune response and enhancing adaptive immunity against invading pathogens (Aderem and Ulevitch, 2000; Medzhitov and Janeway, 2000). Less efficient activation of the TLR response may not evoke potent anti-infection immunity; however, excessive activation of TLR may induce immunopathological processes, such as endotoxin shock (Oda et al., 2014; Zhang et al., 2014) and autoimmune diseases (Hamerman et al., 2016; Wu et al., 2015). How to
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Corresponding author. E-mail address: [email protected] (Y. Shan). These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.molimm.2017.04.015 Received 26 January 2017; Received in revised form 28 March 2017; Accepted 23 April 2017 0161-5890/ © 2017 Elsevier Ltd. All rights reserved.
manipulate or control the TLR response for the prevention and treatment of inflammatory and immunological diseases largely depends on understanding the molecular basis for TLR responses. Currently, the molecular mechanisms for the initiation and regulation of TLR responses remain to be fully elucidated. The Rho GTPases are a family of small signaling G proteins, which are a subfamily of the Ras superfamily (Fritz and Kaina, 2006). The members of the Rho GTPase family (including RhoA, RhoB and RhoC) play important roles in organelle development, cytoskeletal dynamics, and cell movement (Ridley, 2001). Apart from roles it has in common with RhoA and RhoC, RhoB is also implicated in a variety of physiological and pathological processes (Vega and Ridley, 2016), including inflammatory responses, particularly in macrophages and endothelial cells (Wojciak-Stothard et al., 2012). It is reported that RhoB is involved in mannose receptor-mediated phagocytosis in human alveolar macrophages (Zhang et al., 2005), and regulates the secretion of tumor necrosis factor α (TNFα) and nitric oxide by macrophages in a model of lipopolysaccharide (LPS)-induced inflammation in mice, possibly through a pathway involving NF-κB (Wang et al., 2013). Furthermore, RhoB depletion in mouse macrophages impairs produc-
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Fig. 1. LPS increases RhoB expression in RAW264.7 cells. The mRNA (A) and protein (B) levels of RhoB in RAW264.7 cells stimulated with LPS for the indicated times. (C) The mRNA levels of RhoB in RAW264.7 cells stimulated with CpG or poly(I:C) for 2 h. Data are the mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01 compared with control cells.
Recombinant vector encoding RhoB was constructed by PCR-based amplification of cDNA from mouse peritoneal macrophages, and then subcloned into the pcDNA3.1-HA eukaryotic expression vector. Antibody to RhoB was from Santa Cruz. Antibodies to IRF3 phosphorylated at Ser396, IRF3, c-Jun N-terminal kinase (JNK) phosphorylated at Thr183 and Tyr185, JNK, phosphorylated Erk, Erk, IκBα phosphorylated at Ser32-36, IκBα, HA-Tag, Myc-Tag, Flag-Tag, anti-GAPDH and anti-β-actin were used as previously described (Chen et al., 2013).
tion of inflammatory cytokines both in normoxic and hypoxic conditions (Huang et al., 2017). Although several studies have investigated the effect of RhoB on the innate immune response, the molecular mechanism remains unclear. RhoB localizes to major histocompatibility complex class II (MHCII) molecules in dendritic cells (DCs) and regulates endosome transport by promoting actin assembly on endosomal membranes (Fernandez-Borja et al., 2005; Ocana-Morgner et al., 2009). Intracellular MHCII can act as an adaptor molecule, interacting with the tyrosine kinase Btk via the costimulatory molecule CD40, and promoting TLR-mediated innate immune responses (Liu et al., 2011). Therefore, we sought to determine whether RhoB is associated with promoting TLR signaling via interaction with MHCII. Here, we demonstrate that RhoB is a positive regulator of TLR-mediated signaling that allows efficient proinflammatory cytokine production in response to several inducers of macrophage activation. We further demonstrate the regulatory mechanism by which RhoB interacts with MHCII in endosomes and then interacts with Btk, promoting TLR-mediated innate immune responses.
2.3. Real-time quantitative RT PCR Total RNA was extracted with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized with qScript cDNA Synthesis kit (Quanta Biosciences). SYBRGreen Detection system (Bio-Rad Laboratories) was used for qPCR as previously described (Lee et al., 2008, 2009). Data were normalized to the expression of β-actin in each sample. 2.4. Luciferase reporter assay
2. Material and methods For luciferase assays, HEK293T cells were co-transfected with luciferase reporter plasmid, Renilla (RL) luciferase reporter vector and RhoB expressing plasmid. 24 h after transfection, cells were treated with LPS for 4 h, and then luciferase activity was measured using the Dual-luciferase reporter assay kit (Promega) according to the manufacturer’s instructions in a Lumat LB9507 luminometer (Berthold, Germany).
2.1. Cell culture and reagents HEK293T and RAW264.7 cells were maintained in Dulbecco's modified Eagle medium (DMEM; high glucose) supplemented with 10% fetal bovine serum (gibco, life technology). Thioglycollate-elicited mouse peritoneal macrophages were obtained and cultured in endotoxin-free RPMI-1640 medium with 10% FCS (Invitrogen). siRNA targeting RhoB (Santa Cruz Biotechnology) was used at a final concentration of 20 nM. Cells were transfected with siRNA using RNAiMAX reagent (Invitrogen) according to the manufacturer’s instruction. C57BL/6 mice (5–6 weeks old) were obtained from the Institute of Zoological Sciences, Chinese Academy of Medical Sciences (Beijing). LPS derived from Escherichia coli 0111:B4 was obtained from Sigma (St Louis, MO). CpG ODN and poly (I:C) were purchased from InvivoGen (San Diego, CA). The pRL-TK-Renilla-luciferase plasmid as obtained from Promega (Madison, WI).
2.5. Immunofluorescence staining and confocal microscopy For the colocalization analysis, cells were visualized after sequential immunostaining with specific antibody. The immunostaining process was performed as described. Briefly, cells were washed with phosphatebuffered saline, fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in phosphatebuffered saline, and then blocked in phosphatebuffered saline containing 5% bovine serum albumin and 5% fetal calf serum. Then samples were incubated with appropriate secondary antibodies. Slides were finally examined under a Zeiss LSM 510 confocal microscope (Carl Zeiss, Heidelberg, Germany). The co-localization coefficient was calculated as described previously (Ozgen et al., 2014). This analysis method gave rise to two
2.2. Plasmids and antibodies The Myc-tagged MHCII-α and V5-tagged MHCII-β expression plasmids were constructed as described previously (Chen et al., 2013). 201
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Fig. 2. Silencing of RhoB expression inhibits production of proinflammatory mediators by macrophages upon LPS stimulation. The effect of si-RhoB on RhoB protein in mouse peritoneal macrophages (A). The TLR4 protein level was analyzed by western blot (B) and cell viability was analyzed by CCK-8 assay (C) in RhoB silenced peritoneal macrophages after LPS treatment. The production of proinflammatory mediators, including IL-6, TNFα and IL-1β, was measured either by q-PCR or by ELISA in RhoB silenced peritoneal macrophages after LPS treatment (3 h and 6 h) (D-F), CpG (6 h) or poly(IC) (6 h) (G). Data are the mean ± SD of three independent experiments.
lysis buffer and boiled in 20 μl of 2 × Laemmli’s buffer. Samples were subjected to 8–12% SDS-PAGE analysis and electro-transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with the indicated primary antibodies, followed by secondary antibodies. Membranes were then washed and visualized with an enhanced chemiluminescence detection system (ECL; GE Healthcare). The relative protein levels were determined using the ImageJ program.
correlation coefficients: the green pixels overlapping with the red channel (M1) or vice versa (M2). In order to calculate the percentage of colocalization of two protein, we used M1 when calculates overlapping red pixels (MHCII) with green pixels (Lamp1), and used M2 when calculates overlapping green pixels (MHCII or RhoB) with red pixels (RhoB or Btk). 100% co-localization gives a value of 1. 2.6. Immunoblotting, and coimmunoprecipitation assay
2.7. ELISA assay
Cells were lysed in lysis buffer and freshly added protease inhibitor cocktail. The cell lysates were mixed with antibodies for 2 h, followed by the addition protein G-Sepharose beads (GE Healthcare) for an additional 2 h at 4 °C. Immunoprecipitates were washed four times with
Cytokines in supernatants were analyzed using ELISA kits for mouse TNF-α, IL-1β and interleukin (IL)-6 (R & D Systems). 202
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Fig. 3. Silencing RhoB expression inhibits TLR ligand-triggered downstream signaling pathways in macrophages. The NF-κB, MAPK and Btk pathways were analyzed by western blot in mouse peritoneal macrophages transfected with si-RhoB or si-ctrl upon LPS treatment (A). NF-κB luciferase activity was measured using the Dual-Luciferase Reporter Assay System in RAW264.7 cells transfected with si-RhoB or si-ctrl with or without LPS treatments (B). Colocalization of MHCII (red) with Lamp1 (green) in RAW264.7 cells with si-RhoB or si-ctrl Nuclei were stained with DAPI (blue). The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient Calculator and displayed as a bar graph. Bars represent the mean + SEM of at least 15 cells (C). Data are the mean ± SD of three independent experiments. ** P < 0.01 compared with cells transfected with si-ctrl but without LPS stimulation; # P < 0.05 compared with cells transfected with si-ctrl upon LPS stimulation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
3.2. RhoB increases the production of proinflammatory mediators in macrophages upon LPS stimulation
2.8. Statistical analysis All the experiments were performed at least three independent experiments. Data are presented as mean ± SD. Significance was determined with Student’s t-test, and P < 0.05 was considered statistically significant
To test whether RhoB was associated with the regulation of innate immune responses, we efficiently silenced RhoB expression in murine peritoneal macrophages by transfection with short interfering RNA (siRNA) duplexes against the mouse RhoB gene (si-RhoB) (Fig. 2A). RhoB silencing did not affect TLR4 expression (Fig. 2B) or cell viability (Fig. 2C). Notably, knockdown of RhoB significantly decreased the production of proinflammatory cytokines, including IL-6, TNFα and IL1β, in response to simulation with LPS at 3 and 6 h, both at the mRNA level and secreted into the cell culture supernatant (Fig. 2D-F). In addition, RhoB silencing also significantly decreased the expression of IL-6 and TNFα in macrophages upon stimulation of poly (I:C) and CpG (Fig. 2G). These data indicate that RhoB increases the production of proinflammatory cytokines in TLR-triggered macrophages.
3. Results 3.1. TLR agonists induce RhoB expression in macrophages To investigate the potential role of RhoB in TLR-mediated innate immunity, we first detected the expression of RhoB in LPS-stimulated macrophages. As shown in Fig. 1A, RhoB mRNA levels gradually increased, reaching a maximum at 1 h of approximately 3.5-fold compared with the control group, then decreased to baseline within 24 h after LPS stimulation. Furthermore, RhoB protein levels increased, mirroring the mRNA levels, after LPS treatment (Fig. 1B). Similar increases in RhoB mRNA were also obtained by stimulation of macrophages with CpG ODN or poly(I:C) (Fig. 1C). These results suggest that RhoB, which is induced by TLR agonists, may play an important role in TLR-mediated innate immunity.
3.3. RhoB induces LPS-initiated signaling pathways in macrophages We further investigated the effect of RhoB on TLR-mediated signaling pathways in macrophages. We observed that knockdown of RhoB markedly decreased the phosphorylation of IκBα (p-IκBα), JNK, Erk and p38 in peritoneal macrophages upon LPS stimulation (Fig. 3A). 203
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Fig. 4. RhoB interacts with intracellular MHCII and Btk. Immunoblot analysis of the interactions of RhoB with MHCII α or β chains in RAW264.7 cells cotransfected with vectors encoding HA-RhoB, Myc-MHCIIα or V5-MHCIIβ chains with LPS treatment (A). Immunoblot analysis of the interaction of Btk, MHCII and RhoB in peritoneal macrophages stimulated with LPS (B). Colocalization of RhoB (red) with MHCII (green) or RhoB (green) with MHCII (red) in RAW264.7 cells with or without LPS treatment. Nuclei were stained with DAPI (blue).The fractional co-localization between detection channels was determined by the Manders Correlation Coefficient Calculator and displayed as a bar graph. Bars represent the mean + SEM of at least 15 cells (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
MHCII in DCs (Kamon et al., 2006; Ocana-Morgner et al., 2009). Moreover, MHCII can promote TLR-mediated innate immune responses by maintaining the activation of Btk (Liu et al., 2011). To determine whether RhoB promotes TLR ligand-triggered innate immune responses via interaction with MHCII, we transfected RAW264.7 cells with expression vectors for HA-RhoB and Myc-tagged MHCII α and β chains, followed by coimmunoprecipitation analysis. We found that RhoB interacted with the MHCII α chain, but not the β chain (Fig. 4A). Furthermore, endogenous RhoB also interacted with MHCII and the downstream Btk protein in macrophages (Fig. 4B). Immunofluorescence imaging demonstrated the colocalization of RhoB and MHCII or Btk, with or without LPS stimulation, in the cytoplasm of mouse peritoneal
Of note, knockdown of RhoB also significantly decreased the level of pBtk (Y222), which is necessary for full activation (Liu et al., 2011) (Fig. 3A). In addition, knockdown of RhoB also decreased NF-κB activity in HEK293T cells stimulated with LPS (Fig. 3B). Of note, RhoB silencing did not affect the colocalization of MHCII with lysosome associated membrane protein 1 (Lamp1, an abundant membrane protein of late endosomes) (Fig. 3C). Collectively, these data suggest that RhoB promotes TLR-triggered signaling pathways in macrophages.
3.4. RhoB interacts with MHCII in endosomes It has been reported that RhoB colocalizes intracellularly with 204
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pathways, upon LPS stimulation. The present study demonstrates that RhoB serves as a potent positive regulator of TLRs signaling and may provide a novel perspective for the design of preventive or therapeutic approaches to inflammatory autoimmune diseases. RhoB is normally expressed at low steady-state levels in cells, but can be rapidly and transiently upregulated in macrophages by several stimuli, including UV irradiation, growth factors, cytokines and LPS (Fritz et al., 1995; Jahner and Hunter, 1991; Kamon et al., 2006; OcanaMorgner et al., 2009). In the present study, we found that TLR agonists (LPS, CpG, poly(I:C)) can induce RhoB expression in macrophages, indicating that RhoB levels are regulated in response to TLR activation, and thus RhoB may be involved in the regulation of TLR-mediated signaling. Several studies have implicated RhoB in inflammatory responses, particularly in macrophages and endothelial cells, however, the detailed mechanisms remain uncharacterized. It was recently shown that a TRIF-mediated pathway activates RhoB, which is colocalized with MHCII in intracellular membrane vesicles and necessary for the alteration of cytoskeletal components to traffic intracellular MHCII-containing vesicles toward the surface of DCs after LPS stimulation (Kamon et al., 2006). MHCII is expressed by professional antigen presenting cells, including DCs, macrophages and B cells and has a crucial role in the development and function of the immune system (Miyake et al., 2017; Roche and Furuta, 2015). The classical function of MHCII is to present peptides processed from extracellular proteins to CD4+ helper T cells and to direct the processes of positive and negative selection, shaping the T cell repertoire during maturation and lineage commitment (Mucida et al., 2013; Weigand et al., 2012). In addition to the classical function of MHCII molecules of presenting antigen to CD4+ T cells, intracellular MHCII molecules can act as adaptors, interacting with the tyrosine kinase Btk via the costimulatory molecule CD40 to maintain Btk activation, and thereby promoting TLR signaling (Liu et al., 2011). Based on the major findings, we sought to determine whether RhoB plays an important role in regulating the function of endosomal MHCII in TLR ligand-triggered innate immune responses. We demonstrated that RhoB is a positive regulator of TLR-mediated signaling that allows efficient proinflammatory cytokine production in response to several macrophage activators. During LPS stimulation, RhoB is required for full initiation of several intracellular signaling pathways including the Btk, NF-κB pathway and three mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38. These signaling pathways induce the expression of many genes encoding proinflammatory mediators, such as IL-6, TNFα and IL-1β. Thus, our findings suggest that RhoB is a regulator of inflammatory signaling pathways in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. MHCII, which is a heterodimeric cell surface protein composed of an α chain and a β chain, is a type I integral membrane protein with short cytoplasmic domains and four large extracellular domains (Liu et al., 2011; Schafer et al., 1995). Various transgenic MHCII knockout mice have been generated including MHCIIAα−/− (I-Aα−/−), MHCIIAβ−/− (I-Aβ−/−) and the double knockout (I-Aαβ−/−) (Hou et al., 1995; Kontgen et al., 1993; Madsen et al., 1999). It follows that disruption of either α or β gene locus prevents the cell surface expression of MHCII molecules. Alsharifi et al. observed that the avirulent strain of Semliki Forest virus (aSFV) caused a very acute and lethal infection in I-Aα−/−, but not in I-Aβ−/− or I-Aαβ−/−, mice. I-Aα−/− mice exhibit a striking susceptibility to viral infections (Alsharifi et al., 2013). However, the detailed mechanism remains unclear. In the present study, we found that the MHCII α chain, but not β chain, associated with RhoB. Confocal microscopy also demonstrated that intracellular MHCII colocalized with RhoB and Btk in the endosomes of macrophages stimulated with LPS. Knockdown of MHCII expression significantly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and IFN-β activity by RhoB upon LPS treatment. These results indicated that RhoB activates TLR ligand-triggered innate immune responses through
Fig. 5. RhoB induces TLR-mediated signaling via interaction with MHCII. Immunoblot analysis of the interactions of RhoB with MHCII and Btk in LPS-treated RAW264.7 cells cotransfected with si-MHCII and HA-RhoB vectors (A). NF-κB and IFN-β luciferase activity were measured using the Dual-Luciferase Reporter Assay System in RAW264.7 cells cotransfected with si-RhoB or si-ctrl and the HA-RhoB vector, with or without LPS treatment (B). ** P < 0.01 compared with cells transfected with si-ctrl but without HARhoB vector; # P < 0.05 compared with cells cotransfected with si-ctrl and HA-RhoB vector.
macrophages (Fig. 4C). These results suggest that RhoB interacts with intracellular MHCII molecules in macrophages. 3.5. RhoB induces TLR-mediated signaling via interaction with MHCII To test whether the interaction of RhoB with MHCII is involved in the regulation of TLR ligand-triggered innate immune responses, we designed siRNA targeting MHCII (si-MHCII) and confirmed that siMHCII significantly decreased the protein level of MHCII (Fig. 5A). Knockdown of MHCII expression significantly reduced the interaction of RhoB with Btk in RAW264.7 cells (Fig. 5A), suggesting that the interaction of RhoB with Btk is dependent on MHCII. In addition, knockdown of MHCII expression in RAW264.7 cells attenuated the induction effect of RhoB on NF-κB and interferon (IFN)-β activity upon LPS stimulation (Fig. 5B). In contrast, overexpression of RhoB significantly increased both activities upon LPS but not without LPS stimulation. However, overexpression of RhoB by pretreatment with si-MHCII for 24 h followed by LPS treatment did not increase the activity of NFκB or IFN-β. And these treatments did not affect the viability or TLR4 expression in cells (Fig. 5B). These results indicate that RhoB activates TLR-triggered innate immune response through interaction with MHCII. 4. Discussion We are interested in whether the interaction of RhoB with endosomal MHCII is involved in TLR-mediated innate immune responses. Here, we demonstrated that RhoB, whose expression is induced by TLR agonists (LPS, CpG, poly(I:C)), can promote TLR-initiated signaling pathways and the production of proinflammatory cytokines by macrophages. Specifically, RhoB interacts with the MHCII α chain in endosomes and then with the downstream protein Btk, thereby inducing intracellular signaling pathways, including the NF-κB and MAPK 205
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binding to MHCII. However, the detailed molecular mechanism remained unclearly. RhoB as a Rho family members controls tubulin and F-actin cytoskeletal rearrangements. However, previous studies demonstrated that in unstimulated DCs, efficient surface localization of MHCII is largely independent of RhoB activity (Ocana-Morgner et al., 2009; Kamon et al., 2006). Upon LPS activation, expression and activation of RhoB in DCs and in B cells may account for the cytoskeletal actin rearrangement necessary for the surface expression of MHCII (Kamon et al., 2006), but they did not demonstrated whether RohB affected MHCII expression at endosomal membranes. Here, we did not find that knockdown of RhoB affected the localization of MHCII molecules on the endosome of macrophages upon LPS treatment. Thus, the mechanism by which the interaction of RhoB with intracellular MHCII induces the activation of Btk may not rely on Rho GTPase activity. The discovery of which region of MHC class II interacts with CD40 and Btk in the endosomes of TLR-activated macrophages, and why Btk can be activated after stimulation of human B lymphocytes with CD40L, could give new insights into thel underlying mechanism of RhoB in promoting MHC activation. In conclusion, our study demonstrated that RhoB serves as a potent positive regulator of TLR signaling pathways by interacting with MHCII in endosomes of macrophages and enhancing the production of proinflammatory cytokines. Therefore, RhoB is involved in the activation of TLR-mediated signaling, and may be a therapeutic target for the treatment of inflammatory and autoimmune diseases. References Aderem, A., Ulevitch, R.J., 2000. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787. Alsharifi, M., Koskinen, A., Wijesundara, D.K., Bettadapura, J., Mullbacher, A., 2013. MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies. PLoS One 8, e68458. Chen, W., Han, C., Xie, B., Hu, X., Yu, Q., Shi, L., Wang, Q., Li, D., Wang, J., Zheng, P., et al., 2013. Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation. Cell 152, 467–478. Fernandez-Borja, M., Janssen, L., Verwoerd, D., Hordijk, P., Neefjes, J., 2005. RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1. J. Cell Sci. 118, 2661–2670. Fritz, G., Kaina, B., 2006. Rho GTPases: promising cellular targets for novel anticancer drugs. Curr. Cancer Drug Targets 6, 1–14. Fritz, G., Kaina, B., Aktories, K., 1995. The ras-related small GTP-binding protein RhoB is immediate-early inducible by DNA damaging treatments. J. Biol. Chem. 270, 25172–25177. Hamerman, J.A., Pottle, J., Ni, M., He, Y., Zhang Bucknere, J.H., 2016. Negative regulation of TLR signaling in myeloid cells–implications for autoimmune diseases. Immunol. Rev. 269, 212–227. Hayden, M.S., Ghosh, S., 2011. NF-kappaB in immunobiology. Cell Res. 21, 223–244. Hou, S., Mo, X.Y., Hyland, L., Doherty, P.C., 1995. Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins. J. Virol. 69, 1429–1434. Huang, G., Su, J., Zhang, M., Jin, Y., Wang, Y., Zhou, P., Lu, J., 2017. RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response. Cell Mol.
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