RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages
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 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 production 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.
Section snippets
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
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
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,
References (31)
- et al.
Toll-like receptors in the induction of the innate immune response
Nature
(2000) - et al.
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
(2013) - et al.
Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation
Cell
(2013) - et al.
RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1
J. Cell Sci.
(2005) - et al.
Rho GTPases: promising cellular targets for novel anticancer drugs
Curr. Cancer Drug Targets
(2006) - et al.
The ras-related small GTP-binding protein RhoB is immediate-early inducible by DNA damaging treatments
J. Biol. Chem.
(1995) - et al.
Negative regulation of TLR signaling in myeloid cells–implications for autoimmune diseases
Immunol. Rev.
(2016) - et al.
NF-kappaB in immunobiology
Cell Res.
(2011) - et al.
Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins
J. Virol.
(1995) - et al.
RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response
Cell Mol. Immunol.
(2017)
The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts
Mol. Cell Biol.
TRIF-GEFH1-RhoB pathway is involved in MHCII expression on dendritic cells that is critical for CD4 T-cell activation
EMBO J.
Targeted disruption of the MHC class II Aa gene in C57BL/6 mice
Int. Immunol.
Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk
Nat. Immunol.
Mice lacking all conventional MHC class II genes
Proc. Natl. Acad. Sci. U. S. A.
Cited by (20)
Differential role for rapid proteostasis in Rho GTPase-mediated control of quiescent endothelial integrity
2023, Journal of Biological ChemistryPattern recognition receptor mediated innate immune response requires a Rif-dependent pathway
2023, Journal of AutoimmunityCitation Excerpt :Also, RhoA and IL-1R-associated kinase are signal transducers for LPS-induced TLR4-mediated proinflammatory cytokine synthesis in human monocytes [24]; RhoA acts as a molecular switch downstream of TLR2/TLR3-Src-initiated pathways to trigger NF-κB-dependent gene transcription [25]. RhoB is found to be a positive physiological regulator of TLR2, 3 and 4 signaling via binding to MHCII in macrophages [26]. Meanwhile, PAMPs in the host cytosol can be detected by NLRs, such as NOD1 could senses cytosolic microbial products by monitoring the activation state of small Rho GTPases, and induce NF-κB-dependent inflammatory responses [6].
Listeria monocytogenes crosses blood brain barrier through Rho GTPases induced migration of macrophages and inflammatory interleukin expression
2021, Microbial PathogenesisCitation Excerpt :RhoB expression was increased with low doses of infection. RhoB promotes the expression of IL-1β, IL-6 and TNF-α by Toll-like receptors (TLRs) mediated activation of NF-κB [46]. Whereas, the expression of RhoB and Cdc42 was not significantly changed in murine macrophage infected with Listeria monocytogenes comparing with the RhoA and Rac1.
A functional polymorphism in the promoter of RhoB is associated with susceptibility to Vibrio anguillarum in turbot (Scophthalmus maximus)
2019, Fish and Shellfish ImmunologyCitation Excerpt :Studies conducted by Huang et al. [25] demonstrated that RhoB promoted the production of proinflammatory cytokines, including IL-1β, IL-6 and TNF-α, through activate NF-κB signaling and enhance macrophage adhesion which sustained the inflammation. Another study in TLR-stimulated macrophages found that RhoB could activate NF-κB signaling by interact with the tyrosine kinase Btk at early time of response [26]. In turbot, RhoB is intensely activated in gill at 1 hpi and in blood at 3 hpi, revealing that immune inflammatory response occurs at the early stage of V. anguillarum stimulation.
Downregulated expression of miR-223 promotes Toll-like receptor-activated inflammatory responses in macrophages by targeting RhoB
2017, Molecular ImmunologyCitation Excerpt :Another study demonstrated that RhoB could enhance the expression of TNF-α, IL-6, and IL-1β in macrophages exposed to hypoxic conditions (Huang et al., 2017). Moreover, work in TLR-stimulated macrophages has suggested that RhoB interacts with endosomal major histocompatibility complex class II α chains and with the tyrosine kinase Btk, which activate NF-κB signaling (Liu et al., 2017). In our study, we found that the 3′-UTR of RhoB mRNA contains two miR-223-binding sites that are highly conserved between species, including mouse and human, and we found that miR-223 could directly regulate RhoB expression via these 3′-UTR sites.
- 1
These authors contributed equally to the work.