ERK-MAP-kinases differentially regulate expression of IL-23 p19 compared with p40 and IFN-β in Theiler's virus-infected RAW264.7 cells
Introduction
The infection of macrophages by viruses initiates an innate anti-viral immune response through the production of cytokines. IFN-β, induces an anti-viral state [1], [2] and increases NK cell cytotoxicity [3]. IL-12, induces NK cell proliferation [4], NK cell production of IFN-γ [5], and macrophage nitric oxide [6], all of which have potent anti-viral properties [7], [8]. However, IL-12 is a dimer of p35 and p40 subunits [9] each of which can dimerize with other subunits. In particular, p40 [10] assembles into homodimers or with p19 to form IL-23 [11]. Recently viral infections were shown to induce p19 expression in macrophages [12]. Therefore viral infection of macrophages could stimulate production of IL-23.
Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages and induction of autoimmune DD in certain strains of mice is an accepted animal model of human MS [13]. In another MS model, experimental allergic encephalomyelitis (EAE), IL-23 p19/p40 is associated with DD while IL-12 p35/p40 and IFN-β are not and may actually be beneficial [14], [15]. TMEV infection of macrophage lineage cells can induce IFN-β and IL-12 p35/p40 [16] but it is unclear if TMEV infection of macrophages also induces expression of IL-23 p19/p40. If this is the case then IL-23 may also contribute to TMEV-DD and a means to therapeutically control IL-23 expression would be desirable.
TMEV induced cytokine expression is likely controlled in part by activation of p38 and p42/p44 extracellular signal-regulated (ERK) Mitogen activated protein kinases (MAPK) and transcription factors like ATF-2, a p38 MAPK target [17]. Virus activated MAPKs catalyze amino terminal phosphorylations of NF-κB activator (TANK)-binding kinase 1 (TBK1) [18], [19]. TBK1-activates NF-κB [20], which assembles with other transcription factors, including ATF-2 [21], to induce expression of IFN-β. However, the potential role of MAPKs in TMEV-induced cytokine expression by macrophages is unknown.
To determine if TMEV infection of macrophages induces IL-23 in addition to IFN-β or IL-12 and if MAPKs are involved in this expression, RAW264.7 macrophage cells were infected with TMEV as previously shown [22], [23]. TMEV infection of RAW264.7 cells induces, in addition to IFN-β, expression of the IL-23 subunits p19 and p40 but not the IL-12 p35 subunit. Also, both p38 and ERK-MAPKs and the p38 MAPK-target ATF-2, are activated within 30 min of infection. Inhibition of the p38 MAPK increased p19, decreased p40 but had no significant effect on IFN-β expression. In contrast, inhibition of ERK-MAPKs increased expression of IFN-β and p40, reduced p19 expression, and prolonged the activation of ATF-2. Thus ERK MAPKs regulate expression of TMEV-induced p19 in an opposite manner from TMEV-induced IFN-β and p40 and may play a role in extinguishing p38-mediated ATF-2 activation.
Section snippets
Cells and viruses
RAW264.7 cells were grown in DMEM cell culture medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen) and 50 μg/ml gentamycin (Invitrogen). The DA strain of TMEV was obtained from Dr. Kristen Drescher, Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE. TMEV was grown in BHK-21 cells to produce stocks with 5 × 106 PFU/ml. The p38 MAPK inhibitor SB203580 and the ERK MAPK inhibitor U0126 were obtained from Promega Corporation (Madison, WI).
RT-PCR
TMEV activates ERK and p38 MAPKs in RAW264.7 cells
Innate expression of cytokines from virus-infected macrophages is triggered by activation of cytoplasmic MAPKs. To determine if TMEV infection of RAW264.7 cells activates MAPKs, phosphorylation of ERK MAPKs p42/p44 and p38 MAPKs was monitored by immunoblot. TMEV infection of RAW264.7 cells induced maximum activation of p38 and ERK by 30 min with substantially lower activation at 3 h (Fig. 1A). As little as 5 and up to 40 μM of the p38 MAPK inhibitor, SB203580, only slightly reduced TMEV-induced
Discussion
Viruses like Epstein Barr virus [27] and human herpes virus-6 [28] have been implicated in the etiology of MS in humans. Therefore, TMEV infection of macrophages and the ensuing DD that occurs in certain strains of mice is a useful model for virus-induced MS [29]. These viruses may cause the development of MS by stimulating the production of specific cytokines that are thought to contribute to MS. It is known that IFN-β and IL-12p35/p40 are produced by virus-infected macrophages [3]. However,
Acknowledgements
This work was support by grants RG3381-A2 from the National Multiple Sclerosis Society and grant P20 RR15635 from the NIH COBRE program of the National Center for Research Resources.
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