In this study, we evaluated the cooperative effect of GCs and NDV in mouse bone marrow-derived DCs on the expression of 89 genes whose products play important roles in the TLR signaling pathways. Among the genes differentially regulated by DEX and/or NDV, we focused on IL-10
, as mRNA expression of this gene showed the most significant change. In addition, DCs are the major immune cells that secrete this cytokine 
. NDV infection enhanced DEX pre-treatment-induced IL-10 mRNA expression and protein production in DCs. This effect of NDV started as early as 3 hours after infection and persisted for 24 hours. MCMV and DEX cooperatively enhanced production of IL-10 in mice. NDV activated ERK1/2, phosphorylated human GR at serine 203 (213 in mice) and ultimately increased the transcriptional activity of GR, not only on the IL-10
gene but also on other TLR-unrelated glucocorticoid-responsive genes.
IL-10 is an anti-inflammatory cytokine, which suppresses inflammation by affecting functions of various immune cells 
. Upon viral infection, circulating levels of IL-10 increase after ~24 hours to 20 days in mice to facilitate resolution of inflammation promoted by pro-inflammatory cytokines, which are secreted into circulation immediately (~3–6 hours) after infection 
. Therefore, IL-10 acts as a negative regulatory factor against the pro-inflammatory immune response, which is beneficial for clearing pathogens from infected organisms but whose excessive and prolonged activation is detrimental to local inflammatory tissues 
. Thus, the levels and duration of IL-10 production are tightly regulated during the course of immune response against pathogens, while dysregulation in this process may result in prolonged/persistent infection and even systemic anergy to infected organisms 
. We found that cooperation between DEX and NDV on the IL-10 production started as early as 3 hours after the viral infection in DCs. Thus, it is quite possible that some viruses distort normal regulation of IL-10 secretion in these cells through cooperation with GCs, and increase their propagation in host tissues. Indeed, several viruses in the Herpesviridae
family, such as the Epstein-Barr virus and the varicella-zoster virus, encode IL-10-like molecules, which share immunosuppressive properties of host IL-10, and increase their infectivity to and latency in their hosts 
. MCMV, which we found to induce IL-10 production cooperatively with DEX, is also a member of this family 
, suggesting that IL-10 and its downstream biologic actions are common targets for some members of this viral family to modulate host immune activity. Further, persistent infection of Mycobacterium tuberculosis
and reactivation of its previous inflammatory sites are also associated with excessive production of IL-10 
. As we found in the original screening, DEX pre-treatment and NDV infection cooperatively altered mRNA expression of several genes including CLEC4E
, in addition to IL-10
. Thus, it is possible that virus also modulates host immune response by changing expression of these genes through cooperation with GCs.
Our results on the cooperation of DEX and virus on IL-10 production may in part explain the previous observation that mental/physical stress increases susceptibility to viral infection and tendency to exacerbate/prolong its disease course 
. This hypothesis may be supported by our results that the NDV-induced enhancement of IL-10 expression was particularly observed with the strong synthetic glucocorticoid DEX as well as with high concentrations of CORT frequently encountered in stressed animals. Further, our results may also provide a potential mechanistic explanation to the exacerbation of bronchial asthma by physical/emotional stress and viral infection, as elevation of IL-10 production and resulting activation of Th-2-directed humoral immunity play important pathogenetic roles in this potentially lethal airway disease 
We demonstrated that NDV infection enhanced GC-stimulated production of IL-10 by phosphorylating GR through ERK in DCs. It is known that several viruses activate ERK in macrophages, alveolar epithelial A549 cells and primary tracheobronchial epithelial cells 
. Since IL-10 promoter does not contain GREs 
, ERK appears to enhance indirect transcriptional activity of GR on this promoter. In preliminary experiments, we observed that addition of some TLR ligands (TLR7/8 and TLR9) increased IL-10 mRNA levels in DCs similar to viral infection (data not shown), thus it is possible that NDV infection activated ERK pathway through stimulation of the specific pattern recognition receptor(s), such as TLR7/8 and 9. In addition to these TLRs, RNA viruses activate p38 MAPK through the cytoplasmic helicase RIG-like receptors 
. Poly(I:C), a synthetic double stranded RNA, stimulates several MAPKs including ERK in neutrophils, although involvement of RIG-like receptors in the activation of ERKs have not been verified as yet 
. These pieces of evidence may suggest that RIG-like receptors also play a role in the activation of ERK in response to infection of RNA viruses in DCs.
Some serine/threonine kinases including p38 MAPK, JNK and the cyclin-dependent kinases (CDKs), phosphorylate several serine residues located in the N-terminal domain of GR and positively or negatively modulate the transcriptional activity of this receptor 
. Activation of GR by ligand is necessary for ERK to phosphorylate this receptor possibly due to their ligand-dependent interaction, although virus activates this kinase independently to glucocorticoids. As reported in the case of CDK5 and p38 MAPK, phosphorylation-mediated alteration in the transcriptional cofactor attraction to the activation function-1 domain of GR may be one of the mechanisms underlying the ERK-mediated enhancement of GR transcriptional activity 
. We found that NDV-induced GR phosphorylation enhanced DEX-induced mRNA expression of several well-known glucocorticoid responsive, GRE-containing genes in addition to IL-10. This result suggests that NDV can potentially modulate expression of many glucocorticoid-responsive genes in addition to this cytokine by phosphorylating GR. Indeed, NDV may also modulate through phosphorylation of GR the expression of other TLR signaling pathway-related genes, which we found to be regulated by DEX and NDV in our screening. It is reported that the respiratory syncytial virus (RSV), which is one of the major causes of lower respiratory tract infection and hospital visits during infancy and childhood, represses the anti-inflammatory action of glucocorticoids through GR 
. Since RSV is in the same Paramyxoviridae
family as NDV, it is possible that RSV modulates the anti-inflammatory action of these hormones by phosphorylating GR through activation of ERK.
In conclusion, we described a novel cooperation between viral infection and GCs on the expression of glucocorticoid-responsive genes in DCs through phosphorylation of GR by ERK. Through this activity particularly on IL-10, viruses may increase their propagation in host organisms by suppressing the latters’ immune activity.