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NF-?B1 inhibits TLR-induced IFN-? production in macrophages through TPL-2-dependent ERK activation.

ABSTRACT: Although NF-?B1 p50/p105 has critical roles in immunity, the mechanism by which NF-?B1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-? expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-? production. Markedly higher serum levels of IFN-? were observed in Nfkb1(-/-) mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-? production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-? production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1(-/-) macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-? secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1(-/-) macrophages, which rescued LPS activation of ERK, also inhibited IFN-? expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.

SUBMITTER: Yang HT

PROVIDER: S-EPMC3070925 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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NF-κB1 inhibits TLR-induced IFN-β production in macrophages through TPL-2-dependent ERK activation.

Yang Huei-Ting HT Wang Yanyan Y Zhao Xixing X Demissie Ezana E Papoutsopoulou Stamatia S Mambole Agnes A O'Garra Anne A Tomczak Michal F MF Erdman Susan E SE Fox James G JG Ley Steven C SC Horwitz Bruce H BH

Journal of immunology (Baltimore, Md. : 1950) 20110107 4

Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and exp ...[more]

PMID: 21217011

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Project description:Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, ?/?/?) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-?B, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

Project description:Marek's disease virus (MDV), which causes T cell lymphomas in chickens, is economically important and has contributed to knowledge of herpesvirus-associated oncogenicity. The DNA-sensing pathway induces innate immune responses against DNA virus infection, and nuclear factor ?B (NF-?B) signaling is critical for the establishment of innate immunity. Here, we report that RLORF4, an MDV-specific protein directly involved in viral attenuation, is an inhibitor of the DNA-sensing pathway. The results showed that ectopically expressed RLORF4 blocked beta interferon (IFN-?) promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). RLORF4 selectively inhibited the activation of NF-?B but not IFN-regulatory factor 7. RLORF4 was found to bind the endogenous NF-?B subunits p65 and p50, and it also bound to the Rel homology domains of these subunits. Furthermore, RLORF4 suppressed the nuclear translocation of p65 and p50 mediated by tumor necrosis factor alpha and interferon-stimulatory DNA. Finally, deletion of RLORF4 from the MDV genome promoted IFN-? and interleukin-6 (IL-6) production in vitro and in vivo In the absence of RLORF4, the host cellular immunity was significantly increased, and reduced viral titers were observed during infection of chickens. Our results suggest that the RLORF4-mediated suppression of the host antiviral innate immunity might play an important role in MDV pathogenesis.IMPORTANCE Marek's disease virus (MDV) RLORF4 has been shown to be directly involved in the attenuation of MDV upon serial passages in vitro; however, the exact function of this protein during viral infection was not well characterized. This study demonstrated that RLORF4 significantly inhibits cGAS-STING-mediated NF-?B activation by binding to the Rel homology domains of the NF-?B subunits p65 and p50, interrupting their translocation to the nuclei and thereby inhibiting IFN-? production. Furthermore, RLORF4 deficiency promoted the induction of IFN-? and downstream IFN-stimulated genes during MDV infection in chickens. Our results suggest that the contribution of RLORF4 to MDV virulence may stem from its inhibition of viral DNA-triggered IFN-? responses.

Dataset Information

NF-?B1 inhibits TLR-induced IFN-? production in macrophages through TPL-2-dependent ERK activation.

Publications

NF-κB1 inhibits TLR-induced IFN-β production in macrophages through TPL-2-dependent ERK activation.

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