Modulation of proinflammatory NF-κB signaling by ectromelia virus in RAW 264.7 murine macrophages

Abstract

Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus Orthopoxvirus--ectromelia virus (ECTV)--in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxviruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

“…First, we found that LPS-induced nuclear translocation of key transcription factors,such as NF-κB, IRF3 and IRF7 that control the transcription of cytokine and chemokine genes was impaired in ECTV-infected GM-BM. ECTV has been demonstrated to modulate NF-κB signaling pathway in other APCs [49], as well as in non-immune cells [50–52]. Inhibition of NF-κB activation can be mediated by ECTV-encoded ankyrin/F-box proteins, such as EVM002, EVM005, EVM159 and EVM170 that impair IĸBα degradation [53].…”

“…Inhibition of NF-κB activation can be mediated by ECTV-encoded ankyrin/F-box proteins, such as EVM002, EVM005, EVM159 and EVM170 that impair IĸBα degradation [53]. Moreover, ECTV encodes counterparts of the following VACV inhibitory proteins altering NF-κB signaling pathway: A46 that disrupts TLR and IL-1β signaling, B14 that targets the IκB kinase (IKK) complex, and N1 that probably acts upstream of IKK complex activation [49,51]. Interestingly, our studies revealed that p65 NF-κB is recruited to ECTV replication sites in GM-BM.…”

“…Interestingly, our studies revealed that p65 NF-κB is recruited to ECTV replication sites in GM-BM. On the contrary, p65 NF-κB has been shown to not accumulate in viral factories in RAW 264.7 macrophages [49] and BALB/3T3 fibroblasts] 52] infected with ECTV and in HeLa cells infected with VACV] 54]. Thus, the presence of cellular proteins within viral factories seems cell type specific.…”

“…Thus, the presence of cellular proteins within viral factories seems cell type specific. The accumulation of NF-κB within viral replication centers in ECTV-infected GM-BM is surprising, since such phenomenon has not been observed earlier [49,52,54]. Poxviruses, such as ECTV and VACV, are capable of binding to specific host proteins [14] and retain certain cellular proteins in the region of cytoplasmic DNA factories in infected cells [54].…”

MicroRNA-322 inhibits inflammatory cytokine expression and promotes cell proliferation in LPS-stimulated murine macrophages by targeting NF-κB1 (p50)

“…The complex process of inflammatory response is initiated by innate immune system receptors, which recognize a lot of pathogens or damage signals and precisely regulate immune response. Cells expressing pathogen-recognition receptors (PRRs) are able to present pathogens, antigens and secrete cytokines and chemokines [3,4]. PRRs, such as toll-like receptors (TLRs), activate cellular signalling pathways that result in NF-κB translocating into nucleus, and lead to the production of inflammatory cytokines and chemokines, which directly contribute to inflammatory lesions and immune diseases [5].…”