TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4-6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P<0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [(3)H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling response in EC that is regulated by TRAF6.

Upon viral infection, the cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA to activate antiviral signaling to induce type I interferon (IFN). RIG-I-like receptors (RLRs) activate antiviral signaling in a tissue-specific manner. The molecular mechanism underlying antiviral signaling in the respiratory system remains unclear. We studied antiviral signaling in the lower respiratory tract (LRT), which is the site of many harmful viral infections. Epithelial cells of the LRT can be roughly divided into two groups: bronchial epithelial cells (BECs) and pulmonary alveolar epithelial cells (AECs). These two cell types exhibit different phenotypes; therefore, we hypothesized that these cells may play different roles in antiviral innate immunity. We found that BECs exhibited higher antiviral activity than AECs. TNF receptor-associated factor 3 (TRAF3) has been shown to be a crucial molecule in RLR signaling. The expression levels of TRAF3 and TRAF5, which have conserved domains that are nearly identical, in the LRT were examined. We found that the bronchus exhibited the highest expression levels of TRAF3 and TRAF5 in the LRT. These findings suggest the importance of the bronchus in antiviral innate immunity in the LRT and indicate that TRAF3 and TRAF5 may contribute to RLR signaling.

Acute pancreatitis is a common threat to human health. Caerulin provokes severe inflammations, causing injuries to surrounding pancreatic cells. TNF receptor-associated factor 3 (TRAF3) is a highly versatile regulator of immune response. The current study aims to understand the potential effect of TRAF3 on caerulin-induced pro-inflammatory responses. In the primary-cultured mouse bone marrow-derived macrophages (BMDMs), caerulin induced TRAF3 protein stabilization, which formed a complex with mitogen-activated protein kinase kinase 3 (MKK3) to mediate downstream p38 activation. Lentiviral shRNA-mediated TRAF3 stable knockdown significantly attenuated caerulin-induced MKK3-p38 activation and production of several key pro-inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-17. Remarkably, TRAF3 knockdown in caerulin-stimulated BMDMs also alleviated cytotoxicity to Panc02 and primary mouse pancreatic cells. Thus, TRAF3 is required for caerulin-induced p38 activation and macrophage-mediated pro-inflammatory responses. TRAF3 expression in macrophages could be a novel therapeutic target protein for the treatment of acute pancreatitis.

TNF receptor-associated factor 6 (TRAF6) plays a critical role in NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, both of which mediate macrophage activation in response to pathogen-associated molecular patterns such as bacterial endotoxin, lipopolysaccharides (LPS). In this study, we investigated whether HLA-B associated transcript-3 (BAT3) regulates LPS-induced macrophage activation. BAT3 physically interacted with TRAF6 in macrophages, and this interaction was enhanced in the cells after LPS treatment. Furthermore, BAT3 inhibited the homo-oligomerization of TRAF6 as well as the interaction between TRAF6 and its downstream kinase transforming growth factor beta-activated kinase 1 (TAK1), thereby suppressing TRAF6-mediated signaling events. Intriguingly, TRAF6 mediated ubiquitination of BAT3 and this ubiquitination was crucial for its inhibitory effect on TRAF6-mediated signaling. Depletion of BAT3 by RNA interference resulted in enhancement of LPS-induced activation of the NF-κB signaling with increasing expression levels of pro-inflammatory cytokines. These findings suggest that BAT3 functions as the negative regulator of LPS-induced macrophage activation.

Atherosclerosis is exacerbated by periodontal pathogens, which induce vascular inflammation after entering the bloodstream. Among oral indigenous bacteria, Streptococcus sanguinis and S. anginosus are related to systemic disorders, such as infective endocarditis and abscess, and are sometimes detected in human atherosclerotic plaques or blood. Thus, these oral streptococci may contribute to the progression of atherosclerosis. To test this hypothesis, apolipoprotein E-deficient spontaneously hyperlipidemic mice were intraorally challenged with S. sanguinis or S. anginosus. Atherosclerotic plaque formation increased significantly in the S. sanguinis-challenged group compared with the carboxymethylcellulose-treated control group. Expression levels of mRNAs of proinflammatory cytokines in the aorta and levels of atherosclerosis-related mediators in blood increased upon S. sanguinis challenge. Adaptor molecule TNF receptor-associated factor 6 was also enhanced in the aorta when mice were challenged with S. sanguinis. Furthermore, challenge with S. anginosus induced systemic inflammation, but inflammation-related mRNA expression levels in the aorta only increased slightly and were accompanied by minimal expansion of the lesion area. By contrast, with the exception of IL-1α, the expression levels of inflammation-related genes did not change in gingival tissues of both bacteria- and sham-challenged groups. These results reveal that S. sanguinis causes aortic inflammation that leads to accelerated progression of atherosclerosis.

The conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) originate from the same common dendritic cell precursor cells in the bone marrow. The pDCs produce large amounts of type 1 interferon in response to foreign nucleic acid and crucially contribute to host defense against viral infection. Tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5) is a pivotal component of various TNF receptor signaling pathways in the immune system. Although the functions of TRAF5 in T and B lymphocytes have been well studied, its roles in pDCs remains to be fully elucidated. In this study, we show that the expression of TRAF5 supports the generation of pDCs in the bone marrow and also critically contributes to the homeostasis of the pDC subset in the periphery in a cell-intrinsic manner. Furthermore, we provide evidence that TRAF5 promotes the commitment of DC precursor cells toward pDC versus cDC subsets, which is regulated by the balance of transcription factors TCF4 and ID2. Together our findings reveal that TRAF5 acts as a positive regulator of pDC differentiation from bone marrow progenitors.