HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.

Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.

IRAK3 is a critical modulator of inflammation in innate immunity. IRAK3 is associated with many inflammatory diseases, including sepsis, and is required in endotoxin tolerance to maintain homeostasis of inflammation. The impact of IRAK3 on inflammatory markers such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell culture models remains controversial.

Chronic subdural hematoma (CSDH) is an angiogenic and inflammatory disease. Toll-like receptors (TLRs) transduce intracellular signals, resulting in the activation of nuclear factor κB (NF-κB), which leads to the production of inflammatory cytokines. High-mobility group box 1 (HMGB1) functions as a mediator of inflammatory responses through TLRs. In this study, we examined the expression of HMGB1 and components of the Toll-like receptor and NF-κB signaling pathways in the outer membrane of CSDH. Eight patients whose outer membrane was successfully obtained during trepanation surgery were included in this study. The expression of TLR4, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 4 (IRAK4), TNF receptor-associated factor 6 (TRAF6), TGFβ-activated kinase 1 (Tak1), interferon regulatory factors 3 (IRF3), IκB kinase β (IKKβ), IKKγ, IκBε, IκBα, NF-κB/p65 and β-actin was examined by Western blot analysis. The expression of TLR4, NF-κB/p65 and interleukin-6 (IL-6) was also examined by immunohistochemistry. The concentrations of HMGB1 and IL-6 in CSDH fluids were measured using ELISA kits. Above-mentioned molecules were detected in all cases. In addition, TLR4, NF-κB/p65 and IL-6 were localized in the endothelial cells of vessels within CSDH outer membranes. The concentrations of HMGB1 and IL-6 in CSDH fluids were significantly higher than that in the CSF and serum. There existed a correlation between the concentrations of HMGB1 and IL-6 in CSDH fluids. Our data suggest that HMGB1 in CSDH fluids produces the inflammatory cytokine IL-6 in endothelial cells through the Toll-like receptor and NF-κB signaling pathways. Anti-HMGB1 therapy might be a useful method to treat the growth of CSDH.

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

Adiponectin, an adipokine predominantly produced from adipose tissue, exhibited potent anti-inflammatory properties. In particular, it inhibits production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In the present study, we investigated the role of autophagy induction in the suppression of Lipopolysaccharide (LPS) -induced TNF-α expression by globular adiponectin (gAcrp) and its potential mechanisms. Herein, we found that gAcrp treatment increased expression of genes related with autophagy, including Atg5 and microtubule-associated protein light chain (LC3B), induced autophagosome formation and autophagy flux in RAW 264.7 macrophages. Similar results were observed in primary macrophages isolated peritoneum of mice. Interestingly, inhibition of autophagy by pretreatment with Bafilomycin A1 or knocking down of LC3B gene restored suppression of TNF-α expression, tumor necrosis factor receptor- associated factor 6 (TRAF6) expression and p38MAPK phosphorylation by gAcrp, implying a critical role of autophagy induction in the development of tolerance to LPS-induced TNF-α expression by gAcrp. We also found that knocking-down of FoxO3A, a forkhead box O member of transcription factor, blocked gAcrp-induced expression of LC3II and Atg5. Moreover, gene silencing of Silent information regulator 1 (SIRT1) blocked both gAcrp-induced nuclear translocation of FoxO3A and LC3II expression. Finally, pretreatment with ROS inhibitors, prevented gAcrp-induced SIRT1 expression and further generated inhibitory effects on gAcrp-induced autophagy, indicating a role of ROS production in gAcrp-induced SIRT1 expression and subsequent autophagy induction. Taken together, these findings indicate that globular adiponectin suppresses LPS-induced TNF-α expression, at least in part, via autophagy activation. Furthermore, SIRT1-FoxO3A axis plays a crucial role in gAcrp-induced autophagy in macrophages.

Mutations in the F-box only protein 7 gene (FBXO7), the substrate-specifying subunit of SCF E3 ubiquitin ligase complex, cause Parkinson's disease (PD)-15 (PARK15). To identify new variants, we sequenced FBXO7 cDNA in 80 Taiwanese early onset PD patients (age at onset ≤ 50) and only two known variants, Y52C (c.155A>G) and M115I (c.345G>A), were found. To assess the association of Y52C and M115I with the risk of PD, we conducted a case-control study in a cohort of PD and ethnically matched controls. There was a nominal difference in the Y52C G allele frequency between PD and controls (p = 0.045). After combining data from China [1], significant difference in the Y52C G allele frequency between PD and controls (p = 0.012) and significant association of G allele with decreased PD risk (p = 0.017) can be demonstrated. Upon expressing EGFP-tagged Cys52 FBXO7 in cells, a significantly reduced rate of FBXO7 protein decay was observed when compared with cells expressing Tyr52 FBXO7. In silico modeling of Cys52 exhibited a more stable feature than Tyr52. In cells expressing Cys52 FBXO7, the level of TNF receptor-associated factor 2 (TRAF2) was significantly reduced. Moreover, Cys52 FBXO7 showed stronger interaction with TRAF2 and promoted TRAF2 ubiquitination, which may be responsible for the reduced TRAF2 expression in Cys52 cells. After induced differentiation, SH-SY5Y cells expressing Cys52 FBXO7 displayed increased neuronal outgrowth. We therefore hypothesize that Cys52 variant of FBXO7 may contribute to reduced PD susceptibility in Chinese.

Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS. However, the precise regulatory mechanisms remain unclear. Here, we show the role of FK506-binding protein 51 (FKBP51) in regulating the dsRNA-dependent expression of type I IFN. The binding of FKBP51 to TRAF6 was first identified by "in vitro virus" selection and was subsequently confirmed with a coimmunoprecipitation assay in HEK293T cells. The TRAF-C domain of TRAF6 is required for its interaction, although FKBP51 does not contain the consensus motif for interaction with the TRAF-C domain. Besides TRAF6, we found that FKBP51 also interacts with TRAF3. The depletion of FKBP51 reduced the expression of type I IFN induced by dsRNA transfection or Newcastle disease virus infection in murine fibroblasts. Consistent with this, the FKBP51 depletion attenuated dsRNA-mediated phosphorylations of IRF3 and JNK and nuclear translocation of RelA. Interestingly, dsRNA stimulation promoted the accumulation of FKBP51 in the mitochondria. Moreover, the overexpression of FKBP51 inhibited RLR-dependent transcriptional activation, suggesting a scaffolding function for FKBP51 in the MAVS-mediated signaling pathway. Overall, we have demonstrated that FKBP51 interacts with TRAF proteins and facilitates the expression of type I IFN induced by cytosolic dsRNA. These findings suggest a novel role for FKBP51 in the innate immune response to viral infection.

Toll-like receptors (TLRs) mediated immune response is crucial for combating pathogens and must be tightly controlled. Tripartite motif (TRIM) proteins are a family of proteins that is involved in a variety of biological and physiological processes. Some members of the TRIM family are important in the regulation of innate immunity. Although it has been shown that TRIM38 negatively regulates innate immunity, the mechanisms by which it does so have not been fully addressed. In this study, we demonstrated that TRIM38 negatively regulates Toll-like receptor 3 (TLR3)-mediated type I interferon signaling by targeting TIR domain-containing adaptor inducing IFN-β (TRIF). We found that overexpression of TRIM38 inhibits TLR3-mediated type I interferon signaling, whereas knockdown of TRIM38 has the reverse effects. We further showed that TRIM38 targets TRIF, a critical adaptor protein downstream of TLR3. TRIF is co-immunoprecipitated with TRIM38, and domain mapping experiments show that PRYSPRY of TRIM38 interacts with the N-terminus of TRIF. Overexpression of TRIM38 decreased expression of overexpressed and endogenous TRIF. This effect could be inhibited by MG132 treatment. Furthermore, the RING/B-box domain of TRIM38 is critical for K48-linked polyubiquitination and proteasomal degradation of TRIF. Collectively, our results suggest that TRIM38 may act as a novel negative regulator for TLR3-mediated type I interferon signaling by targeting TRIF for degradation.

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor kappaB (NF-kappaB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-kappaB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forests. MED has been shown to be able to induce cell cycle arrest and cancer cell apoptosis. However, its effects on inflammatory response are unclear. Herein we showed that MED exhibited inhibitory effect on inflammatory response induced by lipopolysaccharide (LPS). MED significantly inhibited LPS-induced expression of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and nitric oxide (NO) in macrophages. MED inhibited LPS-induced nuclear translocation of nuclear factor (NF)-κB (NF-κB) p65, IκB degradation, IκB kinase (IKK) phosphorylation, and the activation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38, suggesting that MED blocks the activation of both NF-κB and mitogen-activated protein kinase (MAPK) pathways. Furthermore, the effects of MED on LPS-induced activation of upstream signaling molecules such as transforming growth factor-β-activated kinase 1 (TAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor associated kinases1 (IRAK1) were investigated. MED significantly inhibited TAK1 phosphorylation and TRAF6 polyubiquitination, but not IRAK1 phosphorylation and TRAF6 dimerization, indicating that MED inhibits LPS-induced inflammatory responses at least in part through suppression of TRAF6 polyubiquitination. Moreover, MED protected mice from LPS-induced endotoxin shock by reducing serum inflammatory cytokines. These results suggest that MED is a potential lead compound for the development of a novel nonsteroidal anti-inflammatory drug.

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.

Synthetic forms of E. coli monophosphoryl lipid A (sMLA) weakly activate the MyD88 (myeloid differentiation primary response protein) branch of the bifurcated TLR4 (Toll-like receptor 4) signaling pathway, in contrast to diphosphoryl lipid A (sDLA), which is a strong activator of both branches of TLR4. sMLA's weak MyD88 signaling activity is apparent downstream of TLR4/MyD88 signaling as we show that sMLA, unlike sDLA, is unable to efficiently recruit the TNF receptor-associated factor 6 (TRAF6) to the Interleukin-1 receptor-associated kinase 1 (IRAK1). This reduced recruitment of TRAF6 explains MLA's lower MAPK (Mitogen Activated Protein Kinase) and NF-κB activity. As further tests of sMLA's ability to activate TLR4/Myeloid differentiation factor 2 (MD-2), we used the antibody MTS510 as an indicator for TLR4/MD-2 heterotetramer formation. Staining patterns with this antibody indicated that sMLA does not effectively drive heterotetramerization of TLR4/MD-2 when compared to sDLA. However, a F126A mutant of MD-2, which allows lipid A binding but interferes with TLR4/MD-2 heterotetramerization, revealed that while sMLA is unable to efficiently form TLR4/MD-2 heterotetramers, it still needs heterotetramer formation for the full extent of signaling it is able to achieve. Monophosphoryl lipid A's weak ability to form TLR4/MD-2 heterotetramers was not restricted to synthetic E. coli type because cells exposed to a biological preparation of S. minnesota monophosphoryl lipid A (MPLA) also showed reduced TLR4/MD-2 heterotetramer formation. The low potency with which sMLA and MPLA drive heterotetramerization of TLR4/MD-2 contributes to their weak MyD88 signaling activities.

Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.

The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4+ and CD8+ T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44high/CD62Llow expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2-3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.

Oral mucositis (OM) is an important side effect of cancer treatment, characterized by ulcerative lesions in the mucosa of patients undergoing radiotherapy or chemotherapy, which has marked effects on patient quality of life and cancer therapy continuity. Considering that few protocols have demonstrated efficacy in preventing this side effect, the aim of this study was to examine the effect of dexamethasone (DEX) on OM induced by 5-fluorouracil (5-FU) in hamsters by studying signaling pathways. OM was induced in hamsters by 5-FU followed by mechanical trauma (MT) on day 4. On day 10, the animals were euthanized. The experimental groups included saline, MT, 5-FU, and DEX (0.25, 0.5, or 1 mg/kg). Macroscopic, histopathological, and immunohistochemical analyses as well as immunofluorescence experiments were performed on the oral mucosa of the animals. The oral mucosal samples were analyzed by enzyme-linked immunosorbent assays, and quantitative real-time polymerase chain reaction (qPCR). DEX (0.5 or 1 mg/kg) reduced inflammation and ulceration of the oral mucosa of hamsters. In addition, DEX (1 mg/kg) reduced the cytokine levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and macrophage migration inhibitory factor (MIF). DEX (1 mg/kg) also reduced the immunoexpression of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-2, transforming growth factor (TGF)-β, MIF, Smad 2/3, Smad 2/3 phosphorylated and NFκB p65 in the jugal mucosa. Finally, DEX (1 mg/kg) increased interleukin-1 receptor-associated kinase 3 (IRAK-M), glucocorticoid-induced leucine zipper (GILZ), and mitogen-activated protein kinase (MKP1) gene expression and reduced NFκB p65 and serine threonine kinase (AKt) gene expression, relative to the 5-FU group. Thus, DEX improved OM induced by 5-FU in hamsters.

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus of the Iridoviridae family. It causes a serious and potentially pandemic disease in wild and cultured fishes. ISKNV infection induces evident apoptosis in mandarin fish (Siniperca chuatsi) and zebrafish (Danio renio). However, the mechanism is still unknown. After a genome-wide bioinformatics analysis of ISKNV-encoded proteins, the ISKNV open reading frame 111L (ORF111L) shows a high similarity to the tumour necrosis factor receptor-associated factor (TRAF) encoded by fish, mice and mammals, which is essential for apoptotic signal transduction. Moreover, ORF111L was verified to directly interact with the zebrafish TNF receptor type 1 associated death domain protein (TRADD). A recombinant plasmid containing the DNA sequence of ORF111L was constructed and microinjected into zebrafish embryos at the 1-2 cell stage to investigate its biological function in vivo. ORF111L overexpression in the embryos resulted in increased apoptosis. ORF111L-induced apoptosis was clearly associated with significant caspase 8 upregulation and activation. The knockdown of zebrafish caspase 8 expression effectively blocked the apoptosis induced by ORF111L overexpression. Significantly, ORF111L overexpression resulted in much stronger effect on caspase 8 and caspase 3 upregulation compared to zebrafish TRAF2. This is the first report of a viral protein similar to TRAF that interacts with TRADD and induces caspase 8-mediated apoptosis, which may provide novel insights into the pathogenesis of ISKNV infection.

Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

Our laboratory reported previously that TNF receptor associated factor 3 (TRAF3) is a positive regulator of TCR signaling and T cell function. In the current study, we present new findings that reveal differential roles for TRAF3 in the regulation of CD4+ and CD8(+) T cells. In response to TCR stimulation in vitro, TRAF3 has greater impact in CD4(+) T cells than in CD8+ T cells. However, T cell-specific TRAF3 deficient mice (CD4Cre TRAF3(fl°x)/(fl°x); T-TRAF3(-/-)) have a greater number of CD4(+)CD44(hi) effector/memory T cells than littermate control (LMC) mice, possibly due to an inefficient suppressive effect of TRAF3 deficient Foxp3+ regulatory T cells. In contrast, CD8(+)CD44(hi)CD62L(hi) central memory (Tcm) cells are markedly reduced in T-TRAF3(-/-) mice in comparison to LMC mice, although CD8(+)CD44(hi)CD62L(l°w) effector memory T (Tem) cells and naïve T cells (CD8(+)CD44(l°w)CD62L(hi)) do not show significant differences in number. Importantly, TRAF3-deficient Tcm cells exhibit defective homeostasis due to impaired IL-15 signaling. These results indicate that the involvement of TRAF3 in IL-15 mediated signaling to T cells plays a previously unappreciated and critical role in CD8(+) Tcm cell regulation and maintenance.

We have demonstrated that the renal endonuclease DNaseI is up-regulated in mesangial nephritis while down-regulated during progression of the disease. To determine the basis for these reciprocal DNaseI expression profiles we analyse processes accounting for an early increase in renal DNaseI expression. Main hypotheses were that i. the mesangial inflammation and secreted pro-inflammatory cytokines directly increase DNaseI protein expression in tubular cells, ii. the anti-apoptotic protein tumor necrosis factor receptor-associated protein 1 (Trap 1) is down-regulated by increased expression of DNaseI due to transcriptional interference, and iii. pro-inflammatory cytokines promote nuclear translocation of a variant of DNaseI. The latter hypothesis emerges from the fact that anti-DNaseI antibodies stained tubular cell nuclei in murine and human lupus nephritis. The present study was performed on human tubular epithelial cells stimulated with pro-inflammatory cytokines. Expression of the DNaseI and Trap 1 genes was determined by qPCR, confocal microscopy, gel zymography, western blot and by immune electron microscopy. Results from in vitro cell culture experiments were analysed for biological relevance in kidneys from (NZBxNZW)F1 mice and human patients with lupus nephritis. Central data indicate that stimulating the tubular cells with TNFα promoted increased DNaseI and reduced Trap 1 expression, while TNFα and IL-1β stimulation induced nuclear translocation of the DNaseI. TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI. IL-1β-stimulation solely induced nuclear DNaseI translocation. Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear translocation. This demonstrates that IL-1β promotes nuclear translocation of a cytoplasmic variant of DNaseI since translocation clearly was not dependent on DNaseI gene activation. Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.