IL-36γ is a crucial proximal component of protective type-1-mediated lung mucosal immunity in Gram-positive and -negative bacterial pneumonia

Interleukin-36γ (IL-36γ) is a member of novel IL-1-like proinflammatory cytokine family that are highly expressed in epithelial tissues and several myeloid-derived cell types. Little is known about the role of the IL-36 family in mucosal immunity, including lung anti-bacterial responses. We used murine models of IL-36γ deficiency to assess the contribution of IL-36γ in the lung during experimental pneumonia. Induction of IL-36γ was observed in the lung in response to Streptococcus pneumoniae (Sp) infection, and mature IL-36γ protein was secreted primarily in microparticles. IL-36γ-deficient mice challenged with Sp demonstrated increased mortality, decreased lung bacterial clearance and increased bacterial dissemination, in association with reduced local expression of type-1 cytokines, and impaired lung macrophage M1 polarization. IL-36γ directly stimulated type-1 cytokine induction from dendritic cells in vitro in a MyD88-dependent manner. Similar protective effects of IL-36γ were observed in a Gram-negative pneumonia model (Klebsiella pneumoniae). Intrapulmonary delivery of IL-36γ-containing microparticles reconstituted immunity in IL-36γ^−/− mice. Enhanced expression of IL-36γ was also observed in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome because of pneumonia. These studies indicate that IL-36γ assumes a vital proximal role in the lung innate mucosal immunity during bacterial pneumonia by driving protective type-1 responses and classical macrophage activation.     

Herpes simplex virus type 2 infection of macrophages impairs IL-4-mediated inhibition of NO production through TNF-α-induced activation of NF-κB

Summary.  Nitric oxide (NO) production in macrophages by the interferon (IFN)-γ-inducible NO synthase has been shown to play a role in clearance of viral infections. We have previously shown that IFN-γ-induced NO production is augmented by herpes simplex virus type 2 (HSV-2) infection through autocrine tumour necrosis factor (TNF)-α secretion and is inhibited by interleukin (IL)-4. Here we investigated the effect of HSV-2 infection on the inhibitory function of IL-4. Virus infection of mouse J774A.1 macrophages strongly reduced the ability of IL-4 to inhibit IFN-γ-induced NO production, even at very high IL-4 concentrations. The effect of HSV-2 infection did not involve the IL-4 signal transduction pathway through STAT6. IL-4 reduced virus-induced TNF-α secretion and nuclear factor (NF)-κB activation significantly, but less in cells concomitantly treated with IFN-γ. Furthermore, neutralisation of residual TNF-α activity or inhibition of NF-κB activation largely restored the inhibitory effect of IL-4. The data show that inhibition of IFN-γ-induced NO production by IL-4 is impaired by HSV-2 infection due to autocrine TNF-α-mediated NF-κB activation. We suggest that the described phenomenon might be beneficial for the host by limiting high and sustained NO production to infectious foci. Accepted September 9, 1999     

IKBKE is over-expressed in glioma and contributes to resistance of glioma cells to apoptosis via activating NF-κB

IκB kinase-ε (IKBKE), a member of the IκB kinase (IKK) family, has been identified as an oncogenic protein and found to be up-regulated in breast cancer, ovarian cancer and prostate cancer. Nonetheless, the expression status and functional significance of IKBKE in human glioma remain unexplored. For the first time, we have demonstrated that mRNA and protein levels of IKBKE were robustly up-regulated in glioma cell lines and human primary glioma tissues. Immunohistochemistry analysis revealed that 53.5% (38/71) paraffin-embedded archived glioma specimens exhibited high levels of IKBKE expression. Intriguingly, there was no significant difference in IKBKE expression among different grades of glioma. To understand the biological function of IKBKE in the development and progression of human glioma, glioma cells lines ectopically over-expressing IKBKE were established and tested for their responsiveness to apoptotic inducers. Our data showed that IKBKE over-expression inhibited cell apoptosis induced by UV irradiation or adriamycin and, in contrast, shRNAi-mediated suppression of IKBKE increased the sensitivity of glioma cells to the apoptotic inducers. Importantly, we found that up-regulated IKBKE could induce the expression of Bcl-2 through activating NF-κB signalling, and that, specifically, we identified IκB as a critical component for this signalling cascade. The current study suggests that up-regulation of IKBKE may represent an important molecular hallmark that is biologically and clinically relevant to the development and progression, as well as the chemo- and radio-resistance, of the disease.     

Roles of TRAF2 and TRAF3 in Epstein-Barr virus latent membrane protein 1-induced alternative NF-κB activation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1)-induced NF-κB activation is essential for EBV-transformed B cell survival. LMP1 has two C-terminal cytoplasmic domains referred to as C-Terminal Activation Regions (CTAR) 1 and 2 that activate the alternative and canonical NF-κB pathways, respectively. While CTAR2 activates TRAF6, IKKβ and IKKγ-dependent canonical NF-κB pathway, CTAR1 interacts with TRAF2 and TRAF3 and activates NIK and IKKα-dependent alternative NF-κB pathway involving p100 processing into functional p52. Using IKKα^−/−, IKKβ^−/−, IKKγ^−/−, TRAF2^−/−, TRAF3^−/−, TRAF6^−/−, and NIK^aly/aly mouse embryonic fibroblasts (MEFs), potential roles of these proteins in LMP1-induced alternative NF-κB activation were investigated. Deficiency in IKKα or functional NIK, but not in IKKβ, IKKγ, or TRAF6, severely impaired LMP1-induced p100 processing. Notably, p100 was constitutively processed in TRAF2^−/− or TRAF3^−/− MEFs independently of LMP1 suggesting that TRAF2 or TRAF3 may play a regulatory role in p100 processing. Subsequently, TRAF2 or TRAF3 over-expression in HEK293 cells significantly blocked LMP1-induced p100 processing. The LMP1 CTAR1 expression in 293HEK cells activated the alternative p65/p52 complex while CTAR2 failed to do so. Taken together, LMP1 activates alternative NF-κB pathway through functional NIK and IKKα that is regulated by TRAF2 or TRAF3.     

Epstein-Barr virus LMP2A utilizes Syk and PI3K to activate NF-κB in B-cell lymphomas to increase MIP-1α production

The incidence of Hodgkin's lymphoma (HL) is growing due to an increase in Epstein-Barr virus (EBV)-associated HL in AIDS patients. The HL tumor microenvironment is vital for the survival of the malignant Hodgkin-Reed Sternberg (HRS) cells of HL, which express the EBV protein latent membrane protein 2A (LMP2A). While previous work shows that LMP2A mimics B-cell receptor (BCR) signaling to promote the survival of HRS cells, the ability of LMP2A to establish and maintain the tumor microenvironment through the production of chemokines remains unknown. Since BCR signaling induces the production of the chemokine macrophage inflammatory protein-1α (MIP-1α), and since LMP2A is a BCR mimic, we hypothesized that LMP2A increases MIP-1α levels. A comparison of multiple LMP2A-negative and -positive cell lines demonstrates that LMP2A increases MIP-1α. Additionally, LMP2A-mutant cell lines and pharmacologic inhibitors indicate that LMP2A activates a Syk/PI3K/NF-κB pathway to enhance MIP-1α. Finally, based on the finding that an NF-κB inhibitor decreased MIP-1α RNA/protein in LMP2A-positive cells, we are the first to demonstrate that LMP2A increases the nuclear localization of the NF-κB p65 subunit using DNA-binding assays and confocal microscopy in human B cells. These findings not only have implications for the treatment of HL, but also other LMP2A-expressing B-cell tumors that overexpress NF-κB.     

Effect of herpes simplex virus type-1 UL41 gene on the stability of mRNA from the cellular genes: β-actin,fibronectin, glucose transporter-1, and docking protein,and on virus intraperitoneal pathogenicity to newborn mice

Infection with HSV-1 is accompanied by the shut-off of cellular gene expression. The virion-associated function is encoded by the viral gene U_L41. An HSV-1 mutant, vhs-1, which has a genomic deletion in the U_L41 gene, is incapable of inducing the shut-off of cellular gene expression. The effect of HSV-1 infection on the shut-off of the cellular genes (or mRNA degradation) was studied specifically with the cellular genes for -actin, fibronectin, glucose transporter-1, and the docking protein. The level of these specific mRNAs was measured in cells infected with several HSV-1 strains and was compared to that of vhs-1- and mock-infected cells. It was possible to demonstrate a marked reduction in the level of the specific mRNA from these cellular genes in cells infected with several HSV-1 strains but not with the vhs-1 mutant. The pathogenicity of the HSV-1 vhs-1 mutant to newborn mice was studied. It was found that the mutant is less pathogenic to newborn mice than its parental strain HSV-1 KOS.     

A key mediator,PTX3, of IKK/IκB/NF-κB exacerbates human umbilical vein endothelial cell injury and dysfunction

Objective: This study was performed to investigate PTX3-mediated iNOS expression and IKK/IκB/NF-κB activation in PA-induced atherosclerotic HUVECs injury model. Methods: The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining. Expression of genes and proteins were analyzed by real-time PCR and western blotting respectively. Cells were transfected with siRNAs as a gene silencing methods. Results: PA induced cell apoptosis in human umbilical vein endothelial cells in a time and dose-dependent manner. PA also induced upregulation expression of PTX3. TPCA-1, an inhibitor of IKK-2, could suppress the expression of PTX3 and phospho-IκB-α in PA-induced endothelial dysfunction cell model. We also found that transfection of cells with PTX3 siRNA reduced the expression of iNOS and NO, and protected PA-induced cell apoptosis in HUVECs. Conclusions: PTX3 could exacerbate endothelial dysfunction, at least partially, through IKK/IκB/NF-κB activation and overexpression of iNOS and NO, and advance the development of atherosclerosis.     

Effects of Trefoil Peptide 3 on Expression of TNF-α, TLR4, and NF-κB in Trinitrobenzene Sulphonic Acid Induced Colitis Mice

The trefoil factor (TFF) peptides are major secretory products of mucus cells of the gastrointestinal tract. There were evidences that administration of recombinant human TFF3 is effective in treatment of models of colitis, but the mechanism of the effects of rTFF3 is not fully understood. The main aims of this study is to evaluate effects of intraperitoneal injection recombinant human TFF3 on the expression of tumour necrosis factor α (TNF-α), toll-like receptor 4(TLR4), and nuclear factor κB (NF-κB) in trinitrobenzene sulphonic acid (TNBS) induced colitis mice. Distal colitis was induced in BALB/C mice by intracolonic administration of TNBS in ethanol. Treated with administration rhTFF3 for treatment group(5 mg/ml; approximately 0.5 mg/mouse), and normal saline for control for 5 consecutive days. Colonic damage score, tissue myeloperoxidase (MPO) activity, TLR4, NF-κB mRNA expression, and tissue TNF-α, TLR4, NF-κB production were determined, respectively. Once daily application of hTFF3 for 5 days after TNBS/ethanol had been injected, both microscopic and macroscopic injury and inflammatory index had been reduced compared with controls. In addition, decreased tissue TNF-α, TLR4, NF-κB production, and TLR4, NF-κB mRNA expression had been found. This study has shown that hTFF3 may have therapeutic potential in the treatment of inflammatory bowel disease, and one of the mechanisms may related to inhibit the TLR4/NF-κB signaling pathways.     

Shrimp NF-κB binds to the immediate-early gene ie1 promoter of white spot syndrome virus and upregulates its activity

The immediate-early gene ie1 carried by white spot syndrome virus (WSSV) exhibits very strong promoter activity and expresses highly throughout the infection cycle. Here we identified a NF-κB binding motif in the ie1 promoter region. Electrophoretic mobility shift assays indicated that the recombinant Rel homology domain (RHD) of shrimp NF-κB homolog LvRelish bound to the putative NF-κB site in the ie1 promoter. A transactivity assay of the WSSV ie1 promoter in Drosophila Schneider 2 cells demonstrated that LvRelish could increase ie1 promoter activity. These results show that shrimp NF-κB homolog LvRelish transactivates WSSV ie1 gene expression and contributes to its high promoter activity. Further transactivation assays showed that WSSV IE1 protein expression upregulated the promoter activities of WSSV ie1 gene and antimicrobial peptide genes regulated by the NF-κB system. We suggested that WSSV may annex the shrimp NF-κB system, which it uses to enhance the expression of viral immediate-early genes.     

The response to TLR ligation of human CD16⁺CD14⁻ monocytes is weakly modulated as a consequence of persistent infection with the hepatitis C virus

Little is known about the frequency and function of CD16(+)CD14(-) monocytes from chronic HCV patients. We observed that the absolute numbers and ratio of CD16(+)CD14(-) to CD14(+)CD16(-) monocytes were similar between chronic HCV patients and healthy individuals. Functionally, we found that CD16(+)CD14(-) monocytes are more responsive to TLR8-ligation and only weakly responsive to LPS stimulation in producing TNF as compared to CD14(+)CD16(-) monocytes. We found no overt impairment of the function of CD16(+)CD14(-) monocytes from patients, except for an augmented induction of MIP-1β-producing CD16(+)CD14(-) monocytes upon TLR4-ligation. However, the increased frequency of MIP-1β-producing CD16(+)CD14(-) monocytes was not associated with viral load, ALT or fibrosis level. Our findings indicate that, different from other infectious diseases, the frequency and function of CD16(+)CD14(-) monocytes are only minimally altered as a consequence of the persistent state of HCV infections, and our findings therefore do not suggest a role for CD16(+)CD14(-) monocytes in HCV pathogenesis.     

Epstein-Barr virus–associated smooth muscle tumor—report of 3 tumors including 1 intracerebral case with a prominent intratumoral B-lymphocytic component and plasma cells

Three Epstein-Barr virus–associated smooth muscle tumors (intracranial, endobronchial, and paraspinal) in 2 patients are presented. The patient with the intracranial tumor had no concurrent or previous manifestation of this neoplasm and was immunosuppressed because of a renal transplant. The other patient had AIDS and radiologic evidence of involvement of multiple other sites. Although mitotic activity was present, none of the tumors displayed any high-grade malignant histologic features. All tumors were strongly positive for smooth muscle actin and variably expressed h-caldesmon. Desmin was negative in all 3 tumors. The intracranial tumor was associated with prominent intracerebral edema and displayed, in addition to a prominent intratumoral T-cell component, a hitherto unreported component of prominent intratumoral B cells and plasma cells.     

PI3K/AKT/mTOR and TLR4/MyD88/NF-κB Signaling Inhibitors Attenuate Pathological Mechanisms of Allergic Asthma

Inflammation - Asthma is an inflammatory airway disease wherein bronchoconstriction, airway inflammation, and airway obstruction during asthma attacks are the main problems. It is recognized that...     

NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug

Background

Endometrial regenerative cells (ERC) and bone marrow stromal cells (BMSC) are being used in clinical trials. While they have been reported to have similar characteristics, they have not been directly compared.

Methods

We compared micro RNA (miRNA) and gene expression profiles, soluble cytokine and growth factor levels and ability to inhibit ongoing mixed leukocyte reaction (MLR) of ERC and BMSC each derived from 6 healthy subjects.

Results

ERC and BMSC miRNA and gene expression profiles were similar, but not identical; more differences were noted in the expression of genes than in miRNAs. Genes overexpressed in ERCs were more likely to be in immune and inflammation pathways and those overexpressed in BMSCs were more likely to be in stem cell and cancer signaling pathways. In addition, the levels of IL-8 and ICAM-1 were greater in ERC supernatants while the levels of HGF, VEGF, IL-6, CXCL12, TGFB1 and TGFB2 were greater in BMSC supernatants. Additionally, ERC demonstrated greater inhibition of the proliferation of mixed leukocyte cultures.

Conclusions

These results suggest that the in vivo effects of ERC and BMSC may differ. Multiple properties of stromal cells are responsible for their in vivo effectiveness and ERC may be more effective for some of the clinical applications and BMSC for others. Studies in animal models or clinical trials will be required to more fully characterize the differences between ERC and BMSC.     

The NS1 protein of H5N6 feline influenza virus inhibits feline beta interferon response by preventing NF-κB and IRF3 activation

Despite the apparent lack of a feline influenza virus lineage, cats are susceptible to infection by influenza A viruses. Here, we characterized in vitro A/feline/Guangdong/1/2015, an H5N6 avian influenza virus recently isolated from cats. A/feline/Guangdong/1/2015 replicated to high titers and caused CPE in feline kidney cells. We determined that infection with A/feline/Guangdong/1/2015 did not activate the IFN-β promoter, but inhibited it by blocking the activation of NF-κB and IRF3. We also determined that the viral NS1 protein mediated the block, and that the dsRNA binding domain of NS1 was essential to perform this function. In contrast to treatment after infection, cells pretreated with IFN-β suppressed viral replication. Our findings provide an example of an H5N6 influenza virus suppressing IFN production, which might be associated with interspecies transmission of avian influenza viruses to cats.     

A Novel Missense Mutation in the Nuclear Factor-κB Essential Modulator (NEMO) Gene Resulting in Impaired Activation of the NF-κB Pathway and a Unique Clinical Phenotype Presenting as MRSA Subdural Empyema

Introduction  

We describe a previously unreported 437 T→G missense mutation producing a V146G substitution in the first coiled-coil (CC1) domain of nuclear factor-κB essential modulator (NEMO) in a 9-month-old boy with ectodermal dysplasia with immunodeficiency who presented with methicillin-resistant Staphylococcus aureus subdural empyema. We performed in vitro experiments to determine if this novel mutation resulted in impaired NF-κB signaling.