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The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. GermlineiNfkb/i2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adultiNfkb2/isup-/-/supmice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis iniNfkb2/isup-/-/supmice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi ofiNfkb2/isup-/-/supmice. This phenotype was even more striking in the small intestinal mucosa ofiRelB/isup-/-/supmice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as theiriRelB/isup+/+/supwild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.bNEWamp; NOTEWORTHY/bNovel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature iniNfkb2/isup-/-/supmice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA–signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Basic research in cardiology 117(1), 30 (2022). doi:10.1007/s00395-022-00937-4, Published by Springer, Berlin

Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1-mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing) proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1β production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1β expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in Rnh1 -/- mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs., Peripheral T cell lymphoma (PTCL) not otherwise specified (NOS) is a subgroup of PTCL, which has no distinctive features and is poorly characterized at the genetic level. Here, the authors identify two fusion transcripts that activate T cell receptor complex signalling and confer therapeutic vulnerability in PTCL-NOS.

Background Diffuse large B-cell lymphoma (DLBCL) comprises at least two main biologically distinct entities: germinal center B-cell (GCB) and activated B-cell (ABC) subtype. Albeit sharing common lesions, GCB and ABC DLBCL present subtype-specific oncogenic pathway perturbations. ABC DLBCL is typically characterized by a constitutively active NF-kB. However, the latter is seen in also 30% of GCB DLBCL. Another recurrent lesion in DLBCL is an 11q24.3 gain, associated with the overexpression of two ETS transcription factors, ETS1 and FLI1. Here, we showed that FLI1 is more expressed in GCB than ABC DLBCL and we characterized its transcriptional network. Methods Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013 and GSE10846. ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNAs) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was done using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. ChIP-Seq and RNA-Seq data from GCB DLBCL cell lines after FLI1 downregulation were integrated to identify putative direct targets of FLI1. Results Analysis of clinical DLBCL specimens showed that FLI1 gene was more frequently expressed at higher levels in GCB than in ABC DLBCL and its protein levels were higher in GCB than in ABC DLBCL cell lines. Genes negatively regulated by FLI1 included tumor suppressor genes involved in negative regulation of cell cycle and hypoxia. Among positively regulated targets of FLI1, we found genes annotated for immune response, MYC targets, NF-κB and BCR signaling and NOTCH pathway genes. Of note, direct targets of FLI1 overlapped with genes regulated by ETS1, the other transcription factor gained at the 11q24.3 locus in DLBCL, suggesting a functional convergence within the ETS family. Positive targets of FLI1 included the NF-κB-associated ASB2 a putative essential gene for DLBCL cell survival. ASB2 gene downregulation was toxic in GCB DLBCL cell lines and induced NF-κB inhibition via downregulation of RelB and increased IκBα. Additionally, downregulation of FLI1, but not ASB2, caused reduction of NF-κB1 and RelA protein levels. Conclusions We conclude that FLI1 directly regulates a network of biologically crucial genes and processes in GCB DLBCL. FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL.

Background: Intercellular communication mediated by cationic fluxes through the Connexin family of gap junctions regulates glucose-stimulated insulin secretion and beta cell defense against inflammatory stress. Rotigaptide (RG, ZP123) is a peptide analog that increases intercellular conductance in cardiac muscle cells by the prevention of dephosphorylation and thereby uncoupling of Connexin-43 (Cx43), possibly via action on unidentified protein phosphatases. For this reason, it is being studied in human arrhythmias. It is unknown if RG protects islet cell function and viability against inflammatory or metabolic stress, a question of considerable translational interest for the treatment of diabetes. Methods: Apoptosis was measured in human islets shown to express Cx43, treated with RG or the control peptide ZP119 and exposed to glucolipotoxicity or IL-1&beta, + IFNɣ. INS-1 cells shown to lack Cx43 were used to examine if RG protected human islet cells via Cx43 coupling. To study the mechanisms of action of Cx43-independent effects of RG, NO, IkB&alpha, degradation, mitochondrial activity, ROS, and insulin mRNA levels were determined. Results: RG reduced cytokine-induced apoptosis ~40% in human islets. In Cx43-deficient INS-1 cells, this protective effect was markedly blunted as expected, but unexpectedly, RG still modestly reduced apoptosis, and improved mitochondrial function, insulin-2 gene levels, and accumulated insulin release. RG reduced NO production in Cx43-deficient INS-1 cells associated with reduced iNOS expression, suggesting that RG blunts cytokine-induced NF-&kappa, B signaling in insulin-producing cells in a Cx43-independent manner. Conclusion: RG reduces cytokine-induced cell death in human islets. The protective action in Cx43-deficient INS-1 cells suggests a novel inhibitory mechanism of action of RG on NF-&kappa, B signaling.

BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies.MethodsTo identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs.ResultsThe screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB)nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF–NF-κBaxis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis.ConclusionOur data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.

Aims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2-/- mice. Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2.

We consider how a signalling system can act as an information hub by multiplexing information arising from multiple signals. We formally define multiplexing, mathematically characterise which systems can multiplex and how well they can do it. While the results of this paper are theoretical, to motivate the idea of multiplexing, we provide experimental evidence that tentatively suggests that the NF-κB transcription factor can multiplex information about changes in multiple signals. We believe that our theoretical results may resolve the apparent paradox of how a system like NF-κB that regulates cell fate and inflammatory signalling in response to diverse stimuli can appear to have the low information carrying capacity suggested by recent studies on scalar signals. In carrying out our study, we introduce new methods for the analysis of large, nonlinear stochastic dynamic models, and develop computational algorithms that facilitate the calculation of fundamental constructs of information theory such as Kullback–Leibler divergences and sensitivity matrices, and link these methods to a new theory about multiplexing information. We show that many current models such as those of the NF-κB system cannot multiplex effectively and provide models that overcome this limitation using post-transcriptional modifications., Author summary Cells use signalling systems to pass on information arising from their ever-changing environment to their processing units. These biochemical networks regulate the transmission of multiple signals within the noisy and complex cellular environment, controlling whether to turn on or off processes of cell defence, death, division, and others. The question of how they actually achieve that becomes particularly critical given that many diseases occur when signalling systems malfunction. In this paper, we develop methodology and computational tools for simulating, measuring and analysing the ability of signalling systems to transmit multi-dimensional signals. We specifically focus on the capacity of signalling systems to simultaneously transmit multiple signals, such as temperature changes, presence and concentration of cytokines, viral and bacterial pathogens or drugs, through a single noisy, dynamic signalling system. We argue that a signalling system can act as an information hub, sending information in a multiplexed fashion rather similar to the way in which telecommunications networks send multiple signals over a shared medium by combining them into one.

Novel anti–PD-1 antibodies (Abs) not blocking the PD-1–PDL-1 interaction are presented with equivalent antagonistic activity to classical blocking anti–PD-1 Abs and have distinct mechanisms of action that synergize in functional recovery of exhausted CD8 T cells and enhancing tumor suppression in an immunogenic mouse tumor model., Classical antagonistic antibodies (Abs) targeting PD-1, such as pembrolizumab and nivolumab, act through blockade of the PD-1–PDL-1 interaction. Here, we have identified novel antagonistic anti–PD-1 Abs not blocking the PD-1–PDL-1 interaction. The nonblocking Abs recognize epitopes on PD-1 located on the opposing face of the PDL-1 interaction and overlap with a newly identified evolutionarily conserved patch. These nonblocking Abs act predominantly through the CD28 coreceptor. Importantly, a combination of blocking and nonblocking Abs synergize in the functional recovery of antigen-specific exhausted CD8 T cells. Interestingly, nonblocking anti–PD-1 Abs have equivalent antitumor activity compared with blocker Abs in two mouse tumor models, and combination therapy using both classes of Abs enhanced tumor suppression in the mouse immunogenic tumor model. The identification of the novel nonblocker anti–PD-1 Abs and their synergy with classical blocker Abs may be instrumental in potentiating immunotherapy strategies and antitumor activity.

Oxidative stress (OS) occurs in brains of patients with epilepsy and coincides with brain inflammation, and both phenomena contribute to seizure generation in animal models. We investigated whether expression of OS and brain inflammation markers co-occurred also in resected brain tissue of patients with epileptogenic cortical malformations: hemimegalencephaly (HME), focal cortical dysplasia (FCD) and cortical tubers in tuberous sclerosis complex (TSC). Moreover, we studied molecular mechanisms linking OS and inflammation in an in vitro model of neuronal function. Untangling interdependency and underlying molecular mechanisms might pose new therapeutic strategies for treating patients with drug-resistant epilepsy of different etiologies. Immunohistochemistry was performed for specific OS markers xCT and iNOS and brain inflammation markers TLR4, COX-2 and NF-κB in cortical tissue derived from patients with HME, FCD IIa, IIb and TSC. Additionally, we studied gene expression of these markers using the human neuronal cell line SH-SY5Y in which OS was induced using H2O2 . OS markers were higher in dysmorphic neurons and balloon/giant cells in cortex of patients with FCD IIb or TSC. Expression of OS markers was positively correlated to expression of brain inflammation markers. In vitro, 100 µM, but not 50 µM, of H2O2 increased expression of TLR4, IL-1β and COX-2. We found that NF-κB signaling was activated only upon stimulation with 100 µM H2O2 leading to upregulation of TLR4 signaling and IL-1β. The NF-κB inhibitor TPCA-1 completely reversed this effect. Our results show that OS positively correlates with neuroinflammation and is particularly evident in brain tissue of patients with FCD IIb and TSC. In vitro, NF-κB is involved in the switch to an inflammatory state after OS. We propose that the extent of OS can predict the neuroinflammatory state of the brain. Additionally, antioxidant treatments may prevent the switch to inflammation in neurons thus targeting multiple epileptogenic processes at once.

Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor β (TGF-β)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex.

Medullary thymic epithelial cells (mTEC) purge the T cell repertoire of autoreactive thymocytes. Although dendritic cells (DC) reinforce this process by transporting innocuous peripheral self-antigens, the mechanisms that control their thymic entry remain unclear. Here we show that mTEC-CD4+ thymocyte crosstalk regulates the thymus homing of SHPS-1+ conventional DCs (cDC), plasmacytoid DCs (pDC) and macrophages. This homing process is controlled by lymphotoxin α (LTα), which negatively regulates CCL2, CCL8 and CCL12 chemokines in mTECs. Consequently, Ltα-deficient mice have increased expression of these chemokines that correlates with augmented classical NF-κB subunits and increased thymic recruitment of cDCs, pDCs and macrophages. This enhanced migration depends mainly on the chemokine receptor CCR2, and increases thymic clonal deletion. Altogether, this study identifies a fine-tuning mechanism of T cell repertoire selection and paves the way for therapeutic interventions to treat autoimmune disorders., Autoreactive T cells are removed during their development in the thymus through the functions of medullary thymic epithelial cells (mTEC) and dendritic cells (DC), a process termed negative selection. Here the authors show that mTEC-T cell crosstalk and lymphotoxin α signalling are essential for the proper recruitment of DCs into the thymus.

Inflammatory β-cell failure contributes to type 1 and type 2 diabetes pathogenesis. Pro-inflammatory cytokines cause β-cell dysfunction and apoptosis, and lysine deacetylase inhibitors (KDACi) prevent β-cell failure in vitro and in vivo, in part by reducing NF-κB transcriptional activity. We investigated the hypothesis that the protective effect of KDACi involves transcriptional regulation of microRNAs (miRs), potential new targets in diabetes treatment. Insulin-producing INS1 cells were cultured with or without the broad-spectrum KDACi Givinostat, prior to exposure to the pro-inflammatory cytokines IL-1β and IFN-γ for 6 h or 24 h, and miR expression was profiled with miR array. Thirteen miRs (miR-7a-2-3p, miR-29c-3p, miR-96-5p, miR-101a-3p, miR-140-5p, miR-146a-5p, miR-146b-5p, miR-340-5p, miR-384-5p, miR-455-5p, miR-466b-2-3p, miR-652-5p, and miR-3584-5p) were regulated by both cytokines and Givinostat, and nine were examined by qRT-PCR. miR-146a-5p was strongly regulated by cytokines and KDACi and was analyzed further. miR-146a-5p expression was induced by cytokines in rat and human islets. Cytokine-induced miR-146a-5p expression was specific for INS1 and β-TC3 cells, whereas α-TC1 cells exhibited a higher basal expression. Transfection of INS1 cells with miR-146a-5p reduced cytokine signaling, including the activity of NF-κB and iNOS promoters, as well as NO production and protein levels of iNOS and its own direct targets TNF receptor associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1). miR-146a-5p was elevated in the pancreas of diabetes-prone BB-DP rats at diabetes onset, suggesting that miR-146a-5p could play a role in type 1 diabetes development. The miR array of cytokine-exposed INS1 cells rescued by KDACi revealed several other miRs potentially involved in cytokine-induced β-cell apoptosis, demonstrating the strength of this approach.

Tumor-associated macrophages (TAMs) represent potential targets for anticancer treatments as these cells play critical roles in tumor progression and frequently antagonize the response to treatments. TAMs are usually associated to an M2-like phenotype, characterized by anti-inflammatory and protumoral properties. This phenotype contrasts with the M1-like macrophages, which exhibits proinflammatory, phagocytic, and antitumoral functions. As macrophages hold a high plasticity, strategies to orchestrate the reprogramming of M2-like TAMs towards a M1 antitumor phenotype offer potential therapeutic benefits. One of the most used anticancer treatments is the conventional X-ray radiotherapy (RT), but this therapy failed to reprogram TAMs towards an M1 phenotype. While protontherapy is more and more used in clinic to circumvent the side effects of conventional RT, the effects of proton irradiation on macrophages have not been investigated yet. Here we showed that M1 macrophages (THP-1 cell line) were more resistant to proton irradiation than unpolarized (M0) and M2 macrophages, which correlated with differential DNA damage detection. Moreover, proton irradiation-induced macrophage reprogramming from M2 to a mixed M1/M2 phenotype. This reprogramming required the nuclear translocation of NFκB p65 subunit as the inhibition of IκBα phosphorylation completely reverted the macrophage re-education. Altogether, the results suggest that proton irradiation promotes NFκB-mediated macrophage polarization towards M1 and opens new perspectives for macrophage targeting with charged particle therapy.

Background The addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line. Methods Effects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using [methyl-14C]-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses. Results Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment. Conclusion We show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development. Electronic supplementary material The online version of this article (10.1186/s12885-018-4346-1) contains supplementary material, which is available to authorized users.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes. Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH) 2D 3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH) 2D 3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH) 2D 3. In addition, 1,25(OH) 2D 3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH) 2D 3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH) 2D 3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH) 2D 3 against the induction of autoimmune diabetes.

Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-β1(TGF-β1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-β1signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

Infections with highly pathogenic avian influenza viruses (HPAIV) in humans lead to systemic disease associated with cytokine storm and multiorgan failure. In this study we aimed to identify the role of monocytes for the host response to HPAIV infection. Using genome-wide microarray analysis, we surprisingly demonstrate a reduced immune response of human monocytes to HPAIV H5N1 compared to human influenza A viruses. In bioinformatic analyses we could reveal a potential role of the Rar-related orphan receptor alpha (RORa) for the gene expression pattern induced by H5N1. RORa is known as an inhibitor of NF-κB signaling. We provide evidence that in monocytes RORa is activated by H5N1, resulting in inhibited NF-κB signaling. Using murine Hoxb8-immortalized RORa-/-, monocytes rescued NF-κB signaling upon H5N1 infection, confirming the biological relevance of RORa as an H5N1-induced mediator of monocytic immunosuppression. In summary, our study reveals a novel RORa-dependent escape mechanism by which H5N1 prevents an effective inflammatory response of monocytes blocking NF-κB-dependent gene expression.

Viruses have evolved a remarkable array of strategies to escape the host's innate immune responses. In this issue of Cell Host & Microbe, Zhao et al. (2016b) reveal a viral strategy to inactivate RIG-I signaling that relies on deamidation of RIG-I.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a critical mediator of tumor necrosis factor-α (TNF-α) signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) to be recruited into TNF receptor 1 (TNFR1) signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK) activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I). Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2.

Pathogens are sensed by Toll-like receptors (TLRs) and a growing number of non-TLR receptors. Integrins constitute a family of signaling receptors exploited by viruses and bacteria to access cells. By gain- and loss-of-function approaches we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against herpes simplex virus (HSV). αvβ3-integrin signaled through two pathways. One concurred with TLR2, affected activation/induction of interferons type 1 (IFNs-1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and a polarized set of cytokines and receptors. The virion glycoproteins gH/gL sufficed to induce IFN1 and NF-κB via this pathway. The other pathway was TLR2-independent, involved sarcoma (SRC)-spleen tyrosine kinase (SYK)-Caspase recruitment domain-containing protein 9 (CARD9)-TRIF (TIR-domain-containing adapter-inducing interferon-β), and affected interferon regulatory factor 3 and 7 (IRF3-IRF7). The importance of αvβ3-integrin-mediated defense is reflected in the observation that HSV evolved the immediate-early infected cellular protein 0 (ICP0) protein to counteract it. We propose that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors, a role likely exerted toward viruses and bacteria that interact with integrins and mount an innate response.

Obesity and type 2 diabetes present partially overlapping phenotypes with systemic inflammation as a common feature, raising the hypothesis that elevated cytokine levels may contribute to peripheral insulin resistance as well as the decreased beta cell functional mass observed in type 2 diabetes. In healthy humans, TNF-alpha infusion induces skeletal muscle insulin resistance. We now explore the impact of TNF-alpha on primary beta cell function and the underlying signaling pathways. Human and rat primary beta cells were sorted by FACS and cultured for 24 h +/- 20 ng/ml TNF-alpha to explore the impact on apoptosis, proliferation, and short-term insulin secretion (1 h, 2.8 mm glucose followed by 1 h, 16.7 mm glucose at the end of the 24-h culture period) as well as key signaling protein phosphorylation and expression. Prior exposure to TNF-alpha for 24 h inhibits glucose-stimulated insulin secretion from primary beta cells. This is associated with a decrease in glucose-stimulated phosphorylation of key proteins in the insulin signaling pathway including Akt, AS160, and other Akt substrates, ERK as well as the insulin receptor. Strikingly, TNF-alpha treatment decreased IRS-2 protein level by 46 +/- 7% versus control, although mRNA expression was unchanged. While TNF-alpha treatment increased MAP4K4 mRNA expression by 33 +/- 5%, knockdown of MAP4K4 by siRNA-protected beta cells against the detrimental effects of TNF-alpha on both insulin secretion and signaling. We thus identify MAP4K4 as a key upstream mediator of TNF-alpha action on the beta cell, making it a potential therapeutic target for preservation of beta cell function in type 2 diabetes.

Besides its classical effects on salt homeostasis in renal epithelial cells, aldosterone promotes inflammation and fibrosis and modulates cell proliferation. The proinflammatory transcription factor NF-kappaB has been implicated in cell proliferation, apoptosis, and regulation of transepithelial sodium transport. The effect of aldosterone on the NF-kappaB pathway in principal cells of the cortical collecting duct, a major physiologic target of aldosterone, is unknown. Here, in both cultured cells and freshly isolated rat cortical collecting duct, aldosterone activated the canonical NF-kappaB signaling pathway, leading to increased expression of several NF-kappaB-targeted genes (IkappaBalpha, plasminogen activator inhibitor 1, monocyte chemoattractant protein 1, IL-1beta, and IL-6). Small interfering RNA-mediated knockdown of the serum and glucocorticoid-inducible kinase SGK1, a gene induced early in the response to aldosterone, but not pharmacologic inhibition of extracellular signal-regulated kinase and p38 kinase, attenuated aldosterone-induced NF-kappaB activation. Pharmacologic antagonism or knockdown of the mineralocorticoid receptor prevented aldosterone-induced NF-kappaB activity. In addition, activation of the glucocorticoid receptor inhibited the transactivation of NF-kappaB by aldosterone. In agreement with these in vitro findings, spironolactone prevented NF-kappaB-induced transcriptional activation observed in cortical collecting ducts of salt-restricted rats. In summary, aldosterone activates the canonical NF-kappaB pathway in principal cells of the cortical collecting duct by activating the mineralocorticoid receptor and by inducing SGK1.

Tle1 (transducin-like enhancer of split 1) is a corepressor that interacts with a variety of DNA-binding transcription factors and has been implicated in many cellular functions; however, physiological studies are limited. Tle1-deficient (Tle1(Δ/Δ)) mice, although grossly normal at birth, exhibit skin defects, lung hypoplasia, severe runting, poor body condition, and early mortality. Tle1(Δ/Δ) mice display a chronic inflammatory phenotype with increased expression of inflammatory cytokines and chemokines in the skin, lung, and intestine and increased circulatory IL-6 and G-CSF, along with a hematopoietic shift toward granulocyte macrophage progenitor and myeloid cells. Tle1(Δ/Δ) macrophages produce increased inflammatory cytokines in response to Toll-like receptor (TLR) agonists and lipopolysaccharides (LPS), and Tle1(Δ/Δ) mice display an enhanced inflammatory response to ear skin 12-O-tetradecanoylphorbol-13-acetate treatment. Loss of Tle1 not only results in increased phosphorylation and activation of proinflammatory NF-κB but also results in decreased Hes1 (hairy and enhancer of split-1), a negative regulator of inflammation in macrophages. Furthermore, Tle1(Δ/Δ) mice exhibit accelerated growth of B6-F10 melanoma xenografts. Our work provides the first in vivo evidence, to our knowledge, that TLE1 is a major counterregulator of inflammation with potential roles in a variety of inflammatory diseases and in cancer progression.

The paracaspase MALT1 has a central role in the activation of lymphocytes and other immune cells including myeloid cells, mast cells and NK cells. MALT1 activity is required not only for the immune response, but also for the development of natural Treg cells that keep the immune response in check. Exaggerated MALT1 activity has been associated with the development of lymphoid malignancies, and recently developed MALT1 inhibitors show promising anti-tumor effects in xenograft models of diffuse large B cell lymphoma. In this review, we provide an overview of the present understanding of MALT1's function, and discuss possibilities for its therapeutic targeting based on recently developed inhibitors and animal models.

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.

Recent studies have uncovered important cross talk between inflammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of cardiovascular aging. Inhibition of the endocannabinoid anandamide metabolizing enzyme, the fatty acid amide hydrolase (FAAH), is emerging as a promising novel approach for the treatment of various inflammatory disorders. In this study, we have investigated the age-associated decline of cardiac function and changes in inflammatory gene expression, nitrative stress, and apoptosis in FAAH knockout (FAAH−/−) mice and their wild-type (FAAH+/+) littermates. Additionally, we have explored the effects of anandamide on TNF-α-induced ICAM-1 and VCAM-1 expression and monocyte-endothelial adhesion in human coronary artery endothelial cells (HCAECs). There was no difference in the cardiac function (measured by the pressure-volume conductance catheter system) between 2- to 3-mo-old (young) FAAH−/− and FAAH+/+ mice. In contrast, the aging-associated decline in cardiac function and increased myocardial gene expression of TNF-α, gp91phox, matrix metalloproteinase (MMP)-2, MMP-9, caspase-3 and caspase-9, myocardial inducible nitric oxide synthase protein expression, nitrotyrosine formation, poly (ADP-ribose)polymerase cleavage and caspase-3/9 activity, observed in 28- to 31-mo-old (aging) FAAH+/+ mice, were largely attenuated in knockouts. There was no difference in the myocardial cannabinoid CB1 and CB2 receptor gene expression between young and aging FAAH−/− and FAAH+/+ mice. Anandamide dose dependently attenuated the TNF-α-induced ICAM-1 and VCAM-1 expression, NF-κB activation in HCAECs, and the adhesion of monocytes to HCAECs in a CB1- and CB2-dependent manner. These findings suggest that pharmacological inhibition of FAAH may represent a novel protective strategy against chronic inflammation, oxidative/nitrative stress, and apoptosis associated with cardiovascular aging and atherosclerosis.

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants., Author Summary Mammary glands are the most characteristic feature of all mammals. The successful growth and function of the mammary glands is vital for the survival of offspring since the secreted milk is the main nutritional source of a new-born. Ectodysplasin (Eda) is a signaling molecule that regulates the formation of skin appendages such as hair, teeth, feathers, scales, and several glands in all vertebrates studied so far. In humans, mutations in the EDA gene cause a congenital disorder characterized by sparse hair, missing teeth, and defects in exocrine glands including the breast. We have previously shown that excess Eda induces formation of supernumerary mammary glands in mice. Here, we show that Eda leads to extra mammary gland formation also in the neck, a region previously not thought to harbor capacity to support mammary development. Using Eda loss- and gain-of-function mouse models and transcriptional profiling we identify the downstream mediators of Eda. The presence of extra nipples is a fairly common developmental abnormality in humans. We suggest that misregulation of Eda or its effectors might account for some of these malformations. Further, the number and location of the mammary glands vary widely between different species. Tinkering with the Eda pathway activity could provide an evolutionary means to modulate the number of mammary glands.

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease., Author Summary Neutrophil infiltration is characteristic of immune-induced pathology in tuberculosis but mechanisms whereby neutrophils cause tissue destruction are not fully understood. In this study, we show that neutrophils secrete the collagenase MMP-8 in response to direct infection with Mycobacterium tuberculosis and via cellular networks. MMP-8 is up-regulated in respiratory samples from TB patients, driving matrix destruction associated with neutrophil activation and reflects disease severity. Neutrophils are present adjacent to the wall of TB cavities in human histology specimens. The metabolic pathway AMP-activated protein kinase (AMPK) regulates neutrophil MMP-8 secretion with data supported by studies in human neutrophils from AMPK-deficient patients. Host-directed therapy against neutrophil MMP-8 may reduce innate-immune mediated tissue damage in TB.

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-alpha decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-alpha and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-alpha, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells.

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as “alternative therapies”. In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kappa B (NF kappa B) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NF kappa B gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NF kappa B signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

The nuclear factor κB (NF-κB) transcription factor is a master regulator of inflammation. Short-term NF-κB activation is generally beneficial. However, sustained NF-κB might be detrimental, directly causing apoptosis of cells or leading to a persistent damaging inflammatory response. NF-κB activity in stressed cells needs therefore to be controlled for homeostasis maintenance. In mildly stressed cells, caspase-3 cleaves p120 RasGAP, also known as RASA1, into an N-terminal fragment, which we call fragment N. We show here that this fragment is a potent NF-κB inhibitor. Fragment N decreases the transcriptional activity of NF-κB by promoting its export from the nucleus. Cells unable to generate fragment N displayed increased NF-κB activation upon stress. Knock-in mice expressing an uncleavable p120 RasGAP mutant showed exaggerated NF-κB activation when their epidermis was treated with anthralin, a drug used for the treatment of psoriasis. Our study provides biochemical and genetic evidence of the importance of the caspase-3-p120-RasGAP stress-sensing module in the control of stress-induced NF-κB activation.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway. We are grateful to Cesare Montecucco (Dipartimento di Scienze Biomediche Sperimentali dell'Úniversità di Padova and Instituto di Neuroscienze del CNR, Padua, Italy) for providing the PA and to Didier Cabanes (Group of Molecular Microbiology, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal) for providing the Listeria monocytogenes. Liliana M. G. Pereira and Daniela S. Silva were funded by Fundação para a Ciência e a Tecnologia (FCT) fellowships SFRH/BD/43501/2008 and SFRH/BD/35865/2007, respectively, financed by POPH-QREN and cofunded by Fundo Social Europeu and MCTES. Ana do Vale was supported by Programa Ciência, funded by POPH–QREN, Tipologia 4.2–Promoção do Emprego Científico, cofunded by Fundo Social Europeu, and national funding from MCTES. This work was supported by FEDER funds through the Operational Competitiveness Programme (COMPETE) and national funds through FCT under projects FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011) and FCOMP-01-0124-FEDER-009501 (PTDC/CVT/099544/2008).

AIMS: Tumour necrosis factor-alpha (TNF-alpha) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-alpha receptors (TNFRs), the mechanism of TNF-alpha signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-alpha signalling in cardiomyocytes. METHODS AND RESULTS: We used RASSF1A knockout (RASSF1A(-/-)) mice and wild-type (WT) littermates in this study. Acute stimulation with a low dose of TNF-alpha (10 microg/kg iv) increased cardiac contractility and intracellular calcium transients' amplitude in WT mice. In contrast, RASSF1A(-/-) mice showed a blunted contractile response. Mechanistically, RASSF1A was essential in the formation of the TNFR complex (TNFRC), where it functions as an adaptor molecule to facilitate the recruitment of TNFR type 1-associated death domain protein and TNFR-associated factor 2 to form the TNF-alpha receptor complex. In the absence of RASSF1A, signal transmission from the TNF-alpha receptor complex to the downstream effectors, such as cytoplasmic phospholipase A2 and protein kinase A, was attenuated leading to the reduction in the activation of calcium handling molecules, such as L-type Ca(2+) channel and ryanodine receptors. CONCLUSION: Our data indicate an essential role of RASSF1A in regulating TNF-alpha signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNFRC and in signal transmission to the downstream targets.

Summary Diabetes is a major risk factor for atherosclerosis. Although atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, the entry of inflammatory leukocytes into lesions fuels disease progression and impairs resolution. We show that diabetic mice have increased numbers of circulating neutrophils and Ly6-C hi monocytes, reflecting hyperglycemia-induced proliferation and expansion of bone marrow myeloid progenitors and release of monocytes into the circulation. Increased neutrophil production of S100A8/S100A9, and its subsequent interaction with the receptor for advanced glycation end products on common myeloid progenitor cells, leads to enhanced myelopoiesis. Treatment of hyperglycemia reduces monocytosis, entry of monocytes into atherosclerotic lesions, and promotes regression. In patients with type 1 diabetes, plasma S100A8/S100A9 levels correlate with leukocyte counts and coronary artery disease. Thus, hyperglycemia drives myelopoiesis and promotes atherogenesis in diabetes.

Background During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear. Results We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria. Conclusions These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

BACKGROUND: Several activities of the transmembrane form of TNF (memTNF) in immune responses to intracellular bacterial infection have been shown to be different from those exerted by soluble TNF. Evidence is based largely on studies in transgenic mice expressing memTNF, but precise cellular mechanisms are not well defined and the importance of TNF receptor regulation is unknown. In addition, memTNF activities are defined for a particular modification of the extracellular domain of TNF but a direct comparison of different mutant memTNF molecules has not been done in vivo. METHODOLOGY: To understand the activities of memTNF we compared two commonly used mouse strains lacking soluble TNF but possessing functional and normally regulated membrane-bound TNF knockin (memTNF KI) for their capacity to generate cell-mediated immune responses and resistance to M. bovis BCG infection, and to regulate TNF receptors. PRINCIPAL FINDINGS: M. bovis BCG infection resulted in similar bacterial loads in one strain of memTNF KI (memTNF(Δ1-9,K11E)) and in wild-type mice, in contrast, the other strain of memTNF KI mice (memTNF(Δ1-12)) showed higher sensitivity to infection with high mortality (75%), greater bacterial load and massive lung pathology. The pattern of cytokines/chemokines, inflammatory cells, pulmonary NF-κB phosphorylation, antigen-dependent IFN-γ response, and splenic iNOS was impaired in M. bovis BCG-infected memTNF(Δ1-12) KI mice. Macrophages expressing TNFR2 were reduced but soluble TNFRs were higher in memTNF(Δ1-12) KI mice during the infection. In vitro, M. bovis BCG-induced NF-κB activation and cytokines were also decreased in memTNF(Δ1-12) KI bone marrow-derived macrophages. CONCLUSION: Our data show that two memTNF molecules exerted very different activities upon M. bovis BCG infection resulting in protection or not to bacterial infection. These results suggest a regulatory mechanism of memTNF and TNF receptors being critical in the outcome of the infection and highlight the role of cell-bound and soluble TNFR2 in memTNF-mediated anti-microbial mechanisms.

Activation of nuclear factor kappaB (NF-kappaB) by interleukin-1beta (IL-1) usually results in an anti-apoptotic activity that is rapidly terminated by a negative feedback loop involving NF-kappaB dependent resynthesis of its own inhibitor IkappaBalpha. However, apoptosis induced by ultraviolet B radiation (UVB) is not attenuated, but significantly enhanced by co-stimulation with IL-1 in human epithelial cells. Under these conditions NF-kappaB remains constitutively active and turns into a pro-apoptotic factor by selectively repressing anti-apoptotic genes. Two different mechanisms have been separately proposed to explain UV-induced lack of IkappaBalpha recurrence: global translational inhibition as well as deactivation of the Ser/Thr phosphatase PP2Ac. Using mathematical modelling, we show that the systems behaviour requires a combination of both mechanisms, and we quantify their contribution in different settings. A mathematical model including both mechanisms is developed and fitted to various experimental data sets. A comparison of the model results and predictions with model variants lacking one of the mechanisms shows that both mechanisms are present in our experimental setting. The model is successfully validated by the prediction of independent data. Weak constitutive IKKbeta phosphorylation is shown to be a decisive process in IkappaBalpha degradation induced by UVB stimulation alone, but irrelevant for (co-)stimulations with IL-1. In silico knockout experiments show that translational inhibition is predominantly responsible for lack of IkappaBalpha recurrence following IL-1+UVB stimulation. In case of UVB stimulation alone, cooperation of both processes causes the observed decrease of IkappaBalpha. This shows that the processes leading to activation of transcription factor NF-kappaB upon stimulation with ultraviolet B radiation with and without interleukin-1 costimulation are more complex than previously thought, involving both a cross talk of UVB induced translational inhibition and PP2Ac deactivation. The importance of each of the mechanisms depends on the specific cellular setting.

International audience; TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

PTEN is inactivated in a subset of lung cancer; therefore, we investigated the involvement of PTEN inactivation in invasiveness of lung cancer cells. AKT at Ser473 was phosphorylated in several lung cancer cell lines with loss of PTEN expression. Therefore, we created a tetracycline inducible expression system of wild-type PTEN (PTEN-WT) as well as catalytically (PTEN-G129R) and lipid phosphatase (PTEN-G129E) inactive PTEN mutants using the PC14, PC9 and PC3 lung adenocarcinoma cell lines, in which endogenous PTEN expression was not detected and AKT at Ser473 was phosphorylated by Western blot analysis. Induction of PTEN-WT reduced phosphorylation of AKT and inhibited the transcriptional activity of NFkB, whereas PTEN mutants did not, suggesting that PTEN inactivation results in the activation of the AKT/NFkB pathway in PC14, PC9 and PC3 cells. Furthermore, overexpression of PTEN-WT suppressed anchorage independent growth in soft agar and reduced invasiveness in a trans-well chamber assay of PC14 cells. Neither PTEN-G129R nor PTEN-G129E had suppressive effects on anchorage independent growth and invasiveness. Augmentation of invasiveness by constitutively active AKT was also shown in mouse NIH3T3 cells. Therefore, it was strongly indicated that activation of the PI3K/AKT/NFkB pathway by PTEN inactivation results in augmented invasiveness in lung cancer cells and lipid phosphatase activity of PTEN plays a key role in this process.

The IκB kinase complex induces nuclear factor kappa B activation and has recently been recognized as a key player of autoimmunity in the central nervous system. Notably, IκB kinase/nuclear factor kappa B signalling regulates peripheral myelin formation by Schwann cells, however, its role in myelin formation in the central nervous system during health and disease is largely unknown. Surprisingly, we found that brain-specific IκB kinase 2 expression is dispensable for proper myelin assembly and repair in the central nervous system, but instead plays a fundamental role for the loss of myelin in the cuprizone model. During toxic demyelination, inhibition of nuclear factor kappa B activation by conditional ablation of IκB kinase 2 resulted in strong preservation of central nervous system myelin, reduced expression of proinflammatory mediators and a significantly attenuated glial response. Importantly, IκB kinase 2 depletion in astrocytes, but not in oligodendrocytes, was sufficient to protect mice from myelin loss. Our results reveal a crucial role of glial cell-specific IκB kinase 2/nuclear factor kappa B signalling for oligodendrocyte damage during toxic demyelination. Thus, therapies targeting IκB kinase 2 function in non-neuronal cells may represent a promising strategy for the treatment of distinct demyelinating central nervous system diseases.