Cardiomyocyte-specific IκB kinase (IKK)/NF-κB activation induces reversible inflammatory cardiomyopathy and heart failure

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b(+) cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.     

The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the IκB kinase subunit IKKα

Listeria monocytogenes is an intracellular pathogen responsible for severe foodborne infections. It can replicate in both phagocytic and nonphagocytic mammalian cells. The infectious process at the cellular level has been studied extensively, but how the bacterium overcomes early host innate immune responses remains largely unknown. Here we show that InlC, a member of the internalin family, is secreted intracellularly and directly interacts with IKKα, a subunit of the IκB kinase complex critical for the phosphorylation of IκB and activation of NF-κB, the major regulator of innate immune responses. Infection experiments with WT Listeria or the inlC-deletion mutant and transfection of cells with InlC reveal that InlC expression impairs phosphorylation and consequently delays IκB degradation normally induced by TNF-α, a classical NF-κB stimulator. Moreover, infection of RAW 264.7 macrophages by the inlC mutant leads to increased production of proinflammatory cytokines compared with that obtained with the WT. Finally, in a peritonitis mouse model, we show that infection with the inlC mutant induces increased production of chemokines and increased recruitment of neutrophils in the peritoneal cavity compared with infection with WT. Together, these results demonstrate that InlC, by interacting with IKKα, dampens the host innate response induced by Listeria during the infection process.     

Mechanisms of Disease: primary Sjögren's syndrome and the type I interferon system

Sj?gren's syndrome is a chronic autoimmune disease of largely unknown etiology and pathogenesis. The salivary and lacrimal glands are the main target organs, and key cells and molecules involved in the autoimmune process have been detected in these glands. Chemokines, expressed by epithelial cells, can attract T cells and dendritic cells that produce proinflammatory cytokines, which stimulate the immune response and induce apoptosis in the acinar and ductal epithelial cells. The autoantigens SSA and SSB are translocated to the apoptotic blebs and trigger infiltrating B cells to produce autoantibodies against SSA and SSB. Germinal-center-like structures can form within glandular lymphocyte foci, facilitating the antigen-driven B-cell activation. Many of the autoimmune mechanisms described above can be induced by type I interferon (IFN), and activation of this system in patients with Sj?gren's syndrome has been described. A possible scenario is that an initial viral infection induces type I IFN production in salivary glands with a subsequent activation of the adaptive immune system. Resultant autoantibodies form nucleic-acid-containing immune complexes that can trigger prolonged type I IFN production, leading to a self-perpetuating autoimmune reaction. Several potential therapeutic targets for Sj?gren's syndrome exist within the type I IFN system.     

Cross-talk between the unfolded protein response and nuclear factor-κB signalling pathways regulates cytokine-mediated beta cell death in MIN6 cells and isolated mouse islets

Aims/hypothesis

Pancreatic beta cell destruction in type 1 diabetes may be mediated by cytokines such as IL-1β, IFN-γ and TNF-α. Endoplasmic reticulum (ER) stress and nuclear factor-κB (NFκB) signalling are activated by cytokines, but their significance in beta cells remains unclear. Here, we investigated the role of cytokine-induced ER stress and NFκB signalling in beta cell destruction.

Methods

Isolated mouse islets and MIN6 beta cells were incubated with IL-1β, IFN-γ and TNF-α. The chemical chaperone 4-phenylbutyric acid (PBA) was used to inhibit ER stress. Protein production and gene expression were assessed by western blot and real-time RT-PCR.

Results

We found in beta cells that inhibition of cytokine-induced ER stress with PBA unexpectedly potentiated cell death and NFκB-regulated gene expression. These responses were dependent on NFκB activation and were associated with a prolonged decrease in the inhibitor of κB-α (IκBα) protein, resulting from increased IκBα protein degradation. Cytokine-mediated NFκB-regulated gene expression was also potentiated after pre-induction of ER stress with thapsigargin, but not tunicamycin. Both PBA and thapsigargin treatments led to preferential upregulation of ER degradation genes over ER-resident chaperones as part of the adaptive unfolded protein response (UPR). In contrast, tunicamycin activated a balanced adaptive UPR in association with the maintenance of Xbp1 splicing.

Conclusions/interpretation

These data suggest a novel mechanism by which cytokine-mediated ER stress interacts with NFκB signalling in beta cells, by regulating IκBα degradation. The cross-talk between the UPR and NFκB signalling pathways may be important in the regulation of cytokine-mediated beta cell death.     

Visfatin regulates the terminal processes of human labour and delivery via activation of the nuclear factor-κB pathway

The inflammatory process plays a pivotal role during the pathogenesis of human labour, both at term and preterm. Visfatin levels increase during normal human pregnancy and in infection associated preterm labour. The effects of visfatin in the processes of human labour and delivery, however, are not known. The aim of this study was to determine the effect of visfatin on the expression and release of pro-labour mediators in human placenta. Samples were obtained from normal pregnancies at the time of Caesarean section. Human placenta was incubated in the absence (basal control) or presence of a 50 ng/ml visfatin for 24 h (n = 6). Inflammatory gene expression was analysed by quantitative RT-PCR (qRT-PCR), the medium was collected and cytokine, prostaglandin and 8-isoprostane (marker of oxidative stress) release was quantified by ELISA, and secretory protease activity by zymography. Visfatin significantly increased IL-6 and IL-8 gene expression and secretion, COX-2 expression and resultant prostaglandin (PG) E₂ and PGF_2α release, and 8-isoprostane release. There was, however, no effect of visfatin on pro MMP-9 enzyme activity. These actions of visfatin were elicited via the nuclear factor-κB (NF-κB) pathway as visfatin induced the degradation of IκB-α (inhibitor of NF-κB) whilst increasing NF-κB p65 DNA binding activity. Further to this, visfatin-induced pro-labour responses were abrogated by treatment with the NF-κB inhibitor BAY 11-7082. Collectively, these data indicate that visfatin activates pro-inflammatory cytokine release and phospholipid metabolism in human placenta via activation of the NF-κB pathway. Thus, visfatin represents a novel cytokine linked to the events of human labour initiation.     

LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER^T fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α₂-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment.Innate immunity is a phylogenetically conserved system that provides a first line of defense against pathogens (1, 2). Pattern recognition receptors (PRRs), including members of the Toll-like receptor (TLR) family, play an important role in innate immunity, binding microorganism-derived molecules, and initiating proinflammatory cell signaling (1, 3, 4). The effector systems of innate immunity, including proinflammatory cytokines and complement, may be very potent and when regulatory mechanisms fail, shock and death may result (3). Diverse diseases are exacerbated by dysregulated innate immunity, including Crohn’s disease, rheumatoid arthritis, asthma, psoriasis, atherosclerosis, and cancer (3, 5, 6). Understanding mechanisms that control innate immunity is a significant problem in medicine.LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor, which is essential for embryonic development (7, 8). In adults, there is increasing evidence that LRP1 regulates inflammation (9). LRP1-deficient macrophages, isolated from mice in which LRP1 is conditionally deleted in myeloid cells (mLRP1^−/− mice), express increased levels of proinflammatory chemokines, including monocyte chemotactic protein/CCL2, MIP-1α/CCL3, and MIP-1β/CCL4 (10–12). These macrophages also migrate more rapidly, due to activation of CCL3-CCR5 signaling (10) and express decreased levels of biomarkers associated with M2 polarization (13).In syngeneic tumors in mLRP1^−/− mice, LRP1-deficient macrophages accumulate in increased number and express increased levels of CCL3 (10). Macrophage infiltration is also increased in atherosclerotic lesions in mLRP1^−/− mice (11). However, mechanisms by which LRP1 regulates macrophage physiology remain incompletely understood. LRP1 deficiency is associated with increased NFκB activity in passaged cell lines (12); however, “loss of function” model systems do not address the role of LRP1 as a receptor for diverse ligands (7, 14). In neurons and neuron-like cells, different LRP1 ligands elicit distinct cell-signaling responses by engaging separate LRP1 coreceptors (15–17). If LRP1 regulates macrophage physiology in a ligand-specific manner, this would represent a powerful mechanism by which macrophages may respond to changes in their microenvironment.In this study, we challenged mLRP1^−/− mice and control mLRP1^+/+ mice with lipopolysaccharide (LPS), which is a major ligand for the PRR, TLR4 (18). The response to LPS was greatly exacerbated in mLRP1^−/− mice. Using a second genetic model system, we confirmed that LRP1 gene deletion in macrophages increases expression of proinflammatory mediators. We then showed that expression of proinflammatory mediators is controlled in macrophages by LRP1 ligands in a ligand-specific manner. LRP1 agonists, such as α₂-macroglobulin (α₂M) and tissue-type plasminogen activator (tPA), suppressed expression of proinflammatory mediators, even in the presence of LPS. Antagonists, such as receptor-associated protein (RAP) and lactoferrin (LF), increased expression of the identical mediators, mimicking the effects of LRP1 gene deletion. The activity of LRP1 ligands was linked to regulation of NFκB and the previously unreported ability of LRP1 to control expression of microRNA-155 (miR-155) (19). LRP1, NFκB, and miR-155 emerge as members of a novel system that may control the macrophage inflammatory response in a microenvironment-sensitive manner.     

Pregnancy complicated by type I, type II and gestational diabetes: experiences from the diabetic-obstetric center of Białystock

The aim of the study was the analysis of pregnancy outcome, newborn status, metabolic control and obstetric failure in 365 pregnant diabetic patients treated in Bia?ystok Diabetic-Obstetric Center. Abortions occurred in 1.64% of pregnancies, intrauterine deaths--in 1.1%, and newborns deaths--in 2.47% cases. Macrosomia was observed in 14.8% of children (from 12% in type 1--up to 25% in gestational diabetes class G2). Congenital malformations were seen in 16 newborns of type 1 diabetic women (9.6%), 2 newborns of type 2 diabetics (22.2%), 6 children of mothers with gestational diabetes class G1 (4.2%) and 4 (8.3%)--class G2. The discussion underlines the role of a long duration of the disease as a key factor increasing the risk of complications and the importance of a good metabolic control before and shortly after conception.     

CaMKIIγ regulates the viability and self-renewal of acute myeloid leukaemia stem-like cells by the Alox5/NF-κB pathway

Acute myeloid leukaemia (AML) is a frequently fatal malignant disease of haematopoietic stem and progenitor cells. The molecular and phenotypic characteristics of AML are highly heterogeneous. Our previous study concluded that CaMKIIγ was the trigger of chronic myeloid leukaemia progression from the chronic phase to blast crisis, but how CaMKIIγ influences AML stem-like cells remains elusive. In this study, we found that CaMKIIγ was overexpressed in AML patients and AML cell lines, as measured by qRT-PCR and Western blot assays. Moreover, CaMKIIγ decreased when the disease was in remission. Using an shRNA lentivirus expression system, we established CaMKIIγ stable-knockdown AML cell lines and found that knockdown of CaMKIIγ inhibited the viability and self-renewal of AML stem-like cell lines. Additionally, the ratio of CD₃₄⁺ AML cell lines decreased, and CaMKIIγ knockdown induced the downregulation of Alox5 levels. We further detected downstream molecules of the Alox5/NF-κB pathway and found that c-myc and p-IκBα decreased while total IκBα remained normal. In conclusion, our study describes a new role for CaMKIIγ as a stem-like cell marker that is highly regulated by the Alox5/NF-κB pathway in AML stem-like cells. CaMKIIγ can participate in the viability and self-renewal of AML stem-like cells by regulating the Alox5/NF-κB pathway.     

Hereditary tyrosinaemia type I: A long-term study of the relationship between the urinary excretions of succinylacetone and δ-aminolevulinic acid

Summary Patients with hereditary tyrosinaemia type I (HT) excrete large amounts of succinylacetone (SA) in urine. Owing to structural resemblance of SA to -aminolevulinic acid (ALA), SA inhibits the second enzyme in the pathway for haeme biosynthesis, porphobilinogen synthase, resulting in increased urinary ALA excretion. We investigated the relationship between urinary SA and ALA excretions of two patients with different forms of HT (late-infantile and juvenile). In both patients the urinary SA and ALA excretions showed a more or less inverse correlation. The patient with the early-infantile form of HT had a relatively greater increase in urinary SA and ALA excretions in comparison to the patient with the juvenile form of HT. A possible explanation for this unexpected inverse correlation between the urinary excretion of SA and ALA might be a lack of intramitochondrial glycine, a substrate for -aminolevulinic acid synthesis. It has been reported previously that high concentrations of SA reversibly and competitively inhibit the transport of glycine through membranes.     

Fatty acids acutely enhance insulin-induced oxidative stress and cause insulin resistance by increasing mitochondrial reactive oxygen species (ROS) generation and nuclear factor-κB inhibitor (IκB)–nuclear factor-κB (NFκB) activation in rat muscle,in the absence of mitochondrial dysfunction

Aims/hypothesis  

Insulin effects reportedly involve reactive oxygen species (ROS) and oxidative stress in vitro, but skeletal muscle oxidative stress is an emerging negative regulator of insulin action following high-fat feeding. NEFA may enhance oxidative stress and insulin resistance. We investigated the acute impact of insulin with or without NEFA elevation on muscle ROS generation and insulin signalling, and the potential association with altered muscle mitochondrial function.     

Nuclear factor-κB activation by type II collagen peptide in articular chondrocytes: its inhibition by hyaluronan via the receptors

Objective

This study aimed to examine nuclear factor-κB (NF-κB) activation by a synthetic peptide from type II collagen fragment (CB12-II) and its inhibition by hyaluronan (HA) via its receptors, CD44, and intercellular adhesion molecule-1 (ICAM-1) in chondrocytes.

Methods

Osteoarthritic cartilage explants or chondrocytes in monolayer were cultured with CB12-II. Secreted levels of matrix metalloproteinase (MMP)-13 in conditioned media and NF-κB activation in chondrocytes were determined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Cultures were pretreated with HA to evaluate the inhibitory effect on CB12-II action, and the role of HA receptors in HA effect was investigated using antibodies to CD44 and ICAM-1.

Results

CB12-II stimulated phosphorylation and nuclear translocation of NF-κB, leading to increased MMP-13 production. HA suppressed NF-κB activation and MMP-13 induction by CB12-II. The individual antibody to CD44 or ICAM-1 partially reversed HA effect on CB12-II action, and both antibodies in combination completely blocked the HA effect.

Conclusions

This study clearly demonstrates that CB12-II activates NF-κB for MMP-13 induction and that HA inhibits CB12-II action through interaction with CD44 and ICAM-1 in chondrocytes. HA administration into osteoarthritic joints could suppress the catabolic action of matrix degradation products such as CB12-II as a potent NF-κB inhibitor.     

Relationships among protein tyrosine phosphatase 1B,angiotensin II,and insulin-mediated aortic responses in type 2 diabetic Goto–Kakizaki rats

Objective

We investigated the relationships among protein tyrosine phosphatase 1B (PTP1B), angiotensin II (Ang II), and insulin signaling in the presence of endothelial dysfunction in type 2 diabetic Goto–Kakizaki (GK) rat aortas.

Methods and results

Aortas isolated from GK or control Wistar rats were examined in the presence or absence of Ang II with or without a selective antagonist of the Ang II type 1 (AT₁) receptor or a PTP1B inhibitor to evaluate vascular functional and molecular mechanisms, such as insulin-induced relaxation, nitric oxide (NO) production, phosphorylation of insulin receptor substrate (IRS)-1, endothelial NO synthase (eNOS), and phosphorylation, and the subcellular localization of PTP1B. GK aortas exhibited reductions of: 1) insulin-induced relaxation, 2) NO production, 3) Ser¹¹⁷⁷-p-eNOS, and 4) Tyr⁶¹²-p-IRS-1. Pre-incubation with a PTP1B inhibitor normalized these reductions. In Wistar aortas, the four above-mentioned parameters were reduced by Ang II, but were completely inhibited by co-treatment with the PTP1B inhibitor. The membrane expression of PTP1B was greater in GK than in Wistar aortas, and it was increased by Ang II in Wistar rats. The membrane PTP1B expression in the presence of insulin + Ang II was reduced by the PTP1B inhibitor or AT₁-receptor antagonist.

Conclusions

These results suggest that the membrane PTP1B suppressed insulin-mediated aortic relaxation, and this was due to the Ang II-AT₁-receptor signaling pathway. The inhibition of PTP1B warrants further investigation as a potential therapeutic target for endothelial dysfunction in type 2 diabetes.