Enikö Kokai

Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

Epstein-Barr Virus-Induced Gene-3 Is Expressed in Human Atheroma Plaques

Atherosclerosis is characterized by a complex immune response in the vessel wall, involving both inflammation and autoimmune processes. Epstein-Barr virus-induced gene 3 (Ebi3) is a member of the interleukin (IL)-12 heterodimeric cytokine family, which has important immunomodulatory functions. To date, little is known about the role of Ebi3 in vascular disease. We examined the expression of Ebi3 in human atheromatous lesions and analyzed its transcriptional regulation in vascular cells. The in situ expression of Ebi3 in human endarterectomy specimens was analyzed by immunohistochemistry. In these lesions, smooth muscle cells expressed Ebi3 as well as the IL-27alpha/p28 and IL-12alpha/p35 subunits. Primary aortic smooth muscle cells up-regulated Ebi3 in response to proinflammatory stimuli like tumor necrosis factor-alpha and interferon-gamma. Interestingly, pretreatment of these cells with the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone strongly reduced Ebi3 induction. Chromatin immunoprecipitation experiments revealed that this inhibition is due to interference with p65/RelA recruitment to the Ebi3 promoter. Our data support a possible role of Ebi3 in atherogenesis either as homodimer or as IL-27/IL-35 heterodimer, and suggest that Ebi3 could be an interesting target for therapeutic manipulation in atherosclerosis.

Analysis of nuclear actin by overexpression of wild-type and actin mutant proteins.

Abstract Compared to the cytoplasmic F-actin abundance in cells, nuclear F-actin levels are generally quite low. However, nuclear actin is present in certain cell types including oocytes and under certain cellular conditions including stress or serum stimulation. Currently, the architecture and polymerization status of nuclear actin networks has not been analyzed in great detail. In this study, we investigated the architecture and functions of such nuclear actin networks. We generated nuclear actin polymers by overexpression of actin proteins fused to a nuclear localization signal (NLS). Raising nuclear abundance of a NLS wild-type actin, we observed phalloidin- and LifeAct-positive actin bundles forming a nuclear cytoskeletal network consisting of curved F-actin. In contrast, a polymer-stabilizing actin mutant (NLS-G15S-actin) deficient in interacting with the actin-binding protein cofilin generated a nuclear actin network reminiscent of straight stress fiber-like microfilaments in the cytoplasm. We provide a first electron microscopic description of such nuclear actin polymers suggesting bundling of actin filaments. Employing different cell types from various species including neurons, we show that the morphology of and potential to generate nuclear actin are conserved. Finally, we demonstrate that nuclear actin affects cell function including morphology, serum response factor-mediated gene expression, and herpes simplex virus infection. Our data suggest that actin is able to form filamentous structures inside the nucleus, which share architectural and functional similarities with the cytoplasmic F-actin.

Canonical NF-κB signaling in hepatocytes acts as a tumor-suppressor in hepatitis B virus surface antigen-driven hepatocellular carcinoma by controlling the unfolded protein response

Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). Efficient suppression of HBV viremia and necroinflammation as a result of nucleos(t)ide analogue treatment is able to reduce HCC incidence; nevertheless, hepatocarcinogenesis can occur in the absence of active hepatitis, correlating with high HBV surface antigen (HBsAg) levels. Nuclear factor κB (NF‐κB) is a central player in chronic inflammation and HCC development. However, in the absence of severe chronic inflammation, the role of NF‐κB signaling in HCC development remains elusive. As a model of hepatocarcinogenesis driven by accumulation of HBV envelope polypeptides, HBsAg transgenic mice, which show no HBV‐specific immune response, were crossed to animals with hepatocyte‐specific inhibition of canonical NF‐κB signaling. We detected prolonged, severe endoplasmic reticulum stress already at 20 weeks of age in NF‐κB‐deficient hepatocytes of HBsAg‐expressing mice. The unfolded protein response regulator binding immunoglobulin protein/78‐kDa glucose‐regulated protein was down‐regulated, activating transcription factor 6, and eIF2α were activated with subsequent overexpression of CCAAT/enhancer binding protein homologous protein. Notably, immune cell infiltrates and liver transaminases were unchanged. However, as a result of this increased cellular stress, insufficient hepatocyte proliferation due to G1/S‐phase cell cycle arrest with overexpression of p27 and emergence of ductular reactions was detected. This culminated in increased DNA damage already at 20 weeks of age and finally led to 100% HCC incidence due to NF‐κB inhibition. Conclusion: The role of canonical NF‐κB signaling in HCC development depends on the mode of liver damage; in the case of HBsAg‐driven hepatocarcinogenesis, NF‐κB in hepatocytes acts as a critical tumor suppressor by augmenting the endoplasmic reticulum stress response. (Hepatology 2016;63:1592‐1607)

Canonical NF‐κB signaling in hepatocytes acts as a tumor‐suppressor in HBV surface antigen‐driven HCC by controlling the UPR

Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). Efficient suppression of HBV viremia and necroinflammation as a result of nucleos(t)ide analogue treatment is able to reduce HCC incidence; nevertheless, hepatocarcinogenesis can occur in the absence of active hepatitis, correlating with high HBV surface antigen (HBsAg) levels. Nuclear factor κB (NF‐κB) is a central player in chronic inflammation and HCC development. However, in the absence of severe chronic inflammation, the role of NF‐κB signaling in HCC development remains elusive. As a model of hepatocarcinogenesis driven by accumulation of HBV envelope polypeptides, HBsAg transgenic mice, which show no HBV‐specific immune response, were crossed to animals with hepatocyte‐specific inhibition of canonical NF‐κB signaling. We detected prolonged, severe endoplasmic reticulum stress already at 20 weeks of age in NF‐κB‐deficient hepatocytes of HBsAg‐expressing mice. The unfolded protein response regulator binding immunoglobulin protein/78‐kDa glucose‐regulated protein was down‐regulated, activating transcription factor 6, and eIF2α were activated with subsequent overexpression of CCAAT/enhancer binding protein homologous protein. Notably, immune cell infiltrates and liver transaminases were unchanged. However, as a result of this increased cellular stress, insufficient hepatocyte proliferation due to G1/S‐phase cell cycle arrest with overexpression of p27 and emergence of ductular reactions was detected. This culminated in increased DNA damage already at 20 weeks of age and finally led to 100% HCC incidence due to NF‐κB inhibition. Conclusion: The role of canonical NF‐κB signaling in HCC development depends on the mode of liver damage; in the case of HBsAg‐driven hepatocarcinogenesis, NF‐κB in hepatocytes acts as a critical tumor suppressor by augmenting the endoplasmic reticulum stress response. (Hepatology 2016;63:1592‐1607)

Myc Regulates Embryonic Vascular Permeability and Remodeling

Previous work has shown that c-Myc is required for adequate vasculogenesis and angiogenesis. To further investigate the contribution of Myc to these processes, we conditionally expressed c-Myc in embryonic endothelial cells using a tetracycline-regulated system. Endothelial Myc overexpression resulted in severe defects in the embryonic vascular system. Myc-expressing embryos undergo widespread edema formation and multiple hemorrhagic lesions. They die between embryonic days 14.5 and 17.5. The changes in vascular permeability are not caused by deficiencies in vascular basement membrane composition or pericyte coverage. However, the overall turnover of endothelial cells is elevated as is revealed by increased levels of both proliferation and apoptosis. Whole-mount immunohistochemical analysis revealed alterations in the architecture of capillary networks. The dermal vasculature of Myc-expressing embryos is characterized by a reduction in vessel branching, which occurs despite upregulation of the proangiogenic factors vascular endothelial growth factor-A and angiopoietin-2. Thus, the net outcome of an excess of vascular endothelial growth factor-A and angiopoietin-2 in the face of an elevated cellular turnover appears to be a defect in vascular integrity.

Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality.

The transcription factor NF-κB has been associated with a range of pathological conditions of the heart, mainly based on its function as a master regulator of inflammation and pro-survival factor. Here, we addressed the question what effects activation of NF-κB can have during murine heart development. We expressed a constitutively active (CA) mutant of IKK2, the kinase activating canonical NF-κB signaling, specifically in cardiomyocytes under the control of the α-myosin heavy chain promoter. Expression of IKK2-CA resulted in embryonic lethality around E13. Embryos showed defects in compact zone formation and the contractile apparatus, and overall were characterized by widespread inflammation with infiltration of myeloid cells. Gene expression analysis suggested an interferon type I signature, with increased expression of interferon regulatory factors. While apoptosis of cardiomyocytes was only increased at later stages, their proliferation was decreased early on, providing an explanation for the disturbed compact zone formation. Mechanistically, this could be explained by activation of the JAK/STAT axis and increased expression of the cell cycle inhibitor p21. A rescue experiment with an IκBα superrepressor demonstrated that the phenotype was dependent on NF-κB. We conclude that activation of NF-κB is detrimental during normal heart development due to excessive activation of pro-inflammatory pathways.