Adrienn Angyal

Tribbles-2 is a novel regulator of inflammatory activation of monocytes

Inflammatory activation of monocytes is an essential part of both innate immune responses and the pathogenesis of conditions such as atherosclerosis. However, the mechanisms which modulate the response of monocytes to inflammatory stimuli are still poorly understood. Here, we report that tribbles-2 (trb-2) is a novel regulator of inflammatory activation of monocytes. Down-regulation of trb-2 levels potentiates LPS-induced IL-8 production via enhanced activation of the extracellular signal-regulated kinase and jun kinase mitogen-activated protein kinase (MAPK) pathways. In keeping with this, the endogenous level of trb-2 expression in human primary monocytes is inversely correlated to the cell’s ability to produce IL-8. We show that trb-2 is a binding partner and a negative regulator of selected MAPKs. The potential in vivo relevance of these findings is highlighted by the observation that modified low-density lipoprotein profoundly down-regulates trb-2 expression, which may, in turn, significantly contribute to the inflammatory processes in the development of vascular disease. Taken together, our results define trb-2 as a potent novel regulator of monocyte biology, controlling the activation of these cells.

Modulation of immune response by combined targeting of complement receptors and low-affinity Fcγ receptors

Immune complexes (ICs) induce effective pathogen-specific innate and humoral immune response via immunecomplex-binding receptors, such as murine complement receptor type 1 and 2 (mCR1/2) and murine low-affinity Fc receptors for IgG (mFcgammaRII and III). The exact function of mCR1/2 in cooperation with mFcgammaRII/III in modulation of humoral immunity has not yet been adequately clarified. The aim of this study was to target these receptors by specific single-chain fragments of antibody (scFv), either individually or in combination, thus modelling the action of IC. For targeting, we used scFv derived from the well-characterized 7g6 and 2.4g2 monoclonal antibodies recognizing mCR1/2 and mFcgammaRII/III, respectively. These scFvs were monobiotinylated and conjugated to streptavidin or streptavidin-coated microspheres. Such complexes were investigated with respect to target receptor recognition and in vivo localization. Antibody response against the constructs was measured by ELISA and ELISPOT. Results show that targeting streptavidin complexes to mFcgammaRII/III induces stronger IgG1 response than targeting to mCR1/2 yet both strategies enhance the antibody response compared to the control group immunized with non-targeted peptide-streptavidin complexes. Moreover, the immunogenicity of coupled antigens increased using microspheres as carrier, instead of using soluble streptavidin. In summery, our in vivo experiments reveal that mFcgammaRII/III is more potent a target than CR1/2 and show that combined targeting of CR1/2 and FcgammaRII/III receptors does not result in cumulative enhancement of the antigen specific immune response. In addition, microparticle-mediated enhancement of immunization can be further improved by FcgammaRII/III targeting.

Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways.

T-bet is a new synergistic meeting point for the BCR and TLR9 signaling cascades

The importance of the BCR and TLR9 in autoimmunity and in the production of auto‐antibodies is well established but the underlying molecular mechanism still needs to be determined. Here, we aim to characterize the BCR‐TLR9 cross‐talk by its effect on T‐bet, as T‐bet is activated and regulated by both receptors and has an important role in class‐switching to pathological IgG2a in mice. Using primary mouse B cells, we demonstrate that T‐bet expression is synergistically elevated by the cross‐talk between the BCR and TLR9. To test the effect of this synergy on IgG2a‐switching, the levels of switched B cells were checked by functional tests. We found that BCR costimulation had no additional effect on TLR9‐induced IgG2a expression, however the expression of Rad51 was synergistically increased. To check the biological significance of the synergy, we compared T‐bet expression in B cells from healthy and collagen‐induced arthritis mice but no differences were found. Taken together, we demonstrate here that signaling cascades driven by the BCR and TLR9 have a newly identified meeting point at T‐bet. The two cascades act synergistically on T‐bet; however additional signals may be needed to induce prolonged functional responses such as class‐switch recombination.

CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin–FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner

Fcgamma receptors (FcgammaRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcgammaRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcgammaRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcgammaRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcgammaRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin-FITC, and tested its effect on the FITC-hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11b(high) and CD11c(high) cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC-dextran) or TD (FITC-keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcgammaRIII might be a key mediator of this mechanism.

Enhanced Macrophage Tribbles-1 Expression in Murine Experimental Atherosclerosis

Development of the atherosclerotic plaque involves a complex interplay between a number of cell types and an extensive inter-cellular communication via cell bound as well as soluble mediators. The family of tribbles proteins has recently been identified as novel controllers of pro-inflammatory signal transduction. The objective of this study was to address the expression pattern of all three tribbles proteins in atherosclerotic plaques from a mouse model of atherosclerosis. Each tribbles were expressed in vascular smooth muscle cells, endothelial cells as well as in resident macrophages of mouse atherosclerotic plaques. The role of IL-1 mediated inflammatory events in controlling tribbles expression was also addressed by inducing experimental atherosclerosis in ApoE−/−IL1R1−/− (double knockout) mice. Immunohistochemical analysis of these mice showed a selective decrease in the percentage of trb-1 expressing macrophages, compared to the ApoE−/− cohort (14.7% ± 1.55 vs. 26.3% ± 1.19). The biological significance of this finding was verified in vitro where overexpression of trb-1 in macrophages led to a significant attenuation (~70%) of IL-6 production as well as a suppressed IL-12 expression induced by a proinflammatory stimulus. In this in vitro setting, expression of truncated trb-1 mutants suggests that the kinase domain of this protein is sufficient to exert this inhibitory action.

Possible therapeutic applications of single-chain antibodies in systemic autoimmune diseases

B cells participate in the induction and maintenance of systemic autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, via production of pathogenic autoantibodies, contributing to the formation of immune complexes. Immune complex deposition in the kidney and joints causes inflammation and organ destruction, and chemokine production enhances T cell activation and tissue damage. The development of the disorder depends on several factors, for example, genetic susceptibility, environmental factors or immune dysregulation. Traditional therapies, which aimed at the alleviation of symptoms, are giving way to biological therapies with the potential of disrupting disease progression. This article focuses on antibody therapies, especially on the applications of single-chain antibodies, as new biological agents for the treatment of systemic autoimmune disorders.

Grb2‐Associated Binder 1 (Gab1) Adaptor/Scaffolding Protein Regulates Erk Signal in Human B Cells

Abstract:  RNA silencing experiments showed that knocking down Gab1 adaptor protein in BL41 human Burkitt lymphoma cells significantly reduced B cell receptor (BCR)–induced Erk phosphorylation, indicating that Gab1 plays a pivotal role in regulating Erk activity in B cells.

157 Myeloid expression of trib1 regulates the polarisation state of tissue resident macrophages that has consequences on plasma lipid and metabolic homeostasis

Introduction Genome wide association studies have identified Tribbles-1 (TRIB1) to be significantly associated with all major plasma lipid traits and as a risk factor for ischaemic heart disease and myocardial infarction. Studies in mice using Trib1 full body KO and liver-specific over-expression and KO models have shown that hepatic expression of TRIB1 reduces circulating lipids. Additionally, Trib1 has been implicated as a regulator of alternatively activated macrophages. However the potential interplay between hepatocytes, macrophages and Trib1 remain unexplored. This study aimed to assess whether myeloid Trib1 regulates tissue macrophage polarisation and investigate its consequences on plasma lipid homeostasis. Methods We developed myeloid specific Trib1 conditional knockout (Trib1 fl/fl x Lyz2Cre; Trib1KO) and over-expressor mice (ROSA26Trib1.Tg x Lyz2Cre; Trib1Tg), thereby deleting or over-expressing Trib1 in myeloid cells. Plasma lipid levels were directly measured by ion mobility. Macrophage phenotype was characterised in the liver (Kupffer cells, KCs), adipose (ATMs) and BMDMs by qPCR and semi-quantitative immunofluorescence analysis. Western blotting was used to assess regulators of macrophage polarisation. Furthermore, microarray analysis of human monocyte derived macrophages (MDMs) was employed to identify potential TRIB1-regulated cytokines. Results Loss of myeloid Trib1 increased levels of plasma triglyceride, VLDL-C (p<0.05) and promoted pro-inflammatory polarisation in KCs (p<0.01), ATMs (p<0.01) and BMDMs (p<0.05), while Trib1Tg mice revealed opposing changes in all parameters assessed. Western blotting showed TRIB1 modulates protein levels of C/EBP-β2 and –β² (p<0.05), both key regulators of macrophage polarisation, via the control of COP1 activity and miR-155 expression. Microarray analysis of MDMs indicated TRIB1 may regulate production of a number of pro-inflammatory cytokines that are implicated in fatty liver disease and adipocyte lipolysis. Reduced expression of these was confirmed in in Trib1Tg BMDMs (p<0.05). Conclusions Myeloid Trib1 is a potent regulator of lipid homeostasis, the loss of which promotes inflammation in metabolic tissues. Our observations uncover a novel mechanism of KC-hepatocyte cross talk mediated through Trib1.

223 MIRNA202 is a Novel Regulator of Tribbles-1 Expression

Introduction Tribbles-1 (trib-1) pseudokinase is a regulatory protein that has been shown to be a protective gene in a number of cell types and processes, relevant to the development of atherosclerosis. These include inhibition of vascular smooth muscle cell proliferation, polarisation of macrophages towards an alternatively activated phenotype and lowering the production and release of LDL in hepatic tissues. Therefore, understanding the molecular mechanisms, which regulate trib-1 expression are of significant interest. Our group have identified miRNA202 as a controller of trib-1 levels. Rationale Trib-1 mRNA is highly unstable with a half-life of less than 1 h; the 3’ UTR encodes for a number of putative binding sites for miRNAs, including miRNA202. This study aimed to experimentally validate the importance of miRNA202 in the control of trib-1 expression. Methods 1. We have used a luciferase reporter to demonstrate the role of trib-1 3’UTR in mRNA stability and to characterise the impact of miR202 on trib-1 3’UTR in hepatic cells (HepG2). 2. qRT-PCR was used to quantify endogenous trib-1 and miRNA202 levels upon stimulation of IL-1 and in high glucose media. 3. Western blotting was used to elucidate the effect of miRNA202 on trib-1 protein levels. Results 1. Overexpression of miR202 reduced luciferase – trib1-3’UTR reporter expression. 2. The endogenous trb-1 mRNA was also modulated by miR202. Furthermore, stimulation of IL-1 in HepG2 cells has shown trb-1 levels to reduce by >70% and miR202 levels to increase by 2 fold. 3. Trib-1 protein levels have shown a reduction upon overexpression of miRNA202. Conversely, miRNA202 inhibitor increased trib-1 protein levels. Discussion miR202 is a novel regulator of trib-1 expression and may represent a target by which trib-1 levels could be raised in vivo, thereby providing a mechanism to augment the anti-atherosclerotic effects of this protein.