Project 3: Exploring the role of T cell ORAI-MCU-NOX2 cross regulation in heart failure

PI Göttingen: I. Bogeski, D.M. Katschinski; PI London: A.M. Shah, A. Ivetic;                                             PhD student: Magdalena Shumanska

Scientific background and preliminary results

Immune cells play a significant role in the progression of cardiovascular remodeling and ultimately in the development of heart failure (Emmerson et al, 2018; Fildes et al, 2009, Liao et al, 2018). Moreover, the subtype composition of the tissue-invading immune cells following acute myocardial infarction strongly determine the disease outcome (Frangogiannis, 2014). However, the molecular mechanisms and players, which regulate immune cell invasion, are not fully understood.

Calcium and reactive oxygen species (ROS) are essential determinants of immune cell function (Simeoni and Bogeski, 2015). In particular, T-cell and monocyte immune responses critically depend on store-operated Ca2+-entry (SOCE) mediated by the ORAI Ca2+ channels as well as on ROS production via the NADPH oxidase (NOX) enzyme family (Hogan et al, 2010). We have studied the interplay between calcium and redox signals and found that oxidation inhibits SOCE; an effect mediated by the modification of a specific cysteine residue present in ORAI1 and ORAI2, but absent in ORAI3 channels (Bogeski et al, 2010). Moreover, we have developed protein biosensors for simultaneous monitoring of T-cell immune synapse formation and hydrogen peroxide (H2O2) production during T-cell activation (Mishina et al, 2012). We have also established red fluorescent H2O2 sensors, which allow combined detection of H2O2 and calcium with a subcellular resolution (Ermakova et al, 2014). Using these tools, we have identified H2O2 hotspots near the immune synapse (IS) during CD4+ T-cell activation. Additionally, we found that T-cell activation induces mitochondrial reprograming and identified mitochondria as one of the sources of the IS-related ROS hotspots. In a recent study, we identified a novel calcium-redox feedback loop between the ORAI Ca2+ channels and the ROS-producing NADPH-oxidase 2 (NOX2) in primary human monocytes (Saul et al, 2016). Published and unpublished data also advocate for involvement of the mitochondrial calcium uniporter (MCU) (Petrungaro et al, 2015) in T-cell and monocyte-based immunity and propose these mitochondrial calcium channels as possible determinants of the immune cell calcium-redox feedback loop.

A recent study identified NOX2 as an important determinant of regulatory T cell-induced cardiovascular remodeling (Emmerson et al, 2018). However, the role of the ORAI-MCU-NOX2 cross regulation in T cells and monocytes in heart failure is not known.

Hypotheses of the PhD project

Our hypothesis is that the plasma membrane-based ORAI channels, the inner mitochondria membrane MCU channel and NOX enzymes form a regulatory complex that determines the role of T cells in heart failure.

Work programme

To test our hypothesis we will use a variety of in vitro and in vivo experimental approaches.

Aim 1: Evaluate the role of ORAI-MCU-NOX2 complex in isolated human T cells

Using high throughput live cell fluorescent microscopy, we will evaluate the calcium and ROS signals in primary human T cells from healthy donors, before and following physiological stimulation. Concretely, we will use fluorescent dyes and genetically encoded protein sensors to quantify calcium and H2O2 signals on a subcellular level i.e. in cellular compartments such as cytosol, mitochondrial intramembrane space and mitochondrial matrix. Moreover, using a set of functional assays, we will gain insights about the immune cell differentiation profiles, proliferation states and effector molecules (e.g. cytokines, chemokines and proteases) production. In addition, T cell migration will be evaluated using 2D and 3D migration/invasion assays. Given that protein expression levels and metabolic activity will depend on the exact immune cell type, we will link patterns of redox and calcium signals and protein expression levels with a certain T cell subset and thus gain insights regarding the specific role of ORAI1, MCU and NOX enzymes and their crosstalk in T cell function.

Aim 2: Examine the role of ORAI-MCU-NOX complex on T cell phospho- and redox-proteome

Calcium and ROS are closely linked with, and determine the posttranslational protein modifications such as oxidation and phosphorylation. In turn, protein oxidation and phosphorylation may directly determine the role of immune cells in heart failure. To examine which molecular determinants might be modified by the ORAI-MCU-NOX regulatory complex, we will perform phospho- and redox proteomic analysis in T-cells lacking or overexpressing ORAI1, MCU and/or NOX2. The immune cells will be of human (siRNA-, shRNA-, CRISPR/Cas9-modified) or of murine origin (knock out animal models, see Aim 3). This unbiased screening approach will help us in identifying novel molecular players under control of the ORAI-MCU-NOX2 complex in T cells. To test if these phospho- and redox patterns are altered during heart failure, the proteomic analysis will also be performed in T cells of heart failure animal models (see below).

Aim 3: Evaluate the role of ORAI-MCU-NOX complex in heart failure animal models

A number of in vivo experimental approaches and assays will be applied to link the findings in aim 1 and 2 with the pathological role of immune cells in heart failure. These include chronic systemic Angioteinsin II stimulation, left anterior descending coronary artery (LAD)-ligation and transverse aortic constriction (TAC)-ligation. T-cell (CD4+) specific ORAI1 (collaboration with Stefan Feske or Cd4-Cre (Jackson Laboratory [JAX] strain 017336 and Orai1fl/fl mice (Amgen)), MCU (collaboration with R. Rizzuto) and NOX2 (Ajay M. Shah Lab) knockout mice are commercially available or through collaborations and members of the IRTG1816 consortium.

Prof. Dr. Ivan Bogeski
Heart Center Göttingen, Department of Cardiovascular Physiology
+49 (0)551-39 65520 or 5896

Research interests: Redox signalling, Ca2+ channels

Prof. Dr. med. Dörthe M. Katschinski
Speaker IRTG 1816
Heart Centre Göttingen, Department of Cardiovascular Physiology
+49 (0)551-39 9778 or 5896

Research interests: Hypoxia sensing, signalling and adaptation

Prof. Dr. Ajay M. Shah
Speaker British Heart Foundation Centre of Research Excellence
BHF Centre of Research Excellence, KCL

Research interests: NADPH oxidases, redox signalling and heart failure

Dr. Aleksandar Ivetic
Speaker British Heart Foundation Centre of Research Excellence
Cardiovascular Division, KCL

Research interests: Mechanisms of inflammation

Magdalena Shumanska
PhD student 3rd cohort

RP 3:3 Exploring the role of T cell ORAI-MCU-NOX2 cross regulation in heart failure