Carsten Kummerow

Differential Redox Regulation of ORAI Ion Channels: A Mechanism to Tune Cellular Calcium Signaling

Redox sensitivity of T cells decreases through ORAI Ca2+ channel subunit switching during T cell differentiation. Adapting to Oxidizing Environments Reactive oxygen species (ROS) were thought for many years to be only detrimental, causing damage to DNA and proteins. However, it has become clear that ROS, particularly H2O2, can act as intracellular signaling molecules that link cellular redox state to such processes as proliferation and differentiation. Bogeski et al. have uncovered a role for ROS in regulating calcium channel activity—and intracellular Ca2+ signals crucial to the immune response—in T lymphocytes. They found that activity of ORAI1 calcium channels was blocked by H2O2, whereas that of the related ORAI3 channels was not. Redox sensitivity decreased as naïve human T helper lymphocytes differentiated into effector T helper lymphocytes, which was associated with an increase in the abundance of mRNA encoding the insensitive ORAI3 protein. The authors suggest that changes in the specific complement of ORAI channels, and thereby sensitivity to ROS, could enable T lymphocytes to fine tune cellular responses in oxidizing environments such as those found during inflammation. Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca2+) influx through Ca2+ release–activated Ca2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca2+ imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H2O2) on ORAI channel activity and human T helper lymphocyte (TH cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H2O2. The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. TH cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector TH cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector TH cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector TH cells may tune cellular responses under oxidative stress.

Calcium microdomains at the immunological synapse: how ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium‐dependent T‐cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium‐dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re‐distributed into areas beneath mitochondria, which prevented PMCA up‐modulation and decreased calcium export locally. This nano‐scale distribution—only induced following IS formation—maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.

Redox regulation of calcium ion channels: chemical and physiological aspects.

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease. Changes in the intracellular free Ca(2+) concentration are key triggers for diverse cellular functions. Ca(2+) homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca(2+) permeable ion channels such as voltage-gated (CaV) Ca(2+) channels, transient receptor potential (TRP) channels and Orai channels.

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance.

Ca(2+) homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca(2+) signaling is how different Ca(2+) signals are translated into specific cell functions. In T cells, Ca(2+) signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca(2+) influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca(2+) influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca(2+) signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca(2+) signaling may be linked to immune suppressive diseases and autoimmune diseases.

Calcium dependence of T cell proliferation following focal stimulation

Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca2+ dependence of proliferation and apoptosis in primary human CD4+ T cells following stimulation with anti‐CD3/anti‐CD28‐coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL‐2 production and proliferation than non‐focal TCR stimuli. Surprising little Ca2+ entry through Ca2+ channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca2+]i) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4+ T cells. We also show that proliferation was very Ca2+ sensitive in the range 90–120 nM, whereas apoptosis was basically constant for [Ca2+]i levels of 90–120 nM. We conclude that very small changes in [Ca2+]i can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

The mitochondrial calcium uniporter regulates breast cancer progression via HIF‐1α

Triple‐negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca2+ uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca2+ uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU‐silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia‐inducible factor‐1α (HIF‐1α) is reduced, suggesting a signaling role for mROS and HIF‐1α, downstream of mitochondrial Ca2+. Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF‐1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca2+ uptake is a potential novel therapeutic target for clinical intervention.

The immunological synapse controls local and global calcium signals in T lymphocytes

Summary:  Cell polarization is a key feature of T‐cell function. The immunological synapse (IS) between T cells and antigen‐presenting cells is a beautiful example of how polarization of cells is used to guide cell function. Receptors, signal transducers, the cytoskeleton, and organelles are enriched at or depleted from the IS after its formation, and in many cases these re‐localizations have already been linked with certain T‐cell functions. One key step for T‐cell activation is a rise in the cytoplasmic calcium concentration. Whereas it is undisputed that the IS initiates and controls calcium signals in T cells, very little is known about the role of T‐cell polarization for calcium signals and calcium‐dependent signal transduction. We briefly summarize the basic commonly agreed principles of IS‐dependent calcium signal generation but then focus on the less well understood influence of polarization on calcium signals. The discussion of the role of polarization for calcium signals leads to a model how the IS controls local and global calcium signals and calcium‐dependent T‐cell functions. We develop a theoretical formalism based on existing spatiotemporal calcium dynamic simulations to better understand the model in the future and allow further predictions which can be tested by fast, high resolution live‐cell microscopy.

Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

Background: The function of formyl peptide receptors (FPRs) is incompletely understood. Results: We report the identification of bacterial signal peptides as potent activators of mammalian FPRs and innate immune responses and define critical features underlying FPR peptide recognition. Conclusion: These findings identify a molecular signature for FPR activation. Significance: Our results define a novel mechanism for sensing bacteria. Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals

Sustained Ca(2+) influx through plasma membrane Ca(2+) released-activated Ca(2+) (CRAC) channels is essential for T cell activation. Since inflowing Ca(2+) inactivates CRAC channels, T cell activation is only possible if Ca(2+)-dependent inactivation is prevented. We have previously reported that sustained Ca(2+) influx through CRAC channels requires both mitochondrial Ca(2+) uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca(2+)-dependent channel inactivation. Here, we show that morphological changes following formation of the immunological synapse (IS) modulate Ca(2+) influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca(2+) entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca(2+) signals. The shape change-dependent mitochondrial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca(2+) influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca(2+) dependent T cell activation.

Docking of Lytic Granules at the Immunological Synapse in Human CTL Requires Vti1b-Dependent Pairing with CD3 Endosomes

Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8+ T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.