Functional multi-organelle units control inflammatory lipid metabolism of macrophages.

Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multi-spectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions. We use this analysis on macrophages, immune cells that undergo rapid metabolic switches upon sensing bacterial and inflammatory stimuli. Our results identify lipid droplets (LDs) as primary inflammatory responder organelle, which forms three- and four-way interactions with other organelles. While clusters with endoplasmic reticulum (ER) and mitochondria (mitochondria-ER-LD unit) help supply fatty acids for LD growth, the additional recruitment of peroxisomes (mitochondria-ER-peroxisome-LD unit) supports fatty acid efflux from LDs. Interference with individual components of these units has direct functional consequences for inflammatory lipid mediator synthesis. Together, we show that macrophages form functional multi-organellar units to support metabolic adaptation and provide an experimental strategy to identify organelle-metabolic signalling hubs.

Targeted Metabolomics on Rare Primary Cells.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming.

Neutrophil swarming is an essential process of the neutrophil response to many pathological conditions. Resultant neutrophil accumulations are hallmarks of acute tissue inflammation and infection, but little is known about their dynamic regulation. Technical limitations to spatiotemporally resolve individual cells in dense neutrophil clusters and manipulate these clusters in situ have hampered recent progress. We here adapted an in vitro swarming-on-a-chip platform for the use with confocal laser-scanning microscopy to unravel the complexity of single-cell responses during neutrophil crowding. Confocal sectioning allowed the live visualization of subcellular components, including mitochondria, cell membranes, cortical actin, and phagocytic cups, inside neutrophil clusters. Based on this experimental setup, we identify that chemical inhibition of the Arp2/3 complex causes cell death in crowding neutrophils. By visualizing spatiotemporal patterns of reactive oxygen species (ROS) production in developing neutrophil swarms, we further demonstrate a regulatory role of the metabolic pentose phosphate pathway for ROS production and neutrophil cluster growth.

Slow integrin-dependent migration organizes networks of tissue-resident mast cells.

Immune cell locomotion is associated with amoeboid migration, a flexible mode of movement, which depends on rapid cycles of actin polymerization and actomyosin contraction1. Many immune cells do not necessarily require integrins, the major family of adhesion receptors in mammals, to move productively through three-dimensional tissue spaces2,3. Instead, they can use alternative strategies to transmit their actin-driven forces to the substrate, explaining their migratory adaptation to changing external environments4-6. However, whether these generalized concepts apply to all immune cells is unclear. Here, we show that the movement of mast cells (immune cells with important roles during allergy and anaphylaxis) differs fundamentally from the widely applied paradigm of interstitial immune cell migration. We identify a crucial role for integrin-dependent adhesion in controlling mast cell movement and localization to anatomical niches rich in KIT ligand, the major mast cell growth and survival factor. Our findings show that substrate-dependent haptokinesis is an important mechanism for the tissue organization of resident immune cells.

LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells.

Metabolism plays a fundamental role in regulating cellular functions and fate decisions. Liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomic approaches provide high-resolution insights into the metabolic state of a cell. However, the typical sample size is in the order of 105-107 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step. Here, we present a comprehensively optimized protocol for targeted metabolomics on rare cell types, such as hematopoietic stem cells and mast cells. Only 5000 cells per sample are required to detect up to 80 metabolites above background. The use of regular-flow liquid chromatography allows for robust data acquisition, and the omission of drying or chemical derivatization avoids potential sources of error. Cell-type-specific differences are preserved while the addition of internal standards, generation of relevant background control samples, and targeted metabolite with quantifiers and qualifiers ensure high data quality. This protocol could help numerous studies to gain thorough insights into cellular metabolic profiles and simultaneously reduce the number of laboratory animals and the time-consuming and costly experiments associated with rare cell-type purification.

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions.

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.

Neutrophils: Amoeboid Migration and Swarming Dynamics in Tissues.

Neutrophils are key cells of our innate immune response with essential roles for eliminating bacteria and fungi from tissues. They are also the prototype of an amoeboid migrating leukocyte. As one of the first blood-recruited immune cell types during inflammation and infection, these cells can invade almost any tissue compartment. Once in the tissue, neutrophils undergo rapid shape changes and migrate at speeds higher than most other immune cells. They move in a substrate-independent manner in interstitial spaces and do not follow predetermined tissue paths. Instead, neutrophil navigation is largely shaped by the chemokine and chemoattractant milieu around them. This highlights the decisive role of attractant-sensing G-protein coupled receptors (GPCRs) and downstream molecular pathways for controlling amoeboid neutrophil movement in tissues. A diverse repertoire of cell-surface expressed GPCRs makes neutrophils the perfect sentinel cell type to sense and detect danger-associated signals released from wounds, inflamed interstitium, dying cells, complement factors or directly from tissue-invading microbes. Moreover, neutrophils release attractants themselves, which allows communication and coordination between individual cells of a neutrophil population. GPCR-mediated positive feedback mechanisms were shown to underlie neutrophil swarming, a population response that amplifies the recruitment of amoeboid migrating neutrophils to sites of tissue injury and infection. Here we discuss recent findings and current concepts that counteract excessive neutrophil accumulation and swarm formation. In particular, we will focus on negative feedback control mechanisms that terminate neutrophil swarming to maintain the delicate balance between tissue surveillance, host protection and tissue destruction.

Positive feedback amplification in swarming immune cell populations.

Several immune cell types (neutrophils, eosinophils, T cells, and innate-like lymphocytes) display coordinated migration patterns when a population, formed of individually responding cells, moves through inflamed or infected tissues. "Swarming" refers to the process in which a population of migrating leukocytes switches from random motility to highly directed chemotaxis to form local cell clusters. Positive feedback amplification underlies this behavior and results from intercellular communication in the immune cell population. We here highlight recent findings on neutrophil swarming from mouse models, zebrafish larvae, and in vitro platforms for human cells, which together advanced our understanding of the principles and molecular mechanisms that shape immune cell swarming.

Neutrophils self-limit swarming to contain bacterial growth in vivo.

Neutrophils communicate with each other to form swarms in infected organs. Coordination of this population response is critical for the elimination of bacteria and fungi. Using transgenic mice, we found that neutrophils have evolved an intrinsic mechanism to self-limit swarming and avoid uncontrolled aggregation during inflammation. G protein-coupled receptor (GPCR) desensitization acts as a negative feedback control to stop migration of neutrophils when they sense high concentrations of self-secreted attractants that initially amplify swarming. Interference with this process allows neutrophils to scan larger tissue areas for microbes. Unexpectedly, this does not benefit bacterial clearance as containment of proliferating bacteria by neutrophil clusters becomes impeded. Our data reveal how autosignaling stops self-organized swarming behavior and how the finely tuned balance of neutrophil chemotaxis and arrest counteracts bacterial escape.

Effect of Radiofrequency Radiation on Human Hematopoietic Stem Cells.

Exposure to electromagnetic fields in the radiofrequency range is ubiquitous, mainly due to the worldwide use of mobile communication devices. With improving technologies and affordability, the number of cell phone subscriptions continues to increase. Therefore, the potential effect on biological systems at low-intensity radiation levels is of great interest. While a number of studies have been performed to investigate this issue, there has been no consensus reached based on the results. The goal of this study was to elucidate the extent to which cells of the hematopoietic system, particularly human hematopoietic stem cells (HSC), were affected by mobile phone radiation. We irradiated HSC and HL-60 cells at frequencies used in the major technologies, GSM (900 MHz), UMTS (1,950 MHz) and LTE (2,535 MHz) for a short period (4 h) and a long period (20 h/66 h), and with five different intensities ranging from 0 to 4 W/kg specific absorption rate (SAR). Studied end points included apoptosis, oxidative stress, cell cycle, DNA damage and DNA repair. In all but one of these end points, we detected no clear effect of mobile phone radiation; the only alteration was found when quantifying DNA damage. Exposure of HSC to the GSM modulation for 4 h caused a small but statistically significant decrease in DNA damage compared to sham exposure. To our knowledge, this is the first published study in which putative effects (e.g., genotoxicity or influence on apoptosis rate) of radiofrequency radiation were investigated in HSC. Radiofrequency electromagnetic fields did not affect cells of the hematopoietic system, in particular HSC, under the given experimental conditions.

TAp73 is a central transcriptional regulator of airway multiciliogenesis.

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

Exploring the Arabidopsis sulfur metabolome.

Sulfur plays a crucial role in protein structure and function, redox status and plant biotic stress responses. However, our understanding of sulfur metabolism is limited to identified pathways. In this study, we used a high-resolution Fourier transform mass spectrometric approach in combination with stable isotope labeling to describe the sulfur metabolome of Arabidopsis thaliana. Databases contain roughly 300 sulfur compounds assigned to Arabidopsis. In comparative analyses, we showed that the overlap of the expected sulfur metabolome and the mass spectrometric data was surprisingly low, and we were able to assign only 37 of the 300 predicted compounds. By contrast, we identified approximately 140 sulfur metabolites that have not been assigned to the databases to date. We used our method to characterize the γ-glutamyl transferase mutant ggt4-1, which is involved in the vacuolar breakdown of glutathione conjugates in detoxification reactions. Although xenobiotic substrates are well known, only a few endogenous substrates have been described. Among the specifically altered sulfur-containing masses in the ggt4-1 mutant, we characterized one endogenous glutathione conjugate and a number of further candidates for endogenous substrates. The small percentage of predicted compounds and the high proportion of unassigned sulfur compounds identified in this study emphasize the need to re-evaluate our understanding of the sulfur metabolome.