Nuclear receptor Nur77 regulates immunomechanics of macrophages.

Nuclear receptor Nur77 plays a pivotal role in immune regulation across various tissues, influencing pro-inflammatory signaling pathways and cellular metabolism. While cellular mechanics have been implicated in inflammation, the contribution of Nur77 to these mechanical processes remains elusive. Macrophages exhibit remarkable plasticity in their morphology and mechanics, enabling them to adapt and execute essential inflammatory functions, such as navigating through inflamed tissue and pathogen engulfment. However, the precise regulatory mechanisms governing these dynamic changes in macrophage mechanics during inflammation remain poorly understood. To establish the potential correlation of Nur77 with cellular mechanics, we compared bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Nur77-deficient (Nur77-KO) mice and employed cytoskeletal imaging, single-cell acoustic force spectroscopy (AFS), migration and phagocytosis assays, and RNA-sequencing. Our findings reveal that Nur77-KO BMDMs exhibit changes to their actin networks compared to WT BMDMs, which is associated with a stiffer and more rigid phenotype. Subsequent in vitro experiments validated our observations, showcasing that Nur77 deficiency leads to enhanced migration, reduced adhesion, and increased phagocytic activity. The transcriptomics data confirmed altered mechanics-related pathways in Nur77-deficient macrophage that are accompanied by a robust pro-inflammatory phenotype. Utilizing previously obtained ChIP-data, we revealed that Nur77 directly targets differentially expressed genes associated with cellular mechanics. In conclusion, while Nur77 is recognized for its role in reducing inflammation of macrophages by inhibiting the expression of pro-inflammatory genes, our study identifies a novel regulatory mechanism where Nur77 governs macrophage inflammation through the modulation of expression of genes involved in cellular mechanics. Our findings suggest that immune regulation by Nur77 may be partially mediated through alterations in cellular mechanics, highlighting a potential avenue for therapeutic targeting.

Probing human immune cell mechanics using acoustic force spectroscopy.

Immune cells continuously adapt their mechanical properties for proper circulation and elicitation of immune responses. Here, we provide a step-by-step protocol for probing the single-cell mechanical properties of primary human monocytes using acoustic force spectroscopy (AFS). We describe steps for the calibration of the AFS chips, the isolation of monocytes from buffy coats, and the probing of monocyte mechanics using AFS. We then detail the data analysis strategy. The protocol is useful for characterizing a wide range of immune cells under various conditions in physiology and pathology. For complete details on the use and execution of this protocol, please refer to Evers et al. 1 and Evers et al.2.

Biochemical reaction network topology defines dose-dependent Drug-Drug interactions.

Drug combination therapy is a promising strategy to enhance the desired therapeutic effect, while reducing side effects. High-throughput pairwise drug combination screening is a commonly used method for discovering favorable drug interactions, but is time-consuming and costly. Here, we investigate the use of reaction network topology-guided design of combination therapy as a predictive in silico drug-drug interaction screening approach. We focused on three-node enzymatic networks, with general Michaelis-Menten kinetics. The results revealed that drug-drug interactions critically depend on the choice of target arrangement in a given topology, the nature of the drug, and the desired level of change in the network output. The results showed a negative correlation between antagonistic interactions and the dosage of drugs. Overall, the negative feedback loops showed the highest synergistic interactions (the lowest average combination index) and, intriguingly, required the highest drug doses compared to other topologies under the same condition.

Topological dynamics of an intrinsically disordered N-terminal domain of the human androgen receptor.

Human androgen receptor contains a large N-terminal domain (AR-NTD) that is highly dynamic and this poses a major challenge for experimental and computational analysis to decipher its conformation. Misfolding of the AR-NTD is implicated in prostate cancer and Kennedy's disease, yet our knowledge of its structure is limited to primary sequence information of the chain and a few functionally important secondary structure motifs. Here, we employed an innovative combination of molecular dynamics simulations and circuit topology (CT) analysis to identify the tertiary structure of AR-NTD. We found that the AR-NTD adopts highly dynamic loopy conformations with two identifiable regions with distinct topological make-up and dynamics. This consists of a N-terminal region (NR, residues 1-224) and a C-terminal region (CR, residues 225-538), which carries a dense core. Topological mapping of the dynamics reveals a traceable time-scale dependent topological evolution. NR adopts different positioning with respect to the CR and forms a cleft that can partly enclose the hormone-bound ligand-binding domain (LBD) of the androgen receptor. Furthermore, our data suggest a model in which dynamic NR and CR compete for binding to the DNA-binding domain of the receptor, thereby regulating the accessibility of its DNA-binding site. Our approach allowed for the identification of a previously unknown regulatory binding site within the CR core, revealing the structural mechanisms of action of AR inhibitor EPI-001, and paving the way for other drug discovery applications.

Single cell force spectroscopy of erythrocytes at physiological and febrile temperatures reveals mechano-modulatory effects of atorvastatin.

RBCs are mechanically active cells and constantly deform as they circulate through vasculature. Their mechanical properties can be significantly altered by various pathophysiological conditions, and the alterations in RBC mechanics can, in turn, have functional consequences. Although numerous mechanical studies have been conducted on RBCs, surprisingly, strain-rate and temperature dependent mechanics of RBCs have not been systematically examined, and current data is primarily based on measurements at room temperature. Here, we have used state-of-the-art single-cell optical tweezers to probe atorvastatin-induced changes of RBC mechanics and its strain-rate dependency at physiologically and medically relevant temperatures. Our data indicate that RBC mechanics is strain-rate and temperature dependent, and atorvastatin treatment softens RBCs at physiological temperature, but not at febrile temperature. The observed mechanical change is a notable side effect of the drug in some therapeutic applications. However, the mechano-modulatory effects of atorvastatin on erythrocytes at physiological temperature might offer new therapeutic possibilities for diseases related to blood cell mechanics.

Single-cell analysis reveals chemokine-mediated differential regulation of monocyte mechanics.

Monocytes continuously adapt their shapes for proper circulation and elicitation of effective immune responses. Although these functions depend on the cell mechanical properties, the mechanical behavior of monocytes is still poorly understood and accurate physiologically relevant data on basic mechanical properties are lacking almost entirely. By combining several complementary single-cell force spectroscopy techniques, we report that the mechanical properties of human monocyte are strain-rate dependent, and that chemokines can induce alterations in viscoelastic behavior. In addition, our findings indicate that human monocytes are heterogeneous mechanically and this heterogeneity is regulated by chemokine CCL2. The technology presented here can be readily used to reveal mechanical complexity of the blood cell population in disease conditions, where viscoelastic properties may serve as physical biomarkers for disease progression and response to therapy.

ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response.

Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells' full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.

Reciprocal regulation of cellular mechanics and metabolism.

Metabolism and mechanics are intrinsically intertwined. External forces, sensed through the cytoskeleton or distortion of the cell and organelles, induce metabolic changes in the cell. The resulting changes in metabolism, in turn, feed back to regulate every level of cell biology, including the mechanical properties of cells and tissues. Here we examine the links between metabolism and mechanics, highlighting signalling pathways involved in the regulation and response to cellular mechanosensing. We consider how forces and metabolism regulate one another through nanoscale molecular sensors, micrometre-scale cytoskeletal networks, organelles and dynamic biomolecular condensates. Understanding this cross-talk will create diagnostic and therapeutic opportunities for metabolic disorders such as cancer, cardiovascular pathologies and obesity.

Deciphering Metabolic Heterogeneity by Single-Cell Analysis.

Single-cell analysis provides insights into cellular heterogeneity and dynamics of individual cells. This Feature highlights recent developments in key analytical techniques suited for single-cell metabolic analysis with a special focus on mass spectrometry-based analytical platforms and RNA-seq as well as imaging techniques that reveal stochasticity in metabolism.