Macrophage-derived foam cells are key regulators of atherogenesis. They accumulate in atherosclerotic plaques and support inflammatory processes by producing cytokines and chemokines. Identifying factors that regulate macrophage lipid uptake may reveal therapeutic targets for coronary artery disease (CAD). Here, we establish a high-throughput screening workflow to systematically identify genes that impact the uptake of DiI-labeled low-density lipoprotein (LDL) into monocyte-derived primary human macrophages. For this, monocytes isolated from peripheral blood were seeded onto 384-well plates, solid-phase transfected with siRNAs, differentiated in vitro into macrophages, and LDL-uptake per cell was measured by automated microscopy and quantitative image analysis. We applied this workflow to study how silencing of 89 genes impacts LDL-uptake into cells from 16 patients with CAD and 16 age-matched controls. Silencing of four novel genes (APOC1, CMTM6, FABP4, WBP5) reduced macrophage LDL-uptake. Additionally, knockdown of the chemokine receptor CXCR4 reduced LDL-uptake, most likely through a G-protein coupled mechanism that involves the CXCR4 ligand macrophage-induced factor (MIF), but is independent of CXCL12. We introduce a high-throughput strategy to systematically study gene function directly in primary CAD-patient cells. Our results propose a function for the MIF/CXCR4 signaling pathway, as well as several novel candidate genes impacting lipid uptake into human macrophages.

Streptococcus pyogenes is a major human pathogen causing a variety of diseases ranging from common pharyngitis to life-threatening soft tissue infections and sepsis. Microbial nucleic acids, especially bacterial RNA, have recently been recognized as a major group of pathogen-associated molecular patterns (PAMPs) involved in the detection of Streptococcus pyogenes via endosomal Toll-like receptors (TLRs) in vitro . However, the individual contribution and cooperation between TLRs as well as cell-type and strain specific differences in dependency on nucleic acid detection during S. pyogenes infection in vitro have not been clarified in detail. Moreover, the role of particularly bacterial RNA for the defense of S. pyogenes infection in vivo remains poorly defined. In this study, we report that in all investigated innate immune cells involved in the resolution of bacterial infections, including murine macrophages, dendritic cells and neutrophils, recognition of S. pyogenes strain ATCC12344 is almost completely dependent on nucleic acid sensing via endosomal TLRs at lower MOIs, whereas at higher MOIs, detection via TLR2 plays an additional, yet redundant role. We further demonstrate that different S. pyogenes strains display a considerable inter-strain variability with respect to their nucleic acid dependent recognition. Moreover, TLR13-dependent recognition of S. pyogenes RNA is largely non-redundant in bone marrow-derived macrophages (BMDMs), but less relevant in neutrophils and bone marrow-derived myeloid dendritic cells (BMDCs) for the induction of an innate immune response in vitro . In vivo , we show that a loss of nucleic acid sensing blunts early recognition of S. pyogenes , leading to a reduced local containment of the bacterial infection with subsequent pronounced systemic inflammation at later time points. Thus, our results argue for a crucial role of nucleic acid sensing via endosomal TLRs in defense of S. pyogenes infection both in vitro and in vivo .

Clonal hematopoiesis of indeterminate potential (CHIP) leads to higher mortality, carries a cardiovascular risk and alters inflammation. All three aspects harbor overlaps with the clinical manifestation of COVID-19. This study aimed to identify the impact of CHIP on COVID-19 pathophysiology. 90 hospitalized patients were analyzed for CHIP. In addition, their disease course and outcome were evaluated. With a prevalence of 37.8%, the frequency of a CHIP-driver mutation was significantly higher than the prevalence expected based on median age (17%). CHIP increases the risk of hospitalization in the course of the disease but has no age-independent impact on the outcome within the group of hospitalized patients. Especially in younger patients (45 – 65 years), CHIP was associated with persistent lymphopenia. In older patients (> 65 years), on the other hand, CHIP-positive patients developed neutrophilia in the long run. To what extent increased values of cardiac biomarkers are caused by CHIP independent of age could not be elaborated solely based on this study. In conclusion, our results indicate an increased susceptibility to a severe course of COVID-19 requiring hospitalization associated with CHIP. Secondly, they link it to a differentially regulated cellular immune response under the pressure of SARS-CoV-2 infection. Hence, a patient’s CHIP-status bears the potential to serve as biomarker for risk stratification and to early guide treatment of COVID-19 patients.

IntroductionThe bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are smaller, their resorptive activity is high which helps in efficient destruction of nasal turbinate bones of pigs.MethodsThe proteome of bone marrow-derived macrophages differentiated into osteoclasts with either RANKL or PMT was analysed. The results were verified by characterizing the metabolic activity using Seahorse analysis, a protein translation assay, immunoblots, real-time PCR as well as flow cytometry-based monitoring of mitochondrial activity and ROS production. A Gαq overexpression system using ER-Hoxb8 cells was used to identify Gαq-mediated metabolic effects on osteoclast differentiation and function.ResultsPMT induces the upregulation of metabolic pathways, which included strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components were expressed more efficiently, cells also displayed increased mitochondrial respiration. The heterotrimeric G protein Gαq plays a central role in this hypermetabolic cell activation as it triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. This seems to be caused by a direct interaction between STAT3 and OPA1 resulting in enhanced mitochondrial respiration. Overexpression of Gαq mimicked the hypermetabolic phenotype observed for PMT-induced osteoclasts and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. In addition, rheumatoid arthritis (RA) patients showed an increase in GNAQ expression, especially in the synovial fluid.DiscussionOur study suggests that Gαq plays a key role in PMT-induced osteoclastogenesis. Enhanced expression of GNAQ at the site of inflammation in RA patients indicates its pathophysiological relevance in the context of inflammatory bone disorders.