Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloidspecific Csn5 deletion. Genetic deletion of Csn5 in Apoe −/− mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proinflammatory gene expression and NF-κB activation while enhancing HIF-1α levels and the expression of M2 marker Arginase 1 in inflammatory-elicited macrophages. MLN4924 further attenuated the expression of chemokines and adhesion molecules in endothelial cells and reduced NF-κB activation and monocyte arrest on activated endothelium in vitro. In vivo, MLN4924 reduced LPS-induced inflammation, favored an antiinflammatory macrophage phenotype, and decreased the progression of early atherosclerotic lesions in mice. On the contrary, MLN4924 treatment increased neutrophil and monocyte counts in blood and had no net effect on the progression of more advanced lesions. Our data show that CSN5 is atheroprotective. We conclude that MLN4924 may be useful in preventing early atherogenesis, whereas selectively promoting CSN5-mediated deNEDDylation may be beneficial in all stages of atherosclerosis.A therosclerosis is the primary cause of cardiovascular diseases. As a chronic inflammatory condition of the vessel wall, atherosclerosis is characterized by endothelial cell activation resulting in the secretion of chemoattractant proteins such as CCL2 and macrophage migration inhibitory factor (MIF) and by increased expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These molecules synergize to sequentially recruit inflammatory cells such as monocytes and T lymphocytes into the vessel wall (1-3).The transcription factor NF-κB plays a crucial role in vascular inflammation and atherogenesis, e.g., by controlling the expression of inflammatory cytokines, chemokines, and adhesion molecules that orchestrate the recruitment and adhesion of leukocytes. Also, numerous genes that regulate differentiation, survival, and proliferation of vascular and immune cells involved in the inflammatory response are targets of NF-κB (4). In resting cells, the NF-κB dimer, p65 and p50, is inactivated by binding to the inhibitor of κB (IκB)-α protein. Inflammatory challenges such as LPS or TNF-α exposure trigg...

Pseudomonas aeruginosa is a Gram‐negative bacterium of the proteobacteria class, and one of the most common causes of nosocomial infections. For example, it causes chronic pneumonia in cystic fibrosis patients. Patient sputum contains 2‐heptyl‐4‐hydroxyquinoline N‐oxide [HQNO] and Pseudomonas quorum sensing molecules such as the Pseudomonas quinolone signal [PQS]. It is known that HQNO inhibits the enzyme activity of mitochondrial and bacterial complex III at the Qi (quinone reduction) site, but the target of PQS is not known. In this work we have shown that PQS has a negative effect on mitochondrial respiration in HeLa and A549 cells. It specifically inhibits the complex I of the respiratory chain. In vitro analyses showed a partially competitive inhibition with respect to ubiquinone at the IQ site. In competing studies with Rotenone, PQS suppressed the ROS‐promoting effect of Rotenone, which is typical for a B‐type inhibitor. Prolonged incubation with PQS also had an effect on the activity of complex III.

Objective: Inflammation is a critical process during the progressive development and complication of abdominal aortic aneurysm. The co-stimulatory dyad CD40-CD40L is a major driver of inflammation and modulates immune responses. This study evaluates the potential of a small molecule inhibitor, which blocks the interaction between CD40 and tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6, referred to as TRAF-STOP, in the early and later phase during AAA progression. Methods and results: AAAs were induced in C57BL/6J mice by infrarenal aortic porcine pancreatic elastase infusion for 7, 14 or 28 days. Inhibition of CD40 signaling by TRAF-STOP resulted in less severe AAA formation and reduced the incidence of AAA development. TRAF-STOP treatment attenuated aortic structural remodeling, characterized by a reduced elastic fiber degradation, lowered expression of matrix metalloproteinase (MMP)-2 and MMP9, as well as preserved collagen type IV content in aneurysmal tissue. Furthermore, this is accompanied by the reduction of key pro-inflammatory genes such as TNFα. Conclusion: Pharmacological inhibition of CD40-TRAF6 signaling protects from adverse aortic structural remodeling during the early phase of AAA progression representing a translational strategy to limit progression of human AAA disease.

Background: Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease, and the pathogenesis is still poorly understood. Recent evidence suggests that AAA displays characteristics of an autoimmune disease and it gained increasing prominence that specific antigen-driven T cells in the aortic tissue may contribute to the initial immune response. Single-cell RNA T- and B cell receptor (TCR and BCR) sequencing is a powerful tool to investigate TCR and BCR clonality and thus to further test this hypothesis. However, difficulties such as very limited numbers of isolated cells must be considered during implementation and data analysis making biological interpretation of the data challenging. Here, we perform a representative analysis of scRNA TCR and BCR sequencing data of experimental murine AAA and show a reliable and streamlined bioinformatic processing pipeline highlighting opportunities and limitations of this approach. Methods: We performed single-cell RNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aortic segment of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham operated mice at day 3 and 28 as well as non-operated mice served as controls. Results: Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed no differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified multiple clones in 11 of 16 AAA samples and in 1 of 8 control samples. Comparison of the immune receptor repertoires indicated that only few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and TRBV12-2+TRBV13-2. Conclusion: In summary, we found no clonal expansion of TCRs or BCRs in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive amount of T and B cells to prevent undersampling and to enable detection of potential rare clones. Using this current scSeq-based approach we did not identify clonal enrichment of T or B cells in experimental AAA.