Perturbation in redox status elicits multiple cellular pathways, including those involved in the inflammatory response. A thiol-based molecule (I-152), releasing N-acetyl-cysteine (NAC) and β-mercaptoethylamine (MEA), was exploited as a redox-modulating agent, and its effects on pro-inflammatory cytokine expression and secretion in lipopolysaccharide (LPS)-stimulated macrophages (MΦ) were investigated. I-152 inhibited cytokine gene expression as well as protein secretion of the most important inflammatory cytokines in three different MΦ models in vitro and ex vivo. It alleviated inflammation via the c-Jun/AP-1 and NF-κB signaling pathways, depending on the dose, and regulated NLRP3 inflammasome expression, leading to decreased IL-1β and IL-18 release and reduced pyroptotic cell death. Consequently, the influence of redox-modulated MΦ secretome on the crosstalk with endothelial cells was evaluated. Co-culture experiments between THP-1 MΦ, that had been pre-treated with I-152 before LPS stimulation, and Human Vascular Endothelial Cells (HUVECs) showed reduced VCAM/ICAM expression in these cells in concomitance with a less oxidized and inflamed MΦ proteomic portrait. Overall, our findings suggest that I-152 redox modulation could target the AP-1/NLRP3 axis, affecting LPS-induced inflammation in MΦ and influencing HUVEC responses, revealing a complex and bidirectional interchange.

Oxysterols are a family of 27-carbon cholesterol oxidation derivatives found in low-density lipoproteins (LDLs) and atherosclerotic plaques where they trigger several biological responses involved in the initiation and progression of atherosclerosis. Several pieces of evidence suggest that oxysterols contribute to endothelial dysfunction (ED) due to their ability to alter membrane fluidity and cell permeability leading to inflammation, oxidative stress and apoptosis. The present study aimed to investigate the molecular events occurring in human microvascular endothelial cells (HMEC-1) in response to autoxidation-generated 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (SEC-B) exposure. Our results highlight that SEC-B rapidly activates HMEC-1 by inducing oxidative stress, nitric oxide (NO) production and pro-inflammatory cytokine release. Exposure to SEC-B up to 24 h results in persistent accumulation of the vasodilator NO paralleled by an upregulation of the endothelial nitric oxide synthase (eNOS) enzyme and downregulation of Caveolin-1 (Cav-1) protein levels. Moreover, reduced expression and extracellular release of the vasoconstrictor factor endothelin-1 (ET-1) are observed. Furthermore, SEC-B stimulates the expression of the cytokines interleukin-6 (IL-6) and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). This proinflammatory state leads to increased monocyte recruitment on activated HMEC-1 cells. Our findings add new knowledge on the role of SEC-B in ED and further support its potential implication in atherosclerosis.

Lymph node architecture and function are markedly altered with aging. We previously demonstrated that the lymph nodes of old C57BL/6 and BTBR male mice contain reduced levels of PA28αβ-capped proteasomes compared to young mice. Here, we examined aged BTBR females relative to aged males and found that proteasome remodeling occurs in a sex-specific manner. Specifically, old female exhibited higher immunoproteasome activity than old male, despite similar levels of proteasome and immunoproteasome subunit expression and proteasome complex assembly. In female mice activity was mostly driven by PA28αβ-capped 20S proteasomes that declined with age in males. These results highlight intriguing differences in proteasome assembly and activity between males and females providing evidence for sex-dependent changes in immunosenescence.

The persistence of P. aeruginosa infections is largely driven by the secretion of several factors during invasion, including the redox-active phenazine pyocyanin (PYO), which promotes biofilm formation and oxidative stress. Biofilms contribute to chronic infections and antibiotic resistance, limiting the efficacy of conventional therapies. We found that a synthetic compound, I-152, a conjugate of N-acetyl-L-cysteine (NAC) and S-acetylcysteamine (also known as S-acetyl-β-mercaptoethylamine; SMEA), effectively restored colistin susceptibility against P. aeruginosa by altering biofilm nanomechanical properties. These perturbations in matrix integrity were associated with I-152’s ability to hinder the phenazine redox cycle, shifting PYO to a reduced state as well as enabling S-conjugate formation. The compound decreased PYO accumulation in bacterial cultures and PYO-generated reactive oxygen species (ROS) in macrophage cells. Together with PYO, LPS is another driver of ROS-dependent inflammatory signaling in the host, which leads to an uncontrolled cytokine response and organ damage, especially in patients with cystic fibrosis. I-152 treatment downregulated the expression of LPS-induced inflammatory cytokines, i.e., IL-6 and TNF-α, in bone marrow-derived macrophages (BMDM) isolated from transgenic CFTR ^-/- and CFTR ^+/+ mice. Consistently, I-152 partially counteracted the inflammatory response in the P. aeruginosa LPS-induced acute lung injury murine model. Taken together, these results support I-152 as an adjunctive treatment for P. aeruginosa respiratory infections through a dual mechanism: combating antimicrobial resistance in biofilms and dampening host inflammation in the respiratory system. Highlights: I-152 potentiates colistin activity against P. aeruginosa by compromising the biofilm surface I-152 rewires the pyocyanin (PYO) redox state and forms covalent adducts with it PYO accumulation and PYO-induced ROS generation in macrophages is impaired by I-152 Ex vivo, I-152 dampens excessive pro-inflammatory response to P. aeruginosa LPS in CFTR ^- / ^- and CFTR ⁺ / ⁺ BM-derived macrophages I-152 (140 mg/Kg) attenuates LPS-driven inflammation and lung damage in CFTR ⁺ / ⁺ mice

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe’s effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAH ^enu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation. Highlights: - Proteasome complex levels and activity were upregulated in PAH ^enu2 mouse brains - Autophagy was also enhanced in PAH ^enu2 mouse brains - Despite increased PDI oxidation, the Unfolded Protein Response was not activated - Redox and energy metabolism were disrupted in both brain and blood

Ataxia-telangiectasia (A-T) is a multisystemic genetic disorder caused by pathogenic mutations in the ATM gene, with effects extending beyond the canonical DNA damage response. We investigated whether two compact ATM transcript variants, ATM 4-53 (exons 4 and 53 joined) and ATM SINT (an in silico-designed ATM variant), could rescue disease associated with cellular alterations and inform construct selection for future delivery strategies. A-T fibroblasts transduced with either ATM variants or an empty vector were compared with wild-type fibroblasts using complementary label-free and tandem mass tag-based quantitative proteomics. Bioinformatic pathway and network analyses were integrated with biochemical and functional validation assays. A-T fibroblasts displayed coordinated alterations in proteostasis, proteasome function, RNA processing pathways, interferon-associated signaling, extracellular matrix organization, cell migration, and nanomechanical properties. Expression of both ATM variants partially or extensively reversed these abnormalities, restoring protein folding and proteasomal components, reducing protein aggregation, attenuating inflammatory signaling, and normalizing extracellular matrix-related phenotypes. ATM SINT generally produced the broader and more consistent rescue, particularly for proteostasis and RNA processing-associated networks. These findings show that compact ATM variants can recover multiple non canonical ATM dependent cellular functions in A-T fibroblasts and identify ATM SINT as the more effective construct in this model. Proteomic profiling combined with orthogonal functional validation may therefore support rational selection of ATM variants for subsequent preclinical delivery studies.