Background: IFNg is a prominent proinflammatory cytokine that holds a pivotal position in both inflammation and autoimmune disorders. STAT1, the central transcription factor to the IFNg response is activated by JAK kinases to mediate the activation of Interferon Stimulated Genes. Dysregulation or mutation of STAT1 results in severe conditions, compromising the body's defense against pathogens or triggering autoimmune responses. This is underscored by Single Nucleotide Polymorphisms (SNPs) of STAT1 gene. Amongst them, SNPs affecting specific Cysteines cause Gain of Function (GOF) phenotype that has been identified as the etiology of Chronic Mucocutaneous Candidiasis Disease (CMCD) rare disease. This suggests a crucial role of Cys in enabling an effective STAT1-dependent response. Regulation of STAT1 activity through different post-translational modifications is well-characterized. Our study focuses on understanding if and how Cysteines reversible oxidative post-translational modifications (ox-PTMs) contribute to the fine-tuning of STAT1 regulation in the context of IFNg stimulation. Methods and results: Using maleimide-derivative bioswitch methods to label Cys ox-PTMs and immunoblotting, we demonstrated that STAT1 is subjected to reversible Cys ox-PTMs. Additionally, using the DCP-Bio1 probe, we demonstrate that STAT1 undergoes Cys sulfenylation in response to IFNg. Analysis of Cys/Ala mutations revealed that mutations in two functional domains are associated with GOF phenotypes, characterized by increased activating phosphorylation, nuclear accumulation, decreased dephosphorylation. RNASeq analysis confirmed increased IFNg-induced gene expression by the C/A mutants. Moreover, oxidant treatment impaired IFNg-induced STAT1 phosphorylation. Conclusion: Our data support a model in which previously unrecognized reversible ox-PTMs of STAT1 Cysteine residues dampen STAT1 activity in part through the promotion of STAT1 dephosphorylation. This opens doors for the future development of drugs targeting Cys residues to control STAT1 activity.

Oxidized low density lipoproteins (oxLDL) interact with the cluster of differentiation 36 receptor (CD36), a scavenger membrane protein, at the surface of macrophages. Internalization of oxLDL by CD36 is implicated in initiation of atherosclerosis pathology. The synthetic cyclic azapeptide MPE-298 binds selectively to CD36 with high affinity and exhibits potent anti-atherosclerotic effects in mice. Aiming to decipher the anti-atherosclerotic mechanism of MPE-298, CD36 internalization and intracellular trafficking was investigated in murine macrophages. After transfection with mouse CD36 conjugated to green fluorescent protein (GFP), J774 macrophages were incubated with either MPE-298 (100 nM) or oxLDL (25 μg/mL) for 5 to 15 minutes. The intracellular trafficking of MPE-298 was also monitored in RAW 264.7 macrophages, which were incubated with MPE-298 tagged with ATTO-465 as a fluorescent probe (5 µM) for 10 to 30 minutes. Colocalization with CD36 was examined using specific antibodies for labeling in different endocytic compartments: early (EEA-1), late (Rab7) and recycling (Rab11) endosomes, as well as lysosomes (LAMP-1). Analysis using the ImageJ software indicated that upon exposure to MPE-298, CD36 is rapidly internalized in the macrophage and colocalized within the early and late endosomes and lysosomes, but not within the recycling endosomes. Similarly, the fluorescent MPE-298 analog is also internalized and colocalized within the late endosomes and lysosomes. In contrast, upon macrophage exposure to oxLDL, CD36 colocalizes mainly within the early endosomes. In conclusion, binding of MPE-298 by CD36 at the macrophage membrane induces rapid endocytosis of the CD36-MPE-298 complex and rapid transfer to lysosomal compartments. In addition to negatively modulating CD36 expression on membranes, MPE-298, compared to oxLDL, causes alternate intracellular localization of CD36 within the macrophages. The latter may explain in part the first steps in the anti-atherosclerotic mechanism of MPE-298. Supported by the Canadian Institutes of Health Research.

Neutrophils play a central role in the host's response to infection, contribute to the inflammatory process and influence cancer development. They communicate with other cells, shaping the immune response through the release of soluble components such as granular proteins, cytokines, and ROS. Additionally, they may release more complex and insoluble components like neutrophil extracellular traps (NETs) or exosomes. Upon deeper investigation, we have identified for the first time the presence of glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) and hyaluronic acid (HA) within NETs. This discovery demonstrates, in an unprecedented manner, that neutrophils are capable of secreting GAGs. Moreover, we have identified a novel DNA-free fibrillar complex, termed proteoglycofili (PGF), which is released by living neutrophils under anoxic conditions, more accurately mimicking the environment of infection and inflammation. We identified that PGF is also composed of granular proteins and both CS and HA. By subjecting PGF and NETs to hyaluronidase treatment, we established that CS and HA are crucial for the maintenance of their filamentous structure and antimicrobial activity. Furthermore, we have demonstrated that the release of these GAGs by neutrophils is dependent on the NADPH oxidase activity, and was shown to be impaired in neutrophils from patients with chronic granulomatous disease (CGD). We have confirmed the secretion of these GAGs by neutrophils, either within PGF or NETs, across various inflammatory disease models, such as mouse colon tumor and mouse prostate adenocarcinoma, and an infectious guinea pig model of shigellosis. Given the broad regulatory role of GAGs in both health and disease, our results strongly indicate that the secretion of GAGs by neutrophils could profoundly influence in the development of many diseases.