Protein arginine deiminases (PADs) are a class of Ca2+ dependent cysteine hydrolases that catalyze a protein post-translational modification known as citrullination, where a positive protein-bound arginine residue is converted into a neutral unconventional citrulline residue. The reaction results in a shift of protein charge from positive to neutral, which dramatically impacts the structure, conformation, and function of proteins as well as intra/intermolecular interactions. Citrullination represents an important regulatory mechanism affecting several physiological cellular processes, such as gene expression, cell differentiation, apoptosis, and inflammatory immune responses. Among the PAD family members, PAD2 and PAD4, which are the only isoforms expressed by immune cells, have attracted considerable interest due to their role in producing neutrophil extracellular traps (NETs) and playing a detrimental role in a wide range of diseases such as Rheumatoid Arthritis, Ulcerative Colitis, Systemic Lupus Erythematosus, Multiple Sclerosis, and Alzheimer's Disease. Pharmacological PAD inhibition has brought beneficial therapeutic results in animal models of these pathologies since it leads to an amelioration of the disease-dependent inflammation and reduces immune cell accumulation and tissue damage. However, how PAD2 and PAD4 isoforms directly affect immune cell trafficking in the context of inflammation has not yet been studied. Hence, this project investigated the role of PAD-dependent citrullination in leukocyte adhesive activity under physiological and pathological conditions. We first treated neutrophils and lymphocytes with different concentrations of BB-Cl-amidine, a pan PAD inhibitor, and GSK199, which induces PAD4 specific blockade, and we studied the effect of cell treatment on 2- integrin-dependent adhesion. Our data showed that PAD inhibitors reduce integrin activation and binding to endothelial ligands upon stimulation in vitro with chemokines, suggesting a role for PADs in rapid leukocyte adhesion mediated by 2 integrins. In addition,, we demonstrated that BB-Cl-amidine also blocked lymphocyte 1-integrin-dependent adhesion on VCAM-1 counterligand, suggesting that PADs are involved in controlling signaling pathways common to both 2 and 1 integrin activation. To exclude potential unspecific effects exerted by PAD inhibitors, we confirmed the data obtained with the pharmacological approach and showed that mRNA silencing of PADs blocks integrin-dependent adhesion in vitro, further confirming the involvement of PADs in leukocyte adhesion. To characterize more in detail the role of PADs in adhesion induction, we studied LFA-1 conformational changes triggered by chemoattractants and found that treatment with BB-Cl-amidine and GSK-199 completely prevented integrin transition to more extended conformations, suggesting a role for PADs in integrin affinity increase leading to leukocyte adhesion. Considering the function of PAD2 and PAD4 in NET release and our previous data demonstrating a role for neutrophils in animal models of Alzheimer's disease, we next investigated the effect of PAD blockade in 3xTg-AD mice, which develop both amyloid and tau pathologies. We treated the 3xTg-AD mice with BB-Cl-amidine, and the results from behavioural tests showed that PAD inhibition significantly rescues spatial working memory and associative learning compared to untreated control mice. Histopathological analysis confirmed the beneficial effects of PAD inhibitors since microglial activation, amyloid accumulation, and tau phosphorylation were strongly reduced after PAD blockade. Finally, we quantified brain-infiltrating leukocytes in BB-Cl-amidine treated mice, reporting that treated animals show reduced accumulation of neutrophils, CD4+ cells, and B lymphocytes compared to untreated mice, suggesting a role for PADs in leukocyte trafficking into the brain in AD mice. In conclusion, this PhD project demonstrated that PAD2 and PAD4 are key elements controlling leukocyte adhesion and that PAD-dependent citrullination is required for integrin-mediated adhesion under physiological and pathological conditions. Moreover, we reported that PADs are involved in disease pathogenesis in 3xTg-AD mice and that therapeutic blockade of PADs ameliorates cognition and reduces neuropathology, suggesting that PAD inhibitors may offer a novel therapeutic strategy for AD.
|Titolo:||Protein Arginine Deiminases: a novel mechanism of leukocyte trafficking under physiological and pathological conditions|
|Data di pubblicazione:||2021|
|Appare nelle tipologie:||07.13 Doctoral Thesis|
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|Protein Arginine Deiminases: a novel mechanism of leukocyte trafficking under physiological and pathological conditions.pdf||Tesi di dottorato||Tesi di dottorato||Accesso ristretto||Open Access Visualizza/Apri|