D'Avino, Danilo (2024) Inflammatory diseases: evaluation of sex dimorphism, social stress and new therapeutic strategies. [Tesi di dottorato]

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Abstract

Inflammation is a non-specific defense mechanism the body puts in place to neutralize a harmful phenomenon (pathogen invasion, cellular and tissue damage, etc.). It is a complex set of interactions between soluble factors and immune cells, in which a delicate balance between pro- and anti-inflammatory mediators regulates the duration and resolution of inflammatory response. This process can be influenced by both endogenous (e.g., sex) and exogenous (e.g., stress) factors. Sex impacts on inflammatory diseases in regard to their incidence, manifestations, gravity, and prognosis, as well as response to pharmacological treatments. In recent years, several preclinical studies have been conducted to elucidate some of the molecular mechanisms that underlie sex differences in some inflammatory diseases, such as respiratory diseases. In this regard, we focused on the different incidence of idiopathic pulmonary fibrosis (IPF), in which men are more affected, whose cellular and molecular mechanisms are still unknown. Thus, the first aim of this project was to investigate the role of sex in the initiation and progression of PF using bleomycin (BLM)-induced PF murine model. To this aim C57BL/6J mice of both sexes received subcutaneous BLM injections (3 times/week for 1-4 weeks) and animals were euthanized at different time points to evaluate PF initiation and progression. At the onset of the disease (1-2 weeks), BLM injection induced a more marked cell infiltration in male mice. Conversely, only a slight increase was found in females, with a physiological resolution of lung inflammation after 2 weeks. Furthermore, we observed higher levels of pro-inflammatory/pro-fibrotic mediators in male mice, such as transforming growth factor (TGF-β), interleukin(IL)-1β, and leukotriene(LT) B4, while females were protected by higher levels of anti-fibrotic mediators, such as interferon(IFN)-γ, prostaglandin(PG) E2 and miRNAs. This sex dimorphism in the balance between pro- and anti-fibrotic mediators well correlated with a more severe PF in BLM-treated males, as evidenced by histological analyses and collagen deposition. Pirfenidone (PRF), the drug used in clinical therapy, exhibited a greater activity in reversing PF hallmarks in males, regulating arachidonic acid (AA) metabolites, pro-fibrotic cytokines and antifibrotic miRNAs (214-3p and 96-5p). These results suggest that in early stage of PF both inflammatory reaction and fibrotic process occur in parallel in males and females, but an exacerbated fibrosis takes place only in male mice. Furthermore, PRF showed major efficacy in males, suggesting a possible sex-tailored therapy. In recent years, several studies have evaluated the impact of stress in its various forms, from social to negative emotions-induced stress, on the immune response. However, the main focus of these studies was the evaluation of specific neuroendocrine biomarkers or changes in gene expression in immune cells. So, the second aim of this thesis was to further investigate the role of an exogenous factor, such as social stress, in influencing the inflammatory response. For this purpose, we used an in vivo acute social stress model, where male CD1 mice were exposed to predator odor (fox urine, FU), following the induction of zymosan (ZYM) peritonitis. Four hours after peritonitis induction, we observed a reduced peritoneal neutrophilic infiltrate in ZYM + FU mice compared to the ZYM group (without FU exposure). Accordingly, FACS analysis revealed different stages of neutrophil degranulation in ZYM + FU mice, while ZYM groups showed intact neutrophils. NETosis is one of the mechanisms by which neutrophil death occurs and involves the release of neutrophil extracellular traps (NETs). ELISA assay revealed lower levels of NETs in ZYM + FU mice than ZYM group, probably due to their degradation by DNase. Additionally, FU exposure reduced ZYM-induced production of pro-inflammatory mediators (NOx, IL-1β, TNF-α, PGE2 and LTB4). In conclusion, our preliminary data suggest that an acute stressor exposure, such as fear, could act as a primer for a rapid immune-inflammatory response. Conventional anti-inflammatory therapy, mainly based on nonsteroidal anti-inflammatory drugs (NSAIDs), anti-LTs drugs, and glucocorticoids, has some limitations, including therapeutic inefficacy, high costs, and several side effects. For this purpose, the third aim of this project was to identify new synthetic and natural compounds for the treatment of both acute and chronic inflammatory diseases, such as acute pancreatitis (AP) and asthma. AP is a potentially life-threatening illness with limited options for pharmacological treatment. A library of compounds with indole and carbazole nuclei highly compatible with the soluble epoxide hydrolase (sEH) enzyme binding site was synthesized in collaboration with the University of Salerno (Italy). In vitro studies identified compound 28 (1-(((3R,5R,7R)-Adamantan-1-yl)methyl)-3-(1-(4-isopropylbenzyl)-1H-indol-5-yl)urea) as the most selective inhibitor of the isolated human recombinant sEH enzyme. Next, we evaluated the effects of the compound in cerulein-induced AP in mice. Pancreas H&E staining showed inter- and intralobular edema as well as immune cell infiltration (a prominent neutrophilia) after cerulein injections. The compound 28 showed a protective effect evidenced by the reduction in pancreatic edema and cell infiltration associated with a substantial increase in epoxyeicosatrienoic acid levels and a decrease in dihydroxyeicosatrienoic acid levels compared to control mice, suggesting an inhibitory effect of sEH activity in vivo. AP causes also multi-organ failure due to the development of systemic inflammation. Indeed, in our experimental conditions, cerulein induced pulmonary edema and inflammatory cell infiltration, which were reduced by administration of the compound. We observed a hepatic damage, evaluated through AST and ALT serum levels, following cerulein injection, and it decreased after treatment with the compound. In conclusion, our data pave the way for the development of potent and selective sEH inhibitor to be used as pharmacological strategy for the treatment of AP. Astragalus membranaceus (AM) root is used as herbal medicine in Chinese medicine for its immunomodulatory activities. Recently, beneficial properties of AM have been proposed for allergic diseases. In this study, we evaluated the role of a commercial extract of AM (16% of polysaccharides) in regulating the immune-inflammatory response in vitro and in vivo and its therapeutic application in asthma. AM extract inhibited the production of PGE2 and LTC4 in stimulated J774 and peritoneal macrophages, respectively. The extract also reduced IL-1β, TNF-α and nitrite production, affecting the expression of inducible nitric oxide synthase. In vivo experiments confirmed the anti-inflammatory properties of AM extract, as evidenced by the reduction of ZYM-induced peritoneal cell infiltration and pro-inflammatory mediator production. The efficacy of AM extract in modulating the immune response was confirmed in a model of allergic airway inflammation. The extract improved lung function by inhibiting airway hyperresponsiveness, airway remodeling, and immune response. In conclusion, our data demonstrated that this commercial extract of AM exerts in vitro and in vivo anti-inflammatory effects, suggesting its use as a food supplement in inflammatory diseases.

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