Raucci, Federica (2022) TH17 AND IL-17 PATHWAY IN INFLAMMATION AND INFLAMMATORY-RELATED DISEASES. [Tesi di dottorato]

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Abstract

IL-17 (IL-17A, originally called CTLA-8) was cloned in 1993, but its functions remained obscure for a decade. In 2005, IL-17 became prominent with the discovery of a new population of CD4+ T helper (Th) cells characterized by the expression of IL-17. This subset became known as Th17 cells, and a plethora of literature has since been devoted to understanding the mechanisms that direct the development, differentiation, and regulation of this lineage. Although Th17 cells are typically considered the principal source of IL-17, CD8+ cells have also been shown to make this cytokine. In addition, several innate immune cells produce this cytokine, including neutrophils, macrophages, innate-acting lymphocytes such as γδ T cells, some natural killer T cells (NKT), and IL-17-expressing type 3 innate lymphoid cells (ILC3s). Collectively, IL-17-producing cells, whether adaptive or innate, are often termed type 17. IL-17 is a cytokine that elicits protection against extracellular bacterial and fungal infections and plays an important role in inflammation. However, when produced in excess, it contributes to chronic inflammation associated with many inflammatory and autoimmune disorders. Indeed, the localized and prolonged release of IL-17 in specific tissues has been associated with increased severity of the inflammatory response that remains sustained over time. The cellular and molecular mechanisms behind these effects are far from being clear. This has made IL-17 an attractive therapeutic target. Antibodies targeting IL-17 (secukinumab and ixekizumab) were approved in 2016 to treat moderate to severe plaque psoriasis. However, the efficacy of IL-17 blockade for other conditions has been less dramatic, although there are promising data from trials in ankylosing spondylitis and psoriatic arthritis. Disappointingly and somewhat surprisingly, trials of secukinumab and brodalumab (anti-IL-17RA) in Crohn’s disease were terminated early due to the worsening of the disease in the treatment group. An explanation for this paradox came from studies in mice showing a dominant protective role for IL-17 in maintaining intestinal barrier integrity that apparently outweighs its tissue-damaging potential in inflammatory bowel diseases. Thus, targeting IL-17 is an effective therapy for specific conditions, but its clinical use has revealed new insights into how Th17 cells function in humans. Since the IL-17 discovery, much attention has been given to mediators and factors responsible for the development of IL-17-producing cells, while very few studies have investigated the inflammatory properties of this cytokine. However, recent biochemical and pharmacological studies have reported that in several tissues and cell types, microsomal PGE2 synthase (mPGES) and peroxisome proliferator-activated receptor γ (PPAR-γ) expression are modulated by a variety of inflammatory factors and stimuli. Considering that very little is known about the biological effects promoted by IL-17 in the context of mPGES-1/PPAR-γ modulation, this PhD thesis reports ours in vitro and in vivo evidence that defines the functional coordinate regulation between these two enzymes at the "crossroads of phlogistic pathway" involved in the induction and resolution of inflammation (the first and the second year of PhD). The latter part of the thesis (the third year of PhD and PhD visiting program in the UK) is dedicated to the design of peptide molecules that could be easily synthesized and used as a stable surrogate of IL-17. Specifically, a 20 amino acid peptide (here referred to as nIL-17TM) that retains the primary biological function of the parental molecule has been identified. The peptide was tested in both in vitro and in vivo models of inflammation, and it has been biologically validated in both murine and human cellular systems (neutrophils and macrophages). In addition, enzymatic binding assays were contextually performed to evaluate the peptide’s binding affinity on IL-17 receptors (both IL-17RA and IL-17RC). Finally, last but not least of importance, we describe the synthesis and main pharmacological properties of a novel patented IL-17 neutralizing antibody (here referred to as Ab-IPL-IL-17™) that displayed significant activity on both murine and human biological systems. Also, we describe the efficacy of Ab-IPL-IL-17™ on fibroblasts from Rheumatoid Arthritis (RA) patients. Taken together, our evidence paves the way for discovering new therapeutic approaches to immune-based and inflammatory-related diseases.

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