Targeting JAK-STAT Signalling Alters PsA Synovial Fibroblast Pro-Inflammatory and Metabolic Function

Abstract

ObjectivesPsoriatic arthritis (PsA) is a chronic inflammatory disease associated with psoriasis. Janus Kinase inhibitors (JAKi) have emerged as an encouraging class of drugs for the treatment of PsA. Here, we compare the effect of four JAKi on primary PsA synovial fibroblasts (PsAFLS) activation, metabolic function, and invasive and migratory capacity.MethodsPrimary PsAFLS were isolated and cultured with JAKi (Peficitinib, Filgotinib, Baricitinib and Upadacitinib) in the presence of Oncostatin M (OSM). pSTAT3 expression in response to OSM was quantified by Western Blot analysis. Pro-inflammatory cytokines/chemokines were quantified by ELISA and cell migration by wound-repair scratch assays. Invasive capacity was examined using Matrigel™ invasion chambers and MMP multiplex MSD assays. PsAFLS bioenergetics was assessed using the Seahorse XFe Extracellular Flux Analyzer, which simultaneously quantifies two energetic pathways- glycolysis (ECAR) and oxidative phosphorylation (OCR). In parallel, inflammatory, invasive, and migratory genes were quantified by RT-PCR.ResultsOSM induces pSTAT3 expression in PsAFLS. OSM-induced secretion of MCP-1 and IL-6 was inhibited by all JAKi with Peficitinib, Baricitinib and Upadacitinib showing the greatest effect. In contrast, JAKi had no significant impact on IL-8 expression in response to OSM. PsAFLS cell invasion, migratory capacity and MMP1, 3, and 9 were suppressed following JAKi treatment, with Peficitinib showing the greatest effect. These functional effects were accompanied by a change in the cellular bioenergetic profile of PsAFLS, where JAKi significantly decreased glycolysis and the ECAR/OCR, resulting in a shift to a more quiescent phenotype, with Peficitinib demonstrating the most pronounced effect.ConclusionThis study demonstrates that JAK/STAT signalling mediates the complex interplay between inflammation and cellular metabolism in PsA pathogenesis. This inhibition shows effective suppression of inflammatory mechanisms that drive pathogenic functions of PsAFLS, further supporting the role of JAKi as a therapeutic target for the treatment of PsA.

Metabolic characterization of human sperm cells using the Seahorse metabolic flux analyzer

“…In fact, sperm cells have a very particular metabolism and can apparently choose different metabolic pathways according to substrates' availability. [19][20][21] Additionally, one of the main challenges with these cells is that besides being different from the other cells in the organism, particularly by the fact of being motile, there are also species-specific differences, making it difficult to make comparisons and drawn conclusions with the metabolic outcomes obtained on other cellular types [43][44][45][46][47][48][49][50][51][52][53] but also on spermatozoa from different species. 37,38,[54][55][56] Previously, the SFA has been used in sperm cells from rodents to study metabolic alterations related to important processes such as sperm capacitation 38,57,58 ; or alternatively to study metabolic details, such as the importance of certain metabolites (e.g., beatine) for male fertility.…”

“…[49][50][51][52] Interestingly, their metabolic metrics seem more similar to the ones from quiescent cells. 45,53 These observations might be explained by the absolute size of the sperm cells, in which the midpieces containing only approximately 50−75 mitochondria and the glycolytic machinery limited to the tail, will result in a comparatively smaller metabolic machinery than many somatic cell types. 66,67 Yet, only a normalization of the metabolic parameters according to the cellular volumes/mass will allow to clarify these issues.…”

“…In fact, sperm cells have a very particular metabolism and can apparently choose different metabolic pathways according to substrates’ availability 19–21 . Additionally, one of the main challenges with these cells is that besides being different from the other cells in the organism, particularly by the fact of being motile, there are also species‐specific differences, making it difficult to make comparisons and drawn conclusions with the metabolic outcomes obtained on other cellular types 43–53 but also on spermatozoa from different species 37,38,54–56 …”

Inside the Joint of Inflammatory Arthritis Patients: Handling and Processing of Synovial Tissue Biopsies for High Throughput Analysis

“…One exception to the high cell number requirement for the implementation of functional studies, is synovial fibroblasts due to available, well-characterized and optimized protocols of in vitro expansion of these cells. As a result fibroblasts from IA patient synovial biopsies can be utilized in invasion and migration assays following response treatment, metabolic characterization and even co-culture with immune and stromal cells, including the recently characterized pro-inflammatory MerkTK − CD206 − synovial tissue macrophages ( 17 , 55 , 61 , 119 ). These studies have significantly improved our understanding of synovial fibroblast involvement in IA pathogenesis, however, once removed from the synovial environment and expanded in vitro transcriptional characteristics of specific synovial fibroblasts are potentially altered ( 120 ).…”

Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways

“…Multiple cytokinesignaling pathways are tightly regulated by the actions of receptor-bound JAKs and the signal transducers and activators of transcription (STATs) (5)(6)(7). Dysfunctional cytokine-JAK/ STAT activities have been demonstrated as hallmarks of numerous autoimmune disorders and inflammatory diseases (6)(7)(8)(9)(10). In the past decade, small molecule JAK inhibitors have been shown to be crucial therapeutic agents in the treatment of autoimmune diseases.…”