Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: Gene expression of monocytes and targeted protein biomarker assessment in patients with polymyalgia rheumatica

ABSTRACT Introduction Polymyalgia rheumatica (PMR) is a chronic inflammatory disease that affects individuals of 50 or more years of age. It is characterised by pain and functional impairment of the girdles. Emerging evidence suggests that peripheral blood mononuclear cells (PBMCs), particularly monocytes, may contribute to disease pathogenesis. Aim To investigate gene expression in monocytes isolated from PBMCs of patients with active PMR, either untreated with glucocorticoids (GCs) or experiencing disease relapse following GC tapering or discontinuation. Methods and results This project was awarded a € 5,000 Research Voucher by EULAR (European Alliance of Associations for Rheumatology) in January 2024. Patients were consecutively enrolled based on the 2012 provisional EULAR classification criteria for PMR. The study population included patients with newly diagnosed disease not previously treated with GCs (group A), and those experiencing a disease flare after a GC tapering or discontinuation (group B). At recruitment (T0), all PMR patients underwent venous blood sampling for PBMC isolation and serum separation, as well as ultrasound (US) scans of the girdles. Demographic, clinical and laboratory data were recorded in an electronic database. Patients with other chronic inflammatory or oncological diseases, or who developed such conditions during the study, were excluded. In all PMR patients, blood samples and US examinations were repeated four months after the baseline assessment. PBMCs were isolated by Lympholyte stratification, and monocytes were purified using immunomagnetic negative selection. Purity was confirmed using CD14 and CD16 staining. Total RNA was extracted from monocytes and subjected to RNA sequencing. Differentially expressed genes (DEGs) were identified, and functional analyses were carried out using Gene Ontology (GO) enrichment and GSEA Hallmark pathway analyses. Flow cytometry and ELISA were performed to investigate the protein level. Thirty-two PMR patients have been enrolled: 20 in Group A and 12 in Group B. RNA of acceptable quality was obtained from 7 paired samples at T0 and T4 in Group A, and from 3 paired samples in Group B. In Group A, transcriptional analysis between T0 and T4 identified 252 DEGs (FDR<0.1). Among these, IL-6 (FDR=0.006) and CTSL (cathepsin L, FDR=0.001) were significantly downregulated at T4, whereas IL-1RL1 (FDR=0.001) and AKAP12 (FDR=0.0001) were the most strongly upregulated at T4. In contrast, patients in Group B displayed minimal and almost all non-significant transcriptional changes over time. GO analysis in GC-naïve patients at T4 demonstrated downregulation of inflammatory processes, including response to lipopolysaccharide and respiratory burst. GSEA further confirmed reduced activation of pro-inflammatory pathways, including IL-6/JAK/STAT3, interferon-γ, and TNF via NF-κB, in line with previous reports. Flow cytometry showed increased expression of IL-33R for monocytes of naïve T0 patients. ELISA assays did not reveal statistically significant differences, but a trend toward higher median ST2 levels at T4 was observed, consistent with the RNA-seq data. Conclusions Our findings indicate that monocyte transcriptional profiles dynamically mirror the clinical state of PMR. In particular, IL-1RL1 and CTSL emerge as promising biomarkers and potential regulators of inflammation in this condition. These results provide new insights into the molecular mechanisms underlying PMR and may provide a preliminary molecular framework to guide future targeted protein investigations and support the discovery of novel diagnostic markers or therapeutic targets in PMR.