Background: Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disease and results from MEFV gene gain-of-function variants that lower the activation threshold of the pyrin inflammasome in myeloid cells, leading to recurrent systemic inflammation and IL-1-driven tissue damage. Pyrin senses RhoA-pathway activation and integrates cytoskeletal and kinase signals. Therefore, FMF monocytes are primed for an exaggerated IL-1β release in response to inconsequential stimuli. Pyrin has been known to be expressed in myeloid cells and fibroblasts. While blood-based functional assays and cytokine profiling have improved FMF diagnosis and subtyping, the cell-type-specific transcriptional and metabolic programs that distinguish individual circulating immune populations in FMF patients on treatment, and how these differ between myeloid cells and T-cell subsets, remain incompletely understood.

Objectives: This study aimed to comprehensively characterize single-cell transcriptional and immunometabolic signatures of circulating myeloid and lymphoid populations in FMF compared with healthy controls (HC), and to identify pathways that may underlie persistent inflammation and treatment refractoriness.

Methods: Peripheral blood mononuclear cells (PBMCs) from colchicine-treated FMF patients (n=10) and age matched HC (n=5) were profiled by single-cell RNA sequencing (Parse Biosciences platform), yielding 60,235 high-quality cells, with a median of 2,500 genes per cell. They were clustered into 13 immune populations, including classical and non-classical monocytes, conventional dendritic cells (cDCs), NK cells, naïve and effector CD4 and CD8 T cells, MAIT cells, Tregs, B cells, and plasmablasts. Differential abundance, pseudobulk differential expression, and gene-set enrichment analyses were performed per subset. The metabolic activity of PBMCs single populations was assessed using Cellular Energetics through Non-canonical Amino acid Tagging (CENCAT), FMF patients (n=18), age matched HC (n=8). Purified CD14 ⁺ monocytes were stimulated with LPS for 24h for bulk RNA-seq. PBMCs were also stimulated with LPS and UCN01 to detect IL-1β production

Results: Single-cell RNA sequencing identified 13 distinct PBMC clusters, corresponding to specific cell types based on marker expression. All cells originating from FMF patients expressed a higher number of genes related to RNA translation. Among these, monocytes showed the greatest transcriptional changes in FMF patients compared with HCs. Classical monocytes downregulated NF-κB signaling-related genes, while upregulated oxidative phosphorylation, respiratory electron transport, and RNA-processing pathways; whereas non-classical monocytes and conventional dendritic cells mainly increased interferon-response and antigen-presentation programs. Within classical monocytes, subclustering revealed distinct FMF subsets enriched for BMAL1/ARNTL circadian regulators and Rho-GTPase/RhoU-cycle pathways. LPS-stimulated CD14 ^-+ - monocytes from FMF patients showed a lower interferon-gamma, and RNA-metabolism related response, together with higher IL-1β production in response to UNC-01 augmented LPS stimulation, in line with a pyrin-inflammasome hyper-responsiveness driven by MEFV mutations.

Of high interest, lymphocytes were also clearly different between FMF patients and healthy controls. In fact, they have an upregulation of transcription factors related to lymphocyte differentiation and activation, with a downregulation of inflammation related ones. CTLs from FMF patients showed higher expression of oxidative phosphorylation, respiratory-chain gene and cytotoxicity compared with controls. This finding prompted further investigation using CENCAT metabolic assays, which confirmed higher baseline translation in CTLs, demonstrating higher ATP consumption in FMF patients, as compared to healthy controls.

Conclusions: This study shows that, even under colchicine treatment, FMF is associated with broad remodeling of circulating immune cells, with classical monocytes and CTLs acquiring distinct signatures, such as altered cytoskeletal and transcriptional control of pyrin inflammasome activation, like Rho-GTPase/RhoU-cycle alterations, that go beyond the MEFV mutation alone. Monocytes combine altered inflammatory transcriptional programs with a specific pyrin-dependent IL-1β response to LPS+UCN-01, while CTLs display increased baseline metabolism, indicating that myeloid and T-cell subsets have different activation and metabolic set-points in FMF, compared to HC. Together, these findings reveal that FMF pathogenesis extends beyond genetic mechanisms alone, with pyrin inflammasome activation differentially affecting distinct immune cell types beyond just myeloid cells, as previously understood. Findings on the effector CTLs may provide insights for the association of FMF with HLA Class I-associated disorders, such as spondyloarthritis, and Behcet disease.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.