Background/Purpose: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a life-threatening systemic disorder characterized by inflammation and increased risk of opportunistic infections. VEXAS results from somatic mutations in the ubiquitin-activating enzyme 1 (UBA1) gene that abolish UBA1 function in hematopoietic stem cells and peripheral myeloid cells. Monocytes from VEXAS patients are numerically and functionally impaired and display an inflammatory signature. However, the relationship between the UBA1 mutation and the clinical manifestations of the disease remains elusive. We investigated the functional consequences of UBA1 inactivation in VEXAS monocytes/macrophages.

Methods: We generated human monocytic THP-1 cells with a conditional Cas9-mediated knockout of endogenous UBA1 in parallel with constitutive expression of wild-type (UBA1WT) or VEXAS-associated (UBA1M41V) UBA1 gene. We assessed spontaneous and TNFα-induced inflammatory cell death using flow cytometry, Western blotting and live cell imaging. We also analyzed inflammatory cell death pathways in VEXAS skin biopsies. Next, we assessed pathogen-driven immune responses by measuring cytokine production after TLR stimulation of both whole blood from VEXAS patients (TruCulture system) and UBA1WT and UBA1M41V THP-1. THP-1-derived macrophages were assessed for differentiation, polarization, cytokine production by Luminex, transcriptome, and efferocytosis process by flow cytometry, Western blot and immunofluorescence.

Results: The UBA1M41V THP-1 monocytes exhibited poly-ubiquitination defects, an inflammatory transcriptional profile and increased spontaneous cell death. Specifically, UBA1M41V THP-1 cells were more susceptible to TNFα-induced cell death via RIPK1 activation, which was rescued by caspase- and RIPK1-specific inhibitors. These observations were confirmed in CD14+ sorted cells from peripheral blood and in CD68⁺ cells in skin biopsies from VEXAS patients. TLR-induced cytokine production was defective in UBA1M41V THP-1 cells, which correlated with impaired NF-κB signaling. Similar results were obtained in whole blood from patients after stimulation with TLR agonists. UBA1M41V THP1-derived macrophages displayed an inflammatory phenotype with increased secretion of pro-inflammatory cytokines (IL-1β, IL-6, IFN-y, TNF-α) and increased spontaneous cell death. They also exhibited defective efferocytosis, with decreased MERTK expression and impaired lysosomal degradation of engulfed cells. This was associated with decreased expression of LAMP-1 and cathepsins.

Conclusion: Our results show that the UBA1M41V mutation disrupts TLR-induced monocyte cytokine production and increases cell death, which may explain the severe monocytopenia and susceptibility to serious infections in VEXAS. In addition, the UBA1M41V mutation skews macrophages toward an inflammatory phenotype with an impaired efferocytosis, which likely contributes to chronic inflammation and susceptibility to intracellular pathogen infections. Overall, we provide new insights into the pathophysiology of VEXAS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inflammatory-cell-death-and-impaired-efferocytosis-drive-monocyte-and-macrophage-dysfunction-in-vexas-syndrome/