Targeting enhanced cell death represents a potential therapeutic strategy for VEXAS syndrome

Abstract Objectives To unravel the mechanisms underlying cell death in the vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome using peripheral blood samples and to assess the clinical value of this knowledge. Methods Nine patients undergoing treatment for VEXAS syndrome at Yokohama City University Hospital were included in this study. Monocytes and neutrophils were isolated from peripheral blood and then monocytes were differentiated into polarized macrophages. Viable cell counts, cell death assays and measurements of various indicators such as high mobility group box 1 (HMGB1) concentration, extracellular adenosine triphosphate (ATP) concentration, annexin V level and caspase 1, 3 and 7 activities were performed. Results Elevated cell death of monocytes and neutrophils was observed in VEXAS syndrome patients, as indicated by cultured cell counts and cell death assays. Annexin V assays and measurements of caspase 1, 3 and 7 activities suggested increased apoptosis and pyroptosis in these cells. Serum HMGB1 levels were significantly elevated in VEXAS syndrome patients and decreased after prednisolone (PSL) dose escalation. Monocytes and neutrophils from the VEXAS group exhibited heightened extracellular ATP secretion, which was significantly reduced by soluble PSL co-culture. Conclusion This study confirms increased cell death of monocytes and neutrophils and damage-associated molecular patterns in VEXAS syndrome, and these findings may be valuable for drug screening, therapeutic strategies and as biomarkers.

Inflammation in VEXAS syndrome is characterized by enhanced innate immunity, marked by the presence of neutrophils in inflamed tissues [14].Corticosteroids are effective in reducing inflammation in VEXAS syndrome, but patients often require a gradual increase in dosage.The administration of various immunosuppressants, biologics including IL-6 receptor antibodies [15,16] and Janus kinase inhibitors has been attempted [17], but no alternatives that conclusively spare the prednisolone (PSL) dose are currently available.Allogeneic haematopoietic stem cell transplantation has the potential to cure VEXAS syndrome [18][19][20], but it is challenging to determine the eligibility for this option given the advanced age of VEXAS syndrome patients, which increases the risk of transplant-related complications.Against this background, alternative drugs to treat VEXAS syndrome are urgently needed.
Single-cell RNA sequencing (sc-RNA-seq) of monocytes from VEXAS syndrome patients has revealed increased signatures of apoptosis, pyroptosis and necroptosis, consistent with PANoptosis, an integrated form of programmed cell death [21].We therefore hypothesized that measuring cell death and damage-associated molecular patterns (DAMPs) might be clinically useful.While RNA-seq analyses on peripheral blood and bone marrow were conducted in previous studies, in vitro experiments using primary monocytes and neutrophils from VEXAS syndrome patients have not been extensively reported.Moreover, detailed investigations into the measurement of DAMPs, crucial in the pathogenesis of various autoimmune diseases [22], have not been documented thoroughly in VEXAS syndrome patients.To address these concerns, we investigated the extent of cell death and DAMPs release in patients with VEXAS syndrome and their clinical significance.

Patients and methods
Patients diagnosed with VEXAS syndrome carrying pathogenic UBA1 variants were enrolled at Yokohama City University (YCU); some of the clinical data from these patients have been reported previously [6,23].The study was approved by the Ethics Committee of YCU Hospital (A121129002) and all participants provided written informed consent before specimen collection.Clinical remission was defined as the absence of symptoms associated with VEXAS syndrome and CRP <5 mg/dl, as modified from a previous study [24].All healthy controls had no VEXAS-related conditions and no family history.During the course of the study, patients stopped attending the hospital due to death or other reasons, and each experiment was conducted so that at least three patients who were able to continue attending could be recruited at the time the experiment was conducted.

Measurement of cytokines and high mobility group box 1 (HMGB1)
Cytokines in serum were quantified using the LEGENDplex Human Inflammation Panel (BioLegend, San Diego, CA, USA).Serum HMGB1 was measured using the ELISA kit Exp (Sinotest, Kanagawa, Japan) before and after treatment enhancement with PSL.

Cell counting
M1 or M2Mφ cells on day 7 of culture were air-dried, fixed with 4% paraformaldehyde and stained with the Diff-Quik staining kit (Sysmex Kokusai Shiyaku, Kobe, Japan).The number of Mφ cells per high-power field (200×) was counted using a BX53 microscope (Olympus, Tokyo, Japan).

Cell death assay
For the cell death assay, 50 000 monocytes and 250 000 neutrophils were seeded in triplicate on a 96-well plate.CellTox Green (CTG) solution (Promega, Madison, WI, USA) was added at 100 μL/well on day 0 and mean fluorescence intensity (MFI) was monitored over time.

Extracellular adenosine triphosphate (ATP) measurement
For the extracellular ATP measurement, 50 000 monocytes and 250 000 neutrophils were seeded in triplicate on a 96well plate.RealTime-Glo Extracellular ATP Assay (Promega) was added at 50 μL/well at the start of the experiment and the luminescence intensity was monitored every hour.Referring to a previous report describing that the maximum concentration of PSL in serum was 481 ng/ml (S.D. 81) after a single intravenous injection of 20 mg of sodium PSL succinate in healthy adults [26], we prepared 'PSL1' with water-soluble PSL at a final concentration of 480 ng/ml.We then prepared 10-fold and 100-fold concentrations and co-cultured them with monocytes.
Caspase 3/7, caspase 1 and annexin V assays Monocytes (50 000/well) and neutrophils (250 000/well) were incubated in 96-well plates for 5 or 24 hours, respectively, and then Caspase-Glo 3/7 or Caspase-Glo 1 (Promega) was added and MFI was measured.The RealTime Glo Annexin V Apoptosis Assay (Promega) was used to obtain data.The time course of annexin V-bound luminescence was evaluated.

Statistical analysis
GraphPad Prism version 10 (GraphPad Software, La Jolla, CA, USA) and SPSS version 29 (IBM, Armonk, NY, USA) was used for statistical analysis.Student's unpaired t-test and two-way analysis of variance (ANOVA) were performed, with a P-value <0.05 being considered statistically significant after adjustment for multiple comparisons.

Characteristics of VEXAS syndrome patients included in the study
Nine patients with VEXAS syndrome were recruited in this study.The mean age was 73.1 years (S.D. 11.1).No established diagnostic criteria for VEXAS syndrome are currently available, but all patients had systemic inflammation along with positivity for a pathogenic UBA1 variant (Table 1).Five patients had MDS, five patients had RP, two patients had large vessel vasculitis and one patient had Sweet's syndrome.At the time of collection of specimens for in vitro experiments, all patients were undergoing treatment with corticosteroids, either alone or in combination with tocilizumab (TCZ) and were considered stable with no apparent systemic symptoms.For patient RP43, blood samples were taken before initiation of PSL or immunosuppressive treatments.Patients and treatments at the time of each experiment are shown in Supplementary Table S1, available at Rheumatology Advances in Practice online.

Increased monocyte, neutrophils and monocytederived M1/M2 Mφ cell death in VEXAS syndrome
Previous reports have shown that monocyte and neutrophil cell death is increased in VEXAS syndrome [18].To confirm Targeting enhanced cell death for VEXAS syndrome these findings, we determined the number of Mφs after a 7day culture period.Consistent with findings in earlier reports, the number of Mφs in the M1 and M2 subsets was decreased in the VEXAS group (Fig. 1A-F).Additionally, cell necrosis was assessed using the CTG assay over time and compared between the VEXAS and healthy control (HC) groups.As depicted in Fig. 1G-I, there were significant increases in the death of both monocytes and neutrophils in VEXAS  syndrome.These findings suggest that monocytes and neutrophils in the peripheral blood of patients with VEXAS syndrome exhibit increased susceptibility to cell death.

Peripheral blood monocytes and neutrophils in VEXAS syndrome are involved in apoptosis and pyroptosis
To compare the roles of apoptosis and necrosis in VEXAS syndrome, the results of the annexin V assay were integrated with those of the CTG assay: in monocytes, the increase in annexin V (apoptosis) occurred prior to the increase in CTG (necrosis) (Fig. 2A).In contrast, necrosis preceded apoptosis in neutrophils (Fig. 2B).Further investigation of cell death was conducted by analysing caspase 3/7 and caspase 1 activities in the HC and VEXAS groups.Both monocytes and neutrophils showed higher caspase 3/7 activity in the VEXAS group than in the HC group (Fig. 2C and D).Both monocytes and neutrophils displayed predominant caspase 1 activity in the VEXAS group compared with that in the HC group (Fig. 2E and F).These findings suggest the involvement of both apoptosis and pyroptosis in the cell death of peripheral blood leucocytes in individuals with VEXAS syndrome.

Increased DAMPs in the serum and cells of VEXAS syndrome patients
The outcomes of the experiments described above confirmed that cell death is elevated in the peripheral blood of patients with VEXAS syndrome.To examine whether DAMPs released due to cell death are indeed elevated in individuals with VEXAS syndrome, the concentration of the cell death marker HMGB1 in patient serum was assessed.The results showed that serum HMGB1 levels in relapsed patients were elevated compared with those in HCs (Fig. 3A).Serum HMGB1 levels decreased following PSL treatment (Fig. 3B and Table 2).Interestingly, serum IL-6 levels decreased rapidly after the increase in PSL dose, while serum IL-18 levels remained high.
To corroborate the increased release of DAMPs in cultured cells, the extracellular ATP concentration was monitored over time.The results indicated significantly higher extracellular ATP concentrations in the VEXAS syndrome group (Fig. 3C and D).Additionally, co-culture with PSL led to a dose-dependent reduction in extracellular ATP concentrations, bringing them to levels comparable to those in the HC group (Fig. 3E).These findings suggest that the assessment of ATP levels provides a quantitative means of ascertaining the efficacy of drug interventions.

Discussion
In this study, culture experiments were conducted using monocytes and neutrophils from patients with VEXAS syndrome, with the aim of establishing a foundation for identifying and exploring therapeutic targets.We verified the accelerated cell death of monocytes and neutrophils, innate immune system cells, consistent with previous reports [18].Furthermore, supplementation with glucocorticoids, which are clinically beneficial in VEXAS syndrome treatment, significantly suppressed the extracellular ATP concentration, suggesting the potential for evaluating the efficacy of new drugs by assessing DAMPs or cell death in UBA1 mutant cells.This underscores the importance of targeting cell death suppression in future therapeutic strategies in VEXAS syndrome.Earlier findings demonstrated a high transcriptomic signature of IL-6 in RNA-seq of CD14 þ monocytes from VEXAS syndrome patients [1].In addition, sc-RNA-seq of monocytes from patients with VEXAS syndrome revealed increased apoptosis, pyroptosis and necroptosis signatures, consistent with PANoptosis [21], which can induce an IL-6 signature.From a clinical perspective, we reported our experience with the IL-6 receptor agonist TCZ in three patients with VEXAS syndrome.Even in patients who appeared to respond to TCZ, high serum levels of inflammatory cytokines such as IL-1 and IL-18 persisted for >4 months after initiating TCZ [15].In the current study, leucocytes from patients with no apparent clinical symptoms were used, but increased cell death and secretion of DAMPs persisted.These observations support the hypothesis that PSL and anti-IL-6 receptor antibodies are insufficient to inhibit the cell death that underlies VEXAS syndrome and such cell death may be the basis of the inflammation found in this condition.
In this study, pyroptosis and apoptosis were identified as modes of cell death in VEXAS syndrome monocytes and neutrophils.In haematopoietic cells from VEXAS syndrome patients, mutations in UBA1 cause abnormal ubiquitination, resulting in the accumulation of proteins that do not fold properly in the endoplasmic reticulum, which is thought to induce the unfolded protein response.The unfolded protein  response is known to increase apoptosis [29], and this study confirms that cell death of monocytes and neutrophils is indeed enhanced in peripheral blood.So, determining why UBA1 mutant haematopoietic cells in peripheral blood rapidly die while those in bone marrow undergo clonal expansion will be important for elucidating therapeutic targets in VEXAS syndrome.Elevated levels of HMGB1 and extracellular ATP, known as DAMPs [30], were previously observed in VEXAS syndrome patients.HMGB1 is a nuclear protein and, after its translocation into the extracellular lumen during cell activation or cell death, it binds to Toll-like receptors (TLRs) such as TLR2 and TLR4, contributing to the development and onset of autoimmune diseases [31].Studies have reported that HMGB1 is upregulated in several autoimmune diseases, including rheumatoid arthritis, SLE, type 1 diabetes and autoimmune thyroid disease [32][33][34][35].Serum HMGB1 level may reflect disease activity in VEXAS syndrome, as it decreased with intensified treatment with PSL and should be tested for potential use as a biomarker of this disease.
ATP is released extracellularly following cellular stress and cell death under inflammation, and extracellular ATP signals through membrane-bound purinergic receptors (e.g.P2X4R and P2X7R) [36].P2X4R and P2X7R are expressed on human macrophages and cause the release of inflammatory cytokines such as IL-1β, IL-6 and TNF-α [37].P2X7R also results in IL-1β production through NLR family pyrin domain containing 3 inflammasomes [38].Treatment of cells in the VEXAS group with PSL, which has been clinically proven to be useful, significantly reduced the extracellular secretion of ATP by monocytes, implying that extracellular DAMP concentrations could be used for in vitro drug screening.
This study had several limitations, including the small sample size, preventing the comparison of results among different UBA1 variants, and the heterogeneity in UBA1 mutant cell frequencies may have affected the extent of cell death.Such variations in UBA1 mutations in VEXAS syndrome may contribute to distinct phenotypes and disease severity.Indeed, it has been reported that differences in UBA1 mutations in VEXAS syndrome lead to differences in the translation of UBA1b, which in turn determine the particular phenotype and level of disease severity [39].Other limitations of this study are the absence of untreated cases and the lack of an analysis of the effects of treatment on cell death in vivo.

Conclusion
This study demonstrates that cell death is enhanced and DAMP levels are increased in patients with VEXAS syndrome and suggests that the in vitro evaluation of these features may be helpful for drug screening, therapeutic strategies and biomarkers.

Figure 1 .
Figure 1.Increased cell death in peripheral monocytes and neutrophils in VEXAS syndrome.Monocytes from four HCs and three patients with VEXAS syndrome stimulated with M-CSF or GM-CSF for 7 days in 96-well dishes to induce M1 and M2 macrophages (Mφ) were stained with Diff-Quik.Representative images of (A) M1Mφ and (B) M2Mφ of the VEXAS group and (C) M1Mφ and (D) M2Mφ of the HC group.Comparison of Mφ counts of (E) M1 and (F) M2 monocytes in HCs and VEXAS syndrome.(G) Amount of cell death of monocytes from three HCs and three VEXAS syndrome patients up to 24 h and (H) three HCs and six patients from day 0 to day 2 by CellTox Green staining.(I) Increased cell death in neutrophils from five HCs and four VEXAS syndrome patients as assessed by CellTox Green staining up to 8 h.� P < 0.05, ��� P < 0.001 by unpaired t-test or two-way ANOVA

Figure 2 .
Figure 2. Evaluation of apoptosis and pyroptosis in peripheral monocytes and neutrophils of patients with VEXAS syndrome.(A, B) CellTox Green cell death data and RealTime Glo Annexin V apoptosis assay results measured simultaneously are displayed in one figure.(C, D) Caspase 3/7 activity and (E, F) caspase 1 activity were evaluated in monocytes and neutrophils from HCs and VEXAS syndrome patients.Error bars indicate the S.E.M. � P < 0.05, �� P < 0.01 by unpaired t-test

Figure 3 .
Figure 3. Increased DAMPs in serum and cells of VEXAS patients.(A) Serum level of HMGB1 was measured in HCs and patients with VEXAS syndrome in Yokohama City University.(B) Relative changes of serum levels of HMGB1 with treatment in relapsed VEXAS syndrome before and after PSL increment.(C) Amount of extracellular ATP secretion from monocytes and (D) neutrophils from HCs and VEXAS syndrome patients by RealTime-Glo Extracellular ATP Assay.(E) Amount of extracellular ATP secretion of monocytes after co-culture with water-soluble prednisolone.Error bars indicate S.E.M. � P < 0.05, �� P < 0.01, ��� P < 0.001 by unpaired t-test or two-way ANOVA or analysis of covariance

Table 2 .
Changes in variables over time in relapsed VEXAS syndrome

Patient ID Variables Prior to PSL increment 2 days after PSL increment 4 days after PSL increment
Patients in whom cytokine and HMGB1 data were measured simultaneously are listed only if they were measured more than once.Serum IL-1β, 6 and 18 concentrations in units of pg/ml, serum HMGB1 concentration in units of ng/ml, serum CRP concentration in units of mg/dl.