Introduction:Systemic Lupus Erythematosus (SLE) exhibits a pronounced sex bias, affecting females approximately nine times more frequently than males; however, males tend to experience a more severe clinical course yet the molecular basis for these differences remains unclear. Methods:Leveraging epigenomic, transcriptomic, and proteomic data from the whole blood of 720 SLE patients (679 females, 41males) and 84 healthy controls (77 females, 7 males), we conducted comprehensive multi-omic analyses to identify sex-specific molecular features of this disease. Specifically, differential expression analysis for each modality was conducted using a factorial design to identify differences between disease and healthy controls (SLE-HC) for each sex, as well as the interaction effects between sex and disease ([Male SLE - Male HC] - [Female SLE - Female HC]). Benjamini & Hochberg false discovery rate (FDR) was used for multiple test correction. Results:The strongest signal differentiating males and females with SLE was the aberrant expression of the long non-coding RNA, XIST, in males. This XIST expression in males with SLE was bimodal, with 54% of males having elevated XIST expression, and correlated with disease severity. Males with SLE also exhibited significant hypermethylation of the X chromosome and transcriptional silencing of X-linked genes - hallmarks of X-chromosome inactivation (XCI), a process typically restricted to females. Conclusion:These results suggest that X-chromosome silencing by XIST may contribute to SLE disease in males.

OBJECTIVES:Giant cell arteritis (GCA) is an immune-mediated vasculitis of large- and medium-sized arteries that can lead to systemic symptoms and irreversible vision loss. Glucocorticoids (GCs) remain the primary treatment but fail to induce sustained remission (SR) in approximately half of patients, resulting in disease relapses and significant treatment-related toxicity. We aimed to identify tissue-based markers at disease onset capable of distinguishing patients who later achieve SR from those who do not (non-remission [NR]) under GC monotherapy. METHODS:Using spatial biology techniques, we performed a comprehensive analysis of GCA-affected arterial tissues obtained at disease onset, correlating molecular profiles with clinical trajectory. We compared gene expression and immune cell populations between SR and NR groups, corroborated key findings by immunohistochemistry, and evaluated the diagnostic performance of identified biomarkers. RESULTS:Patients with NR exhibited an upregulation of extracellular matrix remodelling (ECM) and T cell activation pathways, reflecting persistent inflammation and fibrotic-like responses. By contrast, SR cases were distinguished by an enrichment of immunoglobulin G-producing plasma cells in the adventitia, which correlated with increased macrophage infiltration in the intima. Quantitative analyses suggested that combining plasma cell and macrophage markers could accurately predict GC responsiveness. CONCLUSIONS:Our findings reveal an immunopathologic signature in GCA at diagnosis, characterised by plasma cell-rich infiltrates associated with favourable outcomes, and ECM- and T cell-associated inflammation enriched in GC-refractory cases. These insights could foster a precision medicine approach to GCA management, enabling patient stratification for GC-sparing therapies and optimising patient outcomes.

Ixekizumab (IXE), an IL-17A antagonist, and guselkumab (GUS), an IL-23p19 antagonist, are common psoriasis treatments. This longitudinal analysis assessed gene expression profiles in IXE- and GUS-treated patients with plaque psoriasis through week 4. In IXORA-R (NCT03573323), a head-to-head phase 4 study, adults with moderate-to-severe plaque psoriasis were assigned 1:1 to receive IXE or GUS. RNA expression was assessed in lesional tissue (n=72) from IXE-treated patients, GUS-treated patients, and healthy control groups (199 samples in total). Empirical Bayes was used to model RNA-sequencing data; differential expression was analyzed by tissue type, treatment, and time point, correcting for random effects. At week 1, lesions from IXE-treated patients had greater numbers of differentially expressed genes (392 upregulated, 696 downregulated) than those from GUS-treated patients (0 upregulated 0 downregulated). By week 4, the numbers of differentially expressed genes increased in both groups (IXE: 1882 upregulated, 1649 downregulated; GUS: 318 upregulated, 131 downregulated). Molecular shifts from baseline to week 4 occurred earlier with greater magnitude in IXE- than in GUS-treated patients. Rapid normalization of transcriptomic changes in patients receiving an IL-17A antagonist reflected downregulation of key inflammatory genes in psoriatic epidermis. Differentially expressed genes involved in epidermal IL-17A and IL-36 responses correlated with PASI 100 response.

PT022 / #742 Topic: AS12 - Genetics, Epigenetics, Transcriptomics POSTER TOUR 05: SLE PATHOGENESIS 24-05-2025 10:00 AM - 10:20 AM Systemic lupus erythematosus (SLE) exhibits a pronounced female-biased imbalance in disease prevalence, with a female-to-male ratio of 9:1. Although the exact mechanisms remain unclear, the significant role of X-chromosome dosage is supported by karyotypic risks associated with SLE. This study utilizes paired transcriptomic (RNAseq), proteomic (Olink), and epigenomic (EMSeq) data from large phase 3 trials to gain mechanistic insights into the drivers behind the sexual bias in SLE. Baseline RNAseq, Olink, and EMseq data were collected from the whole blood of 722 SLE patients (680 female, 42 male) and 84 healthy controls (77 female, 7 male) from 2 phase 3 clinical trials (NCT03616964, NCT03616912). Differential expression analysis was performed using a factorial design to calculate the following comparisons for each modality: i) SLE vs healthy controls in females, ii) SLE vs healthy controls in males, and iii) interaction between sex and disease. Gene set enrichment analysis (GSEA) of patient subsets was conducted using Gene Ontology (GO) biological process terms. To ensure our cohort did not contain erroneous results due to Klinefelter’s males, we inferred X-chromosome heterozygosity by calculating the read depth of the X-chromosome from the EMseq bam files. To validate our results, we performed similar differential expression analysis on EMseq data from an independent cohort of SLE patients (241 females, 13 males) from 2 additional phase 3 clinical trials (NCT01205438, NCT01196091). The strongest changes differentiating how sexes respond to disease were observed in the expression of lncRNA XIST (X-inactive specific transcript) and epigenetics. Specifically, we noted an increased expression of XIST in males with SLE compared to healthy controls (Figure 1A), with this expression exhibiting a bimodal distribution (Figure 1B). GSEA indicated that males with high XIST expression exhibit enrichment in biological processes (GO) related to metabolism and immunoglobulin production, such as oxidative phosphorylation, B cell mediated immunity, and immunoglobulin production compared to XIST low males. Consistent with XIST’s known role in X-chromosome inactivation, we observed corresponding hypermethylation of the X-chromosome and downregulation of X-linked genes in males with SLE (Figure 1C). Lastly, we observed similar patterns of X-chromosome hypermethylation using EMseq data from an independent cohort of SLE patients. A) Violin plots of XIST expression (RNAseq) in women (left) and men (right). B) Density plot of the bimodal expression of XIST in men with SLE. Dashed red line indicates the separation between XIST high and XIST low groups. C) Distribution plots of significant changes (|FC| > 1.5; FDR < 0.1) in gene methylation (top), promoter methylation (middle), and gene expression (bottom) on the X chromosome in females (pink), XIST high males (green), and XIST low males (blue) compared to healthy controls. Figure 1. Partial X-chromosome inactivation in males with SLE. This study, which includes the most comprehensive and largest dataset of male SLE patients to date, shows that males with SLE express significantly higher levels of XIST, accompanied by hypermethylation of the X-chromosome and downregulation of X-linked genes compared to healthy controls, suggesting partial X-chromosome inactivation in males with SLE. Importantly, we have confirmed that these changes are not artifacts of Klinefelter’s patients within the cohort. We hypothesize that this X-chromosome inactivation may be driving SLE via several mechanisms, including: i) inactivation of immunoregulatory molecules, ii) inducing development of SLE autoantibodies, and/or iii) driving interferon production via TLR7.

O026 / #740 Topic:AS22 - SLE Heterogeneity Late-Breaking Abstract ABSTRACT CONCURRENT SESSION 04: ADVANCING LUPUS THERAPIES AND INSIGHTS 22-05-2025 1:40 PM - 2:40 PM Integrative omics approaches offer a powerful strategy to dissect the complex biological networks and pathways involved in disease pathophysiology. However, integrating diverse data types with varying scales, biological contexts, and feature numbers poses significant challenges. This study aims to: i) identify molecular subtypes of SLE patients via a multi-omic integrative approach, ii) characterize these clusters using molecular and clinical data, and iii) identify the most discriminant subset of features for patient classification. Similarity Network Fusion (SNF) was used to integrate baseline transcriptomic (RNAseq), proteomic (Olink), and epigenomic (EMseq) data from the whole blood of 722 SLE patients that were randomized to placebo plus standard of care in phase 3 clinical trials (NCT03616964,NCT03616912), and 84 healthy controls. Patient subgroups were identified via spectral clustering, and cluster robustness was determine using a bootstrapping approach (n = 30). Clusters were characterized with omics data via differential expression and gene set enrichment analysis (GSEA). Clusters were also characterized with clinical metrics via t-tests and random forest analysis. Finally, we utilized Data Integration Analysis for Biomarker Discovery using Latent cOmponents (DIABLO) modeling to identify potential biomarkers associated with these SLE classes. Integration of the omics datatypes identified 3 distinct clusters of individuals: cluster 1 (n = 176), cluster 2 (n = 299), and cluster 3 (n = 331). All 84 healthy controls were grouped within cluster 2 (Figure 1A). Notably, clustering based on individual datatypes failed to reproduce these distinct clusters. Clinical data revealed that SLE patients in cluster 2 exhibited significantly lower dsDNA, IFI44L, and SLEDAI scores, along with higher complement (C3) levels compared to the other clusters (Figure 1B), indicating a milder form of SLE. This is consistent with the fact that these SLE patients clustered with the healthy controls. Additional clinical measurements (Figure 1C) and pathway enrichment analysis (Figure 1D) revealed distinct signatures in the other 2 clusters: cluster 3 displayed an elevated adaptive immunity signature, while cluster 1 was characterized by innate immunity signatures. Finally, integrating all 3 datatypes using the DIABLO modeling approach identified 3 latent components with a minimal set of discriminating biomarkers for each cluster. A) Spectral clustering of patient-patient similarities calculated from the SNF-fused data. B) Distribution of conventional SLE metrics among the 3 SNF clusters (excluding healthy patients). Significant differences assessed with a t-test and false discover rate (FDR). C) Significant clinical differences between Cluster 1 and Cluster 3 using t-test and FDR. Fold change (C1/C3) greater than 1 indicates increased measurements in Custer 1 compared to Cluster 3. D) Normalized Enrichment Score (NES) of select GO terms from a GSEA analysis comparing C1 vs C3 using RNAseq. Positive NES indicates increased activity in C1, and vice-versa. Figure 1. SLE patient clusters and characterization. Multi-omic integration revealed 3 molecularly distinct clusters of SLE patients. Using orthogonal datatypes (clinical and omics), we characterized these clusters into 3 novel classifications: mild, innate-driven, and adaptive-driven immunity. This work helps our understanding of the complex heterogenous nature of SLE and will guide targeted treatment approaches with innate or adaptive involvement.

A wide variety of electrophilic derivatives of itaconate, the Kreb’s cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1β in response to these innate activators. In contrast, the production of interferon (IFN)β, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects up to one in five children and millions of adults in developed countries. Clinically, AD skin lesions manifest as subacute and/or chronic lichenified eczematous plaques, which are often intensely pruritic and prone to secondary bacterial and viral infections. Despite the emergence of novel therapeutic agents, treatment options and outcomes for AD remain suboptimal. An improved understanding of AD pathogenesis may help improve patient outcomes. Dysregulated Th2-polarized skin inflammation and impaired skin barrier function interact to drive AD pathogenesis; however, much remains to be understood about the molecular mechanisms underlying this interplay. The current study used published clinical trial datasets to define a skin-related AD gene signature. This meta-analysis revealed significant reductions in IL1F7 transcripts (encodes IL-37) in AD patient samples. Reduced IL1F7 correlated with lower transcripts for key skin barrier function genes in the epidermal differentiation complex. Immunohistochemical analysis of normal (healthy) human skin specimens and an in vitro three-dimensional human skin model localized IL-37 protein to the epidermis. In comparison with normal human skin, IL-37 levels were decreased in AD patient skin. Addition of Th2 cytokines to the aforementioned in vitro three-dimensional skin model recapitulates key aspects of AD skin and was sufficient to reduce epidermal IL-37 levels. Image analysis also indicated close relationship between epidermal IL-37 and skin epidermal differentiation complex proteins. These findings suggest IL-37 is intimately linked to normal keratinocyte differentiation and barrier function and implicates IL-37 as a potential biomarker and therapeutic target for AD.

Aims and methods Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. Results Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42–0.72); inverse relationships were detected with cell junction targets (R2=0.49–0.71) and β-catenin (R2=0.51–0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. Conclusions Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

The core protease (CP) sub-complex of the 26S proteasome houses the proteolytic active sites and assumes a barrel-shape comprised of four co-axially stacked heptameric rings formed by structurally related α- and β-subunits. CP biogenesis typically begins with the assembly of the α-ring, which then provides a template for β-subunit integration. In eukaryotes, α-ring assembly is partially mediated by two hetero-dimeric chaperones, termed Pba1-Pba2 and Pba3-Pba4 in yeast. Pba1-Pba2 initially promotes orderly recruitment of the α-subunits through interactions between their C-terminal HbYX/HbF motifs and pockets at the α5-α6 and α6-α7 interfaces. Here, we identified PBAC5 as a fifth α-ring assembly chaperone in Arabidopsis that directly binds the Pba1 homolog PBAC1 to form a trimeric PBAC5-PBAC1-PBAC2 complex. PBAC5 harbors a HbYX motif that docks with a pocket between the α4 and α5 subunits during α-ring construction. Arabidopsis lacking PBAC5, PBAC1, and/or PBAC2 are hypersensitive to proteotoxic, salt, and osmotic stresses, and display proteasome assembly defects. Remarkably, while PBAC5 is evolutionarily conserved among plants, sequence relatives are also dispersed within other kingdoms, including a scattered array of fungal, metazoan, and oomycete species.

Ex vivo skin culture holds promise as a preclinical model for understanding pathogenic mechanisms and testing therapeutics for skin diseases. This project studied the impact of ex vivo culture on the transcriptome profile and cytokine/chemokine production of nonlesional and lesional skin from adult atopic dermatitis (AD) patients (n=19) with moderate to severe disease. Punch biopsy (2mm) specimens were immediately processed for RNA (baseline) or cultured for 48 hours (37°C, 5% CO2, DMEM-10% FBS), followed by RNA isolation and supernatant collection. Transcriptome analyses were performed with HTA2.0 arrays (Affymetrix), and supernatants were analyzed with multiplex immunoassays. Microarray data analysis (t-SNE) revealed cultured samples clustered separately from baseline samples, which suggested culturing the skin had a dominant effect on gene expression and obscured AD transcriptome profiles. Relative to baseline samples, cultured AD skin exhibited increased inflammatory cytokine and chemokine transcripts. Proteins encoded by these transcripts were also detected in culture supernatants. These data demonstrated AD skin culture activates an inflammatory response distinct from that seen in vivo. Ex vivo culture of healthy donor skin also activated this response, which was determined to be interleukin-1 alpha (IL-1α)-dependent. In ex vivo skin culture, this IL-1α-induced inflammatory response obscured the ability of selected recombinant cytokines to induce known target genes. In conclusion, IL-1α activates a defined inflammatory response during ex vivo skin culture, which can obscure the native (in vivo) transcriptome/secretome of skin specimens. These results suggest including IL-1α neutralization as part of ex vivo skin culture systems would create a better preclinical skin disease model for evaluating therapeutic compounds.

Systemic Sclerosis (SSc) is a chronic autoimmune disease whose origin and pathogenesis are not yet well known. Recent studies are allowing a better definition of the disease. However, few studies have been performed based on metabolomics. In this way, this study aims to find altered metabolites in SSc patients in order to improve their diagnosis, prognosis and treatment. For that, 59 SSc patients and 28 healthy volunteers participated in this study. Urine and plasma samples were analysed by a fingerprinting metabolomic approach based on HPLC-ESI-QTOF-MS. We observed larger differences in urine than plasma metabolites. The main deregulated metabolic families in urine were acylcarnitines, acylglycines and metabolites derived from amino acids, specifically from proline, histidine and glutamine. These results indicate perturbations in fatty acid beta oxidation and amino acid pathways in scleroderma patients. On the other hand, the main plasma biomarker candidate was 2-arachidonoylglycerol, which is involved in the endocannabinoid system with potential implications in the induction and propagation of systemic sclerosis and autoimmunity.

The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not construct distinct proteasome sub-types. We also detected a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP-RP association. Proteasomes from seedlings exposed to the proteasome inhibitor MG132 accumulated assembly intermediates, reflecting partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Genetic analyses of Arabidopsis UMP1 revealed that, unlike in yeast, this chaperone is essential, with mutants lacking the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single ump1 mutants were hypersensitive to conditions that induce proteotoxic, salt and osmotic stress, and also accumulated several proteasome assembly intermediates, consistent with its importance for CP construction. Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more subunits.