Autoantibody hotspots reveal origin and impact of immunogenic XIST ribonucleoprotein complex

Abstract

Four out of five patients with autoimmune diseases are women. The XIST ribonucleoprotein (RNP) complex, comprising the female-specific long noncoding RNA XIST and over 100 associated proteins, may drive several autoimmune diseases that disproportionately affect women, who have elevated levels of autoantibodies against the XIST RNP. However, the structural distribution, potential origin, and clinical significance of XIST RNP autoantibodies remained unexplored. Here, we find that XIST RNP is associated with autoantigens associated with six female-biased autoimmune conditions. Mapping autoantibody targets to their occupancy sites on XIST shows that these autoantigens are concentrated at discrete "hotspots" along the XIST lncRNA, notably the A-repeat. Cell type-specific protein expression data nominated neutrophils as a predominant source of hotspot antigens, and we confirmed the presence of both XIST and hotspot antigens in neutrophil extracellular traps during NETosis, an immunogenic programmed cell death pathway triggered by neutrophil activation upon which neutrophils extrude their nuclear content. Furthermore, we found that levels of autoantibodies against a top hotspot antigen, SPEN, that binds the A-repeat, correlate with severe digital ischemia in systemic sclerosis in two independent cohorts. Together, these data show a plausible mechanism for the origin of AXA, guided by RNA structure and RNA-protein interactions, and show that antibodies to XIST RNP holds promise for disease endotyping and prognostication in femalebiased autoimmune conditions.

Fig. 1.. Autoantigens in female-biased autoimmune diseases are enriched for XISTassociated proteins.

(A) Flowchart for this study. (B) Numbers of XIST-associated proteins (XAPs) that are autoantigens among all XAP. (C) Numbers of autoimmune diseases that have XAP as autoantigens among all autoimmune diseases in the Human Autoantigen Atlas. (D) Number of autoantigens in autoimmune diseases. XAP are highlighted in red. (E) The association between antigenic XAP and autoimmune diseases. Association confidence scores obtained from (3) are displayed as heatmap. -log10(p-value) from hypergeometric tests are displayed as bar graphs with p < 0.05 as red bars. Female to male ratios for each autoimmune disease are displayed as bar graph, red bars indicate autoantigens in these diseases are enriched in XAP, blue bars indicate autoantigens in these diseases are not enriched in XAP; **p < 0.01, Mann-Whitney U test.

Fig. 2.. Immunogenicity of XAPs is associated with XIST functional domains.

(A) Overlap of reactivity quantified antigenic proteins with XIST-associated proteins with available location information. Number of reactivity-quantified antigenic proteins with available location information, displayed as intersection from the Venn diagram. (B) Workflow for assigning and inferring the location of antigenic proteins in XIST RNA by protein-protein interaction. (C) Binding profile for XIST-associated proteins (XAP). Row normalized binding intensity for each XAP are displayed as heatmap in each row within 100-bp windows across XIST RNA. XIST secondary structure was determined by psoralen crosslinking in living cells and deep sequencing(4); each arc on top represents a RNA duplex along XIST RNA. Clusters of XAP are indicated by the bar on the right-hand side. (D) Reactivity of XAP derived autoantigens across general population and autoimmune disease patients. Disease annotations and sex annotations are on the top of the heatmap. Annotations for XAP-derived autoantigens in different XIST functional domains are shown on two sides of the heatmap. (E) Functional annotations for XAP-derived autoantigens. Panels from left to right are, 1) Pairwise co-occurrence of autoantigens (black curves indicate intra-domain co-occurrence, colored curves indicate inter-domain co-occurrence with color assignment established from the upper domain into another domain); 2) Significantly elevated autoantigens in any autoimmune disease from(2); 3) Numbers of samples that have MFI > 100 reactivity; 4) Immunogenic hotspots: hotspots that are significant elevated in any autoimmune disease are shown in red, others are shown in blue; 5) Blood profile annotation from the human protein atlas; 6) Immunogenic hotspots in pristane-induced SLE mouse samples.

Fig. 3.. NETosis releases XIST-associated proteins to a cell-free environment.

(A) Cell of origin for antigenic proteins. Proportions for each cell type are displayed as a pie chart. (B) Twenty-two neutrophil-associated antigenic proteins are displayed on the y-axis, and the degree of positive association with autoimmune disease are displayed as heatmaps. The number of human samples with MFI > 100 for each protein (sera reactivity) are shown as a bar graph. (C) dHL-60 cells were stimulated with ionomycin to induce NETosis and stained with DAPI (blue), anti-SPEN (green), and XIST FISH probe (red). (D) Human neutrophils were stimulated with PMA to induce NETosis and stained with DAPI (blue), anti-RBM15 (green), and XIST FISH probe (red). (E) Model of the XIST RNP complex acting as an immunogenic scaffold. Created in BioRender.

Fig. 4.. An AXA hotspot is associated with high-risk vasculopathy in systemic sclerosis patients.

(A) Schematic showing the location of the protein fragment used in the fluorescent bead-based antigen array to assess sera reactivity against SPEN. RRM, RNA recognition motif. RID, receptor interaction domain. SPOC, Spen paralog and ortholog C-terminal. IDRs, intrinsically disordered regions. (B) Distribution of mean fluorescence intensity (MFI) values for sera reactivity against SPEN in the Stanford scleroderma cohort. (C) Calculated difference in proportion for categorical variables of interest in the Stanford scleroderma cohort as defined by Stanford investigators, with horizontal lines indicating the 95% confidence intervals, and a dotted vertical line at 1.0 signifying no association. * p < 0.05. (D) Calculated difference in proportion for digital ulcers and severe Raynaud phenomenon (i.e. digital pitting scars, digital tip ulceration, digital gangrene) in the JHU validation scleroderma cohort, as defined by JHU investigators. * p < 0.05. (E) Representative clinical photos of severe digital ulceration and gangrene in three patients with high anti-SPEN sera reactivity.