An NT5E loss-of-function variant permits tissue inflammation and hypertension in systemic lupus erythematosus

Figures

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  Figure 1. Elevated NT5E and ENTPD1 expression in lupus kidney and skin (A) Log2 fold differential expression values for NT5E and ENTPD1 from lupus whole blood, PBMCs, B cells, T cells, LN kidney, and skin. (B) Individual sample gene expression from the blood, kidney, skin, or synovium was analyzed by GSVA for the enrichment of NT5E and ENTPD1. Asterisks (∗) indicate p < 0.05 using a Welch's t test comparing SLE to control where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and p < 0.0001.

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  Figure 2. NT5E expression in skin cell populations from SLE, DLE, and healthy donors (A and B) Major cell populations (A) and more granular subpopulations (B) were defined based on differentially expressed transcripts characteristic of each cell type. (C) Normalized NT5E expression is visualized in the UMAP plot. (D) NT5E expression levels in the defined major populations are shown as violin plots, with FDR-corrected p values indicating significant differences between donor groups. (E) NT5E expression in subpopulations is shown only for those with significant differences between donor groups. <5% false detection rate was considered significant, with level of significance in (D) and (E) indicated by ∗∗∗p < 0.0005, ∗∗p < 0.005, or ∗p < 0.05. scRNA-seq data from GSE179633, derived from skin biopsy samples of patients with SLE, DLE, and healthy donor controls, were analyzed.

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  Figure 3. Adenosine signaling gene expression is elevated in lupus tissues and is associated with immunological activity (A) Pathway representation of adenosine (ADO) formation by the activity of the ecto-enzymes CD39 and CD73. (B and C) GSVA enrichment score for the ADO and additional immune processes were generated in SLE patients and healthy control samples from kidney (B, left; GSE32591) and skin (right; GSE81071) and whole blood (C; GSE88884 and GSE45291). Asterisks (∗) indicate a p < 0.05 using Welch’s t test comparing SLE to control. (D) Heatmap showing correlation coefficients between ADO and various cell-type-specific GSVA scores. N.d. (gray boxes), cell type not detected. Asterisks (∗) indicate p values with significantly non-zero slopes; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.00001.

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  Figure 4. Adenosine function is uncoupled from downstream immunomodulatory activity (A) Pathway representation showing the generation of intracellular cAMP from extracellular ADO and its downstream immunomodulatory effects. (B and D) GSVA enrichment scores representing the cAMP-mediated immunosuppressive gene signature were generated in kidney (B; GSE32591) and skin (D; GSE81071) datasets from SLE patients and healthy controls. Asterisks (∗) indicate a p < 0.05 using Welch’s t test comparing SLE to control where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C and E) Linear regression analysis showing the relationship between GSVA scores for the ADO signature and the cAMP-mediated immunoregulatory signature in the kidney (C) and skin (E). R2 predictive values and p values are listed. N.s., not significant.

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  Figure 5. Elevated immune activity in patients harboring the rs2229524 risk genotype (A) The M379T mutation is rendered in multicolored spheres, with a critical Arg395 residue in sticks and the required zinc atoms in silver spheres. (B) Molecular dynamics simulations of the active state showing local opening and closing of the catalytic site in the wild-type and the M379T NT5E mutant as measured by Arg395 nitrogen-zinc minimum distances over the simulations. (C) Three-dimensional rendering of the open, Zn-accessible conformation in the WT compared to the locally closed, Zn-inaccessible mutant enzyme in the simulations (Zn, arrowhead). Trp381, located on the same loop as residue 379, plays a critical role in closing access to the binding site (indicated by arrows). (D) Volcano plot visualization of DEGs between SLE patients homozygous for the reference allele (T/T) and patients with at least one risk allele (T/C + C/C). Red dots indicate significant DEGs (p value < 0.05); select genes of interest are labeled. (E) Pathway enrichment results using GO terms for Biological Processes and IPA canonical pathways of significant up- and downregulated genes. (F) GSVA enrichment scores for ADO…

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  Figure 6. Key pathways associated with the risk genotype are enriched in immune and metabolic activity (A and B) Metastructures using up- and downregulated DEGs from SLE patients with (T/C + C/C) and without (T/T) the rs2229524 risk allele were generated based on PPI networks, clustered using MCODE and visualized in Cytoscape. Cluster size indicates the number of genes per cluster, edge weight indicates the number of inter-cluster connections, and color indicates the number of intra-cluster connections. Enrichment for biological function and immune cell type was determined by BIG-C, GO terms, IPA, and I-Scope. (C and D) GSVA enrichment scores were generated in kidney and skin datasets using genes in each metacluster. PPI cluster enrichment is summarized for each cluster-tissue pair and visualized in the heatmap. Increasing and decreasing enrichment are indicated by the red and blue gradient, respectively. Asterisks (∗) indicate p < 0.05 using Welch’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (E) Schematic illustration showing how suboptimal levels of ADO linked to deleterious mutations in exon 6 affecting NT5E enzymatic activity may contribute to the elevated frequency…