Population-level single-cell genomics reveals conserved gene programs in systemic juvenile idiopathic arthritis

Systemic autoimmune and autoinflammatory diseases are characterized by genetic and cellular heterogeneity. While current single-cell genomics methods provide insights into known disease subtypes, these analysis methods do not readily reveal novel cell-type perturbation programs shared among distinct patient subsets. Here, we performed single-cell RNA-Seq of PBMCs of patients with systemic juvenile idiopathic arthritis (SJIA) with diverse clinical manifestations, including macrophage activation syndrome (MAS) and lung disease (LD). We introduced two new computational frameworks called UDON and SATAY-UDON, which define patient subtypes based on their underlying disrupted cellular programs as well as associated biomarkers or clinical features. Among twelve independently identified subtypes, this analysis uncovered what we believe to be a novel complement and interferon activation program identified in SJIA-LD monocytes. Extending these analyses to adult and pediatric lupus patients found new but also shared disease programs with SJIA, including interferon and complement activation. Finally, supervised comparison of these programs in a compiled single-cell pan-immune atlas of over 1,000 healthy donors found a handful of normal healthy donors with evidence of early inflammatory activation in subsets of monocytes and platelets, nominating possible biomarkers for early disease detection. Thus, integrative pan-immune single-cell analysis resolved what we believe to be new conserved gene programs underlying inflammatory disease pathogenesis and associated complications.


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Cell counts.Individual cell counts for each patient in each disease group or healthy individual in the control group, depicted separately for each cell type. of cellular gene expression inpacts in each SJIA subtype.Gene summary for cellHarmony comparisons, showing overview of up-and downregulated genes for each cell type (cut-off for Contr vs MAS rawp ≤ 0.005, all other comparisons adj.p-value ≤ 0.05) .
Variation in the cellular source for different IFN mediated genes.A-C) Heatmaps of IFN induced genes organized by cell type.A) Module M1.2, B) Module M3.4,C) Module 5.12 as previously described inBanchereau et al., 2016.
Supplementary Figure 6.T-cell population markers in all cells.Top 10 Seurat defined marker genes for each of the 12 T-cell annotated cell populations.

UMAP
Figure 7. T cell polarization markers expressed in the T cell populations.Expression of CCL4, IFNG and TBX21 gene expression indicating Th1 lineage, CCR3 or CCR6 as Th2 markers and STAT3 displaying Th17 polarization.
specific marker gene expression in distinct B-cell subsets.A) UMAP representing 5 B cell populations identified by scRNA-seq.B) Matrix representing cell frequency per individual SJIA patient or control for 5 B cell populations.C) Feature plots representing expression levels of selected B cell population marker genes.
cell populations in SJIA.A) UMAP representing 2 DC cell populations and 1 HSPC population identified by scRNA-seq.B) Matrix representing cell frequency per individual SJIA patient or control for selected populations.C) Feature plots representing marker gene expression levels of 2 DC cell populations and 1 HSPC population.
cell populations in SJIA.A) UMAP representing 2 NK cell populations identified by scRNA-seq.B) Matrix representing cell frequency per individual SJIA patient or control for 2 NK cell populations.C) Feature plots representing expression levels of selected NK cell population marker genes.
-megakaryocytic cell populations in SJIA.A) UMAP representing 2 Erythrocyte and 2 Platelet cell populations identified by scRNA-seq.B) Matrix representing cell frequency per individual SJIA patient or control for selected populations.C) Feature plots representing marker gene expression levels of Erythrocyte and Platelet marker genes.cluster enriched pathway genes.Genes and biological pathways (PathwayCommons) enriched in UDON analysis of SJIA patient pseudo-bulk fold clusters (GO-Elite).

Fraction
. Comparison of CNA NAM-PC gene sets to UDON cluster marker genes.A) Fraction of variance explained by each neighborhood adjacency matrix principal component (NAM PC) identified by CNA.B) Heatmap from the software GO-Elite, displaying gene-set enrichment z-scores for all queried NAM-PC positive and negative loading genes (n=100).C) Enrichment of cell types in CNA's expanded populations associated with clinical phenotypes and UDON subtypes in the SJIA patient cohort.Bar plot indicates the percentage of CNA expanded cells by the total number of cells per cell type.Associations also identified by SATAY-UDON are colored in green.
. UDON reproducibly finds novel SJIA patient subtypes.A) The SJIA patient cohort was split into 19 training and 7 test samples.B) Heatmap pseudobulk-fold heatmap of the excluded test samples reclassified into re-derived UDON clusters on the 19 sample training dataset.C) UDON-SATAY enriched analysis results comparing associations specifically idenfitied in the training dataset association (blue check mark), those detected in both the training and original (all samples) UDON analysis (green check mark) and those only detected in the original UDON analysis (red check mark).activation pathway serum protein evaluation in SJIA.TCC Average, C1q, MBL (Mannose binding lectin) and C4 levels were measured by LUMINEX.Significant differences were calculated by one-way ANOVA and depicted as *= adj.p-value ≤ 0.05.
analysis of SATAY-UDON associations.A) Marker-Finder analysis and B) DEG network (p≤0.05)comparing gene programs of Eryth-Immature Pseudobulks of U2 vs. Eryth-Immature in other UDON Clusters.C) MarkerFinder analysis and D) DEG network (p≤0.05)comparing gene programs of CD8 Mixed Pseudobulks of U4 vs. CD8 Mixed in other UDON Clusters.Enrichment of PathwayCommons gene sets from GO-Elite are indicated on the left of the heatmaps in panels A and C.