Additional file 1 of Age-stratification reveals age-specific intestinal microbiota signatures in juvenile idiopathic arthritis
Creators: name: Budzinski, Lisa
name: Sempert, Toni
name: Lietz, Leonie
name: Maier, René
name: Kang, Gi-Ung
name: von Stuckrad, Anne Sae Lim
name: Goetzke, Carl Christoph
name: Roth, Maria
name: Shah, Aayushi
name: Abbas, Amro
name: Lehman, Katrin
name: Necke, Kathleen
name: Bartsch, Stefanie
name: Hoffmann, Ute
name: Mashreghi, Mir-Farzin
name: Biesen, Robert
name: Kallinich, Tilmann
name: Chang, Hyun-Dong
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material: Figure S1. Phenotyping of intestinal microbiota by multi-parameter microbiota flow cytometry and identification of disease-specific signatures. (A) Human intestinal bacteria from stool samples were stained with monoclonal antibodies specific for the human immunoglobulins IgA1, IgA2, IgM and IgG and with the lectins peanut agglutinin (PNA), wheat germ agglutinin (WGA), Solanum tuberosum lectin (STL) and Concanavalin A (ConA). Each staining panel also included the cell wall/membrane-permeable DNA dye Hoechst 33342. After data acquisition in a flow cytometer, the cells of each staining panel were clustered according to a previously defined self-organizing map (SOM) into 2025 clusters representing a set of phenotypic features and the abundance of cells that display those. The clusters for both panels are combined to compute the microbiota fingerprint out of 4050 clusters. The abundance of cells per cluster in the total of 4050 clusters represented the overall microbiota phenotype of a sample. (B) R-Pipeline to select cohort-specific features from the microbiota phenotype (B1) to obtain a microbiota biosignature (B2-B4). The clusters were filtered by (B2) Wilcoxon statistical evaluation and (B3) recursive feature elimination to select the significant and most robust clusters defining the specific microbiota phenotype signature represented for all samples by their beta-diversity (Bray-Curtis dissimilarity) projected by a Principal component Analysis (PCoA) (B4). In a PCoA the differences between samples correlate with their distance as more similar samples are closer to each other than very distinct samples (B4). The same analysis approach was used to identify taxonomic signatures for the cohort comparisons using a taxonomic count table as input. Figure S2. Taxonomic and phenotypic signature to identify JIA from pediatric controls before feature selection. PCoA of the beta-diversity by Bray-Curtis index of all samples of the JIA cohort (n=54 individuals, purple) and healthy controls (n= 38 individuals, grey) for the entire taxonomic signature(A) or phenotypic signature (B, 4050 clusters) of the intestinal microbiome for the comparison JIA vs. healthy pediatric controls. R2 and ANOVA test for each group. Figure S3. Detailed overview of selected taxonomic features to identify JIA from pediatric healthy controls.List of selected 28 taxonomic features ordered by generalized fold change. Features enriched in JIA (generalized fold change &gt; 0) are shown in purple, features enriched in pediatric controls (generalized fold change &lt; 0) are depicted in grey. The feature abundance is represented as box plot showing the 25th percentile, median and 75th percentile. Figure S4. Detailed overview of selected phenotypic clusters to identify JIA from healthy controls. (A) List of selected phenotypic clusters of both panels, ordered by generalized fold change and described for their marker composition by a heatmap (signal intensities normalized for each marker). (B) Representation of all selected phenotypic clusters for their location in 2D dot plot of all markers included in the panels (y-axis) against forward scatter (FSC, x-axis). All clusters enriched in JIA are depicted in purple, clusters enriched in controls are depicted in grey. Figure S5. Disease severity is no confounder of the taxonomic or phenotypic intestinal microbiota signatures in this JIA cohort. (A, B) Box plots for the distance of each patient Bray-Curtis beta-diversity index to the mean beta-diversity index of all healthy controls, evaluated for the disease severity (inactive/mild vs. high disease activity) intestinal microbiome signature in taxonomy (A) or microbiota phenotype (B). Grouped for disease severity: inactive/mild disease activity (IDa/MiDa, blue): cJADAS10 &lt;4, moderate disease activity (MoDa, grey): cJADAS &gt;4 and &lt;13, high disease activity (HDa, red): cJADAS10&gt;13. Box plot show median, upper hinge: 75th percentile, lower hinge 25th percentile of data. Wilcoxon test, unpaired. Figure S6. Detailed overview of selected taxonomic features to identify JIA from pediatric controls by age-wise comparison. (A, B, C) Box plots represent the abundance of all selected features for age group 1 (A, 9 taxa), age group 2 (B, 18 taxa) and age group 3 (C, 6 taxa) ordered by generalized fold change. Taxa enriched in JIA (generalized fold change &gt; 0) are depicted in magenta (age group 1), blue (age group 2), dark green (age group 3) and taxa enriched in pediatric controls are depicted in pink (age group 1), light blue (age group 2), light green (age group 3). Box plot show median, upper hinge: 75th percentile, lower hinge 25th percentile of data. Figure S7. Representation of the selected phenotypic clusters for age-wise comparisons of JIA and pediatric controls. (A, B, C) Representation of all selected phenotypic clusters for their location in 2D dot plot of all markers included in the panels (y-axis) against forward scatter (FSC, x-axis). All clusters enriched in JIA are depicted in magenta (age group 1), blue (age group 2), dark green (age group 3) and clusters enriched in controls are depicted in pink (age group 1), light blue (age group 2), light green (age group 3). Figure S8. Multi-dimensional representation of the phenotypic clusters representing JIA by t-SNE. Combination of selected phenotypic clusters from all comparisons: all JIA vs. all pediatric controls plus age-matched comparisons (524 clusters). T-SNE shown for all markers normalized fluorescence intensities: blue = low intensity; red = high intensity. Clusters referring to immunoglobulin panel: rectangles. Clusters referring to lectin panel: circles. Figure S9. Microbiota signature of RA patients. (A, B) PCoA of the beta-diversity by Bray-Curtis index of all samples of the RA cohort (n=16 individuals, orange) and adult healthy controls (n= 18 individuals, beige) for the selected taxonomic signature (A, 20 features) or phenotypic signature (B, 72 clusters) of the intestinal microbiome for the comparison RA vs. adult controls. R2 and ANOVA test for each group. (C) Box plots represent the abundance of all 20 selected taxonomic features of the intestinal microbiome RA signature ordered by generalized fold change. Taxa enriched in RA (generalized fold change &gt; 0) are depicted in orange and taxa enriched in adult controls are depicted beige. Box plot show median, upper hinge: 75th percentile, lower hinge 25th percentile of data. (D) Representation of all selected phenotypic clusters for their location in 2D dot plot of all markers included in the panels (y-axis) against forward scatter (FSC, x-axis). All clusters enriched in RA are depicted in orange, clusters enriched in controls are depicted in grey. Figure S10. Microbiota signatures of JIA therapeutics. (A, B) PCoA of the beta-diversity by Bray-Curtis index for all JIA patients (n=54) based on therapy for (A) 16S rRNA sequencing and (B) microbiota phenotyping. R2 and ANOVA test for therapy and ellipses indicate the 95 % confidence interval for each group. Figure S11. Microbiota signatures of JIA subtypes. (A, B) PCoA of the beta-diversity by Bray-Curtis index for all JIA patients (n=54) regarding subtype for (A) 16 SrRNA sequencing and (B) microbiota phenotyping. R2 and ANOVA test for subtype and ellipses indicate the 95 % confidence interval for each group.