Supplementary Materials for

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advances.sciencemag.org/cgi/content/full/7/2/eabd0515/DC1

Supplementary Materials for

Critical role of synovial tissue–resident macrophage niche in joint homeostasis and

suppression of chronic inflammation

Qi-Quan Huang, Renee Doyle, Shang-Yang Chen, Qicong Sheng, Alexander V. Misharin, Qinwen Mao, Deborah R. Winter*, Richard M. Pope*

*Corresponding author. Email: rmp158@northwester.edu (R.M.P.); deborah.winter@northwestern.edu (D.R.W.)

Published 6 January 2021, Sci. Adv. 7, eabd0515 (2021) DOI: 10.1126/sciadv.abd0515

The PDF file includes:

Figs. S1 to S8

Legends for data files S1 and S2

Other Supplementary Material for this manuscript includes the following:

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SUPPLEMENTARY FIGURES

Fig. S1. Gating strategy for synovial tissue macrophage subsets and monocytes during homeostasis defined by flow cytometry. (A), Gating Strategy for identifying CD11b+F4/80+CD64+ total synovial tissue macrophages and the 5 distinct subsets, named as FI1, FI2, FI3, FH1 and FH2. (B), Representative (of 3 independent experiments) expression of cell surface markers on CM and FI1 and FI2 macrophages by fluorescent intensity. (C), Gating strategy for characterizing blood monocytes.

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Fig. S2. Relationship of synovial macrophages with arthritis activity, differentiation and age, and reduced alveolar macrophages. (A), The correlation of FH1 macrophages, granulocytes and B cells to arthritis clinical score and arthritis duration in HUPO mice as indicated in each panel, from 22-37 week old HUPO mice without or with arthritis.

Correlations were determined by Pearson’s linear correlation with Bonferroni correction (pc). (B), Differential expression of MHCII and F4/80 on HUPO and control FH1 macrophages. (C), Alveolar macrophages, defined as

SiglecF+CD64+CD11c+F4/80+CD11b-, from 22-37 week old control and HUPO mice (B-C). (D), The number of total ankle synovial macrophages (STM) from 4-37 week old control mice, and the FH1 to FH2 ratio for the indicated age groups. Statistical analyses were determined by Pearson’s linear correlation for (left panel of D) or by 2-tailed Student's

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t-Fig. S3. Targeting Flip in STMs did not alter the apoptosis and proliferation of FH subsets: (A), cFlar (Flip) gene expression in monocytes and each STM subset from HUPO and control mice (n=3/group). (B),Expression of CD11c by flow cytometry on control and HUPO CM, NCM and each STM subset (age 4 to 37 weeks, n= 8-13 for monocytes and 18-19 for STM). The middle panel is a representative histogram of CD11c and F4/80 from a control mouse. (C), Percent apoptotic cells in each of STM subset from HUPO and control mice (n=4-5, ages 22-37 weeks). (D), The expression of Mif and Spp1 (n=3). (E), BrdU incorporation at 30 minutes into FH2 and FH1 STMs from control and HUPO mice and (F), bone marrow macrophage-dendritic cell precursors (MDP) and monocyte progenitors (cMoP) from control and HUPO mice (n=5-6). Statistical analyses were performed by Student’s 2-tailed t-test (A, left panel of B, C-D, and F) and by one-way ANOVA plus Tukey for (right panel of B and E). Individual values and the mean ±1SE are presented. *p< 0.05, ** p<0. 01 and *** p< 0.001 between indicated groups.

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Fig. S4. Increased circulating monocytes in HUPO mice exhibit arthritogenic potential. (A), Analysis of circulating monocytes. From left to right:the representative flow cytometry, the numbers of total monocytes, the frequency of CM and NCM (n= 11-21) and the MHCII+ CM and NCMs (n=5-9). (B), Scatterplot showing fold change of genes expressed in HUPO or control, CM or NCM. DEGs are presented in colors, and the numbers of genes in each sector are identified. (C), The top GO terms for DEGs in red from panel B. All GO terms for the genes in other sectors were not significantly enriched. (D), DCs identified as CD45+CD64-MHCII+CD11b+, in HUPO compared with control joints (n=8-21). Data in panels A-D from mice 22-37 weeks of age. (E), Cartilage proteoglycans were identified by Safranin-O staining of ankle sections from 4 week old control or HUPO mice. The areas in black boxes were enlarged in panels on the right. Data presented as loss of proteoglycan on a 0-4 scale. B= bone, C and arrows indicate cartilage. Values are mean ±1SE. Statistical analyses by one-way ANOVA plus Tukey (A, D) and by 2-tailed Student’s t-test (E). ** p< 0.01 and *** p<

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Fig. S5. Reduction of the HUPO FH1 subset is associated with arthritis. (A), Experimental design for bone marrow transplant (BMT). Donor BM from pools of CD45.2 control or HUPO mice (5-8 weeks old) were intravenous

administrated into lethally irradiated CD45.1 control or HUPO recipients (8-18 weeks old), as indicated. (B-D), Analysis of recipient ankle synovial macrophages (STM) 15 weeks post-BMT. (B), The percent of donor macrophages identified in recipients; (C), the total number of STMs and their subset distribution (%); (D), The incidence of arthritis in recipients. Values are mean ± SE for (B) and counts in (C). The % of each subset in the pie graphs represents the mean of 3-5 mice

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Fig. S6. B cells, monocytes and synovial macrophages in parabiosis. (A), B cells in blood and ankles from parabionts, presented as the percent from self (recipient) or from paired partner (donor) (n=3-6 pairs) harvested at 5 and /or 10 weeks. (B), Percent of donor classical (CM) and non-classical monocytes (NCM) identified in recipients’ blood of parabionts. The total numbers of donor and recipient monocytes are indicated at the bottom (n = 3-6 pairs, harvested at 5 and/or 10 weeks). (C), Apoptosis of CD45+ cells in the joints of HUPO and control mice (age 22-37 weeks). (D), Comparison of chimerism between control-control and HUPO-HUPO parabionts, employing data from Fig 4B and E. Statistical analyses

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Fig. S7. Distinct transcription factor (TF) binding motifs are enriched in genes affected in HUPO. (A), The percent of monocyte genes that contain an instance of the given TF binding motif that were either up-regulated in at least 3 HUPO synovial tissue macrophage subsets (n=128) or not up-regulated in any subset (n= 1912). (B), Mef2c gene expression in HUPO or control FH1 or FH2 subsets. (C), The percent of genes containing an instance of the MEF2 binding motif in HOMER recognized genes that were either down-regulated in both HUPO FH1 and FH2 (from dark blue

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Fig. S8. t-Distributed Stochastic Neighbor Embedding (tSNE) of HUPO differentially expressed genes in human synovial monocytes from patients with RA or OA. (A) tSNE plot of human synovial monocytes categorized by disease types (23). (B-C). Module scores of top 20 up- and down-regulated FH1/2 genes in HUPO, for the OA and RA synovial tissue monocytes.

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SUPPLEMENTARY DATA FILES

Data file S1. RNA-seq gene expression and ontology (GO) processes. Clustering of gene expression values and GO process pathways for control mice presented in the heatmap of Figure 1C. The 1936 differentially expressed genes across CM and the synovial tissue macrophage subsets were clustered by K-means of GENE-E software. Values are presented in FPKM, as the mean of 3 replicates from each cell type. GO enrichment analyses were performed at

http://cbl-gorilla.cs.technion.ac.il.

Data file S2. RNA-seq gene expression and ontology (GO) processes. Clustering of gene expression values and GO process pathways for HUPO compared with control mice presented in heatmap of Figure 5C. The 2021 differentially expressed genes comparing HUPO and control mice, across CM and the synovial tissue macrophage subsets was clustered by K-means of GENE-E software. Values are presented in FPKM, as the mean of 3 replicates from each cell type. GO enrichment analyses were performed at http://cbl-gorilla.cs.technion.ac.il.

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