Pro-Inflammatory Cytokine Blockade Attenuates Myeloid Expansion in a Murine Model of Rheumatoid Arthritis

Hematopoiesis SUPPLEMENTARY APPENDIX Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis

Giovanny Hernandez, 1 Taylor S. Mills, 1 Jennifer L. Rabe, 1 James S. Chavez, 1 Susan Kuldanek, 1 Gregory Kirkpatrick, 2 Leila Noetzli, 2 Widian K. Jubair, 3 Michelle Zanche, 4 Jason R. Myers, 4 Brett M. Stevens, 1 Courtney J. Fleenor, 5,6 Biniam Adane, 1 Charles A. Dinarello, 7 John Ashton, 4 Craig T. Jordan, 1 Jorge Di Paola, 2 James R. Hagman, 5,6 V. Michael Holers, 3 Kristine A. Kuhn 3 and Eric M. Pietras 1,6 1Division of Hematology, University of Colorado Anschutz Medical Campus, Aurora, CO; 2Department of Pediatrics, University of Col - orado Anschutz Medical Campus, Aurora, CO; 3Division of Rheumatology, University of Colorado Anschutz Medical Campus, Aurora, CO; 4Genomics Research Center, University of Rochester, Rochester, NY; 5Department of Biomedical Research, National Jewish Health, Denver, CO; 6Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO and 7Division of Infectious Disease, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

Received: May 6, 2018. Accepted: April 17, 2019. Pre-published: May 30, 2019. Correspondence: ERIC M. PIETRAS - [email protected] Data 4 A C 4 4 CIA mice BM Ctrl CIA 3 3 11 8 2 2 FSC Lin 1 1 Kit - Mean arthritis score arthritis Mean

c 30 25 0 0 4 7 0 7 14 21 Sca-1 0 7 14 21 Day post boost 53 43 MPP4 B BM Flk2 Ctrl CIA CD150 38 47

1 MPP3 MPP2 - Gr 17 19 37 12 54 18 CD48 Mac 37 44 HSC Gr1 CD150 HSCST 23 26 12 12 HSCLT MPP1 Mon 38 42 63 61 23 26 FSC Ly6C Pre Gr 51 48 Mac-1 CD34 22 10 Flk2 CD19 Pre- 0.3 0.2 2 pro B 3 B220 IL7R IgM+ 47 37 Kit - CLP 75 66 c IgM IgM- 48 58 B220 Sca-1

3 2 MkP 3 2 CD19 CD150 97 97 CD3 CD41 + CD8 60 52 R

γ GMP 50 53 Fc CD8 CD CD4 4+ 26 21 CD150 46 45 I II 2 34 4 13 CFU- Pre- 50 9 46 8 E CFU-E CD71 III 1 0.3 CD105 Pre- Pre- GM MkE Ter119 IV 15 8 CD150 19 15 25 15 Supplementary Figure S1: Disease score and gating strategies for mature and HSPC BM populations. (A) Clinical arthritis score of CIA mice (n = 11). (B-C) Representative FACS plots and frequencies of parent gates for BM (B) mature and erythroblast populations, and (C) hematopoietic stem and progenitor populations in Ctrl and CIA mice. A B C Ctrl Ctrl CFU-GM Ctrl Ctrl CIA 18018 ** CIA 505 ) * CIA 7

) CIA 1 404 10

Spleen: 10 × 12012 5104 BM cells × Ctrl CIA 303 202 606 Methylcellulose 101 Colonies ( Colonies Cell Cell number ( 00 00 Spleen1 CFU assays Spleen1

D E BM Ctrl CIA

Lin 25000025 CtrlCtrl ) CD45 31 300 7 4 20000020 CIACIA 10 EC 26 28 × 200 15000015 CD31 10000010 Sca-1 73 70 Cells/million 100 1 * - MSC 11 5 500005 Cells/million ( Cells/million

Sca 00 0 OBC 9 14 EC OBC MSC CD51 1 EC OBC MSC

Supplementary Figure S2: Spleen and BM stroma changes in CIA mice. (A) Representative image showing sizes of Ctrl and CIA spleens; (B) Total cellularity of RBC-depleted spleens (N = 5/grp); (C) CFU- GM colony number from splenocytes of Ctrl and CIA mice (N = 4/grp). (D) Representative FACS plots and (E) number of the indicated BM stromal populations in Ctrl and CIA mice, expressed as number per million stromal cells. (N = 4/grp); *p < 0.05, **p < 0.01, as determined by Mann-Whitney u-test. Data are compiled from one experiment. A B Peripheral blood (PB) Ctrl CFU-GM Ctrl CIA 606

CD45.1 Donor CIA ) 1

CD45.2 31 4010 4 CtrlCtrl 300 100 HSC × 1 - Myeloid CIACIA

Mac 202 200 Methylcellulose Gr-1 ( Colonies 100 CFU assays 00 HSC1

CD3 T B B220 0 1 C D Donor-derived LSK Flk2- 6060 CtrlCtrl

300 (%) * Ctrl CIA CIACIA MPP MPP 23 19 50 11 4040 200 3 2

derived LSK LSK derived 2020 % Donor LSK 100 - CD48 HSC 39 9

Donor 00 CD150 0 HSCHSC MPP2MPP MPP3MPP MPP4MPP 1 2 3 4 E F Donor-derived LSK Flk2- 6060 CtrlCtrl 300 Legend (%) CIACIA Ctrl CIA Legend MPP 23 17 26 17 40 MPP 5 200 4 40 * 3 2

20 derived LSK LSK derived 20

100 - % Donor LSK CD48 HSC 36 25

CD150 0 Donor 00 1 HSCHSC MPP2MPP MPP3MPP MPP4MPP 2 3 4 Supplementary Figure S3: CFU assay and HSC and progenitor chimerism in Ctrl and CIA transplant experiments. (A) Representative FACS plots showing CD45.2+ donor chimerism and lineage analysis in PB from recipient mice. (B) CFU-GM colony number from HSC of Ctrl and CIA mice (N = 2/grp). (C) Representative FACS plots and (D) composition of donor-derived LSK compartment in recipient mice transplanted with purified HSC from Fig 3A. (E) Representative FACS plots and (F) composition of donor- derived LSK compartment in recipient mice transplanted with unfractionated BM cells from Fig 3C. Data in C-F are from recipient mice with >5% donor LSK chimerism to ensure sufficient events for analysis. *p < 0.05 as determined by Mann-Whitney u-test. Data are representative of two independent experiments. A

HSC

Ctrl CIA

Sca-1 CD34 CD150 CD48 Flk2

B C Ctrl Ctrl 2 2 5000 5 CtrlCtrl 300 CIAHSC CIA ) CIACIA Ctrl 40003 4 gMFI CIA 10 200 3000× 3 1 1 2000 2 (vs. Ctrl) gMFI

100 Number 1000 1 Fold change change Fold

ROS ROS ( 0 0 0 0 0 Sca-1 CD34 CD150 CD48 Flk2 HSC1 1 Flk2 Sca-1 CD48

Supplementary Figure S4: HSC surface markers and ROS levels. (A) Representative FACS plots and (B) levels of the indicated surface markers, expressed as geometric mean fluorescence intensity (gMFI) (N = 4 Ctrl and 8 CIA). (C) Representative FACS plots and ROS levels in HSC from Ctrl and CIA, expressed as geometric mean fluorescence intensity (MFI) of CellROX dye. (N = 4 Ctrl and 4 CIA). Data are compiled from one experiment. A B MPP2 MPP3 MPP4 MyPro MPP2 MPP3 MPP4 MyPro 100 100 100 100 Ctrl G2/S/M S/G2/M S/G2/MCell cycle S/G2/M CIA G1 G1 G1phase G1 G0 G0 G0 S/GS/G2/M2/M G0 MPP2-4 5050 50 50 50 GG11

MyPro GG00 % Total cells Total % Freq. of Parent Gate (%) Freq. of Parent Gate (%) Cell cycle Freq. of Parent Gate (%) analysis 00 0 0 0 Ctrl CIA Ctrl CIA Ctrl CIA Ctrl CIA CIA Ctrl CIA Control CIA CIA Control Control

Supplementary Figure S5: Hematopoietic progenitor cell cycle analysis. (A) Experimental design and (B) cell cycle distribution of the indicated populations ( N = 8 Ctrl and 6 CIA). *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Mann-Whitney u-test. Data are compiled from two independent experiments. Serum G-CSFG-CSF ** RANTES

IL-12p70 IL-10IL-10 ENA-78 Eotaxin MIP-1 MIP-1 M-CSF MCP-1 MCP-3 G-CSF GRO

MIP-2 IL-3IL-3 TNF IP-10 IL-1 IL-1 IL-18 IL-28 IL-23 IL-27 IL-22 IL-10 IFN IL-9 IL-6 IL-5 IL-4 IL-3 LIF LIFLIF 500 500 α α α α β β γ Ctrl IL-1β 400 ctrl1 IL-1β 400 300 ctrl2 CIA M-CSFM-CSF 300 ctrl3 IP-10IP-10 200 200 ctrl4 IL-4IL-4 100 100 ctrl5 IL-5IL-5 0 ctrl6 IL-6IL-6 ctrl7 Serum IL-22IL-22 CIA1 IL-9IL-9 CIA2

CIA3 IL-27IL-27

CIA4 Luminex IL-23IL-23 Array IFNIFNγγ ** IL-12p70IL-12p70 GROGROαα RANTESRANTES TNFTNFαα * MIP-1MIP-1αα MCP-3MCP-3 MCP-1MCP-1 pg/ml MIP-2MIP-2 0 500 IL-1IL-1αα

100 200 300 400 500 ENA-78ENA-78 EotaxinEotaxin IL-28IL-28 IL-18IL-18 MIP-1MIP-1ββ Ctrlctrl1Ctrlctrl2CtrlCIActrl3CIActrl4CIActrl5ctrl6ctrl7CIA1CIA2CIA3CIA4 Ctrl CIA

Supplementary Figure S6: Cytokine array analysis of serum. Experimental strategy and heatmaps from Luminex cytokine arrays showing cytokine levels for serum (N = 7 Ctrl and 4 CIA) collected from mice from two independent experiments. Statistically significant differences are indicated to the right of each row in the heatmaps. *p < 0.05; **p < 0.01 as determined by Mann-Whitney u-test. A Hematocrit B10 Hb B10 Hct Hemoglobin ** * 60 60 15 15 *

40 40 10 10 g/dl 20 20 5 5 RBC mass (%) mass RBC

0 0 0 0 Ctrl CIA CIA Ctrl CIA CIA +Ana +Ana Ctrl CIA Ana B MPP2 MPP3 MPP4 MyPro MPP2 MPP3 MPP4 MyPro 100 100 100 100 100 S/G2/M S/G2/M S/G2/MCell cycle S/G2/M G1 G1 G1 phase G1

G0 G0 G0 S/GS/G2/M2/M G0 50 50 50 50 50 GG11

GG00 % Total cells Total % Freq. of Parent Gate (%) Freq. of Parent Gate (%) Freq. of Parent Gate (%) 0 0 0 0 0 Ctrl CIA CIA Ctrl CIA CIA Ctrl CIA CIA Ctrl CIA CIA B10 CIA +Ana B10 CIA +Ana B10 CIA +Ana +Ana 1 2 3 Anakinra Anakinra Anakinra

Supplementary Figure S7: Impact of IL-1 blockade on RBC and hematopoietic progenitor cell cycle distribution. (A) Complete blood count (CBC) analysis. (B) cell cycle analysis of MPP and MyPro populations in Ctrl, CIA and CIA +Anakinra (Ana) mice. (N = 5 Ctrl, 5 CIA and 5 CIA + Ana for all assays). **p < 0.05, **p < 0.01 as determined by one-way ANOVA with Tukey’s test for multiple comparisons. Data are representative of two independent experiments. Supplementary Table Legends

Supplementary Table S1: Up-and downregulated genes in CIA HSC. Data are expressed as log2 fold change in CIA HSC versus Ctrl HSC. Genes highlighted in bold are significantly differentially expressed based on padj <0.05.

Supplementary Table S2: Ingenuity Pathway analysis of CIA HSC. Upstream regulator analysis of significantly differentially expressed genes in CIA HSC versus Ctrl HSC, including activation z-score, p-value and pathway target molecules significantly enriched in each pathway.

Items highlighted in bold are shown in Figure 4.

Supplementary Table S3: Gene ontology (GO) analysis of downregulated genes in CIA HSC.

GO analysis of significantly differentially expressed genes in CIA HSC versus Ctrl HSC, showing p-value, number and percentage of genes enriched in each GO category, p-value and genes significantly enriched in each category. Items highlighted in bold are shown in Figure 4.

Supplementary Table S4: Gene ontology (GO) analysis of upregulated genes in CIA HSC.

Supplementary Table S5: GSEA analysis downregulated genes in CIA HSC. Gene set enrichment analysis (GSEA) of RNA-seq data showing gene set names, gene set sizes, enrichment scores and p-values for each enriched gene set.

Supplementary Table S6: GSEA analysis upregulated genes in CIA HSC. Gene set enrichment analysis (GSEA) of RNA-seq data showing gene set names, gene set sizes, enrichment scores and p-values for each enriched gene set.

Supplementary Table S7: Antibodies used in this study. Antibody list includes clones, manufacturer, fluorochrome and dilution.

Supplementary methods

Collagen emulsion preparation, CIA induction and clinical scoring

CIA induction protocols are based on published, detailed methodologies for induction in

C57BL/6J mice1and in B10.RIII mice2. Due to differences in strain susceptibility to CIA and disease course, protocols for C57BL/6 and B10.RIII mice are distinct. For C57BL/6 mice, collagen was prepared by making a 2mg/ml solution of chicken collagen in 0.01N acetic acid (Sigma) and mixed with an equal volume of Complete Freund’s adjuvant (Sigma). CFA/collagen emulsions also contained 2mg/ml heat-killed Mycobacterium tuberculosis strain H37Ra (InVivoGen). For

B10.RIII mice, a 2mg/ml solution of bovine collagen in 0.01N acetic acid (Sigma) was mixed with an equal volume of Complete Freund's adjuvant without additional heat-killed Mycobacterium tuberculosis. Both emulsions were produced by hand mixing components in 5 ml glass emulsifying syringes joined with 22G micro-emulsifying needles (Thomas Scientific). Syringes were kept partially submerged in ice water for 10 minutes, and complete emulsion was verified by dropping a bead of the material in water; completely emulsified beads hold their shape in water. 150 µl of emulsion was injected intradermally near the base of the tail in mice anesthetized with isoflurane.

Booster injections were performed at day 15 (B10.RIII) or day 21 (C57BL/6J).

Induced C57BL/6J animals developed disease with approximately 40-80% penetrance.

Animals failing to develop disease following the booster injection according to clinical scoring criteria were not included in final experimental cohorts. Induced B10.RIII animals developed disease with 100% penetrance. Arthritis was scored using a 5-point scale: 0 = normal; 1 = 1 hind or fore paw joint affected or minimal diffuse erythema and swelling; 2 = 2 hind or fore paw joints affected or mild diffuse erythema and swelling; 3 = 3 hind or fore paw joints affected or moderate diffuse erythema and swelling; 4 = marked diffuse erythema and swelling, or 4 digit joints affected; and 5 = severe diffuse erythema and severe swelling of entire paw, unable to flex digits. Animals were analyzed 21 days after the second collagen type II injection. Peripheral blood parameters were determined via post-euthanasia retro-orbital bleeding and complete blood count (CBC) analysis using a HemaVet analyzer (Drew Scientific).

Detailed flow cytometry methods

Antibody clones and dilutions used are listed in Supplementary Table 7. All surface staining was performed as previously described3 on ice in staining medium (SM; HBSS +2% heat- inactivated FBS). Following RBC depletion, cellularity was determined using a ViCell automated counter (Beckman Coulter) To identify HSPC, RBC-depleted cells were incubated on ice for 30 min. with the following antibodies: PE-Cy5-conjugated anti-CD3, CD4, CD5, CD8, B220, Gr-1,

Mac-1 and Ter119 (Lineage), plus CD34-FITC, CD150-PE, FcgR-PerCP/eFluor710, IL-7R-

PE/Cy7, Sca-1-BV421, CD41-BV510, Flk2-Biotin, CD105-BV786, ESAM-APC, CD48-

AlexaFluor 700, and c-Kit-APC/Cy7 (Biolegend). 50ug/ml Rat IgG (Sigma) was included in all live staining incubations to block nonspecific antibody interactions. Cells were subsequently washed in SM, spun at 1200 ´ g, stained with streptavidin-BV605, and dead cells counterstained with SM containing 1µg/ml propidium iodide (PI). Mature BM and spleen populations were identified by staining with the following antibodies: CD4-FITC, CD8-PE, Mac-1-PE/Cy7, Gr-1-

Pacific Blue, Ly6C-BV605, B220-BV786, IgM-APC, CD3-A700 and CD19-APC/Cy7

(Biolegend). For analysis of intracellular ROS, cells were surface stained as above, followed by incubation for 30 min in HBSS without FBS at 37oC with 5µM CellROX Deep Red dye (Thermo

Fisher) according to manufacturer's instructions. Following incubation, cells were washed in SM, counterstained with PI and analysed by flow cytometry. BM stroma were analyzed as previously described3,4. Briefly, leg bones were crushed in HBSS without FBS, bone chips were collagenase- I digested at 37oC while shaking for 1hr, and stained with Lineage-PE/Cy5, Sca-1 Pacific Blue,

CD45-APC/Cy7, CD31-FITC, and CD51-Biotin, washed and stained by SA-APC. Cells were analyzed using a FACSCelesta or FACS LSRII (Becton-Dickenson). For cell cycle analysis,

Ki67/DAPI staining was performed as previously described5,6. BM cells were stained with the aforementioned lineage cocktail, plus CD34-FITC, Flk2-PE, Sca-1-PE/Cy7, ESAM-APC, CD48-

A700, c-Kit APC/Cy7, and CD150-BV785. Following staining, cells were washed in SM, fixed at room temperature (RT) for 30 min. with Cytofix/Cytoperm (BD Biosciences), washed and spun down at 1200 x g for 5 min. in 1x PermWash Buffer (BD Biosciences), permeablized in Cytoperm

Plus (BD Biosciences) for 10 min. at RT, washed and spun again in 1x PermWash, and re-fixed in

Cytofix/Cytoperm for 5 min. Fixed and permeablized cells were subsequently stained with anti-

Ki67-PerCP/Cy5.5 for 30 min at RT, washed in 1x PermWash Buffer, and resuspended in SM containing 2µg/ml DAPI. For IL-1b intracellular staining, 1 x 106 BM cells were stained with the aforementioned mature cell panel, with CD8-BV510. Cells were subsequently fixed as described above, and stained with anti-IL-1b-PE for 30 min. at RT. Positive staining was determined using fluorescence minus one (FMO) controls. For cell sorting, c-Kit enriched BM cells were subsequently stained with the aforementioned lineage cocktail, plus CD150-PE, CD48-AlexaFluor

700, c-Kit-APC/Cy7, Sca-1-BV421, and Flk2-Biotin followed by SA-BV605. Cells were then counterstained with SM containing PI before sorting.

Methylcellulose cultures

For granulocyte-macrophage (GM) colony forming assays, 1´104 BM cells, 1´105

splenocytes or 100 purified HSC were cultured in a 3cm plate containing 1ml Methocult (Stemcell

Technologies, M3231) supplemented with penicillin (50U/ml), streptomycin (50 µg/ml) (Life

Technologies), SCF (25 ng/ml), IL-11 (25ng/ml), Flt3L (25ng/ml), GM-CSF (10ng/ml), IL-3 (10ng/ml), Epo (4U/ml), and Tpo (25ng/ml) (Peprotech). For CFU-E assays, 1´105 BM cells were plated in 1ml Methocult supplemented with recombinant human insulin, transferrin and EPO

(Stemcell Technologies, M3334). For CFU-B assays, 1´104 BM cells were plated in 1ml

Methocult supplemented with IL-7 (Stemcell Technologies, M3630). Plates were incubated in a

o humidified air-jacket incubator (Thermo Fisher) with 5% CO2 at 37 C. Colonies were enumerated

2 (CFU-E), 7 (CFU-B) or 7-10 days (CFU-GM) after plating.

RNA-seq sample preparation, quality control and analysis

RNA was isolated from cell pellets using an RNEasy Micro kit (Qiagen) per manufacturer’s

recommendations. 150pg of cDNA was used to generate Illumina compatible sequencing libraries

with the NexteraXT library preparation kit (Illumina) per manufacturer’s protocols. RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent). 1ng of total RNA was pre-amplified with the SMARTer Ultra Low Input kit v4 (Clontech) per manufacturer’s recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Flourometer (Life

Technnologies) and the Agilent Bioanalyzer 2100 (Agilent). The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina). Single end reads of 100nt were generated for each sample using an Illumina HiSeq2500v4 high-throughput sequencer. Raw reads generated from the Illumina HiSeq2500 sequencer were demultiplexed using bcl2fastq version 1.8.4. Quality filtering and adapter removal were performed using

Trimmomatic version 0.32 with the following parameters: "LEADING:13 TRAILING:13

SLIDINGWINDOW:4:20 ILLUMINACLIP:adapters.fasta:2:30:10 MINLEN:15".

Processed/cleaned reads were then mapped to the GRCm38.p4 (mm10/mg38) with STAR_2.4.2a with the following parameters: "--twopassMode Basic --runThreadN ${CPUS} --runMode alignReads --genomeDir ${STARREF}--readFilesInclt_${SAMPLE}_R1.fastq-- outSAMtypeBAM SortedByCoordinate—outSAMstrandFieldintronMotif --outFilterIntronMotifs

RemoveNoncanonical --outReadsUnmapped Fastx". Raw read counts were obtained using htseq- count v0.6.1 and Gencode-M12 gene annotations. Additional analysis was performed using

Ingenuity Pathway Analysis software with default user settings (Qiagen) and the GSEA tool

(Broad Institute). GO analyses of significantly up- or downregulated genes were performed using

DAVID 6.8 (https://david.ncifcrf.gov/).

Fluidigm gene expression analysis

Sample preparation and data analysis using the Fluidigm platform was performed as previously described3,7. Briefly, eight pools of 100 cells per well were sorted into 96-well PCR plates containing 5µl CellsDirect buffer (Invitrogen) and subjected to 18 rounds of specific target amplification using DeltaGene assay primer pairs commercially designed by Fluidigm. Genomic

DNA contamination was removed using exonuclease 1 (New England Biolabs) digestion. Samples were mixed with SsoFast EvaGreen Supermix (BioRad), and sample and DeltaGene primer mixes were loaded and run on Fluidigm 96.96 or 48.48 IFC gene expression arrays in a Biomark instrument (Fluidigm). Ct values were obtained using Fluidigm Biomark analysis software and exported for expression was using the DCt method, normalized to Actb.

Serum collection for cytokine analysis

For serum collection, blood was obtained from mice via cardiac puncture with a 28.5G needle and 0.5ml syringe (Becton Dickenson). Blood was allowed to coagulate in a 1.5ml microcentrifuge tube (Eppendorf) at room temperature for 30 minutes. Samples were subsequently spun at 10,000 ´ g for 10 minutes to separate blood cells and serum was stored frozen at -80oC until analysis. Cytokine arrays were analyzed on a MAGPIX analyzer running xPONENT

4.2 software (Luminex).

Supplementary Methods References

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