Supplementary Materials for

Synovial fibroblast-neutrophil interactions promote pathogenic

adaptive immunity in rheumatoid arthritis

Carmelo Carmona-Rivera, Philip M. Carlucci, Erica Moore, Nithya Lingampalli,

Hannes Uchtenhagen, Eddie James, Yudong Liu, Kevin L. Bicker, Heidi Wahamaa,

Victoria Hoffmann, Anca Irinel Catrina, Paul R.Thompson, Jane H. Buckner,

William H. Robinson, David A. Fox, Mariana J. Kaplan*

*Corresponding author. Email: mariana.kaplan@nih.gov

Published 14 April 2017, Sci. Immunol. 2, eaag3358 (2017)

DOI: 10.1126/sciimmunol.aag3358

The PDF file includes:

Materials and Methods

Fig. S1. ACPAs recognize NET peptides.

Fig. S2. Skin fibroblasts from patients with psoriasis internalize NETs.

Fig. S3. NETs up-regulate type I IFN-inducible genes in FLS.

Fig. S4. Endogenous expression of RAGE and TLR9 in FLS.

Fig. S5. Up-regulation of MHC II in OA FLS incubated with NETs.

Fig. S6. IFN-γ is not detected in NETs or FLS supernatants.

Fig. S7. Detection of IL-17A and IL-17B in neutrophil lysate and NETs.

Fig. S8. NETs are internalized by a TLR9-dependent and IL-17R–independent

mechanism.

Fig. S9. FLS do not express HC-gp39 or MPO.

Fig. S10. Characterization of vimentin epitope binding to HLA-DRB1*04:04 and

T cell clone specificity.

Fig. S11. Cytokine profile of cocultures of Aggrecan 225 CD4+ T cells and

haplotype-matched FLS.

Fig. S12. GK1.5 antibody depletes specifically CD4 T cells but not CD8 T cells.

Fig. S13. DRB1*04:01 transgenic mice immunized with FLS loaded with NETs

develop in vivo antibody responses to citrullinated peptides.

Table S1. Mass spectrometry analysis demonstrates multiple citrullinated peptides

in NETs induced with IgM rheumatoid factor.

immunology.sciencemag.org/cgi/content/full/2/10/eaag3358/DC1

Reference (56)

Other Supplementary Material for this manuscript includes the following:

(available at immunology.sciencemag.org/cgi/content/full/2/10/eaag3358/DC1)

Source data Excel file

Source data blots (.pptx format)

Supplementary Materials

Materials and Methods

Reagents

Commonly used reagents and their source were as follows: cytochalasin D, chloroquine, mouse

monoclonal anti-tubulin clone DM1A (all from Sigma), goat anti-EEA1 clone N-19 (Santa

Cruz), mouse monoclonal neutralizing anti-CD28 (clone CD28.6, eBioscience), mouse

monoclonal neutralizing anti-HLADR (L243)(Abcam), BODIPY558/568 Phalloidin

(ThermoFisher), rabbit anti-TLR9 (Abcam), mouse monoclonal anti-MHC class II (Abcam),

Hoechst and Sytox green (Life technologies), rabbit anti- IFN-γ (Abcam), rabbit anti-

myeloperoxidase (MPO) (DAKO), goat anti IL-17A, goat anti-gp-39 ( S-18, Santa Cruz) and

goat anti-IL17B (R&D System), human recombinant histone H3 and histone H4 (BioLabs),

human recombinant IL-17A and IL17B (BioVision) and human recombinant IFN-γ (Millipore).

Secondary antibodies coupled to fluorophores were from Life Technologies. Mouse IL-2, human

IFN-γ and human IL-8 ELISA kits were from eBioscience and human IL-6 from BD Bioscience.

Rheumatoid factor was a kind gift from Dr. Pojen Chen, UCLA and has been previously

described12.

Proteomic analysis for detection and quantification of citrullinated proteins

NET samples (~20 µg) generated from control neutrophils that had been stimulated with

rheumatoid factor were mixed with 10 mM Dithiothreitol and heated at 96ºC for 10 min.

Samples were digested with Trypsin (Promega) overnight at 37C. Digested peptide samples was

desalted by C18 ZipTip (Millipore), lyophilized and re-suspended in 0.1% formic acid for LC-MS

analysis. Samples were loaded on an Easy nLC II nano-capillary HPLC system (Thermo

Scientific) with a C18 Nano Trap Column, (Acclaim PepMap100 C18, 2 cm, nanoViper, Thermo

Scientific) and an analytical column (Acclaim PepMap RSLC C18, 15 cm, nanoViper, Thermo

Scientific) connected with a stainless steel emitter, coupled online with a Q Exactive hybrid

OrbiTrap mass spectrometer (Thermo Scientific) for µRPLC-MS/MS analysis. The MS data

were searched against the UniProt Homo sapiens database downloaded from the European

Bioinformatics Institute website (http://www.ebi.ac.uk/integr8, January, 2015) utilizing

Proteome Discoverer 1.4 (ThermoFisher). Oxidation (+15.9949 Da) of methionyl residue, and

citrullination (+0.9840 Da) on arginine residue were included as dynamic modifications. The

data was searched with a precursor ion tolerance of 20 ppm and a fragment ion tolerance of 50

ppm. The peptide identifications are filtered through protein percolator with the cutoff of a false

peptide discover rate (FDR) less than 1% for all peptide identified.

Assessment of NET internalization by FLS

OA and RA FLS were cultured in 6-well plates at a density of 100,000/well or using coverslips.

In specific experiments, cells were pretreated with 1 µg/mL cytochalasin-D (CytoD), 40 µg/mL

of chloroquine (CQ), 4 µM of the specific human TLR9 inhibitor (ODN TTAGGG, 5’-

tttagggttagggttagggttaggg-3’)(InvivoGen), or 4 µM of ODN negative control for 30 min in

complete media, followed by addition of 50 µg of spontaneously generated RA-NETs and

further incubation for 6 or 72 h. Supernatants were then harvested and centrifuged at 300xg for 5

min at RT. FLS were washed twice with PBS, then either lysed or fixed for additional

experiments.

For experiment to inhibit the effects of RAGE, FLS were pre-incubated with 2-4 µM of RAGE

antagonist peptide, RAP (Calbiochem) for 30 min at 37C. Fifty micrograms of RA NETs were

added to the FLS and incubated for 1 hour. Cells were either lysed for co-immunoprecipitation

and Western blot analysis or fixed with 4% PFA for immunofluorescence analysis.

Fluorescent microscopy

FLS were washed twice with PBS and fixed in 4% paraformaldehyde in PBS overnight at 4C.

FLS were permeabilized with 0.2% (wt/vol) Triton-x-100 in PBS for 10 min at RT. Cells were

blocked with 0.2% porcine skin gelatin (Sigma) in PBS for 30 min and incubated in a humid

chamber for 1h at 37C with primary antibody diluted in blocking buffer. Coverslips were

washed three times and incubated with secondary antibodies coupled to Alexa-488 or Alexa-555

fluorophores for 30 min at 37C. DNA was stained with 1:1000 Hoechst for 10 min. Samples

were washed three more times with PBS and mounted on glass slide using Prolong-gold solution.

Images were acquired on a Zeiss LSM780 confocal laser-scanner microscope.

RNA isolation and real-time PCR

Following various culture conditions mentioned above, RNA was isolated from OA and RA FLS

using DNA-Free RNA kit (Zymo Research). Total RNA (500 ng) was reverse transcribed using

iScript RT single strand cDNA (BioRad). qPCR was performed using TaqMan Gene Expression

Master Mix (Applied Biosystem), human GAPDH primers(Hs99999905_m1) as internal control,

and sequence-specific primers for HLA-DRA (Hs00219575_m1), HLA-DRB

(Hs03027795_uH), TLR9 (Hs00152973_m1), IFNA1(Hs00256882_s1), and IFNB1

(Hs01077958_s1). To assess the interferon stimulated genes we utilized SsoAdvancedTM

Universal SYBR Green Supermix (BioRad). Primers for SYBR Green were GAPDH primers

forward: 5’-ACCACAGTCCATGCCATCAC-3’, reverse: 5’-TCCACCACCCTGTTGCTGTA-

3’; MX1 primers forward: 5’-TACCAGGACTACGAGATTG-3’, reverse: 5’-

TGCCAGGAAGGTCTATTAG-3’; IFI44 primers forward: 5’-

CTCGGTGGTTAGCAATTATTCCTC-3’, reverse: 5’-AGCCCATAGCATTCGTCTCAG-3’;

and IFI44L primers forward: 5’-GAACTGGACCCCATGAAGG-3’, reverse: 5’-

ACTCTCATTGCGGCACACC-3’. Assay was performed in duplicate.

Flow cytometry

To quantify MHC on the plasma membrane, stimulated RA FLS cells were stained in FACS

buffer with PE-anti-human HLA- A, B C (Biolegend) as well as Brilliant Violet 650tm anti-

human HLA-DR (Biolegend). Cells were washed in phosphate-buffered saline and fixed with

4% paraformaldehyde. For intracellular quantification of MHC, RA FLS cells were first fixed

and permeabilized with BD Cytofix/cytopermTM (BD Biosciences) and washed with BD

Perm/washTM (BD Biosciences) according to the manufacturer’s instructions. Cells were stained

with PE-anti-human HLA A, B C (Biolegend) as well as Brilliant Violet 650tm anti-human HLA-

DR (Biolegend) for 30 min at 4°C. Cells were washed and flow cytometry was performed.

Co-immunoprecipitation

FLS were incubated in the presence or absence of 50 µg of spontaneously generated RA-NETs,

or NETs + RAGE inhibitor for 1 hour at 37C. Cells were washed twice with cold PBS and lysed

with 300 µL of lysis buffer (50mM Tris-HCl pH 7.4, 300 mM NaCl, 0.5% w/v Triton X-100,

5mM EDTA) supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN). Lysates

were centrifuged at 16,000 xg for 10 min at 4C. Supernatants were precleaned by incubation

with G-Sepharose beads (Amersham Pharmacia Biotech) for 60 min at 4C. Pre-cleared lysates

were subsequently incubated with mouse monoclonal anti-RAGE clone A-9 (Santa Cruz) or

mouse monoclonal anti-GFP (Santa Cruz) and G-sepharose beads overnight at 4C. Beads were

washed 5 times with cold lysis buffer. Bound proteins were eluted by boiling in 30 µL of

Laemmli buffer at 95C for 5 min. Samples were resolved in a SDS-PAGE and TLR9 quantified

by Western blot.

Western blot

Neutrophils and treated FLS were lysed as described (14, 56). Proteins were separated in a 4-

12% gradient Bis-Tris gel (Invitrogen), transferred onto a nitrocellulose membrane and blocked

with 10% BSA for 30 min at room temperature. After overnight incubation with primary

antibodies, membranes were washed three times with PBS-Tween (PBS-T) and incubated with

secondary antibody coupled to horseradish-peroxidase for two hours at room temperature, or

with secondary antibody coupled to IRDye 800CW. Membrane was washed three times with

PBS-T and incubated in ECL for 1 min. Membrane was developed using c-Digit scanner (Li-

COR). Membranes incubated with IRDye secondary antibodies were developed using Li-COR

Odyssey Clx scanner (Li-COR).

Splenocyte isolation

Spleens from DRB1*04:01 mice were homogenized into a single cell suspension using a 100

µm cell strainer. Cells were resuspended in FACS buffer (2% FBS in PBS) and centrifuged at

300 x g at RT. Cells were washed once with PBS and red blood cells were lysed with ACK

buffer (Quality Biological), washed with PBS and resuspended in RPMI containing 10% FBS.

Splenocytes were seeded in 48-well plates and incubated with a cocktail of citrullinated proteins

(100 ng each of cit-H3, cit-H4, cit-MPO, cit-vimentin) or naïve proteins (100 ng each of H3, H4,

MPO and vimentin) for 5 days. Supernatants were collected and mouse IL-2 was quantified

using ELISA Ready-SET-Go (eBioscience), following manufacturer’s instructions.

In vitro citrullination and Dot blot

One microgram of recombinant histone H3 (Abcam), histone H4, (Abcam), myeloperoxidase and

vimentin (MyBioSource) were citrullinated in vitro with 0.2 µM PAD4 in 100 mM Tris-HCl pH

7.6, 10 mM CaCl2, 50mM NaCl, 2mM DTT for 3 h at 37°C. An aliquot of 100 ng of each

citrullinated protein was loaded onto a nitrocellulose membrane. Membrane was blocked with

10% BSA for 30 min at room temperature, incubated with 1:250 mouse serum in 5% BSA

overnight at 4°C. After incubation with IRDye 800CW-conjugated anti-mouse secondary

antibody, membrane was developed in a Li-COR Odyssey Clx infrared imaging system (Li-

COR).

CD4+ T cells depletion: twelve week- old female DRB1*04:01 were injected every other

day with 1.0 x 105 of FLS loaded with NETs. The day after, animals were injected weekly with

1mg/mL of mAbGK1.5 or rat-IgG2b isotype control antibody (BioCell) intraperitoneally for 6

weeks. Blood was collected after 3 or 6 weeks of injections and ACPAs was assessed. T cell

depletion was confirmed by flow cytometry.

In vivo tracking of FLS: 1 x 10 5 DRB1*04:01 FLS were labeled with CellTrace™ Violet

(Thermo Fisher Scientific) and injected into the synovial space using a 27-gauge needle. Mice

were euthanized 2-4 days after injection and lymph nodes and spleen were harvested. Single cell

suspensions of lymph nodes and spleen were prepared and FACS was performed. The

CellTrace™ Violet-positive FLS were calculated by their percentage on FACS analysis.

Assessment of cartilage damage

Knee joints were decalcified, dehydrated and embedded in parafilm. Tissue sections (5 m) were

stained for Safranin-O to assess cartilage integrity and histology and this was scored by a

veterinary pathologist blinded to the treatment conditions. Scores included % of cartilage loss,

presence of clefts or fibrillation, presence of pannus and synovial thickness.

Fig. S1. ACPAs recognize NET peptides. Spontaneously generated NETs from RA peripheral

blood neutrophils were incubated with ACPAs and with MPO antibodies. Immunofluorescence

analysis demonstrates that ACPAs recognize NET-peptides and colocalize with MPO. MPO was

used as surrogate marker of NETs. Ctrl IgGs were used as control. Results are representative of

three independent experiments performed with confocal microscope.

Fig. S2. Skin fibroblasts from patients with psoriasis internalize NETs. (a) Primary skin

fibroblasts from psoriasis patients were incubated in the presence or absence of spontaneously

generated RA NETs and in the presence or absence of 4 µM TLR9 inhibitor for 2 h. Western blot

analysis demonstrates that NETs (MPO) are internalized by psoriasis FLS and that this is

abolished in the presence of a TLR9 inhibitor. Tubulin was used as loading control.

Fig. S3. NETs up-regulate type I IFN-inducible genes in FLS. OA and RA FLS were

incubated in the presence or absence of spontaneously generated RA NETs from peripheral

blood neutrophils for 24 h. RNA was isolated and qPCR was performed to detect type I

Interferons. Results represent mean ± s.e.m. of 3 independent experiments. Mann-Whitney U test

was used,**P< 0.01.

Fig. S4. Endogenous expression of RAGE and TLR9 in FLS. (a) OA and RA FLS were

incubated in the presence or absence of spontaneously generated RA NETs for 24 h. Western

blot analysis demonstrates that the levels of RAGE remain stable under various experimental

conditions. (b) RA FLS were preincubated with 2-4 µM of RAGE inhibitor for 30 min, then

incubated with spontaneously generated RA NETs for 1 hour. Cell lysate was resolved in a SDS-

PAGE and Western blot analysis demonstrates the presence of TLR9, cleaved TLR9 and RAGE.

Fig. S5. Up-regulation of MHC II in OA FLS incubated with NETs. OA FLS were incubated

with NETs for 5 days. Immunofluorescence analysis shows upregulation of MHCII when

compared to OA FLS without NETs or isotype control. IFN gamma incubation was used as

positive control. Results are representative of three independent experiments performed with

confocal microscope. Scale bar m.

Fig. S6. IFN-γ is not detected in NETs or FLS supernatants. (a) Isolated NETs from LPS-

stimulated control neutrophils or spontaneously generated RA- NETs were analyzed by Western

blot, using antibodies against IFN-γ, MPO and HC-gp39. Each lane depicts and individual

isolated NETs. Recombinant IFN-γ was used as positive control (b) ELISA analysis

demonstrates that IFN-γ is absent from NETs, n=3 per group. (c) OA or RA FLs were incubated

with NETs for up to 12 days (12d). ELISA analysis fails to detect IFN-γ secretion in all

conditions tested.

Fig. S7. Detection of IL-17A and IL-17B in neutrophil lysate and NETs. IL-17B is detected

in isolated NETs by Western Blot analysis. Each lane depicts independent NET isolation per

group (Ctrl and RA).

Fig. S8. NETs are internalized by a TLR9-dependent and IL-17R–independent mechanism.

RA-FLS were incubated in presence or absence of NETs and/or in the presence of TLR9

inhibitor or IL17R/IL17B neutralizing antibodies for 2h. Western blot analysis demonstrate that

NETs are internalized through a TLR9 dependent pathway and IL17R does not mediate NET

internalization in FLS. Tubulin was used as loading control.

Fig. S9. FLS do not express HC-gp39 or MPO. RA FLS treated with IFN gamma for 5 days.

Immunofluorescence analysis shows that RA FLS do not express HC-gp-39 nor MPO, while the

cells were capable of upregulation of MHCII in the plasma membrane when compared to isotype

control. Results are representative of three independent experiments performed with confocal

microscope. Scale bar m.

Fig. S10. Characterization of vimentin epitope binding to HLA-DRB1*04:04 and T cell

clone specificity. (A) Competitive binding of the Vim and citrullinated vimentin (CitVim)

epitopes to recombinant DRB1*04:04 molecules revealed strong binding of citVim, while the

native version of the epitope showed no interpretable binding at the range of concentrations

tested. (B) The immunogenicity of citVim was validated in peptide-stimulated PBMC cultures at

day 14 using DRB1*0404 citVim tetramers (left). T-cell clones generated from tetramer positive

T-cells were also re-stained with tetramer to confirm specificity (right). (C) Proliferation of the T

cell clone in response to the citrullinated but not the native Vim epitope as measured by

thymidine incorporation and validated through the addition of the DR-blocking antibody L243.

Fig. S11. Cytokine profile of cocultures of Aggrecan 225 CD4+ T cells and haplotype-

matched FLS. DRB1-04:01 RA FLS were co-cultured with DRB1-04:01-restricted cit-aggrecan

225 specific T-cells for 5 days in the presence or absence of RA NETs. Supernatants were

collected and cytokine multiplex analysis was performed to detect (a) IFN-, (b) TNF- and (c)

IL-1ra. Results represent mean ± s.e.m. of 2-4 independent experiments.

Fig. S12. GK1.5 antibody depletes specifically CD4 T cells but not CD8 T cells.

DRB1*04:01 transgenic mice were immunized with FLS loaded with NETs in the presence of

1mg/kg of CD4+ depleting antibody (GK 1.5) or isotype control for 7 weeks. Flow analysis

demonstrate that GK 1.5 antibody significantly depletes CD4+ T but not CD8+ T cells. 3609-

3612 are animals injected with isotype control; 3613-3616 are animals injected with GK 1.5.

Results represent mean ± s.e.m. of 4 mice/group. Mann-Whitney U test was used, *P< 0.05.

a

Fig. S13. DRB1*04:01 transgenic mice immunized with FLS loaded with NETs develop in

vivo antibody responses to citrullinated peptides. Heat map assessing presence of serum

antibodies recognizing specific epitopes when comparing animal immunized with FLS loaded

with NETs versus mice immunized with FLS alone (n=5 mice/group).

Table S1. Mass spectrometry analysis demonstrates multiple citrullinated peptides in NETs

induced with IgM rheumatoid factor. List of peptides that are citrullinated in NETs and

sequences.