Research ArticleRHEUMATOID ARTHRITIS

Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis

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Science Immunology  14 Apr 2017:
Vol. 2, Issue 10, eaag3358
DOI: 10.1126/sciimmunol.aag3358
  • Fig. 1 ACPAs recognize multiple citrullinated peptides in NETs and induce NETosis.

    (A) Rh-PG probe against citrulline was used to detect specific citrullinated proteins in purified NETs generated by stimulating control neutrophils with IgM rheumatoid factor. (B) ACPAs differentially recognize citrullinated autoantigens in NETs when compared with control IgG. Spontaneously generated NETs from peripheral blood neutrophils from two RA patients (RA01 and RA02) were isolated and resolved in SDS-PAGE. Western blot was performed using ACPAs or control IgG. (C) ACPAs bind to NETs and (D) enhance NETosis. Red, control IgG (ctrl-IgG) or ACPAs; blue, Hoechst stain. (E) Representative peaks processed by MaxQuant 1.6.3.4 of citrullinated peptides (vimentin) detected in NETs using PEAKS’ software (Thermo-Fisher Scientific). (F) Mass spectrometry analysis demonstrates multiple citrullinated peptides in NETs. (G and H) ELISA analysis of synovial fluid from OA (n = 10) and RA (n = 17) patients to detect autoantibodies recognizing citrullinated or noncitrullinated forms of MPO and neutrophil elastase, respectively. (I and J) ELISA analysis of IgGs isolated from synovial fluid (SF) of OA (n = 6) and RA (n = 11) patients showing recognition of native or cit-MPO or elastase. (A) to (C) are representative of three independent experiments. Scale bars, 10 μm. Results are the means ± SEM of n = 5 to 6. For statistical analyses, Mann-Whitney U test was used. m/z, mass/charge ratio; OD, optical density.

  • Fig. 2 NETs are internalized by FLS into EEA1-positive compartments.

    RA-NETs were incubated with OA or RA FLS or skin fibroblasts for 2 hours. (A) Internalized NETs colocalize with EEA1 compartments in FLS. Red, MPO; green, EEA1; blue, DNA. Results are representative of three independent experiments performed with a confocal microscope, Scale bars, 10 μm. (B) Western blot analysis of fibroblasts incubated in the presence or absence of RA-NETs shows that MPO bound to NETs is internalized by OA and RA FLS but minimally by healthy control skin fibroblasts. Results are representative of three independent experiments. (C) Percentage of MPO-positive cells (FLS that internalized NETs) decreased after CQ but not cyto D exposure. (D) Representative confocal images after treatment with cyto D and CQ. Red, MPO or F-actin; green, MPO; blue, DNA. Scale bars, 10 μm. (E) Western blot analysis confirms that MPO internalization is impaired in FLS preincubated with CQ but not in FLS preincubated with cyto D. (F) IL-6 release by FLS is dependent on NET internalization. Results are the means ± SEM of n = 4. For statistical analyses, Mann-Whitney U test was used. ns, not significant; hIL, human IL.

  • Fig. 3 RAGE-TLR9 axis mediates internalization of NETs by FLS.

    OA and RA FLS were incubated in the absence or presence of TLR9 inhibitors or control oligos and NETs for 72 hours. (A) Western blot analysis shows intracellular MPO when FLS were incubated in the presence or absence of TLR9 inhibitor. (B) IL-6 and (C) IL-8 quantification on FLS supernatants. (D) Quantitative polymerase chain reaction (qPCR) analysis shows TLR9 mRNA expression in OA and RA FLS and skin fibroblasts in the presence or absence of NETs. Results for (B) to (D) are the means ± SEM of four independent experiments. (E) FLS were pretreated with or without 2 to 4 μM RAGE inhibitor and incubated with NETs for 1 hour. Coimmunoprecipitation (IP) was performed against RAGE, and TLR9 was detected by Western blot. Anti–green fluorescent protein (GFP) was used as negative control. (F) Plasma membrane (top) and intracellular (bottom) detection of RAGE and TLR9 were performed on FLS pretreated with or without RAGE inhibitor. Red, TLR9; green, RAGE; blue, DNA of three independent experiments; white arrows highlight areas of colocalization of RAGE and TLR9. Scale bars, 5 μm. Mann-Whitney U test was used.

  • Fig. 4 IL-17B present in NETs up-regulates MHC II in OA and RA FLS.

    OA and RA FLS were incubated in the presence or absence of spontaneously generated RA-NETs or IFN-γ (1000 U/ml). (A) Detection of MHC II in FLS by immunofluorescence. Green, MHC II; blue, DNA. Results are representative of three independent experiments. Scale bars, 10 μm. (B) Plasma membrane and intracellular MHC I and II were quantified by flow cytometry in RA FLS treated with NETs, IFN-γ, or IFN-α for 5 days. (C) IL-17B (red) is externalized in control (Ctrl) NETs generated with LPS (1 μg/ml) and in spontaneously generated RA-NETs. (D) IL-17B is detected in isolated NETs by Western blot analysis. Each lane depicts independent NET isolation per group (Ctrl and RA). Mann-Whitney U test was used. Results are the means ± SEM of 10 independent experiments,*P < 0.05. (E) OA FLS were incubated with RA-NETs in the presence or absence of IL-17B (1 μg/ml) neutralizing antibodies for 48 hours. Quantification of HLA-DRA and HLA-DRB mRNA was performed by real-time PCR. Bars are the means ± SEM of four independent experiments. (F) RA FLS were incubated with 100 ng of human recombinant IL-17A or IL-17B for 72 hours. qPCR and immunofluorescence analyses assessed MHC II mRNA expression and protein localization, respectively. ANOVA with Bonferroni’s test was performed for (B), (E), and (F). Results are the means ± SEM of four independent experiments.

  • Fig. 5 Arthritogenic peptides contained in NETs internalized by FLS are loaded into their MHC II compartment and presented to antigen-specific CD4+ T cells.

    (A) Internalized NET-bound proteins colocalize with MHC II compartments in OA and RA FLS. (B) Plasma membrane detection of MHC II and NET proteins in unpermeabilized FLS assessed by immunofluorescence. Red, MPO or HC-gp39; green, MHC II; and blue, DNA. (C) Detection of mouse IL-2 (mIL-2) after DRB1*04:01 RA FLS (with and without NETs) were incubated in the presence or absence of HC-gp39-specific CD4+ T cell hybridomas for 5 days. Peptide is HC-gp39 263 to 275 (RSFTLASSETGVG). (D) Detection of various cytokines after DRB1*04:04 RA FLS (with or without NETs) were incubated with haplotype-matched cit-vimentin–specific CD4+ T cells in the presence or absence of neutralizing antibodies (Ab) against CD28 or MHC II. Mann-Whitney U test was used. Results are the means ± SEM of four to six independent experiments. Scale bars, 10 μm.

  • Fig. 6 DRB1*04:01 humanized mice that receive intra-articular injections of mouse FLS loaded with RA-NETs develop ACPAs and cartilage damage.

    DRB1*04:01 FLS were isolated and incubated with human RA-NETs for 3 days before intra-articular injection. (A) Internalization of NETs by FLS was assessed by immunofluorescence using an antibody against MPO (red). (B) Serum ACPA levels at various time points in animals immunized with FLS alone or FLS loaded with NETs (n = 5 per group). Results are the means ± SEM. Mann-Whitney U test was used. (C) Sera from three mice immunized for 7 weeks with FLS or with FLS loaded with NETs were analyzed by dot blot against citrullinated proteins. (D) Western blot analysis to detect serum autoantibodies recognizing human NET proteins (arrows) in DRB1*04:01 animals that received FLS alone or FLS-NETs; each lane depicts two independent NET isolations. (E) IL-2 synthesis by DRB1*04:01 mouse splenocytes incubated with a cocktail of native (pep; H3, H4, MPO, and vimentin) or citrullinated peptides (cit-pep; a cocktail of cit-H3, cit-H4, cit-MPO, and cit-vimentin), when comparing animals immunized with FLS loaded with NETs (DR4 + NETs) with animals immunized with FLS alone (DR4). PHA is used as positive control. Results are the means ± SEM of six independent experiments. Mann-Whitney U test was used. (F) CD4+ T cell–depleted animals immunized with FLS loaded with NETs demonstrate significantly lower titers of ACPAs (n = 4), as measured by ELISA, when compared with non–T cell–depleted mice. Results are the means ± SEM. Mann-Whitney U test was used. (G) Percentage of FLS that migrate to the spleen and lymph nodes (LNs) after intra-articular injection of FLS loaded with NETs or untreated mice (n = 4 to 5). Results are the % means ± SEM of FLS positive for CellTraceViolet (% CTV). Mann-Whitney U test was used. (H) Epitope chip analysis to quantify antibodies recognizing specific epitopes of RA-relevant autoantigens in animals immunized with FLS loaded with NETs when compared with animals immunized with FLS alone. Heat map represents the average of mean fluorescent units of five animals per group. (I) Sagittal sections of cartilage of the injected tibiofemoral compartment were stained with safranin O demonstrating impaired cartilage integrity (arrowheads) in animals immunized with FLS loaded with NETs when compared with animals immunized with FLS alone. (J) Percentage of cartilage loss of the femurs and tibias of animals immunized with FLS or FLS loaded with NETs. Results are the means ± SEM of two to three independent experiments. Mann-Whitney U test was used.

Supplementary Materials

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

    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.

    Source data Excel file

    Source data blots

    Reference (56)

  • Supplementary Materials

    Supplementary Material 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{at}nih.gov

    Published 14 April 2017, Sci. Immunol. 2, eaag3358 (2017)
    DOI: 10.1126/sciimmunol.aag3358

    This 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.
    • Reference (56)

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    Other Supplementary Material for this manuscript includes the following:

    • Source data Excel file
    • Source data blots (.pptx format)

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