IL-6-PAD4 axis in the earliest phase of arthritis in knock-in gp130F759 mice, a model for rheumatoid arthritis

Abstract

Objective: Animal models for human diseases are especially valuable for clarifying molecular mechanisms before or around the onset. As a model for rheumatoid arthritis (RA), we utilise knock-in mice gp130F759. They have a Y759F mutation in gp130, a common receptor subunit for interleukin 6 (IL-6) family cytokines. Definitive arthritis develops around 8 months old and the incidence reaches 100% around 1 year old. Careful examination in the clinical course revealed very subtle resistance in flexibility of joints at 5 months old. Therefore, pathophysiological changes in gp130F759 were examined to dissect molecular mechanisms for preclinical phase of RA.

Methods: Severity of arthritis in gp130F759 was evaluated with a clinical score system and histological quantification. Serum cytokines, autoantibodies and C reactive protein (CRP) were measured. Changes in the synovium were analysed by real-time PCR, flow cytometry and immunohistochemistry.

Results: Around 5 months old, various types of cytokines, rheumatoid factor (RF), anti-circular citrullinated peptide IgM and CRP increased in the sera of gp130F759. Enhancement of neovascularisation, synovial hyperplasia and fibrosis was observed. Also, increases in haematopoietic cells dominated by innate immune cells and gene expression of Il6 and Padi4 were detected in the joints. Il6 was expressed by non-haematopoietic synovial cells, whereas PAD4 protein was detected in the synovial neutrophils. Padi4 is induced in neutrophils in vitro by IL-6. Increases of phospho-STAT3 and PAD4 protein were detected in the synovium. Deletion of IL-6 in gp130F759 normalised the amount of PAD4 protein in the joints.

Conclusion: The IL-6-PAD4 axis operates in the earliest phase of arthritis in gp130F759, implicating it in early RA.

Keywords: anti-CCP; cytokines; early rheumatoid arthritis; inflammation; synovitis.

Figure 2

The earliest pathological changes of arthritis in gp130F759 develop at 5 months of age. (A) Sera were collected from WT (n=5) and gp130F759 (n=6), and concentrations of cytokines were measured using Bio-Plex. Black bald line shows averages and open circle shows values for individual mice. *p<0.05. (B) Histological changes in the synovium of the hindlimb joint with score 0 from wild type (WT) (n=7) and gp130F759 (n=6) at 5–5.5 months old were estimated by degrees of neovascularisation (Nv), hyperplasia (Hp) and fibrosis (Fb) as described in Materials and methods section. The average and individual scores are indicated as horizontal bars and circles, respectively. *p<0.05. (C) Haematopoietic cell numbers in the synovium obtained by collagenase treatment and the spleen from WT (open bars) and gp130F759 (black bars) were analysed by flow cytometry. (D) Titres of CRP, RF-IgM, anti-circular citrullinated peptide (CCP)-IgM and anti-CCP-IgG antibodies in the sera of WT (open bars) and gp130F759 (black bars) were measured from 4 to 12 months old. *p<0.05. CRP, C reactive protein; Fb, fibrosis; Hp, hyperplasia; Nv, neovascularisation.

Figure 1

The clinical and histological changes of arthritis in gp130F759. (A) Incidence and severity of clinical arthritis of gp130F759 (n=13; seven of male and six of female) is shown. The severity score for each mouse, the sum of scores for four limbs, was determined as described in Methods section and online supplementary figure 1. Onset of clinical arthritis was certified by the score higher than two. Severity scores are expressed as the mean±SD. (B) Representative arthritic joint sections from the mice with distinct severity scores. Slices of the joints were stained with H&E. (C) Results of Sirius red staining of the slice specimens obtained from the same mice used in (B) are indicated. WT, wild type.

Figure 3

Padi4 gene expression was increased in the ankle of gp130F759. (A) Expression levels of Padi4 gene were quantified by real-time PCR in the ankle joints, bone marrow or spleen of wild type (WT) and gp130F759 at 5 months old (n=6 each) are shown. The black bar shows average and the open circle shows individual value. (B) Gene expression levels of Mmps, IL-6 family cytokines and chemokines attracting neutrophils, in the joints of wild type (n=6) and gp130F759 (n=6) at 5 months old were quantified. The average and SD are indicated as bar graph. Open bars and black bars indicate WT and gp130F759, respectively. *p<0.05.

Figure 4

PAD4⁺ cells in the synovium of gp130F759 are neutrophils. (A) Amounts of PAD4 protein and STAT3 phosphorylation in the wrist joints from wild type (WT) and gp130F759. Lysates of the joints from three mice of each genotype were pooled and subjected to western blotting. (B) Frozen sections of the synovium from WT and gp130F759 were stained with anti-PAD4 and pSTAT3 antibodies and Cy3-labelled F(ab’)₂ donkey anti-rabbit IgG (H+L). Representative pictures from four independent experiments are shown. Nuclei were stained with Hoechst 33342. Colocalisation was examined with anti-PAD4 and biotinylated pSTAT3 antibodies, which were visualised with Cy3-labelled anti-Rabbit IgG and Alexa488-streptavidin, respectively. A PAD4 producing cell whose STAT3 is phosphorylated (inset). The bars indicate 20 µm. (C) Gene expression levels of Padi4 and Il6 in haematopoietic (CD45⁺) or non-haematopoietic (CD45⁻) synovial cells which were separated with rat anti-mouse CD45 antibody and sheep anti-rat IgG magnetic beads. Summarised data from three independent experiments are shown. (D) IL-6 concentration in supernatant of primary culture of CD45⁻ synovial cells from WT and gp130F759 at 5 months old (n=7). *p<0.05. (E) Morphology of synovial cells producing PAD4. The photos of representative cytospin specimens from WT and gp130F759 are shown. Synovial cells cytospun onto the slide glass were incubated with anti-PAD4 antibody (red) and Hoechst 33342 for nuclear staining (blue). In the graph, black bar shows average and the open circle shows individual value for each mouse. (F) IL-6 produced by CD45⁻ synovial cells induced Padi4 expression in neutrophils. WT bone marrow neutrophils were stimulated for 6 hours with culture supernatant fluid (CSF) from gp130F759 pretreated with anti-IL-6 antibody or control IgG1. Then RNA from the neutrophils were prepared and transcription of Padi4 was estimated by real-time PCR using specific primers and SYBR green. Relative expression levels compared with Actb are shown. (G) IL-6 in the CSF from gp130F759 induced activation of STAT3 in neutrophils. WT bone marrow neutrophils were stimulated for 30 min with CSF from gp130F759 pretreated with anti-IL-6 antibody or control IgG1. The neutrophils were cyto-spun, air-dried and stained with anti-pSTAT3 antibody and Cy3-labelled donkey anti-rabbit IgG antibody. Pictures were taken with LSM700 confocal microscope. The bars indicate 10 µm.

Figure 5

IL-6-dependent changes in preclinical phase of arthritis in gp130F759. Multiple parameters of WT, Il6KO (abbreviated as KO), gp130F759 (F759) and Il6KO/gp130F759 (KO/F759) at 5–6 months old (n=4 each) were examined. As systemic changes, (A) weight of the spleen, (B) serum titres of CRP, RF-IgM, and anti-CCP IgM are indicated at top. As a local changes in the joints, (C) Il6 gene expression by real time PCR, (D) the relative amounts of PAD4 protein by western blot {the average ratio of band densities (PAD4/β-actin) of WT=1.0} and (E) synovial cell numbers are indicated at bottom. Statistical significance was estimated by the Kruskal-Wallis test and Dunn’s method. ND; not detectable. CCP, circular citrullinated peptide; CRP, C reactive protein.

Figure 6

The IL-6-PAD4 axis in the synovium of preclinical phase of rheumatoid arthritis (RA)-like arthritis in gp130F759. anti-citrullinated peptide autoantibodies (ACPAs) are detectable much earlier than onset of RA. Presence of ACPA indicates that tolerant self-antigens have been modified by citrullination and that the modified self-antigens become recognised as foreign by adaptive immunity. Thus, ACPA and anti-circular citrullinated peptide (CCP) antibody, which is adapted for clinical examination to detect ACPA as a whole, are useful biomarkers to predict development of RA. In other words, anti-CCP antibody can be used to define the preclinical phase of RA by the criteria: anti-CCP antibody positive and lack of clinical symptoms of arthritis. Since the natural course of RA is now appreciated as (1) preclinical RA, (2) early ‘clinically evident’ RA and (3) chronic ‘established’ RA, the molecular events and causal relationships between them in preclinical RA are gathering great attention. But the information of synovial microenvironment in preclinical RA is hardly obtainable from human. Using the mutant gp130 knock-in mice, gp130F759, we report here that gp130F759 around 5 months old could be an ideal model for the pathological phases spanning preclinical to early clinically evident RA. During this time window, the IL-6-PAD4 axis is generated by interaction of non-haematopoietic cells, most likely fibroblast-like synoviocytes, and neutrophils, representative of innate immunity, attracted by CXCL7. ERK, Extracellular Signal-regulated Kinase; MAPK, Mitogen-activated Protein Kinase; M.O.; months old.