Positive association between serum thymic stromal lymphopoietin and anti-citrullinated peptide antibodies in patients with rheumatoid arthritis

Abstract

Thymic stromal lymphopoietin (TSLP) has been suggested recently to play an important role in the pathophysiology of rheumatoid arthritis (RA). However, there is little information on serum TSLP concentrations in RA and its clinical significance. The present study investigated whether serum TSLP concentrations were affected in patients with RA. Using an enzyme-linked immunosorbent assay (ELISA), we measured TSLP concentrations in the serum obtained from 100 patients with RA, 60 patients with osteoarthritis (OA) and 34 healthy volunteers. We also investigated the correlation between serum TSLP concentrations and clinical parameters of disease activity in RA [disease activity score using 28 joint counts (DAS28)-C-reactive protein (CRP), DAS28-erythrocyte sedimentation rate (ESR), Clinical Disease Activity Index (CDAI]), patient’s/-physician’s Visual Analogue Scale (VAS), swollen joints count, tender joints count, CRP, ESR and matrix metalloproteinase-3 (MMP-3) concentrations]. In addition, we investigated the correlation between serum TSLP concentrations and anti-citrullinated peptide antibody (ACPA) and serum tumour necrosis factor (TNF)-α. Serum TSLP levels in patients with RA were significantly higher than those in patients with OA and in healthy volunteers. Interestingly, serum TSLP concentrations were correlated significantly with ACPA titres, but not with other clinical parameters. There was a significant increase in serum TSLP concentrations in patients with RA, which was correlated positively with serum ACPA titres. These findings suggest that in patients with RA, TSLP may play a role in ACPA production by B cells.

Introduction

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine that triggers dendritic cell (DC)-mediated T helper type 2 (Th2) inflammatory responses, and is considered to play a role as a master switch of allergic inflammation in conditions such as atopic dermatitis and asthma 1. However, it is becoming increasingly clear that TSLP may impact multiple disease states within multiple organ systems.

TSLP concentrations in synovial fluid samples obtained from patients with rheumatoid arthritis (RA) were reported to be higher than those from patients with osteoarthritis (OA), and that tumour necrosis factor (TNF)-α significantly up-regulated TSLP expression in RA synovial fibroblasts 2. In addition, blockade of TSLP inhibited arthritis severity in an anti-collagen type II antibody-induced arthritis (CAIA) mouse model 2, and TSLP receptor-deficient mice had less severe arthritis than wild-type mice in T cell-mediated arthritis models 3. Furthermore, TSLP-primed CD11c⁺ myeloid DCs induced Th1 and Th17 activity in addition to Th2 activity in patients with RA 4. These findings suggest that TSLP plays an important role in the pathophysiology of RA. However, there is little information on serum TSLP concentrations in RA and its clinical significance.

The present study investigated whether serum TSLP concentrations were affected in RA patients and, if so, whether serum TSLP concentrations were correlated with any clinical parameters of disease activity in RA.

Materials and methods

Patients

One hundred patients with RA diagnosed according to the American College of Rheumatology criteria 5, 60 patients with OA and 34 healthy volunteers were recruited to this study at University of Yamanashi Hospital (Yamanashi, Japan). All subjects who had atopic dermatitis, allergic rhinitis or asthma were excluded from the study, as these conditions could affect serum TSLP concentrations. Written informed consent was obtained from all patients before enrolment. Patient profiles are summarized in Table 1. All patients with RA had already undergone specific treatment, and had received steroids, methotrexate and/or biologicals by the time they were tested for TSLP; therefore, untreated RA patients were not enrolled into this study. Protocol approval for this study was provided by the Institutional Research and Ethics Review Board of the University of Yamanashi, Japan.

Table 1.

Patient demographics and treatment status

Patient background
	RA	OA	NA
Number	100	60	34
Age (years)	65·2 (34–87)	74.1 (49–92)	32.1 (19–46)
Male/female	22/78	15/45	18/16
RA patient background
Disease duration (years)		7·56 (0·5–46)
Swollen joints count (0–28)		0·98 (0–9)
Tender joints count (0–28)		2·49 (0–16)
Patient’s VAS (range 0–10)		2·24 (0–8)
Physician’s VAS (range 0–10)		2·15 (0–9)
CRP (mg/dl)		0·73 (0·01–6·16)
ESR (mm/h)		29·0 (3–130)
DAS28-ESR		3·15 (0·77–5·81)
DAS28-CRP		2·56 (0·99–5·59)
CDAI		7·69 (0–33)
SDAI		8·42 (0·01–36·3)
Prednisone use (%)		21
Methotrexate use (%)		87
Biologicals use (%)		20
Infliximab (ACPA+)		6 (6)
Etanercept (ACPA+)		14 (12)

RA = rheumatoid arthritis; OA = osteoarthritis; NA = healthy control; VAS = visual analogue scale; CRP = C-reactive protein; ESR = erythrocyte sedimentation; DAS28 = disease activity score using 28 joint counts; CDAI = clinical disease activity index; SDAI = simplified disease activity index; ACPA = anti-citrullinated protein autoantibodies.

Serum TSLP, TNF-α and anti-citrullinated peptide antibody (ACPA) levels

Blood samples were collected from study participants and stored at −80 °C until assays were performed. Serum concentrations of TSLP and TNF-α were measured using a human TSLP enzyme-linked immunosorbent assay (ELISA) kit and a human TNF-α ELISA kit (RD Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. ACPA was measured using a second-generation ELISA kit (Euroimmun AG, Luebeck, Germany).

Clinical parameters of disease activity in RA

RA Disease Activity Score using 28 joint counts (DAS28)-C-reactive protein (CRP), DAS28-erythrocyte sedimentation (ESR), Clinical Disease Activity Index (CDAI), patient’s/-physician’s Visual Analogue Scale (VAS), swollen joints count (SJC), tender joints count (TJC) and concentrations of inflammatory markers CRP, ESR and matrix metalloproteinase-3 (MMP-3) were evaluated or measured by conventional methods using the serum collected for TSLP.

Statistical analysis

The collected data were sorted, coded and entered into a Microsoft Excel spreadsheet for analysis using GraphPad Prism for Windows, version 6.0 (GraphPad Software, San Diego, CA, USA). Receiver operating characteristics (ROC) curve analysis was used to evaluate the diagnosis accuracy of RA. The statistical differences between serum TSLP concentrations were examined using the χ² test. Mean concentrations of serum TSLP were compared using Student’s t-test. Pearson’s correlation coefficient was employed for evaluating parametric data and Spearman’s rank correlation coefficient was used for the analysis of non-parametric data. The statistical differences between ▵TSLP concentrations and European League Against Rheumatism (EULAR) response were examined using the Mann–Whitney U-test. P < 0·05 was considered statistically significant.

Results

The mean range serum TSLP concentrations in patients with RA was 34·85 (1·2–137·4) pg/ml, which was significantly higher than it was for patients with OA at 5·17 (0–34·2) pg/ml and healthy volunteers at 5·05 (0–42·06) pg/ml; all P < 0·0001 (Fig. 1a). A statistically significant [odds ratio (OR) = 28·3, 95% confidence interval (CI) = 11·83–67·85; P < 0·0001] increase in serum TSLP concentrations was observed in patients with RA compared with OA based on cut-point value of 11·05 pg/ml (sensitivity 85%; specificity 83·4%) (Fig. 1b). Interestingly, serum TSLP concentrations were correlated significantly with ACPA titers; r = 0·38, P = 0·011 (Fig. 1c). In addition, RA patients with high concentrations of serum TSLP (>11·05 pg/ml; n = 83) had higher ACPA-positive rates than RA patients with low concentrations of serum TSLP (<11·05 pg/ml; n = 17) (OR = 5·18, 95% CI = 1·55–17·28; P < 0·05). Conversely, ACPA-positive RA patients (n = 54) had significantly higher mean serum TSLP concentrations at 41·7 pg/ml compared with ACPA-negative RA patients (n = 46) at 26·21 pg/ml; P < 0·01 (Supporting information, Fig. S1).

Figure 1.

(a) Comparison of serum TSLP concentrations among patients with rheumatoid arthritis (RA) (n = 100), osteoarthritis (OA) (n = 60) and healthy volunteers (NA) (n = 34). *P < 0·05. (b) Receiver operating characteristics (ROC) curve analysis for presence of RA. (c) Correlation of serum thymic stromal lymphopoietin (TSLP) concentrations with anti-citrullinated protein autoantibodies (ACPA) concentrations (n = 44).

In contrast, serum TSLP concentrations were not correlated significantly with DAS28-CRP (r = −0·02, P = 0·855), DAS28-ESR (r = 0·013, P = 0·907), CDAI (r = −0·174, P = 0·439), CRP (r = −0·004, P = 0·972), ESR (r = 0·031, P = 0·774), MMP-3 (r = −0·013, P = 0·907), SJC (r = −0·104, P = 0·304), TJC (r = −0·065, P = 0·521), VAS (r = 0·047, P = 0·658), or TNF-α (r = 0·083, P = 0·447) (Supporting information, Fig. S2).

We also determined the changes in serum TSLP concentrations in patients with RA over time. Serum samples were collected from 41 patients at time-points 6 months apart. No significant difference in the mean serum TSLP concentration was observed between the two time-points (33·75 pg/ml versus 34·13 pg/ml, P = 0·933) (Supporting information, Fig. S3). Next, we determined the correlation between changes in serum TSLP (▵ TSLP: before–after) and changes in DAS28-CRP, DAS28-ESR, CDAI, CRP, ESR, MMP-3, SJC, TJC and VAS. ▵ TSLP was not correlated significantly with ▵ DAS28-CRP (r = −0·207, P = 0·357), ▵ DAS28-ESR (r = −0·262, P = 0·24), ▵ CDAI (r = −174, P = 0·439), ▵ CRP (r = 234, P = 0·295), ▵ ESR (r = −0·147, P = 0·513), ▵ MMP-3 (r = −0·252, P = 0·406), ▵ SJC (r = −0·326, P = 0·139), ▵ TJC (r = −0·274, P = 0·218), or ▵ VAS (r = −0·066, P = 0·772) (Supporting information, Fig. S4). In addition, there were only small differences in ▵ TSLP among patients based on their response to treatment, which was classified as good, moderate or no response based on the EULAR response to the treatment (P = 0·313) (Supporting information, Fig. S5), suggesting that serum TSLP concentrations were not affected by treatment.

Discussion

This study showed that serum TSLP concentrations increased significantly in patients with RA versus patients with OA or in comparison with healthy volunteers. Importantly, serum TSLP concentrations were correlated positively with ACPA titres, but were not correlated with systemic markers of RA disease activity. ACPA is an important biomarker and has been used for RA diagnosis and prognosis in patients; it was also shown to be associated with structural damage 6–9. The current findings suggest that TSLP may be a key cytokine linked to this important biomarker for RA and could be implicated in the pathophysiology of RA.

A mechanistic link between TSLP and ACPA remains unclear. TSLP can be produced by RA synovial fibroblasts or many other cell types 1,2. A recent study suggested that TSLP stimulates DCs to produce more B cell activating factor (BAFF), a cytokine that plays a pivotal role in B cell survival, differentiation and activation, all of which constitutes a Th2-independent pathway for antibody production 10,11. Thus, we have also speculated that TSLP-stimulated DCs might promote the survival or differentiation of ACPA-producing B cells via BAFF production and, as a result, a correlation between serum TSLP and ACPA might be observed. Whether or not TSLP affects survival or differentiation of ACPA-producing B cells in RA is currently under investigation.

Alternatively, because ACPA-positive RA patients had higher serum TSLP concentrations than ACPA-negative RA patients (Supporting information, Fig. S1), we have also speculated that TSLP (or TSLP-stimulated DCs) might not only affect survival or differentiation of ACPA-producing B cells, but may preferentially induce ACPA production in B cells. This issue warrants further investigation in future studies.

Serum TSLP concentrations were not correlated with disease activity in this study. In agreement with these data, a recent study demonstrated that serum TSLP concentrations were not correlated with disease severity in atopic dermatitis patients 12. It is thus possible that TSLP is associated only with initiation, but not the duration or magnitude of RA and atopic dermatitis.

Recently, an anti-TSLP blocking antibody was shown to significantly attenuate most measures of allergen-induced early and late asthmatic responses 13,14. Furthermore, another study showed that blocking TSLP in patients with psoriasis decreased DC activation 15. The present study suggests that a TSLP blockade could also have therapeutic potential for patients with RA. If TSLP has become a therapeutic target, we think that measurement of serum TSLP might be useful for determination of drug doses in a manner similar to infliximab 16.

A major limitation of this study is that all the patients with RA had already undergone specific treatment for their condition and had received steroids, methotrexate and/or biologicals by the time they were tested for TSLP. Given that we do not know if specific treatment for RA may modify serum TSLP concentrations, an untreated population would, theoretically, be ideal. However, recruiting patients already diagnosed with RA who are not receiving some form of treatment for their chronic condition would pose numerous challenges, key among them being a dramatically diminished sample size. Thus, we would like to address this study design issue more thoroughly in a future investigation.

In summary, this study demonstrated the significant increase in serum TSLP concentrations in patients with RA, which was correlated positively with serum ACPA concentrations. The current results suggest that TSLP may play an important role in the pathophysiology of RA.

Author contributions

K. K. and T. O. and H. H. designed the study, K. K. performed the experiments, K. K. and T. O. and A. N. wrote the paper. We thank Ms Tomoko Tohno, Yuko Ohnuma and Mutsuko Hara for general assistance.

Disclosure

None.

Supporting Information

Additional Supporting information may be found in the online version of this article at the publisher’s Web site: