Oral dextran sulfate sodium administration induces peripheral spondyloarthritis features in SKG mice accompanied by intestinal bacterial translocation and systemic Th1 and Th17 cell activation

Abstract

Background: Spondyloarthritis (SpA) is an autoimmune and autoinflammatory musculoskeletal disease characterised by systemic enthesitis. Recent research has focused on subclinical inflammatory bowel disease (IBD) in SpA pathogenesis. SKG mice, harbouring the Zap70 W163C mutation, increase autoreactive Th17 cells intrinsically, and in a conventional environment, they exhibit spontaneous arthritis with fungal factors. Under SPF conditions, they show SpA features, including enteritis, after peritoneal injection of β-1,3-glucan. This study aimed to clarify whether oral dextran sulfate sodium (DSS) administration, utilised in IBD model mice, can provoke SpA features in SKG mice under SPF conditions, focusing on the relationship between gut microorganisms and SpA pathogenesis.

Methods: BALB/c and SKG mice were administered oral DSS, and their body weights, arthritis, and enthesitis scores were recorded. In another cohort, antibiotics (meropenem and vancomycin) or an anti-fungal agent (amphotericin B) was administered orally before DSS administration. The splenic Th1 and Th17 cell populations were examined before and after DSS administration using flow cytometry. Furthermore, the amount of circulating bacterial DNA in whole blood was measured by absolute quantitative polymerase chain reaction (qPCR), and the number and characteristics of bacterial species corresponding to these circulating DNA were analysed by next-generation sequencing (NGS).

Results: Ankle enthesitis as a peripheral SpA feature was elicited in half of DSS-administered SKG mice, and none of the BALB/c mice. Pre-administration of antibiotics suppressed enthesitis, whilst an anti-fungal agent could not. Th1 and Th17 cell levels in the spleen increased after DSS administration, and this was suppressed by pre-administration of antibiotics. SKG mice have a larger amount of bacterial DNA in whole blood than BALB/c mice before and 1 day after the initiation of DSS administration. The number of bacterial species in whole blood increased after DSS administration in BALB/c and SKG mice. Some genera and species significantly specific to the DSS-treated SKG mouse group were also detected.

Conclusion: Oral DSS administration alone elicited peripheral enthesitis in SKG mice with bacterial translocation accompanied by increased splenic Th1 and Th17 cell levels. Pre-administration of antibiotics ameliorated these DSS-induced SpA features. These findings suggest that intestinal bacterial leakage plays a pivotal role in SpA pathogenesis.

Fig. 1

Oral dextran sodium sulfate (DSS) administration elicits SpA features in SKG mice. a Experimental scheme of oral DSS administration during the experiment. Mice were given 1% DSS in their drinking water for 2 weeks, followed by regular water. Ten mice were used in each group. Weeks are indicated by w. b The body weight change in BALB/c and SKG mice over the experiment. c–f Arthritis incidence rates and scores and enthesitis incidence rates and scores were recorded every 2 weeks. Values are expressed as mean ± standard error of the mean (SEM). g Representative photographs of hind paws for the enthesitis evaluation method. h Representative haematoxylin and eosin-stained sections of the peripheral enthesis (Achilles tendon) and the axial enthesis (caudal vertebrae). Only DSS-treated SKG mice showed marked cell infiltration around the Achilles tendon and plantar aponeurosis (arrows), and slight cell infiltration around but not inside the annulus fibrosus of the intervertebral disk (arrows). Scale bar = 200μm. Statistical analyses were performed by using the Mann-Whitney U test (*P <0.05)

Fig. 2

Oral DSS activates Th1 and Th17 immunity in the spleens of SKG mice. a Representative flow cytometry (FCM) plots of splenic CD4⁺T cells at weeks 0 and 12. b, c IFN-γ (b) and IL-17A (c) positivity of splenic CD4⁺ T cells in BALB/c and SKG mice at weeks 0 and 12. Statistical analyses were performed by using the Mann-Whitney U test (*P <0.05, **P <0.01)

Fig. 3

The antibiotics, MEPM and VCM, but not the anti-fungal agent AMPH-B, ameliorate DSS-induced peripheral SpA of SKG mice. a Experimental scheme of mouse treatment with oral antibiotics (MEPM (1 g/L)+VCM (0.5 g/L)) or the anti-fungal agent (AMPH-B (0.3 g/L)) and DSS. Agents were administered in their drinking water with 6% DMSO for 1 week, followed by regular water for 1 week. Then, 1% DSS was administered in their drinking water for 2 weeks, followed by regular water. Ten mice were used in each group. b The body weight change in each group over the experiment. c–f Arthritis incidence rates and scores, enthesitis incidence rates and scores were recorded every 2 weeks. Values are the mean ± standard error of the mean (SEM). g, h FCM analysis of IFN-γ (g) and IL-17A (h) positivity of splenic CD4⁺ T cells in SKG mice administered with DMSO and DMSO plus antibiotics (MEPM+VCM). Five mice per group were used. Statistical analyses were performed by using the Mann-Whitney U test (*P <0.05, **P <0.01)

Fig. 4

Oral DSS administration increases the amount of circulating bacterial DNA and the number of common species in the groups. a The concentration of bacterial DNA in whole blood measured by absolute qPCR, 0, 1, 7, and 14 days after oral DSS treatment initiation. Five mice per group were used. Values are presented as mean ± SEM. b The concentration of bacterial DNA measured by qPCR in whole blood after DSS treatment with and without antibiotics. Five mice per group were used. c Venn diagrams showing the number of bacterial species in circulating bacterial DNA on days 0 and 14. Five mice were examined per group. Identical numbers of mice are shown by #. Twenty mice were examined as a whole. d. The Shannon index before and after DSS administration. Five mice were examined per group. Statistical analyses were performed using the Mann-Whitney U test (*P <0.05, **P <0.01)

Fig. 5

LEfSe analysis of circulating bacterial DNA in the whole blood of BALB/c and SKG mice with and without oral DSS administration. The cutoff was set as LDA score > 4.0