Increased gut permeability and intestinal inflammation precede arthritis onset in the adjuvant-induced model of arthritis

Abstract

Background: Intestinal inflammation, dysbiosis, intestinal permeability (IP), and bacterial translocation (BT) have been identified in patients with spondyloarthritis but the time at which they appear and their contribution to the pathogenesis of the disease is still a matter of debate.

Objectives: To study the time-course of intestinal inflammation (I-Inf), IP, microbiota modification BT in a rat model of reactive arthritis, the adjuvant-induced arthritis model (AIA).

Methods: Analysis was performed at 3 phases of arthritis in control and AIA rats: preclinical phase (day 4), onset phase (day 11), and acute phase (day 28). IP was assessed by measuring levels of zonulin and ileal mRNA expression of zonulin. I-inf was assessed by lymphocyte count from rat ileum and by measuring ileal mRNA expression of proinflammatory cytokines. The integrity of the intestinal barrier was evaluated by levels of iFABP. BT and gut microbiota were assessed by LPS, soluble CD14 levels, and 16S RNA sequencing in mesenteric lymph node and by 16S rRNA sequencing in stool, respectively.

Results: Plasma zonulin levels increased at the preclinical and onset phase in the AIA group. Plasma levels of iFABP were increased in AIA rats at all stages of the arthritis course. The preclinical phase was characterized by a transient dysbiosis and increased mRNA ileal expression of IL-8, IL-33, and IL-17. At the onset phase, TNF-α, IL-23p19, and IL-8 mRNA expression were increased. No changes in cytokines mRNA expression were observed at the acute phase. Increased CD4⁺ and CD8⁺ T cell number was measured in the AIA ileum at day 4 and day 11. No increase in BT was observed.

Conclusion: These data show that intestinal changes precede the development of arthritis but argue against a strict "correlative" model in which arthritis and gut changes are inseparable.

Keywords: Animal models; Dysbiosis; Intestinal inflammation; Intestinal permeability; Microbiota.

Fig. 1

Intestinal inflammation in AIA rats. RT-qPCR analyses of pro-inflammatory cytokines mRNA expression in the ileum. At day 4 (preclinical), 11 (onset), and 28 (acute), analyses of IL-8 (A, B, C), IL-33 (D, E, F), IL-17A (G, H, I), TNF-α (J, K, L), and IL-23p19 (M, N, O) mRNA expression were performed (n = 14–15/group). At day 4 (preclinical), 11 (onset), and 28 (acute), immunohistochemical analyses of CD3 + (P, Q, R), CD4 + (S, T, U), and CD8 + (V, W, X) were performed (n = 10/group). The number of cells/mm² was evaluated as described in supplementary methods. Results are expressed as means ± SEM. *(p < 0.05), **(p < 0.005). n.s., not significant

Fig. 2

Intestinal permeability in AIA rats. Plasma zonulin levels were measured in controls and AIA rats at the preclinical (A), onset (B), and acute (C) stages. At day 4 (preclinical), 11 (onset), and 28 (acute) RT-qPCR analyses of zonulin (D, E, F), ZO-1 (G, H,I), and occludin (J, K, L) gene expression were performed. Values expressed as means ± SEM (n = 14/15 per group). **(p < 0.01), **(p < 0.05), n.s.: not significant

Fig. 3

Ileal epithelial intestinal integrity in AIA rats. Plasma iFABP levels were measured in control and AIA rats at the preclinical (A), onset (B), and acute (C) stages. Values expressed as means ± SEM (n = 14/15 per group). ***(p < 0.0005), n.s.: not significant

Fig. 4

Dysbiosis in AIA rats. Relative abundance between AIA and taxonomic level controls of differentially expressed classes for samples at the preclinical (A), onset (B), and acute (C) stages. n = 10 per group. *(p < 0.05), **(p < 0.005)

Fig. 5

Systemic bacterial translocation in AIA rats. Plasma LPS levels were measured in control and AIA rats at the preclinical (A), onset (B), and acute (C) stages. At day 4 (preclinical), 11 (onset), and 28 (acute) Q-RT-PCR analyses of TLR-4 (D,E,F) gene expression were performed. Serum-soluble CD14 levels were measured at the preclinical (G), onset (H), and acute (I) stages. Values are expressed as means ± SEM (n = 14/15 per group). ***(p < 0.0001). n.s., not significant