Aberrant expression of microRNAs in T cells from patients with ankylosing spondylitis contributes to the immunopathogenesis

Abstract

Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by dysregulated T cells. We hypothesized that the aberrant expression of microRNAs (miRNAs) in AS T cells involved in the pathogenesis of AS. The expression profile of 270 miRNAs in T cells from five AS patients and five healthy controls were analysed by real-time polymerase chain reaction (PCR). Thirteen miRNAs were found potentially differential expression. After validation, we confirmed that miR-16, miR-221 and let-7i were over-expressed in AS T cells and the expression of miR-221 and let-7i were correlated positively with the Bath Ankylosing Spondylitis Radiology Index (BASRI) of lumbar spine in AS patients. The protein molecules regulated by miR-16, miR-221 and let-7i were measured by Western blotting. We found that the protein levels of Toll-like receptor-4 (TLR-4), a target of let-7i, in T cells from AS patients were decreased. In addition, the mRNA expression of interferon (IFN)-γ was elevated in AS T cells. Lipopolysaccharide (LPS), a TLR-4 agonist, inhibited IFN-γ secretion by anti-CD3(+) anti-CD28 antibodies-stimulated normal T cells but not AS T cells. In the transfection studies, we found the increased expression of let-7i enhanced IFN-γ production by anti-CD3(+) anti-CD28(+) lipopolysaccharide (LPS)-stimulated normal T cells. In contrast, the decreased expression of let-7i suppressed IFN-γ production by anti-CD3(+) anti-CD28(+) LPS-stimulated AS T cells. In conclusion, we found that miR-16, miR-221 and let-7i were over-expressed in AS T cells, but only miR-221 and let-7i were associated with BASRI of lumbar spine. In the functional studies, the increased let-7i expression facilitated the T helper type 1 (IFN-γ) immune response in T cells.

Figure 1

Comparison of microRNAs (miRNAs) expression in T cells from patients with ankylosing spondylitis (AS) and healthy controls. (a) The expression profile of 270 miRNAs measured by real-time polymerase chain reaction (PCR). Each scatter-spot represents the average of normalized miRNA level in T cells from five AS patients and five healthy controls for each miRNA. The threshold cycle (Ct) is defined as the cycle number at which the change of fluorescence intensity crosses the average background level of the fluorescence signal. The normalized miRNA level was defined as [39 – Ct after normalization with the internal control (U6 small nuclear RNA)]. (b) Eight miRNAs were found potentially over-expressed and five mRNAs were under-expressed in AS T cells (fold change >4·5; P-value < 0·05). (c) The five most potentially differentially expressed miRNAs (defined as fold change >6) were validated further by real-time PCR in T cells from another 22 AS patients and 18 compatible healthy controls. Increased expression of miR-16, miR-221 and let-7i in T cells from AS patients was confirmed after validation.

Figure 2

The correlation of the three over-expressed miRNAs in ankylosing spondylitis (AS) T cells with Bath Ankylosing Spondylitis Radiology Index (BASRI) of lumbar spine. (a) miR-16, (b) miR-221 and (c) let-7i.

Figure 3

The protein targeted by miR-16 (Bcl-2) and let-7i [Toll-like receptor (TLR)-4] in T cell lysates were detected by Western blotting. (a) Protein expression of Bcl-2, TLR-4 molecules in T cell lysates from three ankylosing spondylitis (AS) patients and three healthy controls were shown as representative. The expression levels of (b) Bcl-2 and (c) TLR-4 in T cell lysates from 11 AS patients and 11 healthy controls normalized to the actin expression are shown.

Figure 4

Effect of miR-221 inhibitor transfection on c-kit protein expression in ankylosing spondylitis (AS) T cells. We transfected miR-221 inhibitor into AS T cells via electroporation. (a) The expression level of miR-221 was decreased dramatically after miR-221 inhibitor transfection for 24 h. (b) The protein expression levels of c-kit were undetectable in AS T cells transfected with miR-221 inhibitor or scrambled oligonucleotides.

Figure 5

The expression level of let-7i in T cells from healthy controls, patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and activated Jurkat cells were compared. (a) Comparison of expression levels of let-7i in T cells from 14 controls, eight SLE and nine RA patients. (b) The expression levels of let-7i were decreased significantly in Jurkat cells after stimulation with 250 ng/ml ionomycin and 10 ng/ml phorbol myristate acetate (PMA) for 24 h.

Figure 6

Effect of let-7i mimic transfection on Toll-like receptor (TLR)-4 mRNA and protein expression. Let-7i mimic was transfected into Jurkat cells via electroporation. (a) After let-7i mimic transfection for 24 h, the expression level of let-7i was increased dramatically in the Jurkat cells compared with the scrambled oligonucleotides transfection group. (b) The TLR-4 mRNA expression levels did not change after let-7i transfection. (c) Jurkat cells were cultured with 250 ng/ml ionomycin and 10 ng/ml phorbol myristate acetate (PMA) and normal T cells were cultured with anti-CD3⁺anti-CD28 antibodies for 24 h after let-7i mimic transfection. The protein expression of TLR-4 was inhibited significantly in Jurkat and normal T cells after let-7i mimic transfection. (d) A representative case showing increased TLR-4 protein expression after let-7i mimic transfection into Jurkat (left) and normal T cells (right).

Figure 7

Effect of let-7i inhibitor transfection on Toll-like receptor (TLR)-4 mRNA and protein expression. We transfected let-7i inhibitor into Jurkat cells via electroporation. (a) After let-7i inhibitor transfection for 24 h, the expression level of let-7i was decreased dramatically in the Jurkat cells compared with the scrambled oligonucleotide transfection groups. (b) The TLR-4 mRNA expression levels did not change after let-7i transfection. (c) The protein expression of TLR-4 was increased significantly in Jurkat and ankylosing spondylitis (AS) T cells after let-7i inhibitor transfection. (d) Representative case showing increased TLR-4 protein expression after let-7i inhibitor transfection into Jurkat (left) and AS T cells (right).

Figure 8

Functional analysis of let-7i in anti-CD3⁺anti-CD28-activated normal and ankylosing spondylitis (AS) T cells via transfection with let-7i mimic and inhibitor. (a) Lipopolysaccharide (LPS) (100 ng/ml) potently suppress interferon (IFN)-γ production in anti-CD3⁺anti-CD28 activated T cells from healthy volunteers but not AS patients. *The IFN-γ concentration was lower in anti-CD3⁺anti-CD28⁺ LPS-stimulated normal T cells compared with AS T cells. (P < 0·05) (b) The transfection of let-7i mimic increased IFN-γ production in anti-CD3⁺anti-CD28⁺ LPS-activated T cells from healthy volunteers, but not AS patients. The transfection of let-7i inhibitor suppressed IFN-γ production by anti-CD3⁺anti-CD28⁺ LPS-stimulated AS T cells, but not normal T cells. *In the scrambled oligonucleotides transfected control groups, the IFN-γ concentration was higher in anti-CD3⁺anti-CD28⁺ LPS-stimulated AS T cells compared with normal T cells (P < 0·05).

Figure 9

Comparison of interferon (IFN)-γ mRNA expression between ankylosing spondylitis (AS) and normal T cells and correlations among IFN-γ mRNA with let-7i mRNA and BASRI of lumbar spine in AS patients. (a) The mRNA expression levels of IFN-γ in AS T cells were significantly higher than normal T cells. (b)Correlation between expression levels of IFN-γ mRNA and let-7i. (c) Correlation between expression levels of IFN-γ mRNA and BASRI of lumbar spine in AS patients.