Anti-tumour necrosis factor therapy enhances mucosal healing through down-regulation of interleukin-21 expression and T helper type 17 cell infiltration in Crohn's disease

Abstract

Anti-tumour necrosis factor (TNF) monoclonal antibody (mAb) (infliximab, IFX) has been shown to be highly effective in the management of Crohn's disease (CD). Herein we investigated the potential role of IFX in inducing clinical remission and regulating interleukin (IL)-21 expression and T helper type 17 (Th17) cell infiltration in the intestinal mucosa of CD patients. Twenty-six CD patients were treated with IFX at weeks 0, 2 and 6. Clinical response, mucosal healing, serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were evaluated at week 10 after IFX administration. Expression of IL-21, IL-17A and retinoic acid-related orphan receptor C (RORC) in intestinal mucosa were analysed by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. Peripheral blood and lamina propria CD4(+) T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of IFX. Cytokine profiles and RORC were determined with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. IL-21 and Th17 cells were found to be expressed highly in inflamed mucosa of active CD patients compared with healthy controls. Ten weeks after IFX infusion, CD activity index, ESR, CRP and intestinal mucosal healing were improved markedly in CD patients, and IL-21 expression and Th17 cell infiltration were decreased significantly compared with those before IFX therapy. In-vitro study demonstrated that IFX treatment could suppress IL-21, IL-17A and RORC expression in cultured CD biopsies. Moreover, IFX was also observed to down-regulate markedly IL-17A, IL-21 and RORC expression by CD CD4(+) T cells. IFX is highly effective in inducing clinical remission and promoting intestinal mucosal healing in CD patients through down-regulation of IL-21 expression and Th17 cell infiltration in intestinal mucosa.

Figure 1

Infliximab (IFX) therapy induces clinical remission and promotes intestinal mucosal healing in Crohn's disease (CD) patients. Twenty-six patients with active CD were treated with IFX at weeks 0, 2 and 6; the CD activity index (CDAI), erythrocyte sedimentation rate (ESR) C-reactive protein (CRP) and endoscopic scores were evaluated before and at week 10 after IFX administration. *P < 0·05 versus values before IFX therapy.

Figure 2

Infliximab (IFX) down-regulates interleukin (IL)-21 and T helper type 17 (Th17) cell infiltration in the inflamed mucosa of Crohn's disease (CD) patients. Intestinal mucosal biopsies were taken from healthy controls (n = 16) and CD patients (n = 26) before and 10 weeks after IFX treatment. Total RNA was extracted and analysed for IL-21, tumour necrosis factor (TNF), interferon (IFN)-γ, IL-17A and retinoic orphan receptor C (RORC) mRNA by quantitative real-time polymerase chain reaction (PCR). *P < 0·005 versus healthy controls; ⁺P < 0·05 versus values before IFX therapy.

Figure 3

Infliximab (IFX) decreases interleukin (IL)-21- and IL-17A-positive cell infiltration in the inflamed mucosa of Crohn's disease (CD) patients. Representative frozen sections were obtained from inflamed mucosa of CD patients before (a,c) and at week 10 after IFX treatment (b,d), and stained for IL-21 (a,b) and IL-17A (c,d) by immunohistochemistry. (e,f) The CD sections were stained with a control isotype antibody for IL-21 or IL-17A, respectively. Original magnification × 200.

Figure 4

Infliximab (IFX) treatment decreases interleukin (IL)-21 expression and T helper type 17 (Th17) cell infiltration in intestinal mucosal biopsies from Crohn's disease (CD) patients in vitro. Inflamed intestinal biopsies were obtained from 10 patients with active CD during endoscopic examination, and cultured (two biopsy samples/well) in the presence of IFX or control human immunoglobulin (IgG (HIg) (both at 50 μg/ml) at 37°C in 5% CO₂ humidified air for 24 h. Total RNA was then extracted and analysed for IL-21, IL-17A and retinoic orphan receptor C (RORC) by quantitative real-time PCR. Gene expression was normalized to β-actin mRNA levels in each sample. *P < 0·05 versus values before IFX therapy.

Figure 5

Infliximab (IFX) suppresses Crohn's disease (CD) lamina propria (LP) CD4⁺ T cell interleukin (IL)-21 and IL-17A mRNA expression and T helper type 17 (Th17) cell differentiation. Purified LP-CD4⁺ T cells (1 × 10⁶/ml) from CD patients and healthy controls (Con) were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) in the absence or presence of IFX (50 μg/ml) and IL-21R/Fc (20 μg/ml). After 48 h of culture, the cultured LP CD4⁺ T cells were harvested, followed by extraction of total RNA, and levels of IL-21 (a), IL-17A (b) and Th17 cell transcription factor retinoic orphan receptor C (RORC) (c) were then analysed by quantitative real-time PCR. Gene expression was normalized to β-actin mRNA levels in each sample. Data represent average fold increases or decreases over baseline levels in healthy controls cultured in medium alone (defined arbitrarily as 1·0). *P < 0·005 versus healthy controls under the same stimulatory conditions. ⁺P < 0·05 versus data from the same group cultured in medium alone. ^ΔP < 0·05 versus data from the same group stimulated with anti-CD3 and anti-CD28 mAbs. ^#P < 0·05 versus data from the same group stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) in the presence of IFX.