Anti tumour necrosis-alpha therapy increases the number of FOXP3 regulatory T cells in children affected by Crohn's disease

Abstract

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Its pathogenesis is not completely understood, though the prevailing model is that the intestinal flora drives a strong intestinal T helper 1 (Th1)/Th17 type immune response and inflammation in the genetically susceptible host. This leads to overly aggressive T-cell responses to normal bacteria causing tissue damage. Intestinal homeostasis and maintenance of tolerance to harmless antigens in the intestine has been shown to be maintained by CD4+ CD25+ T regulatory cells (Treg) in animal models of inflammatory bowel diseases. Here we investigated whether Infliximab, a chimeric monoclonal antibody directed against tumour necrosis factor (TNF)-alpha shown to be highly effective in the treatment of CD, has any effect on mucosal CD4+ CD25+ (FOXP3+) Tregs. Colonic mucosal biopsies from children with active Crohn's disease treated in vivo with Infliximab and controls were analysed to determine FOXP3 expression by immunofluorescence and reverse transcription-polymerase chain reaction. We observed that FOXP3+ T cells were significantly reduced in mucosa of CD patients with active disease compared with controls and restored to normal following Infliximab treatment. The frequency of FOXP3+ cells and mRNA expression was significantly increased in CD mucosa from patients treated in vivo with Infliximab compared with CD patients treated with conventional therapies. In conclusion, we show that Infliximab treatment does not solely neutralize soluble TNF-alpha, but also affects activation and possibly expansion of mucosal regulatory T cells. We suggest that anti TNF-alpha immunotherapy can also restore mucosal homeostasis in Crohn's disease.

Figures

Figure 1

FOXP3+ cells are decreased in CD mucosa compared with controls. Colon mucosa from CD patients treated with conventional therapies (n = 4) and controls (n = 4) were stained for FOXP3 expression and analysed by immunohistochemistry. The quantification is based on the number of positive nuclei per area (mm2) of lamina propria. All the data reported in this figure were reproduced in at least five separate experiments. Statistical significance was analysed by Student's t-test.

Figure 2

Expression of FOXP3 in colon mucosa lamina propria of controls, active and Infliximab treated Crohn's patients. Colon biopsies from normal controls, Crohn's patients with active disease or treated in vivo with Infliximab were analysed. FOXP3 staining (green) was visualized by immunofluorescence and analysed using fluorescence microscopy. Arrows show nuclear transcription factor FOXP3 expression. A representative example of FOXP3 expression. All patients were tested and similar results were obtained from the different groups. Data are representative of at least five separate slides. Original magnification ×100.

Figure 3

FOXP3+ nuclei expression in Lamina propria of colon mucosa from controls, active and Infliximab-treated Crohn's patients. The quantification is based on the approximate number of positive nuclei per area of lamina propria. Results from seven CD patients treated in vivo with Infliximab, five CD patients treated with conventional therapies and four controls are shown. Statistical significance was analysed by Student's t-test. Plots show mean values with SD bars.

Figure 4

FOXP3 mRNA transcript was increased in the mucosa of Crohn's patients treated in vivo with Infliximab. Colonic biopsy specimens from Crohn's patients treated in vivo with Infliximab or conventional therapy were analysed. mRNA isolated from intestinal biopsies was subjected to RT–PCR and analysed for FOXP3 transcript. FOXP3 mRNA was increased in the mucosa of CD patients treated in vivo with Infliximab. FOXP3 mRNA expression was normalized to the housekeeping gene GAPDH. A representative example of Foxp3 mRNA determination in a patient treated with Infliximab and a patient treated with conventional therapy is shown. Similar results were obtained from all other patients treated with or without Infliximab. One out of four independent experiments is shown.