Neutralizing IL-15 Inhibits Tissue-Damaging Immune Response in Ex Vivo Cultured Untreated Celiac Intestinal Mucosa

Abstract

In celiac disease (CeD), interleukin 15 (IL-15) affects the epithelial barrier by acting on intraepithelial lymphocytes, promoting interferon γ (IFN-γ) production and inducing strong cytotoxic activity as well as eliciting apoptotic death of enterocytes by the Fas/Fas ligand system. This study investigates the effects of a monoclonal antibody neutralizing the effects of IL-15 (aIL-15) on tissue-damaging immune response in untreated CeD patients by using an organ culture system. Jejunal biopsies from 10 untreated CeD patients were cultured ex vivo with or without aIL-15. Epithelial expressions of CD95/Fas, HLA-E and perforin were analyzed by immunohistochemistry. Apoptosis was detected in the epithelium by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Additionally, the surface epithelium compartment of ex vivo cultured biopsy samples was isolated by laser capture microdissection (LCM). RNA from each LCM sample was extracted and the relative expression of IFN-γ was evaluated by quantitative reverse transcriptase-PCR (qRT-PCR). Biopsies cultured with the aIL-15 antibody showed a reduction in Fas, HLA-E and perforin epithelial expression, as well as a decrease in epithelial TUNEL+ cells compared to biopsies cultured without the aIL-15 antibody. Moreover, downregulation of epithelial IFN-γ expression was recorded in biopsies incubated with aIL-15, compared to those cultured without aIL-15. Our findings suggest that neutralizing the effects of IL-15 in ex vivo cultured untreated CeD intestinal mucosa could block apoptosis by downregulating Fas and HLA-E expression and the release of cytotoxic proteins, such as perforin. Furthermore, it can dampen the hyperactive immune response by reducing IFN-γ expression. More generally, our study provides new evidence for the effects of anti-IL-15 neutralizing monoclonal antibodies in preventing or repairing epithelial damage and further supports the concept that IL-15 is a meaningful therapeutic target in CeD, or inflammatory diseases associated with the upregulation of IL-15.

Keywords: IL-15; celiac disease; cytotoxicity; therapy.

Figure 1

Upper left panel: number of perforin cytotoxic granules analyzed by immunohistochemistry, in mucosal explants from untreated CeD cultured ex vivo without (w/o) or with aIL-15 antibody. Perforin cytotoxic granules were counted in 1 mm epithelium from at least four different fields. The mean value is reported for each subject, and dashes indicate the mean values. Statistical significance was evaluated by comparing responses without or with aIL-15 antibody (* p < 0.01). Upper right panel: perforin cytotoxic granules (brown) in the epithelium of jejunal mucosa from untreated CeD patient cultured ex vivo without or with aIL-15 antibody. In the latter, a decrease in perforin cytotoxic granules (brown) in the epithelial compartment is evident. The image is representative of ten separate experiments in which biopsies taken from ten patients with untreated CeD cultured with or without the antibody aIL-15 were analyzed. Original magnification ×63; scale bar 10 µm.

Figure 2

Upper left panel: Fas epithelial expression is decreased in the surface epithelium of duodenal mucosa of untreated CeD cultured with aIL-15 antibody. Fas expression in intestinal surface epithelium was evaluated in terms of staining intensity and graded on an arbitrary scale of staining from 1 to 3. The criteria for epithelium staining were as follows: weak staining (+) = 1, moderate staining (++) = 2, and strong staining (+++) = 3. Circles represent the response from individual patients. * p < 0.01, ** p < 0.001. Upper right panel: Fas expression in the epithelium of jejunal mucosa from n = 10 untreated CeD cultured ex vivo without (w/o) or with aIL-15 antibody. In the latter, lower staining is detected, particularly in almost all the epithelial cells. Original magnification ×63, scale bar 10 µm.

Figure 3

Relative levels of IFN-γ in the surface epithelium (Ep) compartment isolated by LCM from jejunal biopsies cultured ex vivo without (w/o) or with aIL-15 antibody. The biopsies were analyzed by RT-qPCR from the untreated CeD. The fold change represents the relative expression of IFN-γ mRNA normalized to GAPDH. Each point on the plot is representative of a distinct patient (n = 5). The lines link the IFN-γ gene expression of each subject in the two different experimental conditions.

Figure 4

A possible model whereby anti-IL-15 may block apoptosis mediated by cytolytic mechanisms in celiac disease. (1) CD8+IELs, activated by IL-15, synthesize cytotoxic molecules such as perforin that cause the cytolysis of enterocytes. (2) Moreover, activated CD8+IELs synthesize IFN-γ, which induces enterocyte HLA-E and Fas expression. HLA-E and Fas can combine with the CD8+ IELs cell-surface receptors CD94/NKG2C and FasL, respectively, contributing to enterocyte apoptosis. (3) Anti-IL-15 may limit cytotoxic T-cell function by inhibiting the expression of perforin, HLA-E, Fas and IFN-γ.