Monocytes differentiated with IL-15 support Th17 and Th1 responses to wheat gliadin: implications for celiac disease

Abstract

Interleukin (IL)-15 contributes to the immunopathogenesis of Celiac disease (CD). However, it is not clear how IL-15 affects APC that shape adaptive immune responses to the dietary antigen, gliadin. Using PBMC from healthy individuals, we show that monocytes differentiated with IL-15 (IL15-DC) produced IL-1beta, IL-6, IL-15, IL-23, TNFalpha and CCL20 in response to pepsin-trypsin digested gliadin (PTG) and activated contact-dependent Th17 and Th1 responses from autologous CD4(+) T cells. Lower concentrations of IL-15 augmented IFNgamma responses to PTG in PBMC from CD patients compared to controls. Thus, IL-15 supports Th17 and Th1 responses to a dietary antigen that is normally well-tolerated in healthy individuals by generating IL15-DC. These potentially pathogenic immune responses may result in CD patients and not healthy individuals as a consequence of IL-15 hypersensitivity. Therefore, genetic and/or environmental factors that control IL-15 expression and responsiveness in the intestine likely participate in the pathogenesis of CD.

Figure 1. IL15-DC exhibit features of DC and their monocyte precursors

Elutriated monocytes were stained immediately or were cultured with GM-CSF plus IL-4 or IL-15 for 72h. Cells were labeled with anti-human mAbs or appropriate isotype controls for flow cytometric analysis. Markers were set by isotype overlay. A, Numbers indicate % positive cells. B, Numbers are MFI because the % of cells staining positive for these markers were similar between IL4DC and IL15-DC. Histograms are representative of 5 independent experiments.

Figure 2. PTG stimulates IL15-DC to secrete IL-1β, IL-6, IL-23, TNFα and CCL20 and produce surface IL-15

IL15-DC or IL4-DC were incubated with and without 100μg/ml PTG for 20h. A, Cell-free culture fluids were collected and analyzed for secreted IL-1β, IL-6, IL-23, TNFα, CCL20 and IL-12 by ELISAs. IL-1β, IL-23, CCL20 and IL-12 data are the means of 3–5 independent experiments tested in duplicate, IL-6 and TNFα data are from 1 of the donors tested. B, Cells were harvested and labeled with anti-human IL-15, CD83 or isotype controls (dashed line) and analyzed by flow cytometry. M.C. maturation cocktail (IL-1β, IL-6, TNFα and PGE2) served as a positive control for CD83 on IL4-DC. Histograms are representative of 3 different donors tested in duplicate and numbers signify % positive cells.

Figure 3. IL15-DC induce IL-17 and IFNγ secretion in cultures with autologous CD4⁺ T cells and PTG

A, CD4⁺ T cells were purified by magnetic cell separation and analyzed by flow cytometry for the presence of Th17 surface markers. Dashed histograms are isotype controls and numbers are % positive cells. Histograms from 1 of 4 independent experiments are shown. B, CD4⁺ T cells were cultured alone or together with autologous IL15-DC or IL4-DC with and without 100μg/ml PTG for 72h. Cell-free supernatants were tested for secreted IL-17, IFNγ IL-21 and IL-22 by ELISA. Data are the means of 4–5 different donors tested in duplicate. C, The levels of IL-1β, IL-6, IL-12, IL-23 and TNFα were analyzed in the culture fluids from IL15-DC and CD4⁺ T cells incubated with and without 100μg/ml PTG for 72h by ELISA. Shown are the means of 4 independent experiments tested in duplicate. *, p<0.05 between T cell cultures with IL15-DC +/− PTG

Figure 4. Th17 and Th1 responses induced by PTG are contact-dependent; IFNγ requires contact with IL-15 on the surface of IL15-DC

IL15-DC were cultured with autologous CD4⁺ T cells in the presence or absence of 100μg/ml PTG for 72h. In addition, IL15-DC and autologous CD4⁺ T cells were cultured using 0.4μm semi-permeable transwell inserts to separate the cell populations with 100μg/ml PTG or were incubated together with PTG in the presence and absence of 10μg/ml anti-human IL-15 or mIgG1 for 72h. All culture fluids were harvested and analyzed for IL-17 and IFNγ production by ELISA. The percent inhibition was calculated for each of the 5 different donors tested. Shown are the means of 5 independent experiments tested in duplicate. *, p<0.05 **, p<0.005 ***, p<0.0005

Figure 5. PBMC from CD patients are hyperresponsive to IL-15 compared to PBMC from healthy individuals

PBMC from 5 individuals with CD and 5 healthy individuals (HD) were incubated with and without 100μg/ml gliadin (PTG) in the presence and absence of 10 or 100ng/ml IL-15 for 72h. Cell-free culture supernatants were collected and analyzed for IFNγ by ELISA. Background levels of IFNγ stimulated by IL-15 without PTG were subtracted from IL-15 conditions with PTG. †, p<0.05 differences between conditions within CD or HD *, p<0.05 differences between CD and HD.