Fig 1.
The percentages of CD45+, lineage (lin)- cells and CD45+, lin-, CD56-, CD127+ ILCs in active celiac disease (ACD) did not differ from those in normal controls (CTR) and inactive celiac disease (ICD).
A. Lamina propria mononuclear cells (LPMC), isolated from 9 CTR, 7 ICD patients and 10 ACD patients, were gated on the lymphocytic area in the FSC/SSC plot, then on the CD45+ population and next on the lin- population. Right panel: percentages of CD45+, lin- cells in CTR, ICD and ACD. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values. B. CD45+, lin- LPMC were stained with CD127 and CD56 antibodies. Left panels: representative dot-plots showing the percentages of CD56-, CD127+ ILCs. Right panel shows the percentages of CD45+, lin-, CD56-, CD127+ ILCs in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values.
Fig 2.
Innate lymphoid cells (ILCs) producing TNF-α and IFN-γ are significantly increased in the inflamed mucosa of celiac disease patients.
A. Lamina propria mononuclear cells (LPMC), isolated from 5 normal controls (CTR), 5 inactive celiac disease (ICD) patients and 6 active celiac disease (ACD) patients, were gated on CD45+/lin- population and stained with CD56, CD127, and TNF-α. Representative dot-plots showing TNF-α expression in CD45+, Lin-, CD56-, CD127+ ILCs in CTR, ICD and ACD. Right panel shows the percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing TNF-α in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values. B. LPMC, isolated from 5 CTR, 5 ICD patients and 5 ACD patients, were gated on CD45+/lin- population and stained with CD56, CD127, and IFN-γ. Representative dot-plots show IFN-γ expression in CD45+, Lin-, CD56-, CD127+ ILCs in CTR, ICD and ACD. Right panel shows the percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing IFN-γ in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values. C. LPMC, isolated from 5 CTR, 5 ICD patients and 5 ACD patients, were gated on CD45+/lin- population and stained with CD56, CD127, and IL-17A. Representative dot-plots show IL-17A expression in CD45+, Lin-, CD56-, CD127+ ILCs in CTR, ICD and ACD. Right panel shows the percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing IL-17A in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values.
Fig 3.
Innate lymphoid cells (ILCs) express toll like receptors (TLRs).
A. Lamina propria mononuclear cells (LPMC), isolated from 7 controls (CTR), 6 inactive celiac disease (ICD) patients and 5 active celiac disease (ACD) patients, were gated on CD45+/lin- population and stained with CD56, CD127, and TLR2. Representative dot-plots show TLR2 expression in CD45+, lin-, CD56-, CD127+ cells in CTR, ICD and ACD. Right panel: percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing TLR2 in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values. B. LPMC, isolated from 8 CTR, 7 ICD patients and 5 ACD patients, were gated on CD45+/lin- population and stained with CD56, CD127, and TLR3. Representative dot-plots show TLR3 expression in CD45+, lin-, CD56-, CD127+ cells in CTR, ICD and ACD. Right panel: percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing TLR3 in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values. C. LPMC, isolated from 6 CTR, 7 ICD patients and 4 ACD patients, were gated on CD45+/lin- population and stained with CD56, CD127, and TLR9. Representative dot-plots show TLR9 expression in CD45+, lin-, CD56-, CD127+ cells in CTR, ICD and ACD. Right panel: percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing TLR9 in CTR, ICD patients and ACD patients. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. The horizontal bars represent the median values.
Fig 4.
Stimulation of lamina propria mononuclear cells (LPMC) with poly I:C significantly increases the production of TNF-α and IFN-γ by innate lymphoid cells (ILCs).
A. LPMC, isolated from jejunal specimens of 4 controls, were stimulated with poly I:C and analyzed by flow cytometry. LPMC were gated on CD45+/lin- population and stained with CD56, CD127, and TNF- α. Representative dot-plots show TNF-α expression in CD45+, lin-, CD56-, CD127+ ILCs in cultures of LPMC either left unstimulated (Unst) or stimulated with poly I:C. Right panel: percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing TNF-α in unstimulated cells or after stimulation with poly I:C. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. Each line connects unstimulated and stimulated wells of a single patient. B. LPMC, isolated from jejunal specimens of 5 controls, were stimulated with poly I:C and analyzed by flow cytometry. LPMC were gated on CD45+/lin- population and stained with CD56, CD127, and IFN-γ. Representative dot-plots show IFN-γ expression in CD45+, lin-, CD56-, CD127+ ILCs in cultures of LPMC either left unstimulated (Unst) or stimulated with poly I:C. Right panel: percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing IFN-γ in unstimulated cells or after stimulation with poly I:C. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. Each line connects unstimulated and stimulated wells of a single patient. C. LPMC, isolated from jejunal specimens of 5 controls, were stimulated with poly I:C and analyzed by flow cytometry. LPMC were gated on CD45+/lin- population and stained with CD56, CD127, and IL-17A. Representative dot-plots show IL-17A expression in CD45+, lin-, CD56-, CD127+ ILCs in cultures of LPMC either left unstimulated (Unst) or stimulated with poly I:C. Right panel: percentages of CD45+, lin-, CD56-, CD127+ ILCs expressing IL-17A in unstimulated cells or after stimulation with poly I:C. Each point in the graph indicates the percentage of positive cells in a single sample of a single patient. Each line connects unstimulated and stimulated wells of a single patient.
Fig 5.
Depletion of innate lymphoid cells (ILCs) attenuates poly I:C-induced small intestinal atrophy.
A. Hematoxilyn & eosin (HE)-stained frozen sections of representative small intestinal tissues taken from control RAG1 deficient (-/-) mice injected with phosphate buffered saline (CTR) and poly I:C-treated RAG1 (-/-) mice. B. Lamina propria mononuclear cells (LPMC) were isolated from the small intestine of control RAG1 (-/-) mice and poly I:C-treated RAG1 (-/-) mice, gated on CD45+, lineage (lin)-, CD90.2+ cells and stained with TNF-αIFN-γ and IL-17A. Graphs show the percentages of CD45+, lin- (CD3-, B220-, CD11b-, TER-119-, GR-1-), CD90.2+ ILCs expressing TNF-αIFN-γ and IL-17A in control RAG1 (-/-) mice (n = 3) and poly I:C-treated RAG1 (-/-) mice (n = 6). Data are indicated as mean and standard deviation of all experiments. C. Representative contour plots of Rag1 (-/-) mice treated with poly I:C after injection with control Rat IgG (n = 3) or anti-CD90.2 depleting antibody (n = 3). LPMC were gated on CD45+, lin-, CD90.2+ ILCs. D. HE-stained frozen sections of representative small intestinal tissues taken from RAG1 (-/-) mice treated with poly I:C after injection with control Rat IgG (n = 3) or anti-CD90.2 depleting antibody (n = 3). In the right panel is represented the percentage of villous height in RAG1 (-/-) mice treated with poly I:C after injection with control Rat IgG or anti-CD90.2 depleting antibody compared to control RAG1 (-/-) mice. Data are indicated as mean and standard deviation of all experiments.