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. 2014 Jun 30;211(7):1393-405.
doi: 10.1084/jem.20130753.

Paneth cell extrusion and release of antimicrobial products is directly controlled by immune cell-derived IFN-γ

Henner F Farin  1 , Wouter R Karthaus  1 , Pekka Kujala  2 , Maryam Rakhshandehroo  3 , Gerald Schwank  1 , Robert G J Vries  1 , Eric Kalkhoven  4 , Edward E S Nieuwenhuis  5 , Hans Clevers  6
Affiliations

Paneth cell extrusion and release of antimicrobial products is directly controlled by immune cell-derived IFN-γ

Henner F Farin et al. J Exp Med. .

Abstract

Paneth cells (PCs) are terminally differentiated, highly specialized secretory cells located at the base of the crypts of Lieberkühn in the small intestine. Besides their antimicrobial function, PCs serve as a component of the intestinal stem cell niche. By secreting granules containing bactericidal proteins like defensins/cryptdins and lysozyme, PCs regulate the microbiome of the gut. Here we study the control of PC degranulation in primary epithelial organoids in culture. We show that PC degranulation does not directly occur upon stimulation with microbial antigens or bacteria. In contrast, the pro-inflammatory cytokine Interferon gamma (IFN-γ) induces rapid and complete loss of granules. Using live cell imaging, we show that degranulation is coupled to luminal extrusion and death of PCs. Transfer of supernatants from in vitro stimulated iNKT cells recapitulates degranulation in an IFN-γ-dependent manner. Furthermore, endogenous IFN-γ secretion induced by anti-CD3 antibody injection causes Paneth loss and release of goblet cell mucus. The identification of IFN-γ as a trigger for degranulation and extrusion of PCs establishes a novel effector mechanism by which immune responses may regulate epithelial status and the gut microbiome.

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Figures

Figure 1.
Figure 1.
PC degranulation in intestinal epithelial organoids after muscarinergic but not after bacterial stimulation. (A) Small intestinal organoids were stimulated with 10 µg/ml PGN, 100 µg/ml poly(I:C), 1 µg/ml LPS, 0.1 µg/ml flagellin, 1 µg/ml CpG ODN, 100 µg/ml MDP, heat-inactivated (h.i.), or live E. coli bacteria or Cch (25 µM) for 12 h, and granules were visualized by UEA-1 lectin staining. Nonstimulated cultures served as controls. PC granules are indicated by blue arrowheads; granule release into the crypt lumen is indicated by the blue arrow. Whole organoids (top) and magnified crypts (bottom) are shown. Bars, 100 µm. (B) Intact organoids in Matrigel (top) were stimulated basolaterally (12 h) or organoid fragments (bottom) were stimulated both apically and basolaterally (2 h), with the indicated bacterial patterns and lysozyme expression assessed by Western blot. Cells were washed to remove luminal lysozyme. α-Tubulin was probed for normalization. Experiments were performed in three parallel cultures. (C) Quantification of lysozyme content from Western Blot data in B. Densitometric analysis of lysozyme signals normalized to α-tubulin expression. Error bars show standard deviation; **, P < 0.01; Student’s t test. Results in A–C were reproduced in two independent experiments. (D) Expression of the indicated microbial pattern receptors; RT-PCR analysis using cDNA from isolated crypts and cultured organoids. Hprt was analyzed for normalization. Representative data of three independent experiments. (E) Intact organoids and organoid fragments were stimulated with the indicated stimuli for 1 h, and Icam1 expression was assessed by qPCR analysis. Mean relative expression ± SD in n = 3 parallel wells; **, P < 0.01; ***, P < 0.001; Student’s t test. Data representative of three independent experiments. (F) Human colon cancer cell lines Caco-2 and HT-29 were stimulated with the indicated stimuli for 1 h, and IL-8 expression was assessed by qPCR. Mean relative expression ± SD (log10 scale) in n = 3 parallel wells; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Experiment has been performed twice.
Figure 2.
Figure 2.
IFN-γ–induced PC degranulation. (A) RT-PCR expression analysis of Cd3e (T cells) and Cd45 (leukocytes) in whole mouse small intestinal RNA or in RNA from freshly isolated crypts and cultured organoids. Hprt expression was used for normalization. Representative results of 2 independent experiments. (B) Morphology of small intestinal organoids 2 d after addition of mouse recombinant TNF, IL-1b, IL-6, IL-22 (all at 20 ng/ml) or IFN-γ (5 ng/ml) to the culture medium. PCs were observed in all cultures (blue arrowheads) except after IFN-γ treatment. Representative images from three independent experiments. (C) Small intestinal organoids were stimulated with IFN-γ (2 d; 5 ng/ml) followed by lysozyme immunostaining (red) and UEA-1 lectin staining (green). Blue arrow shows accumulation of UEA-1+ mucus in the crypt lumen. 3D projected confocal images show nuclei (gray) stained with TO-PRO3. Representative images from two independent experiments. (D) Organoids were stimulated for 2 d with indicated concentrations of IFN-γ and epithelial lysozyme content was analyzed by Western blot; results are representative for two independent experiments. (E) Quantification of Western blot data in D by densitometric analysis. Mean normalized lysozyme signal ± SD of triplicate wells is shown; **, P < 0.01; Student’s t test. (F) Histological analysis of organoids 2 d after addition of 5 ng/ml IFN-γ by PAS and alkaline phosphatase (AP) staining. Black arrows show loss of mucus-filled goblet cells. Images are representative from two independent experiments. Bars, 50 µm.
Figure 3.
Figure 3.
Reduced organoid growth and progressive loss of PCs after IFN-γ exposure. (A) Organoid number after prolonged culture in presence of indicated concentrations of IFN-γ. Mean of n = 3 independent cultures ± SD is shown, experiment was performed once. All concentrations caused a significantly reduced growth compared with controls (passage 1, P < 0.05; passage 2, P < 0.001, as determined by Student’s t test). (B–D) Time-course analysis of organoids stimulated with 1 ng/ml IFN-γ for the indicated times. (B) Dissociated organoid cells were analyzed by FACS after cleaved Caspase-3 staining. Mean percentage of positive cells from n = 3 independent experiments ± SD is shown; **, P < 10−2; ***, P < 10−4; Student’s t test. See Fig. S1 A for original FACS data. (C) Quantification of epithelial lysozyme content by Western blot. Experiment was performed in n = 3 parallel cultures; mean normalized signals ± SD are shown; *, P < 0.05; **, P < 0.01; Student’s t test. Results were reproduced twice independently. (D) qPCR analysis for marker gene expression of PCs (Lyz1, Defa6), stem cells (Lgr5, Olfm4) and the IFN-γ target gene Irf1. Mean normalized expression values (n = 3 parallel cultures ± SD) are shown. Results are representative for two independent experiments. (E) In situ hybridization analysis of Defa6 expression after IFN-γ treatment (5 ng/ml for 2 d). Overview (top) and magnified images (bottom) are shown that are representative for 3 independent experiments. (F) Immunostaining of membrane markers: CD24 on PCs (red) and the IFN-γ target MHC2 (green) after IFN-γ stimulation (1 d, 1 ng/ml). Note the ubiquitous MHC2 induction also on degranulated PCs (white arrowheads). Images are representative for two independent experiments. (G) Organoids were stimulated for the indicated times (1 ng/ml IFN-γ) before quantification of CD24-positive cells by FACS. Mean cell number (of n = 2 independent experiments ± SD) is shown. *, P < 0.05 as determined by Student’s t test. See Fig. S1 B for the original FACS data. (H) Morphology 1 d after treatment with 5 ng/ml IFN-γ and subsequent culture for 4 d in normal medium. Blue arrowheads show reformation of PC granules. Bars, 50 µm. Experiment was repeated three times.
Figure 4.
Figure 4.
IFN-γ induces rapid granule release that is caused by PC extrusion. (A) Time-lapse analysis of PC degranulation. Granules were labeled with the fluorescent Zn2+ chelator ZP-1, followed by live microscopy (still images of Videos 1–3). Untreated controls (top row) or cultures stimulated with 5 ng/ml (middle row) or 20 ng/ml IFN-γ (bottom row). Time after addition of IFN-γ is indicated on top (hours:min). Blue arrowheads show unaffected PCs; white arrowheads mark PCs that release their granules during the next time interval, as labeled by asterisks. Note that the fluorescent signal decreases due to bleaching. Experiment was repeated twice with at least 5 organoids per condition. Bars, 50 µm. (B) Transmission electron microscopy analysis of control organoids (left) and cultures stimulated with IFN-γ for 6 h (middle) or 12 h (right). Magnified regions (red boxes) are shown in the bottom row. Blue arrows mark PC granules; residual small granules are marked with a white arrow. The label V shows a large apical vacuole; P marks extruded cell fragments with densely packed granules; cell membranes are marked with dashed lines. Representative images from 1 experiment with n > 10 organoids analyzed per condition. Bars, 5 µm. (C) Cell-fate analysis of PCs after degranulation using live reporters (still images of Video 4). BAC transgenic organoids expressing Lyz2-RFP in PC granules (red signal) and lentiviral Histone 2B (H2B)-Dendra in nuclei (green signal). Time after addition of 1 ng/ml IFN-γ is indicated on top (hours:min). White arrowheads mark individual PCs that extrude both granules and cell nucleus during the next time interval (asterisks). After 24 h the epithelium is devoid of Lyz2-RFP and only few labeled nuclei remain (black arrowheads). Note that long-lived PCs accumulate H2B-Dendra. Results were reproduced in two independent experiments. (D) Quantification of PC fate from time-lapse data in C. In untreated control cultures (top) and after treatment (1 ng/ml IFN-γ; bottom), the status of >100 PCs each was tracked for 36 h. PCs were scored as unaffected (red bars), and then degranulated with nucleus present (orange bars) or extruded (gray bars).
Figure 5.
Figure 5.
Lipid antigen stimulation of iNKT cells triggers PC degranulation in an IFN-γ–dependent manner. (A) Experimental strategy. Cell culture SNs were collected from activated iNKT cells and transferred to the culture medium of small intestinal organoids. iNKT activation in the presence of the lipid antigen α-GC and HeLa-CD1D antigen presentation. SNs were preincubated with neutralizing anti–IFN-γ antibody or control IgG. (B) ELISA quantification of IFN-γ levels in SNs of control iNKT cells and after α-GC stimulation (both in presence of HeLa-CD1D cells). Means of quadruplicate measurement is shown (±SD; ***, P < 10−4; Student’s t test). (C) Small intestinal organoids were cultured for 2 d in presence of iNKT SNs, followed by UEA-1 lectin staining; blue arrowheads label unaffected PCs and blue arrows show discharged PCs. Bars, 100 µm. (D) Western blot analysis of epithelial lysozyme content; conditions as in C. Organoid lysates from three parallel cultures; α-tubulin was probed for normalization. (E and F) Quantification of lysozyme expression from Western blot data shown in D by densitometric analysis. Mean normalized signals ± SD are shown; *, P < 0.05; ***, P < 0.001; Student’s t test. Data (B–F) are from one representative experiment of two independent experiments.
Figure 6.
Figure 6.
T cell activation in vivo causes IFN-γ dependent Paneth loss. (A–G) Wild-type and IFN-γ KO mice were injected i.p. with anti-CD3 antibody (50 µg/mouse); as a control PBS was injected in wild-type mice. 24 h after injection, mice were sacrificed for histological analysis. Groups of n = 7 animals per condition were used and results were reproduced in 2 independent experiments. (A) Lysozyme immunostaining shows reduced PC granules after T cell activation in wild-type but not in IFN-γ KO mice. (B) Quantification of lysozyme-positive cells per crypt section in 45 crypts/animal/region. Mean number ± SD is shown. *, P < 0.05; **, P < 0.01; ***, P < 10−4; Student’s t test compared with control-injected mice. (C) Defa6 in situ hybridization. (D) Quantification of Defa6+ cells per crypt section; mean cell number ± SD is shown. **, P < 10−3; ***, P < 10−5; Student’s t test compared with control-injected mice. (E) Cleaved Caspase-3 immunostaining shows pronounced induction of apoptosis in both wild-type and IFN-γ KO crypts. (F) Quantification of cleaved Caspase-3+ cells per crypt section. Mean cell number ± SD is shown. ***, P < 10−6; Student’s t test compared with control-injected mice. (G) Co-staining of cleaved Caspase-3 (red) and PC granules (UAE-1; green) shows broad induction of apoptosis, also in non-PCs. Nuclei stained with DAPI (gray). Bars, 50 µm.
Figure 7.
Figure 7.
IFN-γ acts as potent secretagogue of goblet cell mucus. (A) PAS staining on histological sections of anti-CD3–injected mice (same experiment as in Figure 6). Black arrows show massive extrusion of goblet cell mucus in wild-type but not in IFN-γ KO mice. Black arrowheads mark unaffected goblet cells on ileal villus (top row); note the IFN-γ–dependent villus damage. For the colon magnified regions (middle row) and overview images (bottom row) are shown. Phenotypes were fully penetrant in n = 7 animals per group and results were reproduced in 2 independent experiments. (B) Mouse colon organoids were treated with IFN-γ for 24 h (5 ng/ml) and stained with UEA-1 lectin (top) or PAS (bottom). Images are representative of two independent experiments. Arrows label secretion of goblet cell mucus. Bars, 100 µm.
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