The absence of interleukin 10 affects the morphology, differentiation, granule content and the production of cryptidin-4 in Paneth cells in mice

Abstract

Paneth cells (PCs) are specialized epithelial cells of the small bowel that contain multiple secretory granules filled with antimicrobial peptides and trophic factors, which are essential for the control of the microorganisms growth and maintaining intestinal integrity. Alterations in their function are associated with an imbalance of the normal microbiota, gastrointestinal infections and inflammatory processes, such as Crohn's disease (CD). One of the most common murine models for studying CD is IL-10-/- mouse. IL-10-/- mice when housed in conventional conditions and take contact with commensal microorganisms develop an acute enterocolitis mediated by a Th1 immune response. Even though, alterations in PCs function are related to CD, they had not been characterized yet in this mouse model. Here we show that in specific pathogen free conditions IL-10-/- mice have aberrant granules and a large number of immature PCs at the bottom of the crypt in the ileum of IL-10-/- mice before developing intestinal inflammation, along with a reduced expression of Indian Hedgehog. In addition, IL-10-/- Paneth cells presented a reduced expression of cryptidin-4, and a heterogeneous distribution of lysozyme+ granules. The alterations in the maturation of the PCs at the bottom of the crypt were not modified after the colonization by the conventional microbiota. On the other hand, depletion of microbiota altered the phenotype, but did not normalize PCs. Our results suggest that IL-10 could be necessary for the integrity of PCs. Moreover, our results help to explain why IL-10-/- mice develop enterocolitis in response to microorganisms.

Fig 1. IL-10^-/- mice have more immature Paneth cells at the bottom of the crypt.

(A) Representative images of Lieberkühn crypts stained with AB-PAS, of C57BL/6 and IL-10^-/- mice. The black arrows show Paneth cells with intermediate cell staining AB+PAS+. Scale bars are 25μm. (B) Number of PC per crypt. n = 120 crypts were analyzed from 4 mice. (C) Quantification of Intermediate Cells per crypt of both strains, according to their location: adjacent to Paneth cells (P1), in the middle zone of proliferation (P2), or in upper positions over differentiated Goblet Cells (P3) (2-way ANOVA, post-hoc Bonferroni *p˂0.05, ***p ˂0.001, n = 660 crypts).

Fig 2. Seven-week-old IL-10^-/- mice do not present more signs of ileum inflammation than WT mice under SPF conditions.

(A) Histopathological analysis of ileum sections of wild-type and IL-10^-/- mice. Left panel: Representative images of ileum sections stained with H&E (Scale bars are 200μm). Right panel: Histologic score. (B) Relative expression of TNF-α and IFN-γ in ileum sections of WT (n = 5) and IL-10^-/- (n = 6) mice.

Fig 3. IL-10^-/- mice present abnormal Paneth cells granules.

Paneth cell (PCs) granules of C57BL/6 and IL-10^-/- mice were evaluated by transmission electron microscopy (n = 3). (A) Representative images of PC granules from both strains (4200X, Scale is 2μm). (B) Number of granules per PC (t-student, ***˂p 0.001). (C) Area of the electrondense core of the PC granules. D) Classification of granules, according to their TEM morphology in: normal, off-center, diffuse, double halo, without halo and amorphous (n = 3 mice, 2-way ANOVA, post-hoc Bonferroni *p˂0.05).

Fig 4. The crypts of IL-10^-/- mice have increased cell proliferation and alterations in secretory lineages allocation.

(A) Ileum crypts of C57BL/6 and IL-10^-/- mice evaluated by transmission electron microscopy (scale bar is 10μm). In both strains were observed basal intestinal stem-cells (ISC) in resting state (yellow arrows) and undifferentiated cells in mitotic phase (red arrows). The black arrow shows a Paneth cell with intermediate granules located in the bottom of the crypt of an IL-10^-/- mouse. (B) Quantitative analysis of the number of basal ISC in resting state and undifferentiated cells in mitotic phase (red arrows) present in the crypts of C57BL/6 (n = 29 crypts) and IL-10^-/- (n = 20 crypts) mice (t-test *p˂0.05). (C) Relative expression of Wnt5a and Ihh in ileum sections of WT (n = 5) and IL-10^-/- (n = 6) mice (U-test Mann Whitney *p˂0.05).

Fig 5. Paneth cells of IL-10^-/- mice had a reduced expression of Cryptdin-4 mRNA transcripts and an abnormal packing of Lysozyme.

(A) Relative expression of cryptdin-1, cryptdin-4, RegIIIγ and lysozyme mRNA in ileum sections of C57BL/6 (n = 9) and IL-10^-/- (n = 6) mice (t-student, *p˂0.05). (B) Representative images of lysozyme immunodetection in Lieberkühn crypts of C57BL/6 and IL-10^-/- mice. Yellow arrows indicate abnormal granules size and distribution. Red arrow indicates diffuse cytoplasmic content.

Fig 6. Conventional microbiota induces enterocolitis in IL-10^-/- mice without affecting the number of intermediate cells at the bottom of the crypt.

(A) Colitis score of IL-10^-/- mice under SPF conditions or in the presence of conventional microbiota (n = 5). (B) Representative images of ileum sections stained with H&E (upper panel, scale bars are 200μm) and with AB-PAS (lower panel, scale bars are 25μm) from WT mice or IL-10^-/- mice under SPF conditions or in the presence of conventional microbiota. (C) Histologic score of ileum, proximal colon and distal colon of IL-10^-/- under SPF conditions or in the presence of conventional microbiota (t-student, *p˂0.05, n = 4). (D) Quantification of Intermediate Cells (AB⁺PAS⁺) per crypt, according to their location: adjacent to Paneth cells (P1), in the middle zone of proliferation (P2), or in upper positions over differentiated Goblet Cells (P3) (n = 1000 crypts).

Fig 7. Depletion of gut microbiota partially improves the Paneth cells phenotype, but does not normalize the state of the crypt.

(A) Ileum crypts of IL-10^-/- mice treated with vehicle (IL-10^-/- VEH) or broad-spectrum antibiotics (IL-10^-/- ATB), evaluated by transmission electron microscopy (scale bar is 10μm). Black arrow shows a PC with double-halo granules, green arrow shows a PC with amorphous granules, yellow arrows show Crypt Base Columnar (CBC) stem cells and red arrow show a mitotic figure. (B) Representative images of PC granules from both groups (scale bar is 5 μm). (C) Crypt Base Columnar (CBC) stem cells of a IL-10^-/- ATB mouse, with large amounts of mitochondria in their cytoplasm (scale bar is 2 μm). D) Classification of granules according to their TEM morphology in: normal, off-center, diffuse, double halo and amorphous (n = 3 mice, 2-way ANOVA, post-hoc Bonferroni *p˂0.05). E) Number of granules per PC (t-student, ***˂p 0.001). F) Number of CBC stem cell per crypt (t-student, ***˂p 0.001). G) Number of undifferentiated cells in mitotic phase per crypt.