Colony stimulating factor-1 dependence of paneth cell development in the mouse small intestine

Abstract

Background & aims: Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development.

Methods: CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine. CSF-1 transgenic reporter mice and mice that express only the membrane-spanning, cell-surface CSF-1 isoform were used to investigate regulation by systemic versus local CSF-1.

Results: Mice deficient in CSF-1 or CSF-1R had greatly reduced numbers of mature PCs. PCs express the CSF-1R, and administration of anti-CSF-1R antibody to neonatal mice significantly reduced the number of PCs. Analysis of transgenic CSF-1 reporter mice showed that CSF-1-expressing cells are in close proximity to PCs. CSF-1/CSF-1R-deficient mice also had reduced numbers of the proliferating epithelial cell progenitors and lamina propria macrophages. Expression of the membrane-spanning, cell-surface CSF-1 isoform in CSF-1-deficient mice completely rescued the deficiencies of PCs, proliferating progenitors, and lamina propria macrophages.

Conclusions: These results indicate local regulation by CSF-1 of PC development, either directly, in a juxtacrine/paracrine manner, or indirectly, by lamina propria macrophages. Therefore, CSF-1R hyperstimulation could be involved in hyperproliferative disorders of the small intestine, such as Crohn's disease and ulcerative colitis.

Figure 1. Abnormal SI crypts and villi in two-week-old Csf1r^−/− and Csf1^op/op mice

(A) Longitudinal sections stained for neutral mucins (PAS-stain) reveal intense staining of goblet cells in the SIs of the mutant mice. (B) Higher power images of the regions boxed in (A) reveal the enlarged mucin-containing deposits in goblet cells (arrows). In addition, the less ordered organization of nuclei in the mutant villi is also evident. Bars = 50 μm. (C) Numbers of cells/crypt length and (D) number of cells/villus length (mean ± SEM; n=3–5/genotype, ANOVA P-values shown).

Figure 2. Lysozyme staining reveals a dramatic reduction of PCs in the SI crypts of two-week-old Csf1r^−/− and Csf1^op/op mice

Low power images of lysozyme⁺ Paneth cells in the SIs of (A) WT, (B) Csf1r^−/− and (C) Csf1^op/op mice. (D) Quantitation of the number of lysozyme⁺ cells/crypt section (mean ± SEM, n = 3, ANOVA P-values shown), revealed significant differences the number of PCs at the crypt base (grey arrow in E) between both mutant and WT, as well as between mutants. (E) Higher power images of WT crypts showing the presence of lysozyme⁺ cells within the lamina propria indicative of macrophages (black arrow). (F) Only background lysozyme staining is evident in the Csf1r^−/− SI. White arrow indicates goblet cell with enlarged mucin body. Bar = 50μm.

Figure 3. Treatment of WT mice with anti-CSF-1R antibody inhibits development of PCs and villus macrophages

Newborn mice were subcutaneously injected with (A) control IgG or (B) anti-CSF-1R antibody for 14 days prior to fixation and sectioning of the SIs and staining with anti-lysozyme antibody and DAPI. Bar = 60μm.

Figure 4. SI crypts of two-week-old Csf1r^−/− and Csf1^op/op mice display hypoproliferative defects

PCNA staining of (A) WT (B) Csf1r^−/− and (C) Csf1^op/op SI reveals proliferation deficits in the mutant crypts. (D) Reduced numbers of PCNA⁺ nuclei/Csf1r^−/− or Csf1^op/op crypt compared with WT littermate crypts (mean ± SEM; n=3/genotype, 25 crypt/villi per mouse counted, ANOVA P-values shown). Bar = 50μm. (E) Quantitative RT-PCR analysis of Csf1r, cyclin D1 and Lgr5 in crypts of Csf1r^−/− and WT littermates (mean ± SEM; ND = no data, n=3, ANOVA P-values shown).

Figure 5. PCs and SI macrophages express the CSF-1R

Immunofluorescence micrography showing: (A) CSF-1R staining (red) of cells within the crypt (PC), below the crypt and within interstitial regions of the villi (macrophages), (B) staining for lysozyme (green) and (C) merged images, showing co-localization of the CSF-1R and lysozyme staining. P, PCs; M, macrophages. Nuclei counterstained with DAPI (blue). Bar = 30μm.

Figure 6. CSF-1 is expressed in close proximity to PCs

Histochemical staining for beta-galactosidase in the SIs of two-week-old (A) control WT and (B–D) TgZ9/TgZ9 mice. (B), Low power and (C) and (D), high power fields, showing the close proximity of β-gal staining (blue, nuclear, arrowed) to the PCs (P). Note: There is spill-over of nuclear beta-galactosidase expression into cytoplasm in some cells.

Figure 7. Expression of the membrane-spanning, cell-surface isoform of CSF-1 is sufficient to rescue the SI defects of two-week-old Csf1^op/op mice

(A) Rescue of proliferating (PCNA), crypt and villus cells. (B) Rescue of PCs. Bar = 50μm.