中科院生物物理所刘志华研究组发表肠道菌与宿主共生研究进展

Nature Immunology: doi:10.1038/ni.3233
Supplementary Figure 3 Deficiency of LRRK2 or Rab2a leads to lysosomal degradation of lysozyme in Paneth cells in cultured organoids. (a) Confocal images of lysozyme and procryptdin in cultured wild-type organoids. The boxed area is shown at higher magnification in the panels below. Scale bar, 10 m. (b) Confocal images of lysozyme and procryptdin in cultured Lrrk2−/− organoids mock treated or treated with 1 g/ml Brefeldin A for 24 h. Boxed areas are shown at higher magnification in the panels below. Scale bar, 10 m. (c) Immunoblotting (upper panels) of indicated proteins and confocal images (lower panels) of lysozyme and procryptdin in cultured Lrrk2−/− crypt organoids transfected with siRNA specific for Rab27a or control. (d) Immunoblotting analyses of LRRK2 and Rab2a in cultured crypt organoids transiently transfected with siRNA specific for Rab2a or control. Actin was used as the loading control. (e) Confocal images of lysozyme and procryptdin in cultured wild-type organoids transfected with siRNA oligos against Rab2a and then treated with 100 M leupeptin or mock treated for 24 h. Boxed areas are shown at higher magnification in the panels below. Scale bars, 10 m. Data are representative of three (a–e) independent experiments.
Nature Immunology: doi:10.1038/ni.3233
Βιβλιοθήκη Baidu
Supplementary Figure 2 LRRK2 deficiency does not affect the expression or secretion of AMPs other than lysozyme in Paneth cells. (a) PAS staining of crypts from wild-type and Lrrk2−/− mice. Scale bars, 50 m. (b) Electron microscopy images of crypts from wild-type and Lrrk2−/− mice. Arrowheads indicate DCVs with enlarged halo regions. Scale bars, 2 m. (c) Quantification of DCVs with enlarged halo regions in wild-type and Lrrk2−/− mice as shown in b. A halo region thickness > 0.2 m was defined as enlarged. A total of approximately 500 DCVs from 2 mice of each genotype were used for quantification. Data are expressed as mean and s.e.m. *P < 0.01, Student’s t-test. (d) Immunoblotting analysis of Reg3 and procryptdin in isolated crypts from indicated animals. Actin was used as a loading control. Quantitation of protein levels by densitometry from three independent experiments, as shown on the left. NS, P > 0.05, Student’s t-test. (e) Whole-mount images taken immediately above the ileal mucosal surface in wild-type and Lrrk2−/− mice stained with Helix pomatia lectin (Lectin-HPA) and anti-procryptdin. Scale bars, 20 m. (f) Schematic diagram of the treatment regimen. Four groups of mice were treated with recombinant lysozyme or PBS (vehicle) by oral gavage once a day for 7 d before being infected with 109 CFU of L. monocytogenes. Fecal samples were collected 10 h after infection. Spleen and liver tissues were harvested 72 h after infection. Data are representative of three (a,b,d,e; mean and s.e.m. in d) independent experiments.
Nature Immunology: doi:10.1038/ni.3233
Supplementary Figure 4 Lysozyme in Paneth cells is degraded by lysosomes in GF mice. (a) Quantitative RT-PCR analysis of mRNA encoding lysozyme (Lyz) among mRNA in isolated crypts from SPF and GF mice. mRNA results were calculated as described in Figure 3c. Data are expressed as the average for three individual mice s.e.m. NS, P > 0.05, Student’s t-test. (b) Immunohistochemical staining of lysozyme in ileal sections from SPF, GF and ex-GF mice. Scale bars, 10 m. (c) RGB analysis of images from Figure 5c. RGB values were determined along the white dotted lines (from basal to apical) indicated in the merged images with the RGB tool in Image J. (d) Immunoblotting of lysozyme in isolated SPF and GF crypts. Actin was used as the loading control. (e) Confocal images of lysozyme and procryptdin in cultured GF organoids treated with 100 M leupeptin or mock treated for 24 h. Boxed areas are shown at higher magnification in the panels below. Scale bars, 10 m. (f) Immunoblotting of lysozyme in SPF and GF organoids treated as in e. Actin was used as the loading control. Data are representative of three (a,b,d–f) independent experiments.
Supplementary Figure 1 LRRK2 deficiency leads to enhanced susceptibility to intestinal infection and altered microbiota. (a) Validation of LRRK2 immunostaining. Confocal images of Lrrk2−/− ileal sections immunostained with an anti-LRRK2 (clone c41-2). Multiple images are aligned to show a complete villus. Horizontal gray lines indicate where the images are aligned. Dashed yellow lines show the base of the crypts. Scale bar, 20 m. (b) Flow cytometry analysis of CD24 and lysozyme in isolated cells from mouse ileal crypts. (c) Immunoblotting analysis of indicated proteins in isolated cell populations. Actin was used as a loading control. (d) Bacterial numbers (CFU) in spleen and liver from wild-type (WT) (n = 5) and Lrrk2−/− (n = 5) mice 24 h (left) and 48 h (right) after they were infected with 104 CFU of L. monocytogenes by tail vein injection. NS, P > 0.05, Mann-Whitney test. (e,f) The relative abundance of dominant phyla (e) and orders/families (f) identified from pyrosequencing data. Families with an average abundance greater than 1% are shown in the graph. The data regarding all the families are presented in Supplementary Table 1. (g) Principal-component analysis plot showing the clustering pattern between wild-type (n = 8) and Lrrk2−/− (n = 8) mice. Blue oval, wild-type mice; red oval, Lrrk2−/− mice. Distances were calculated using OTU abundance data. Mice of different genotypes were housed in separate cages. Numbers 1–5 of one genotype shared a cage. Numbers 6–8 of one genotype were from three different cages. Each symbol (d,g) represents an individual animal; small horizontal lines indicate the median values. Data are representative of three (a–d) independent experiments.