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Paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine

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Science  08 Sep 2017:
Vol. 357, Issue 6355, pp. 1047-1052
DOI: 10.1126/science.aal4677
  • Fig. 1 Large LC3+ vesicles in S. Typhimurium–infected mice contain lysozyme.

    (A) Immunofluorescence of LC3 in intestinal crypts. Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 10 μm. (B) Quantification of LC3+ puncta. Each data point represents one mouse. (C) Immunofluorescence of LC3 in intestinal crypts. Scale bars, 5 μm. (D) LC3+ vesicle diameter measurements. (E) Immunofluorescence of LC3 and lysozyme in S. Typhimurium–infected intestinal crypts. A Paneth cell is outlined. Arrows indicate a lysozyme-filled LC3+ vesicle; arrowheads indicate an autophagosome that does not contain lysozyme. Scale bars, 5 μm. (F) Colocalization of LC3 and lysozyme in intestinal crypts from S. Typhimurium–infected mice. Each point represents one lysozyme granule. (G) Coimmunoprecipitation (IP) of intestinal lysates using the indicated antibodies. Immunoblot (IB) was performed with anti-lysozyme antibody. IgG, immunoglobulin G. (H) Transmission electron microscopy of Paneth cells from uninfected (-S. Tm) and infected (+S. Tm) mice. Asterisks indicate secretory granules; arrowheads indicate surrounding membranes. (I) Immunofluorescence of lysosomes (cathepsin D+), LC3, and lysozyme in S. Typhimurium–infected intestinal crypts. Arrows indicate a lysozyme-filled LC3+ vesicle with no lysosome (cathepsin D) signal; arrowheads indicate lysosomes that are not coincident with lysozyme-filled LC3+ vesicles. Scale bars, 5 μm. (J) Quantification of lysosome (cathepsin D), LC3, and lysozyme colocalization in (I). Each data point represents one lysozyme-containing granule. Two points connected by a line represent the same granule. The dashed line denotes the limit of strong colocalization. (K) Immunofluorescence of LC3 and lysozyme in intestinal crypts. Scale bars, 10 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; one-way analysis of variance (ANOVA) [(B) and (D)]. S. Tm, Salmonella Typhimurium; LYZ, lysozyme.

  • Fig. 2 Lysozyme is secreted via secretory autophagy during bacterial infection.

    (A) Immunoblot of intracellular and secreted fractions of ex vivo small intestinal crypts. Crypts were treated as indicated, and blots were detected with an anti-lysozyme antibody. (B) Quantification of data in (A). (C) Bacterial killing assay against S. Typhimurium using the secreted fraction from (A). (D) Immunoblot of intracellular and secreted fractions of ex vivo small intestinal crypts from wild-type and Atg16L1T300A mice. Crypts were treated as indicated, and blots were detected with an anti-lysozyme antibody. (E) Quantification of data in (D). P values are relative to control group. (F) Bacterial killing assay against S. Typhimurium using the secreted fraction from (D). (G) Immunofluorescence of LC3 and lysozyme in intestinal crypts of S. Typhimurium–infected wild-type and T300A mice. Scale bars, 5 μm. (H) Quantification of LC3 and lysozyme colocalization in (G). Each data point represents one lysozyme-containing granule. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; one-way ANOVA [(B), (C), (E), and (F)]; Student’s t test (H). BFA, brefeldin A; Chloro, chloroquine; 3-MA, 3-methyladenine; T300A, Atg16L1T300A mice; WT wild type.

  • Fig. 3 ER stress caused by invasive bacteria triggers secretory autophagy.

    (A) Immunofluorescence of LC3 and lysozyme in intestinal crypts of germ-free (GF) mice inoculated with the indicated bacterial strains. (B) Quantification of LC3 and lysozyme colocalization in (A). Each data point represents one lysozyme-containing granule. PBS, phosphate-buffered saline. (C) Representative immunoblot of small intestines from mice treated as indicated, with detection of CHOP. (D) Immunofluorescence of LC3 and lysozyme in intestinal crypts of mice treated as indicated. (E) Quantification of LC3 and lysozyme colocalization in (D). Each data point represents one lysozyme granule. (F) Representative immunoblot of small intestines from infected and uninfected mice, with detection of PERK and eIF2α. (G) Immunofluorescence detection of LC3 and lysozyme in crypts of uninfected mice treated with vehicle or salubrinal. (H) Quantification of LC3 and lysozyme colocalization in (G). Each data point represents one lysozyme granule. (I) Bacterial burdens [colony-forming units (CFU)] in intestinal contents, MLNs, liver, and spleen of mice infected with S. Typhimurium and treated as indicated. Each data point represents one mouse, and geometric means are shown. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant; one-way ANOVA [(B), (C), (E), and (I)]; Student’s t test (H). Scale bars [(A), (D), and (G)], 5 μm. CHOP, C/EBP homologous protein; TUDCA, tauroursodeoxycholic acid; PERK, protein kinase RNA-like endoplasmic reticulum kinase; eIF2α, elongation initiation factor 2α; MLN, mesenteric lymph nodes.

  • Fig. 4 A DC-ILC3 circuit controls secretory autophagy in Paneth cells.

    (A) Immunofluorescence of LC3 and lysozyme in intestinal crypts of S. Typhimurium–infected mice. (B) Quantification of LC3 and lysozyme colocalization in (A). Each data point represents one lysozyme granule. (C) Quantification of intestinal crypts displaying a diffuse lysozyme signal. P values in black are relative to the wild-type group; the P value in red is relative to MyD88−/− and Myd88ΔDC mice. (D) Immunofluorescence of LC3 and lysozyme in small intestinal crypts of S. Typhimurium–infected mice. (E) Quantification of LC3 and lysozyme colocalization in (D). Each data point represents one lysozyme granule. (F) Immunofluorescence of LC3 and lysozyme in intestinal crypts of S. Typhimurium–infected mice. (G) Quantification of LC3 and lysozyme colocalization in (F). Each data point represents one lysozyme granule. (H) Quantification of small intestinal crypts displaying a diffuse lysozyme signal. Error bars represent SEM. *P < 0.05; ****P < 0.0001; Student’s t test (E); one-way ANOVA [(B) and (G)]; two-way ANOVA [(C) and (H)]. Scale bars [(A), (D), and (F)], 5 μm.

Supplementary Materials

  • Paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine

    Shai Bel, Mihir Pendse, Yuhao Wang, Yun Li, Kelly A. Ruhn, Brian Hassell, Tess Leal, Sebastian E. Winter, Ramnik J. Xavier, Lora V. Hooper

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S14
    • References

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