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

Abstract

Intestinal Paneth cells limit bacterial invasion by secreting antimicrobial proteins, including lysozyme. However, invasive pathogens can disrupt the Golgi apparatus, interfering with secretion and compromising intestinal antimicrobial defense. Here we show that during bacterial infection, lysozyme is rerouted via secretory autophagy, an autophagy-based alternative secretion pathway. Secretory autophagy was triggered in Paneth cells by bacteria-induced endoplasmic reticulum (ER) stress, required extrinsic signals from innate lymphoid cells, and limited bacterial dissemination. Secretory autophagy was disrupted in Paneth cells of mice harboring a mutation in autophagy gene Atg16L1 that confers increased risk for Crohn's disease in humans. Our findings identify a role for secretory autophagy in intestinal defense and suggest why Crohn's disease is associated with genetic mutations that affect both the ER stress response and autophagy.

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 1. Large LC3+ vesicles in S. Typhimurium-infected mice contain lysozyme

(A) Immunofluorescence of LC3 in intestinal crypts. Nuclei are stained with DAPI. Scale bars=10 μm. (B) Quantification of LC3+ puncta. Each 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. Arrowhead indicates an autophagosome that does not contain lysozyme. Scale bars=5 μm. (F) Co-localization of LC3 and lysozyme in intestinal crypts from S. Typhimurium-infected mice. Each point represents one lysozyme granule. (G) Co-immunoprecipitation of intestinal lysates using the indicated antibodies. Immunoblot was performed with anti-lysozyme (LYZ) antibody. (H) Transmission electron microscopy (TEM) 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 co-localization in I. Each point represents one lysozyme-containing granule. Two points connected by a line represent the same granule. Dotted line represents limit of strong co-localization. (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 ANOVA (B,D); S. Tm, Salmonella Typhimurium; LYZ, lysozyme.

Figure 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 secreted fraction from A. (D) Immunoblot of intracellular and secreted fractions of ex vivo small intestinal crypts from wild type and Atg16L1T300A (T300A) 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. (H) Quantification of LC3 and lysozyme colocalization in G. Each 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). S. Tm, Salmonella Typhimurium; LYZ, lysozyme; BFA, Brefeldin A; Chloro, chloroquine; 3-MA, 3-methyladenine; T300A, Atg16L1T300A mice.

Figure 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 point represents one lysozyme-containing granule. (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 point represents one lysozyme granule. (F) Representative immunoblot of small intestines from infected and uninfected mice, with detection of PERK and eF2α. (G) Immunofluorescence detection of LC3 and lysozyme in crypts of uninfected mice treated with vehicle or salubrinal. (H) Quantification of LC3 and lysozyme co-localization in G. Each point represents one lysozyme granule. (I) Bacterial burdens (CFU) in intestinal contents, MLNs, liver, and spleen of mice infected with S. Typhimurium and treated as indicated. Each 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; One-way ANOVA (B,C,E and I); Student’s t-test (H). Scale bars=5 μm. S. Tm, Salmonella Typhimurium; LYZ, lysozyme; TUDCA, tauroursodeoxycholic acid; MLN, mesenteric lymph nodes.

Figure 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 co-localization in A. Each point represents one lysozyme granule. (C) Quantification of intestinal crypts displaying a diffuse lysozyme signal. p values in black are relative to WT group. 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 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 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,G) Two-way ANOVA (C,H). Scale bars=5 μm. S. Tm, Salmonella Typhimurium; LYZ, lysozyme.