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.
The mammalian intestine is home to a diverse population of bacteria, which includes pathogens that can disrupt host cellular functions. The intestinal epithelium defends against bacterial encroachment through multiple mechanisms, including antimicrobial protein secretion and destruction of invading bacteria through autophagy (
Invasive bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), trigger autophagy in intestinal enterocytes. This is indicated by abundant epithelial cell autophagosomes, marked by LC3 (microtubule-associated protein
1 light chain 3), that capture and eliminate invading bacteria (
To characterize the contents of the LC3+ vesicles, we performed immunofluorescence, transmission electron microscopy, and coimmunoprecipitation assays. These assays revealed that the large LC3+ vesicles contained lysozyme (Fig. 1, E to G) and were absent in Paneth cells of uninfected and fasted mice, where lysozyme was packaged into LC3– vesicles (fig. S2). Ultrastructure analysis showed that the large granules in Paneth cells of infected mice contained lysozyme (fig. S3) and were surrounded by a double membrane (Fig. 1H), a hallmark of autophagosomes (
Canonical autophagy targets the cargo in LC3+ autophagosomes for degradation in lysosomes (
During conventional protein secretion, proteins are transported through the ER–Golgi complex, packaged in secretory granules, and released to the extracellular space. There are various alter- native secretory pathways, including one that utilizes components of the autophagy pathway and is known as secretory autophagy (
To further test this idea, we isolated Paneth cell–containing crypts, infected them in vitro while treating with chemical inhibitors of conventional secretion and autophagy, and analyzed the supernatants for lysozyme secretion. Inhibiting ER-Golgi trafficking with brefeldin A (BFA) did not affect lysozyme secretion in infected or uninfected crypts (Fig. 2, A and B), indicating that lysozyme secretion can bypass the ER-Golgi pathway. Lysozyme secretion was also not altered by treatment with chloroquine (Fig. 2, A and B), which prevents lysosome acidification (
(Fig. 2, A and B). Accordingly, secretions from BFA-treated but not 3-MA–treated crypts killed bacteria (Fig. 2C), indicating that secretory autophagy is essential for antibacterial defense in infected crypts.
We next studied mice in which autophagy is perturbed by a mutation in the Atg16L1 (autophagy related 16–like 1) gene. A mutation in the Atg16L1 gene [Thr300→Ala300 (T300A)] confers an increased risk of developing Crohn’s disease in humans (
We next sought to identify the cellular signals that trigger secretory autophagy. S. Typhimurium disrupts the ER-Golgi complex in infected cells and thus interferes with conventional secretion (
We next investigated the intracellular signaling pathways that link ER stress to secretory autophagy in Paneth cells. When cells sense ER stress, PERK (protein kinase RNA-like endoplasmic reticulum kinase) is activated by phosphorylation. p-PERK then phosphorylates elongation initiation factor 2a (eIF2a), which inactivates eIF2a and attenuates translation (
We next tested whether inhibiting secretory autophagy would compromise intestinal defense against oral S. Typhimurium infection. Inhibiting secretory autophagy by TUDCA treatment of S. Typhimurium–infected mice led to increased numbers of S. Typhimurium in the intestine, mesenteric lymph nodes (MLNs), liver, and spleen (Fig. 3I). Lysozyme gavage of TUDCA-treated infected mice rescued the increased bacterial burden (Fig. 3I), suggesting that the increased bacterial numbers were not due to other effects of TUDCA. Thus, secretory autophagy is essential for host defense against invasive bacteria.
Activation of antibacterial autophagy in intestinal enterocytes requires epithelial cell expression of the Toll-like receptor (TLR) signaling adaptor MyD88 (
The requirement for DC Myd88 suggested the involvement of a known cellular relay in which DC TLRs capture bacterial signals and relay them to epithelial cells via type 3 innate lymphoid cells (ILC3) and their secretion of interleukin-22 (IL22) (
Our results illuminate how the intestine preserves antimicrobial function in the face of a pathogenic bacterial infection (fig. S13) and suggest how simultaneous disruption of both ER stress and autophagy pathways leads to severe inflammation in mice (
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ACKNOWLEDGMENTS
We thank B. Levine for discussions and C. L. Behrendt-Boyd for assistance with mouse experiments. This work was supported by the NIH (grant DK070855 to L.V.H.; grants AI118807 and AI128151 to S.E.W.), the Burroughs Wellcome Foundation (Investigators in the Pathogenesis of Infectious Diseases Award to L.V.H.), the Welch Foundation (grant I-1874 to L.V.H.; grant I-1858 to S.E.W.), and the Howard Hughes Medical Institute (L.V.H.). S.B. was supported by a Gruss-Lipper postdoctoral fellowship.
SUPPLEMENTARY MATERIALS
Materials and Methods
Figs. S1 to S14
References (20–27)
25 November 2016; resubmitted 12 April 2017 Accepted 14 July 2017
Published online 27 July 2017
10.1126/science.aal4677
DIAGRAM: 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 Atg16L1 T300A mice. Crypts were treated as indicated, and blots were detected with an antilysozyme 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 mm. (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.
PHOTO (COLOR): 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 mm. (B) Quantification of LC3+ puncta. Each data point represents one mouse. (C) Immunofluorescence of LC3 in intestinal crypts. Scale bars, 5 mm. (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 mm. (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 mm. (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 mm. *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.
PHOTO (COLOR): 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 eIF2a. (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 mm. CHOP, C/EBP homologous protein; TUDCA, tauroursodeoxycholic acid; PERK, protein kinase RNA-like endoplasmic reticulum kinase; eIF2a, elongation initiation factor 2a; MLN, mesenteric lymph nodes.
PHOTO (COLOR): 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 Myd88DDC 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 mm.