Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection

Abstract

Background & aims: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice.

Methods: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells.

Results: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3β (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1.

Conclusions: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

Keywords: AMP; ATG; Autophagy; Discosoma sp. red fluorescent protein; EGFP; FAE; GFP; IL; Intestinal Barrier; LC3; MEF; MLN; Map1lc3b; Mouse Model; Mucosa; SCV; Salmonella-containing vacuole; TFP; antimicrobial peptide; autophagy-related gene; dsRed; enhanced green fluorescent protein; follicle-associated epithelium; green fluorescent protein; interleukin; light chain 3; mRNA; mesenteric lymph nodes; messenger RNA; microtubule-associated protein 1 light chain 3b; mouse embryonic fibroblast; trifluoperazine.

Figure 1. Systemic and cell-specific deletion of Atg16l1 expression

(A) Schematic of Atg16l1 gene targeting as designed by the International Knockout Mouse Consortium. (B) Western blot of MEFs isolated at E13 showing expression of Atg16l1, p62, LC3B, and actin. (C) Atg16l1 conditional knockout (CKO) mice were generated by crossing Atg16l1^f/f with Villin-cre or CD11c-cre mice. Western blots demonstrate expression levels of Atg16l1, p62, LC3B, and actin in intestinal epithelial cells (left) and CD11c⁺ cells (right).

Figure 2. LC3 expression is induced following in vivo S Typhimurium infection

(A–B) Mice were infected with S Typhimurium and tissues were harvested after 24 hours. Terminal ileum (A) and cecum (B) were immunostained with DAPI (blue) and anti-LC3 (green) and imaged by confocal microscopy. Uninfected wild-type tissues are shown on the left. (C) LC3B mRNA levels from isolated wild-type ileal and cecal epithelial cells were determined by qPCR. Numbers shown are relative to streptomycin-only-treated wild-type mice. Bars indicate mean plus S.D. *P ≤ .01. Data are representative of at least 3 independent experiments (n = 9).

Figure 3. S Typhimurium colocalization with LC3 in intestinal epithelial cells requires Atg16l1

(A) GFP-LC3 mice were infected with dsRed-expressing S Typhimurium and cecal tissues were imaged in vivo by confocal microscopy after 24 hours. Colocalization (arrow) of S Typhimurium (red) and GFP-LC3 (green) is shown. LC3-positive vacuoles contained single (upper panels) or multiple (lower panels) Salmonella. (B–C) Wild-type (B) and Atg16l1^f/fx Villin-cre (C) mice were infected with S Typhimurium and cecal tissues were harvested after 24 hours. Cecal patches were stained with DAPI (blue), anti-Salmonella (red), and anti-LC3 (green). Colocalization of LC3 and Salmonella shown in yellow (arrows). Upper panels show FAE; bottom panels focus deep within the cecal patch/follicle. Data are representative of at least 3 independent experiments (n ≥ 4).

Figure 4. Mice lacking epithelial Atg16l1 have Paneth cell abnormalities

(A) Terminal ileum from wild-type mice was stained with DAPI (blue), anti-Salmonella (red), and anti-LC3 (green) 24 hours after mock or S Typhimurium infection. (B) Gene expression in terminal ileum 72 hours after streptomycin treatment (Strep only) or after 24 hours streptomycin and 48 hours S Typhimurium infection (Infected). Numbers shown are relative to streptomycin-only treated wild-type mice. Bars indicate mean plus S.D. * P ≤ .01. (C) Terminal ileum of uninfected (top) and S Typhimurium-infected (bottom) mice were immunostained with DAPI (blue) and Lysozyme (red) 48 hours post-infection. Dotted lines denote the laser capture zone; arrows indicate presence of lysozyme-positive cells outside this zone. Scale bars = 100 μm. (D) Lysozyme-positive cells per crypt within the laser capture zone were quantified 48 hours post-infection (n=2 mice per group; 25 crypts per mouse). (E) AMP expression by qPCR from laser capture zone shown in C. Numbers shown are relative to streptomycin-only treated wild-type mice. (F) Light microscopy images of Periodic acid-Schiff-stained sections of terminal ileum from uninfected (top) and S Typhimurium-infected (bottom) mice (60X magnification) 48 hours post-infection. Scale bars = 50 μm. Data are representative of at least 2 independent experiments (n ≥ 2).

Figure 5. Mice lacking epithelial Atg16l1 are more susceptible to S Typhimurium infection

(A) Cecal tissues were harvested from uninfected (top) and S Typhimurium-infected (bottom) mice 48 hours post-infection. Representative H&E-stained sections are shown (20X magnification). (B) Cecal tissue sections were blindly scored for inflammation 48 hours post-infection with S Typhimurium. (C) Cytokine levels in cecum and terminal ileum tissues were assessed via qPCR in S Typhimurium-infected mice 48 hours post-infection. Fold expression shown relative to wild-type. (D) Chemokine levels in isolated ileal epithelial cells were assessed via qPCR in S Typhimurium-infected mice 48 hours post-infection. Fold expression shown relative to wild-type. (E) S Typhimurium translocation to MLN and spleen was quantified 48 hours post-infection. Total CFUs per organ are displayed. In B–E, bars indicate mean plus S.D. * P = .002; ** P ≤.001. Data are representative of at least 3 independent experiments (n ≥ 12).

Figure 6. Trifluoperazine induces antibacterial autophagy in epithelial cells

(A) Left: HeLa cells infected with Salmonella were incubated with indicated compound and gentamycin for 16 hours and CFUs were quantified. Right: Percent viability of HeLa cells 16 hours after compound treatment was assessed by luminescence measurement following addition of an equal volume of CellTiter-Glo to cells in media. Error bars represent mean plus S.D. of triplicate samples. (B) HeLa cells infected with S Typhimurium were incubated with 10 μM TFP and 20 μg/ml gentamicin for 16 h and CFUs were quantified. Bars indicate means plus S.D. of three independent experiments in triplicate. (C) HeLa cells transfected with indicated siRNA were treated for 8 hours with TFP in the presence or absence of lysosomal inhibitors. Western blot demonstrates expression levels of Atg16l1, actin, and LC3B. (D) HeLa cells stably expressing EGFP-LC3 were treated with TFP for 3 hours. Cells were then infected with S Typhimurium, followed by incubation with gentamycin sulfate and TFP. Numbers of EGFP-LC3-positive bacteria were calculated as a percentage of total bacteria. (E) Bioluminescent Salmonella-infected HeLa cells were treated with TFP. Intracellular bacteria were quantified by luminescence 24 hours post-infection. Error bars represent means plus S.D. from two independent experiments in quadruplicate. (F) Intracellular bioluminescent Salmonella were quantified by luminescence at indicated times following infection and treatment with 10 μM TFP. Error bars represent means plus S.D. from two independent experiments in quadruplicate.