ATG16L1 governs placental infection risk and preterm birth in mice and women

Abstract

The placenta is a barrier against maternal-fetal transmission of pathogens. Placental infections can cause several adverse pregnancy outcomes, including preterm birth (PTB). Yet, we have limited knowledge regarding the mechanisms the placenta uses to control infections. Here, we show that autophagy, a cellular recycling pathway important for host defense against pathogens, and the autophagy gene Atg16L1 play a key role in placental defense and are negatively associated with PTB in pregnant women. First, we demonstrate that placentas from women who delivered preterm exhibit reduced autophagy activity and are associated with higher infection indicators. Second, we identify the cellular location of the autophagy activity as being in syncytial trophoblasts. Third, we demonstrate that higher levels of autophagy and ATG16L1 in human trophoblasts were associated with increased resistance to infection. Accordingly, loss of autophagy or ATG16L1 impaired trophoblast antibacterial defenses. Fourth, we show that Atg16l1-deficient mice gave birth prematurely upon an inflammatory stimulus and their placentas were significantly less able to withstand infection. Finally, global induction of autophagy in both mouse placentas and human trophoblasts increased infection resistance. Our study has significant implications for understanding the etiology of placental infections and prematurity and developing strategies to mitigate placental infection-induced PTB.

Figure 1. Autophagic activity is increased in preterm placentas.

(A) Representative images of immunohistochemical staining of LC3 and P62 in placentas of early preterm (n = 10), late preterm (n = 10), and term deliveries (n = 20). Scale bar: 200μm. (B) Quantification of LC3 and P62 immunohistochemical staining. Staining images were examined and scored in a blinded fashion. Intensity of staining was scored from 1 (low) to 5 (high, P62) or 6 (high, LC3). (C) Western blot analysis of LC3-II, P62, ATG7, ATG16L1, BECLIN-1, and ACTIN from human placental samples from the indicated groups. (D–H) Quantification of indicated autophagy proteins normalized to ACTIN. (I) White blood cell (WBC) counts of patients in indicated groups. Data are expressed as mean ± SEM in B–I. *P < 0.05, **P < 0.01 using Kruskal-Wallis test with Dunnett’s post-test.

Figure 2. The baseline level of autophagy is higher in syncytialized than nonsyncytialized human trophoblasts.

(A) Immunohistochemical staining of LC3 in a representative human term placental villus sample. Scale bar: 100 μm. Inset bar: 10 μM. (B) Western blot detection and quantification of LC3 levels in BeWo CTBs and STBs. (C) Immunofluorescent localization of the autophagy marker LC3 (green punctae) (arrow). Dashed line outlines STBs, solid lines outline CTBs. Blue, DAPI. Scale bar: 10 μm. Data are expressed as mean ± SEM.*P < 0.05 using Kruskal-Wallis test with Dunnett’s post-test.

Figure 3. Autophagy contributes to STB resistance to bacterial colonization.

(A) Immunofluorescence microscopy of CTBs (BeWo) and STBs (forskolin-treated BeWo) exposed to E. coli (red). STBs are marked by expression of human chorionic gonadotropin β subunit (hCG b, green), and cell membranes were marked by E-cadherin (magenta). The number of intracellular E. coli per trophoblast cell nucleus is shown. n = 200 nuclei/group. ***P < 0.001 by Mann-Whitney U test. (B) Number of intracellular E. coli determined by counting colony-forming units in STBs relative to CTBs (set at 100%). ***P < 0.001 by Mann-Whitney U test. (C) Western blot detection of LC3 in DMSO-, rapamycin- (Rap-), and 3-MA–treated CTBs and STBs. Bafilomycin (Baf) treatment indicates that enhanced LC3 activity is due to autophagic flux. (D) CFU analysis of intracellular bacteria number in CTBs upon rapamycin and 3-MA treatment. CFU counts were compared with DMSO-treated CTBs. Scale bar: 50 μm. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 using Kruskal-Wallis test with Dunnett’s post-test.

Figure 4. ATG16L1 in STBs regulates resistance to infection.

(A) Western blot detection of autophagy proteins ATG16L1, ATG7, BECLIN-1 and LC3 in BeWo CTBs and STBs and quantification of ATG16L1 protein levels in CTBs and STBs. Mean values ± SEM of 3 independent experiments. (B) Western blot detection of ATG16L1 in STBs after exposure to control or ATG16L1-specific siRNA. (C) Quantification of bacterial load in STBs treated with control or ATG16L1-specific siRNA. The graph shows mean values ± SEM of 4 independent experiments. *P < 0.05, **P < 0.01 by Mann-Whitney U test.

Figure 5. Atg16l1-deficient mice are susceptible to placental infection and LPS-induced preterm birth.

(A) Schematic representation of mouse placental explant infections. (B) Western blot detection of ATG16L1 in placentas of the indicated genotype. (C) Quantification of intracellular E. coli load in placental explants of the indicated genotypes normalized to WT at 100%. *P < 0.05, **P < 0.01 by Kruskal-Wallis test with Dunnett’s post-test. (D) Western blot detection of LC3 and P62 in DMSO- and rapamycin-treated placental explants. (E) Quantification of intracellular E. coli load in DMSO- and rapamycin-treated WT, HET, and HM placental explants standardized to DMSO-treated WT. *P < 0.05 by Kruskal-Wallis test with Dunnett’s post-test. (F) Day of delivery in WT, HET, and HM mice injected with PBS or LPS on day 15.5 of pregnancy. Data are expressed as mean ± SEM. *P < 0.001 by 2-way ANOVA with a Bonferroni post-test.