Autophagy Limits Inflammasome During Chlamydia pneumoniae Infection

Abstract

Autophagy can either antagonize or promote intracellular bacterial growth, depending on the pathogen. Here, we investigated the role of autophagy during a pulmonary infection with the obligate intracellular pathogen, Chlamydia pneumoniae (CP). In mouse embryonic fibroblasts (MEFs) or macrophages, deficiency of autophagy pathway components led to enhanced CP replication, suggesting that autophagy exerts a bactericidal role. However, in vivo, mice with myeloid-specific deletion of the autophagic protein ATG16L1 suffered increased mortality during CP infection, neutrophilia, and increased inflammasome activation despite no change in bacterial burden. Induction of autophagy led to reduced CP replication in vitro, but impaired survival in CP-infected mice, associated with an initial reduction in IL-1β production, followed by enhanced neutrophil recruitment, defective CP clearance, and later inflammasome activation and IL-1β production, which drove the resulting mortality. Taken together, our data suggest that a delicate interplay exists between autophagy and inflammasome activation in determining the outcome of CP infection, perturbation of which can result in inflammatory pathology or unrestricted bacterial growth.

Keywords: Chlamydia pneumoniae; IL-1β; autophagy; inflammasome; macrophages.

Figure 1

Autophagy plays a role during CP growth. (A,B) WT and Atg16l1^+/− MEFs were infected with CP (MOI 10) and 68 h later assessed for inclusions (A) and IFUs (B). (C,D) Atg16l1^fl/fl and Atg16l1^fl/fl Lysm^cre BMDM were infected with CP (MOI 1) and 68 h later assessed for inclusions (C) and IFUs (D). (E–G) WT and Atg5^−/− MEFs were infected with CP (MOI 10) and 68 h later assessed for inclusions (E), IFUs (F), and inclusion size (G). (H) Atg5^fl/fl and Atg5^fl/fl Lysm^cre BMDM were infected with CP (MOI 1) and 68 h later assessed for inclusions (C). All panels are representative of three experiments. Significance was assessed by students t-test. ^**p < 0.01, ^***p < 0.001.

Figure 2

CP growth requires adequate nutrition available to the host. (A) WT, Atg16l1^+/−, and Atg5^−/− MEFs were seeded at 5 × 10⁴ in a 24-well plate. Total cell numbers were counted after 3 days. (B,C) Atg5^−/− MEFs were infected with CP (MOI 10) and treated with 1 μg/ml cycloheximide. Inclusions were counted 68 h post infection (B), as was inclusion size (C). (D) WT, Atg16l1^+/−, and Atg5^−/− MEFs were seeded at the indicated amounts in a 24-well plate and infected with CP (MOI 10). Inclusions were counted 68 h post infection. (E) Atg5^−/− MEFs were infected with CP (MOI 10) and the media was supplemented with either MEM amino acids (AA), Na-pyruvate (NaPyr), or both. Inclusions were counted 68 h post infection. All panels are representative of three experiments. Data are mean ± SEM. Significance was assessed by students t-test (B,C) or one-way ANOVA (A,E). ^*p < 0.05, ^***p < 0.001, ^**** p < 0.0001.

Figure 3

Loss of autophagy in myeloid cells leads to neutrophilia and mortality during CP infection. (A) Atg16l1^fl/fl and Atg16l1^fl/fl Lysm^cre mice were infected with 2.5 × 10⁶ IFU CP i.t. and mortality was assessed (n = 5–6). (B–L) Atg16l1^fl/fl and Atg16l1^fl/fl Lysm^cre mice were infected with 1.0 × 10⁶ IFU CP i.t. and mice were sacrificed 3, 5, and 9 days after infection (n = 9–13, pooled from two experiments). (B) Bacterial burden in the lungs. (C) Total cells in the BALF. (D) BALF Alveolar macrophages. (E) BALF T-cells. (F) BALF neutrophils. (G) BALF DCs. (H) BALF IFN-γ. (I) BALF IL-6. (J) BALF IL-12p40. (K) BALF CXCL1. (L) BALF CXCL2. Data are mean ± SEM. Significance was assessed by students t-test. Survival curve was compared using the Log-Rank (Mantel-Cox) Test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 4

Loss of autophagy in myeloid cells leads to an increase in inflammasome activation and IL-1β during CP infection. (A,B) Atg16l1^fl/fl and Atg16l1^fl/fl Lysm^cre peritoneal macrophages were infected with CP at indicated MOI and (A) IL-1β was measured in the supernatants by ELISA 20 h later. (B) % FLICA positive cells. (C) IL-1β in BALF 1 day after CP infection in Atg16l1^fl/fl and Atg16l1^fl/fl Lysm^cre mice (n = 9–11 pooled from two separate experiments). (D) Caspase-1 activity measured by FLICA in frozen lung sections 1 day after CP infection in Atg16l1^fl/fl and Atg16l1^fl/fl Lysm^cre mice (n = 7). Data are mean ± SEM. Significance was assessed by students t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

Autophagy induction fails to rescue mice from CP infection. WT MEFs were infected with CP (MOI 10) and treated with 10 μM Tamoxifen during the first 2 h of infection. (A) CP inclusions. (B) CP progeny. (C) C57Bl/6 mice were infected with 2 × 10⁶ IFU CP. Six hour prior to infection, mice received either sham or 3 mg/kg rapamycin i.p., and again every other day. Mortality was assessed (n = 5–9). (D–J) C57Bl/6 mice were infected with 1 × 10⁶ IFU CP. Six hour prior to infection, mice received either sham or 3 mg/kg rapamycin i.p., and again every other day (n = 8–12, pooled from two separate experiments). Mice were sacrificed on days 3, 6. (D) Total cells in the BALF. (E) BALF Alveolar macrophages. (F) BALF neutrophils. (G) BALF DCs. (H) BALF T-cells. (I) Bacterial burden in the lungs. (J) IL-1β in the BALF after CP infection and rapamycin treatment. (K) Lung inflammation score on day 6. Significance was assessed by students t-test. Survival curves was compared using the Log-Rank (Mantel-Cox) Test. Data are mean ± SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.