Critical role for IL-18 in spontaneous lung inflammation caused by autophagy deficiency

Abstract

Autophagy is an important component of the immune response. However, the functions of autophagy in human diseases are much less understood. We studied biological consequences of autophagy deficiency in mice lacking the essential autophagy gene Atg7 or Atg5 in myeloid cells. Surprisingly, these mice presented with spontaneous sterile lung inflammation, characterized by marked recruitment of inflammatory cells, submucosal thickening, goblet cell metaplasia, and increased collagen content. Lung inflammation was associated with increase in several proinflammatory cytokines in the bronchoalveolar lavage and in serum. This inflammation was largely driven by IL-18 as a result of constitutive inflammasome activation. Following i.p. LPS injection, autophagy-deficient mice had higher levels of proinflammatory cytokines in lungs and in serum, as well as increased mortality, than control mice. Intranasal bleomycin challenge exacerbated lung inflammation in autophagy-deficient mice and produced more severe fibrotic changes than in control mice. These results uncover a new and important role for autophagy as negative regulator of lung inflammation.

Fig. 1. Atg7^Δ mice presents with spontaneous lung inflammation

Bone marrow-derived macrophages (BMDM) from wild type (Atg7^f/f) or Atg7^Δ mice were analyzed by immunoblotting (A). Lung tissues from two (B), six (C) or ten (D) month old mice were analyzed by hematoxylin and eosin (H&E) staining. Flow cytometry analysis of single cell suspensions of the lungs was done using the pan leukocytic marker anti-CD45 (E) or anti-Ly6G (F). Quantification graphs are shown. Bronchoalveolar lavage (BAL) from 2–4 months old Atg7^Δ or control Atg7^f/f mice was analyzed for total cell count (G) or differential cell count (H). Mϕ, macrophages; Lymph, lymphocytes; PMN, polymorphonuclear neutrophils. Data are mean ± SEM, n=3 to 8 mice/genotype, *p<0.05, ** p<0.01 Scale bar, 50 μm.

Fig. 2. Mucus hypersecretion and goblet cell metaplasia in Atg7^Δ mice

Periodic acid Schiff staining was performed on lungs from two (A), six (B) or ten (C) months old control Atg7^f/f or Atg7^Δ mice. Quantitative analyses of results are shown (D). Lung tissues from six-month-old mice were immunostained for muc5ac, and the quantitation of positive cells is shown (E). Data are shown as mean ± SEM, n=4 to 8 mice/genotype, *p<0.05 **p<0.01. Scale bar, 50 μm.

Fig. 3. Cytokine upregulation in Atg7^Δ mice

Lungs from Atg7^f/f mice or Atg7^Δ mice were analyzed by ELISpot. Representative images and quantitative analyses for IL-1β (A) or IL-17 (B) are shown. BAL (C) and serum (D) from 2–4 month old Atg7^Δ or control Atg7^f/f mice were analyzed for IL-1β, IL-18, TGF-β and IL-6. Data are shown as mean ± SEM, n = 4–12 mice/genotype, *p<0.05 **p<0.01.

Fig. 4. IL-18 is required for lung inflammation in Atg7^Δ mice

(A–B) Representative images from Atg7^Δ mice treated with saline or Anakinra (upper panels), isotype control or IL-17 antibody treatment (middle panels), or isotype control or IL-18 antibody (lower panels). (A) Lung tissues were analyzed by hematoxylin and eosin staining. (B) BAL was analyzed by HEMA-3 staining. Quantification of the inflammatory score is shown in (C) Total cell count (D) and deferential count (E) were determined. Data are shown as mean ± SEM n=3–13 mice/treatment, *p<0.05 **p<0.01. Scale bar, 50 μm

Fig. 5. Increased cytokine production and reduced survival in Atg7^Δ mice. in response to LPS

(A–C) Following intraperitoneal LPS injection in mice, serum levels of IL-1β (A) IL-18 (B) and IL-17 (C) were evaluated. (D–F) Kaplan-Meier survival curves of mice were recorded following injection of LPS (D–E) or LPS plus anti IL-18 antibody or isotype control (F) and LPS plus interleukin 1 receptor antagonist Anakinra (G). Data are mean ± SEM, n=3 to 6 mice/genotype for each time point *p<0.05, **p<0.01.

Fig. 6. Atg7^Δ mice exhibit exaggerated lung inflammation after LPS challenge

Atg7^f/f or Atg7^Δ mice were injected intraperitoneally with LPS. At the time of death, lung tissues were collected and stained with H&E (A), periodic acid Schiff (B), or Masson’s trichrome (C). The latter was quantified as mean area (MA) per μm of basement membrane (BM). Soluble collagen in lung homogenates was quantified using Sircol assay at zero and six hr following LPS injection (D). IL-1β (E), IL-18 (F), and IL-17 (G) levels were measured in BAL using ELISA at the indicated time points. Data are mean ± SEM from 3 to 6 mice per genotype at each time point.*p<0.05 **p<0.01. Scale bar, 50 μm.

Fig. 7. Atg7^Δ mice exhibit increased lung responses to bleomycin

Atg7^f/f control mice or Atg7^Δ mice were intranasally instilled with PBS or bleomycin for 7 or 14 days. Percent loss of body weight was recorded (A). Lung tissues were analyzed by H&E (B). Ashcroft scoring was used to evaluate the extent of fibrosis (C). Soluble collagen in lung homogenates (D) and BAL (E) was quantified using Sircol assay. Levels of TGF-β (F) and IL-6 (G) were measured using ELISA. Total count of leukocytes was determined (H). Data are shown as mean ± SEM, n= 4–16 mice/genotype, *p<0.05 **p<0.01. Scale bar, 200 μm.