Targeting Inhibitor of Apoptosis Proteins Protects from Bleomycin-Induced Lung Fibrosis

Abstract

Accumulation of apoptosis-resistant fibroblasts is a hallmark of pulmonary fibrosis. We hypothesized that disruption of inhibitor of apoptosis protein (IAP) family proteins would limit lung fibrosis. We first show that transforming growth factor-β1 and bleomycin increase X-linked IAP (XIAP) and cellular IAP (cIAP)-1 and -2 in murine lungs and mesenchymal cells. Functional blockade of XIAP and the cIAPs with AT-406, an orally bioavailable second mitochondria-derived activator of caspases (Smac) mimetic, abrogated bleomycin-induced lung fibrosis when given both prophylactically and therapeutically. To determine whether the reduction in fibrosis was predominantly due to AT-406-mediated inhibition of XIAP, we compared the fibrotic response of XIAP-deficient mice (XIAP(-/y)) with littermate controls and found no difference. We found no alterations in total inflammatory cells of either wild-type mice treated with AT-406 or XIAP(-/y) mice. AT-406 treatment limited CCL12 and IFN-γ production, whereas XIAP(-/y) mice exhibited increased IL-1β expression. Surprisingly, XIAP(-/y) mesenchymal cells had increased resistance to Fas-mediated apoptosis. Functional blockade of cIAPs with AT-406 restored sensitivity to Fas-mediated apoptosis in XIAP(-/y) mesenchymal cells in vitro and increased apoptosis of mesenchymal cells in vivo, indicating that the increased apoptosis resistance in XIAP(-/y) mesenchymal cells was the result of increased cIAP expression. Collectively, these results indicate that: (1) IAPs have a role in the pathogenesis of lung fibrosis; (2) a congenital deficiency of XIAP may be overcome by compensatory mechanisms of other IAPs; and (3) broad functional inhibition of IAPs may be an effective strategy for the treatment of lung fibrosis by promoting mesenchymal cell apoptosis.

Keywords: X-linked inhibitor of apoptosis protein; fibroblast; fibrocyte; mesenchymal; second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein–binding protein with low pI.

Figure 1.

Transforming growth factor (TGF)-β treatment increased the expression of X-linked inhibitor of apoptosis proteins (XIAPs) and cellular inhibitor of apoptosis proteins (cIAPs) in fibroblasts and fibrocytes. Fibroblasts and fibrocytes from wild-type mice were treated with TGF-β (2 ng/ml) for 48 hours. Total RNA was isolated, and we measured the expression of XIAP (A and D), cIAP-1 (B and E) and -2 (C and F), and β-actin in fibroblasts (A–C) and fibrocytes (D–F) by real-time RT-PCR. Data represent cells collected from n = 3 mice total from two different experiments. ***P < 0.001, **P < 0.01, and *P < 0.05.

Figure 2.

Blockade of IAPs with AT-406 inhibits lung collagen accumulation on Day 21 after bleomycin (Bleo) treatment and decreases lung levels of chemokine (C-C motif) ligand-12 (CCL12). (A) Wild-type (WT) mice were given 1.15 U/kg of Bleo or PBS intratracheally on Day 0. AT-406 (100 mg/kg by oral gavage) was administered daily through Day 20 to half of the mice, and vehicle control (PBS) was administered to the other half. Lungs were harvested on Day 21 for hydroxyproline quantification. (B) Histochemical staining showing representative lungs of mice treated with vehicle control (PBS), Bleomycin (Bleo) or Bleo with AT-406. Shown are hematoxylin and eosin (H&E) and Picrosirius red (PS). Magnification was via 4×, 10×, and 20× objectives from left to right. (C) On Day 7, lungs were harvested, digested, and total lung leukocytes were enumerated. (D) Differential analysis of leukocytes was done to determine the percentage of monocytes (Mono)/macrophages (Macs), lymphocytes (Lymphs), neutrophils (PMNs), and eosinophils (Eos). Data shown represents mean ± SEM; n = 3 animals/group; ns, not significant. (E) Leukocytes were plated at 3 × 10⁶ cells/ml in serum-free media overnight. Cell-free supernatants were analyzed by ELISA for IL-1β, (F) CCL12, (G) TNF-α, and (H) IFN-γ. Data shown are pooled from two independent experiments, with n = 4–6 mice per group in each experiment. ****P < 0.0001, **P < 0.01, *P < 0.05.

Figure 3.

Therapeutic administration of AT-406 limits lung fibrosis. (A) WT mice were given 1.15 U/kg of Bleo or PBS intratracheally on Day 0. Half of each group received AT-406 (100 mg/kg) by oral gavage daily starting on Day 10 through Day 20, and lungs were harvested on Day 21 for hydroxyproline quantification. (B) Histochemical staining showing representative lungs of mice treated with intratracheal PBS and vehicle control (PBS), intratracheal Bleo and AT-406, intratracheal Bleo and vehicle control, or intratracheal Bleo and AT-406. Shown are H&E and PS staining. Magnification was via 20× objective. Data shown represent mean ± SEM, n = 4–6 mice/group. ****P < 0.0001 and *P < 0.05.

Figure 4.

AT-406 augments myofibroblast apoptosis in vivo after Bleo injury. WT mice were given 1.15 U/kg of Bleo or PBS intratracheally on Day 0. On Days 11, 12, and 13, half of each group was administered AT-406 (100 mg/kg via oral gavage), and the lungs were harvested on Day 13. (A) Whole-lung homogenates were assessed for activation of caspase 3/7. P < 0.01 for Bleo/AT-406 compared with controls. (B–H) Lung sections were costained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (C and F) and α-smooth muscle actin (SMA; D and G). Cells that were positive for both TUNEL and α-SMA were quantified using merged images (E and H) in four random 20× objective fields by an investigator who was blinded to the treatment groups. This quantification (B) demonstrated significantly more copositive cells in Bleo/AT-406 lung sections compared with Bleo/PBS lung sections. P = 0.01. Costained cells in representative merged images (E and H) are indicated by arrows. Data shown represent mean ± SEM.

Figure 5.

XIAP-deficient mice are not protected from Bleo-induced pulmonary fibrosis and show elevated levels of IL-1β. WT littermate (XIAP^+/+) or XIAP-deficient (XIAP^−/y) mice were given Bleo or PBS intratracheally on Day 0. Lungs were harvested on Day 7. Lung minces were then digested in collagenase, total lung leukocytes were enumerated, and lung mesenchymal cells were cultured. (A) XIAP mRNA expression was assessed in cultured lung mesenchymal cells by RT-PCR. (B) On Day 21, lungs were harvested for hydroxyproline quantification. (C). Representative H&E staining from Day 21. Magnification was through the 10× (top) and 20× (bottom) objectives. Representative of n = 4 mice examined. (D) Total lung leukocytes from Day-7 lung minces were enumerated. (E) Differential analysis was done to determine the percentage of Mono/Macs, Lymphs, PMNs, and Eos. Data shown represent n = 6–10 mice per group pooled from three independent experiments. Cells were plated at 3 × 10⁶ cells/ml in serum-free media overnight, and cell-free supernatants were analyzed by ELISA for IL-1β (F), TNF-α (G), CCL12 (H), and IFN-γ (I). Data shown represent mean ± SEM; n = 4–6 mice/group. ****P < 0.0001, **P < 0.01.

Figure 6.

XIAP^−/y mesenchymal cells have increased cIAP expression associated with decreased susceptibility to Fas-mediated apoptosis, and inhibition of cIAPs enhances their apoptosis. (A) Lung fibroblasts from WT and XIAP^−/y mice were treated with/without Fas-activating antibody (250 ng/ml) along with Cellplayer kinetic caspase 3/7 reagent. Plates were loaded into the IncuCyte incubator (Essen Bioscience, Ann Arbor, MI) and photographed every 2 hours for 24 hours. Apoptosis was quantified by automated counting “objects per well” (object = green fluorescence indicating cleavage of a fluorogenic substrate by caspase 3/7). (B and C) Total RNA was isolated from fibroblasts cultured from the lungs of XIAP^+/+ and XIAP^−/y mice treated with/without Bleo and expression of cIAP-1 (A), cIAP-2 (B), and β-actin was measured by real-time RT-PCR. Data represent n = 3 per group pooled from multiple mice. **P < 0.01. (D and E) RNA isolated from lung homogenates of untreated XIAP^+/+ and XIAP^−/y mice were assessed for cIAP-1 and -2. Data represent n = 3 per group pooled from multiple mice. *P < 0.05. (F) Lung fibroblasts from XIAP^−/y were treated with/without Fas-activating antibody (250 ng/ml) and/or AT-406 (1.0 μM). Apoptosis was evaluated as described above with n = 3 wells/group. *P < 0.05 versus control and AT-406 and **P < 0.01 versus Fas treatment. Data represent mean ± SEM; n = 3 wells/treatment per group, with nine images per well at each time point.

Figure 7.

AT-406 decreases Bleo-induced lung fibrosis in XIAP^−/y mice. (A–I) XIAP^+/+ or XIAP^−/y mice were given Bleo or PBS intratracheally on Day 0. Lungs were harvested and assessed for inflammation on Day 7 and fibrosis on Day 21, as described in Figure 5. (A) Hydroxyproline quantification at Day 21. Data shown represent mean ± SEM; n = 7–11 mice/group. ****P < 0.0001, *P < 0.05. (B–E) Leukocyte differentials in lung collagenase digest on Day 7 for macrophages (B), lymphocytes (C), neutrophils (D), and eosinophils (E); n = 3 lungs per group. *P < 0.05. (F–G) Cytokines in cell-free supernatants from Day-7 leukocytes for IL-1β (F) and CCL12 (G).