Acute lung injury and fibrosis in a baboon model of Escherichia coli sepsis

Abstract

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-β and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.

Figures

Figure 1.

Histological and physiological changes of the lung during the acute exudative phase of acute respiratory distress syndrome (ARDS) in the baboon model of Escherichia coli sepsis. (A and B) Hematoxylin and eosin (A) and phosphotungstic acid\x{2013}hematoxylin (PTAH) (B) staining show the presence of protein-rich edema fluid filling the alveoli (A and B, asterisks), spotty intra-alveolar hemorrhage (A, arrowheads), and intra-alveolar fibrin deposition (B, blue staining, arrows). (C and D) Electron micrographs, showing (C) neutrophil (N) and macrophage (M) accumulation, and (D) the presence of interstitial fibrin (Fib), suggest the activation of coagulation. (E and F) Immunostaining of elastase-stained neutrophils (E) and CD68-positive macrophages (F) in the lung of healthy control and septic baboons after 24 hours after challenge. Nuclei are shown in blue. (G) Quantitation of neutrophils and macrophages in the lung during the time course of the study. Histogram data are shown as means ± SEM (n = 10 microscopic fields collected from at least three different animals per condition per time point; one-way ANOVA with Dunnett’s multicomparison test, **P < 0.01, ***P < 0.001). (H) Time course changes of plasma myeloperoxidase suggest gradual neutrophil activation and degranulation. (I–K) Biochemical and functional tests demonstrate that E. coli–challenged baboons showed clinical signs of ARDS: abrupt decrease of blood oxygenation (I), increased respiratory rate (tachypnea) (J), and gradual increase in plasma lactate (K). Data are shown as means ± SEM (n = 4). Scale bars, 200 μm (A and B), 10 μm (C and D), 100 μm (E and F).

Figure 2.

Time course changes of cytokine production during acute sepsis and the long-term follow-up in baboons challenged with E. coli. Ethylenediaminetetra-acetic acid plasma samples were collected before challenge (T0), at 2 hours (T + 2 h) and 8 hours (T + 8 h), 7 days (T + 7 d), and 6 months (T + 6 mo) after sepsis challenge. Data are shown as means ± SEM.

Figure 3.

Sepsis-induced cell death in the lung during the first 2 days after E. coli challenge in baboons. (A–F) Immunofluorescence confocal imaging and quantitative analysis of the lung of healthy control and septic baboons after 24–48 hours after challenge, stained for active caspase 3 (A–C; green) or DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]; D–F; green). Nuclei are shown in blue. Data are presented as mean ± SEM (n = 10 microscopic fields collected from at least three different animals per condition per time point; unpaired t test, ***P < 0.001). (G and H) Electron microscopy images show extensive vacuolization of the type 1 alveolar epithelial cells (arrow), intra-alveolar accumulation of cell detritus (arrowheads; G, inset enlarged in H). and chromatin condensation in a presumably epithelial cell (asterisk). Av, alveolae; N, neutrophil; RBC, red blood cells. Scale bars, 100 μm (A and B), 200 μm (D and E), 10 μm (G and H).

Figure 4.

Main pathophysiological features of the lung during the fibroproliferative phase of ARDS after 7 days after E. coli challenge. (A) Double immunostaining for proliferation marker, Ki67 (red), and cytokeratin 18 (green, type 2 epithelial cell marker) shows a robust cell proliferation of type 2 epithelial cells in the animals challenged with E. coli (upper row) versus healthy controls (lower row). (B) Staining for procollagen 3 using an antibody against the N-terminal domain that detects only the nascent collagen and for chaperon, heat shock protein 47, shows focal induction of collagen synthesis within the lung of septic animals (upper row) as compared with healthy control animals (lower row). (C–F) E. coli sepsis induces the expression of markers of epithelial-to-mesenchymal transition: S100A4 (C, red), Notch (D, green), β-catenin (E, green), and SLUG + SNAIL (F, green). Procollagen 1 (C, green) or fibroblast activation protein (FAP) (D and E, red) were used as fibroblast markers. High-magnification insets of the areas marked with dashed lines are shown in (D–F). Throughout this figure, nuclei are shown in blue. Colocalization of green and red staining is shown as yellow; green, red, and blue as white (A–E), and green and blue as cyan (F). Scale bars, 100 μm.

Figure 5.

E. coli sepsis leads to long-term fibrosis. (A) E. coli sepsis induces decreased lung elasticity in baboon no. 2,509 that survived 27 months after experimental E. coli sepsis as compared with a group of five healthy control animals. This reflects a stiffer organ, as shown by the changes in lung compliance (C), expressed as the pressure difference between the interior of the alveoli (PA) and the pleural cavity (PPl) (transpulmonary pressure) required to affect a given change in the volume of air (ΔVL) in the lungs (C = ΔVL / Δ(PA-PPI). (B–D) Pseudocolor-encoded computerized tomography (CT) images of fibrotic lungs from the baboon that survived 27 months after experimental E. coli sepsis (B) as compared with a healthy control animal (C). Changes in mean gray values in 15 regions-of-interest from three representative CT scan images per experimental condition (fibrotic and control) are shown as a scatter plot (D). Data are presented as mean ± SEM (two-tailed Student’s t test; ***P < 0.001). (E–G) Staining with Mallory’s trichrome Masson (E; collagen in blue) or Sirius red (F and G; collagen appears as bright birefringent deposits under epipolarized light) detects extensive fibrosis within the lung parenchyma (F) of challenged baboons, as compared with healthy control animals (G). (H–J) Staining for procollagen 1 (H and I, red) and 3 (H and I, green), and matrix protein osteopontin (J) shows active collagen production and enhanced matrix deposition in the alveolar wall of challenged baboons. Procollagen production in healthy control animals is low and restricted to the adventitia of the large vessels (I, arrow) or airways (I, arrowhead). Nuclei are shown in blue. Overlay of green and red staining is shown as yellow. (K) Quantitative RT-PCR (qRT-PCR) of mRNA encoding the collagen 1α2 and collagen 3α1 chains shows strong increased production of these two major matrix proteins in the lung of sepsis survivors as compared with healthy control animals. Data are shown as fold ratio of challenged versus healthy control animals. (L and M) Biochemical determination of the collagen content of the lung using Sirius red (L), represented as percentage of total protein in lung homogenates, shows almost double-fold increase in the baboons that survived sepsis (n = 6) versus age matched control animals (n = 3). Procollagen 3 activation peptide levels are significantly increased in plasma of the six animals that survived sepsis for over 6 months (n = 6), as compared with the plasma of the same animals before experimental sepsis challenge (n = 6). Procollagen 3 levels in nonchallenged controls (9,989 ± 923 pg/ml, n = 3; not shown on the graph) were not statistically different from the prechallenge values of the experimental groups (10,025 ± 823 pg/ml, n = 6). Data are presented as mean ± SEM (two-tailed Student’s t test; ***P < 0.001. (N–P) Representative electron micrographs of the lung from an animal that survived sepsis challenge for 6 months (N and O) show extensive collagen (coll) deposition within the interalveolar walls as compared with a nonchallenged control animal (P). av, alveolae; RBC, red blood cells. Scale bars, 200 μm (E–G), 100 μm (H–J), 10 μm (N and O).

Figure 6.

E. coli sepsis leads to persistent inflammation in the lung. (A–C) Immunostaining for neutrophil elastase (green) and macrophage marker, CD68 (red), showed significant accumulation of interstitial macrophages in foci within the lung parenchyma (A) of an animal killed after 6.5 months (A) and 27 months (B) after experimental sepsis as compared with a healthy control animal (C). The inflammatory infiltrates were frequently associated with the periphery of the organ or with the airway ducts, as shown in (B). Quantification of these data is shown in Figure 1G. Although not statistically significant, neutrophil count was also increased, particularly in the areas surrounding macrophage accumulation. (D) Staining with Prussian blue showed accumulation of iron-positive (blue) hemosiderin-laden macrophages. (E) Costaining for CD68 (green) and CD163 (red; colocalization, yellow) suggests that the macrophages accumulated may belong to the M2 subtype. (F) Immunostaining for IL-17 shows presence of IL-17–positive cells (red) with macrophage morphology. Scale bars, 200 μm (A, B, and D), 100 μm (E and F).

Figure 7.

Expression of transforming growth factor (TGF)-β signaling proteins in the lung of long-term sepsis–surviving baboons. (A and B) Staining for M2 macrophage marker CD163 (green) and TGF-β (red) in the lung of a baboon killed at 6.5 months after challenge (A) shows marked increased accumulation of cells that costain for TGF-β and CD163 (yellow; presumably M2 macrophages) in the challenged baboons (A) versus unchallenged control animals (B). Insets show higher magnification of the areas marked with dashed borders. Colocalization of the two antigens is shown in yellow and nuclei in blue. (C) Biochemical analysis shows significantly increased TGF-β1 in the plasma of animals that survived E. coli sepsis for longer than 6 months as compared with their prechallenge levels. Data are presented as mean ± SEM; 2-tailed Student t test; ***P < 0.001; n = 6 animals. (D and E) Staining for FAP (green; D) and phospho-Smad2/3 (red; E) demonstrates profibrotic TGF-β signaling in activated fibroblasts. Nuclei are shown in blue. Insets show higher magnification of the areas marked with dashed borders to highlight the nuclear staining pattern of phospho-Smad 2/3 (red and blue overlay appears purple). (F) Immunostaining for connective tissue growth factor (CTGF; green) within the alveolar walls of the lung from a sepsis survivor killed after 6 months after challenge. Lung samples from healthy control animals did not stain for FAP, phospho-Smad, or CTGF (not shown). (G) Quantitative RT-PCR analysis shows increased mRNA expression of TGF-β signaling–positive regulators (SMADs 2, 3, and 4, TGF-β, and TGF-β receptor) and thrombospondin 1 (THBS1) transcripts, and down-regulation of the negative regulator, latent TGF-binding protein 1 (LTBP1), in the septic animals killed over 6 months after challenge (fibrosis group) as compared with healthy control animals. Data are shown as average fold change (n = 6 animals for fibrosis and n = 3 animals for control groups). Scale bars, 200 μm (A, B, D, and E), 100 μm (F).