Steroid-resistant lymphatic remodeling in chronically inflamed mouse airways

Abstract

Angiogenesis and lymphangiogenesis participate in many inflammatory diseases, and their reversal is thought to be beneficial. However, the extent of reversibility of vessel remodeling is poorly understood. We exploited the potent anti-inflammatory effects of the corticosteroid dexamethasone to test the preventability and reversibility of vessel remodeling in Mycoplasma pulmonis-infected mice using immunohistochemistry and quantitative RT-PCR. In this model robust immune responses drive rapid and sustained changes in blood vessels and lymphatics. In infected mice not treated with dexamethasone, capillaries enlarged into venules expressing leukocyte adhesion molecules, sprouting angiogenesis and lymphangiogenesis occurred, and the inflammatory cytokines tumor necrosis factor and interleukin-1 increased. Concurrent dexamethasone treatment largely prevented the remodeling of blood vessels and lymphatics. Dexamethasone also significantly reduced cytokine expression, bacterial burden, and leukocyte influx into airways and lungs over 4 weeks of infection. In contrast, when infection was allowed to proceed untreated for 2 weeks and then was treated with dexamethasone for 4 weeks, most blood vessel changes reversed but lymphangiogenesis did not, suggesting that different survival mechanisms apply. Furthermore, dexamethasone significantly reduced the bacterial burden and influx of lymphocytes but not of neutrophils or macrophages or cytokine expression. These findings show that lymphatic remodeling is more resistant than blood vessel remodeling to corticosteroid-induced reversal. We suggest that lymphatic remodeling that persists after the initial inflammatory response has resolved may influence subsequent inflammatory episodes in clinical situations.

Figure 1

Time course of airway vessel changes during 42-day course of M. pulmonis infection. A–F: Overview of microvascular arcades stained for blood vessels (PECAM-1, green) and lymphatic vessels (LYVE-1, red) in a tracheal whole mount of pathogen-free mice and in mice infected for 7, 10, 14, 28, and 42 days. Asterisks indicate cartilage rings. G and J: In pathogen-free mice, capillaries of uniform caliber (G, arrows) transverse the cartilage rings but lymphatics do not (J). Some resident leukocytes stained for LYVE-1 (J, arrowheads). At 7 days of infection (H and K), blood vessels are enlarged (H, arrows) and accompanied by an influx of leukocytes, many showing PECAM-1 immunoreactivity (H, arrowheads). Initial lymphatics sprouts are present (K, arrows). I: By 28 days, blood vessels show both enlargement (arrows) and sprouting (arrowheads) and newly grown lymphatics fuse to a continuous network. L: Enlarged box in I shows the angiogenic sprouts (arrowheads). Scale bars: 200 μm in (A–F); 50 μm (G–K); 20 μm (L).

Figure 2

Prevention of M. pulmonis induced-vessel changes by dexamethasone. A–C: Confocal micrographs showing tracheal blood vessels (green) and lymphatics (red) in pathogen-free mice (A) and in 28-day infected mice treated concurrently with vehicle (B) or dexamethasone (C) for 28 days. D and E: Capillary diameters (D) and area density of lymphatics (E) showing prevention of infection-induced vessel changes by dexamethasone. F–K: Dexamethasone prevents transformation of capillary to venule phenotype. Venular endothelial markers EphB4 and ICAM-1 are shown in red and PECAM-1 is shown in green. In pathogen-free mice, EphB4 (F) and ICAM-1 (I) are restricted to the venules and neither is present in capillaries (arrows). EphB4 (G) and ICAM-1 (J) extend into remodeled capillaries (arrows) overlying cartilage rings after 28 days of infection. Scattered staining for EphB4 and ICAM-1 in other mucosal cells is also seen in the more remodeled whole-mount tracheas. The expansion is prevented by concurrent dexamethasone treatment (H and K, arrows). Lymphatics in G are indicated by asterisks. Scale bar: 200 μm in A–C; 60 μm in F–K. L–O: qRT-PCR measurement of TNF-α, IL-1β, IL-1Ra, and the ratio of IL-1β expression to IL-1Ra in tracheas of pathogen-free mice and mice infected and treated with vehicle or dexamethasone for 28 days. Values are shown as fold increase compared with pathogen-free levels. *P < 0.05 versus pathogen-free; **P < 0.05 versus corresponding infected, vehicle-treated group (n = 5 to 8 mice per group). Dex, dexamethasone.

Figure 3

Prevention of M. pulmonis burden and leukocyte influx by dexamethasone. Bar graphs of expression of M. pulmonis 16S rRNA in tracheas by qRT-PCR (A), total RNA yield per trachea (B), and lung (C) and bronchial lymph node weights (D) from pathogen-free mice and mice infected for 7, 14, or 28 days with concurrent treatment with vehicle or dexamethasone. Lung and bronchial lymph node weights are normalized to the corresponding final body weights. *P < 0.05 versus pathogen-free; ^†P < 0.05 versus the corresponding infected, vehicle-treated controls (n = 5 to 8, mice per group). E–J: H&E-stained sections of mouse tracheas (E–G) and mouse left lungs (H–J) in pathogen-free mice (E and H) or in infected mice with concurrent treatment with vehicle (F and I) or dexamethasone (G and J) for 28 days. Arrows indicate leukocyte influx in tracheal epithelial layers, and arrowheads mark leukocyte influx in tracheal mucosa. Dex, dexamethasone; B, bronchiole; V, blood vessel. Scale bars: 100 μm in (E–G); 400 μm (H–J).

Figure 4

Differential prevention of leukocyte influx into infected airways by dexamethasone. Confocal microscopic images of tracheas stained for markers of macrophages (Iba1, red in A–C) and neutrophils (S100A8, green in A–C) or T cells (CD3e, red in D–F) and B cells (B220, green in D–F) in pathogen-free mice (A and D) and mice infected for 28 days with concurrent treatment with vehicle (B and E) or dexamethasone (C and F). Asterisks indicate cartilage rings in A and D. Boxes in B and E are enlarged in G and H, respectively. Scale bars: 200 μm in (A–F); 50 μm (G–H). I: Time course of influx of S100A8-immunoreactive cells during infection. J: Number of S100A8-immunoreactive cells and qRT-PCR analysis of S100A8 mRNA expression in pathogen-free mice and after infection for 28 days with concurrent treatment with vehicle or dexamethasone. K–M: qRT-PCR measurement for Iba1, CD3e, and CD19 (B-cell marker). *P < 0.05 versus pathogen-free; **P < 0.05 versus the corresponding infected, vehicle-treated controls (n = 4 to 9 mice per group).

Figure 5

Reversibility of M. pulmonis-induced vessel changes by dexamethasone. A–C: Confocal images of tracheas stained for LYVE-1 (red) and PECAM-1 (green) immunoreactivity in mice infected for 14 days (A) and infected for 42 days and treated with vehicle (B) or with dexamethasone (C) for the last 28 days. Lymphatic islands (arrowheads) are enlarged in inset in C. D–G: Bar graphs show changes of blood vessels and lymphatics in mucosa overlying cartilage rings in reversal studies, as reflected by capillary diameter (D), number of capillaries crossing cartilage midline (E), area density of lymphatics (F), and number of lymphatic islands (G). H–M: Effects of delayed dexamethasone treatment on distribution of venular endothelial markers EphB4 and ICAM-1 (red) and PECAM-1 (green). Remodeled capillaries (arrows) overlying cartilage rings in 14-day (H and K) and 42-day (I and L) infected mice are strongly stained for EphB4 and ICAM-1. Scattered staining for EphB4 and ICAM-1 in other mucosal cells is also seen in the more remodeled whole-mount tracheas. Some EphB4 and ICAM-1 immunoreactivity is still present in the capillaries (arrows) after 28 days of dexamethasone treatment (J and M). Asterisks indicate EphB4-positive lymphatics in H–J. Scale bars: 200 μm in (A–C); 60 μm (H–M). N–Q: qRT-PCR measurement of TNF-α, IL-1β, IL-1Ra, and the ratio of IL-1β expression to IL-1Ra. *P < 0.05 versus pathogen-free mice; **P < 0.05 versus 14-day infected baseline group before treatment; ***P < 0.05 versus corresponding infected, vehicle-treated controls (n = 5 to 8 mice per group).

Figure 6

Reversibility of M. pulmonis burden and leukocyte influx by dexamethasone. Bar graphs of expression of M. pulmonis 16S rRNA in tracheas by qRT-PCR (A), total RNA yield per trachea (B), lung weight (C), and bronchial lymph node weight (D) from pathogen-free mice and mice infected for 14 days and untreated or infected for 14 days followed by treatment with vehicle or with dexamethasone for 14 days or 28 days. Lung and bronchial lymph node weights are normalized to the corresponding final body weights *P < 0.05 versus pathogen-free mice; **P < 0.05 versus 14-day infected baseline group before dexamethasone treatment; ***P < 0.05 versus corresponding infected, vehicle-treated controls (n = 10 to 15 mice per group). E–J: H&E-stained sections of mouse tracheas (E–G) and mouse left lungs (H–J) in mice infected for 14 days (E and H) and in mice infected for 42 days and treated with vehicle (F and I) or dexamethasone (G and J) for the last 28 days. Arrows mark leukocyte influx in tracheal epithelial layers, and arrowheads mark leukocyte influx in tracheal mucosa. Asterisk in J marks leukocyte clusters remaining in lungs after 28 days of dexamethasone treatment. Dex, dexamethasone; B, bronchiole; V, blood vessel. Scale bars: 100 μm in (E–G); 400 μm (H–J).

Figure 7

Differential reversal of leukocyte influx into infected airways by dexamethasone. Confocal microscopic images of tracheas stained for macrophages (Iba1, red in A–C) and neutrophils (S100A8, green in A–C) or T cells (CD3e, red in D–F) and B-cells (B220, green in D–F) in 14-day infected untreated mice (A and D) and mice infected for 14 days followed by treatment with vehicle (B and E) or dexamethasone (C and F) for 28 days. G–H: Boxed areas enlarged in B and E, respectively. Scale bars: 200 μm in (A–F); 50 μm (G and H). I: qRT-PCR analysis of S100A8 mRNA and number of S100A8-immunoreactive cells in pathogen-free mice, mice infected for 14 days, and mice infected for 14 days followed by 28-day treatment with vehicle or dexamethasone. J–L: qRT-PCR measurements for Iba1, CD3e, and CD19 (B-cell marker) in tracheas of pathogen-free mice, 14-day infected baseline group, and mice infected for 14 days followed by treatment with vehicle or dexamethasone for 28 days. *P < 0.05 versus pathogen-free; **P < 0.05 versus 14-day infected baseline before dexamethasone treatment; ***P < 0.05 versus corresponding infected, vehicle treated controls (n = 4 to 9 mice per group).

Figure 8

Schematic diagram of prevention and reversibility of blood vessel and lymphatic remodeling in M. pulmonis-infected airways. Blood vessels are shown in green, lymphatics in red, and leukocytes in blue in three conditions: infected and untreated (A); dexamethasone-prevention studies (B); and dexamethasone-reversal studies (C). In pathogen-free airways, vessels show normal baseline appearance with little or no leukocyte influx. A: In infected and untreated airways, uniform, thin capillaries enlarge into venules that support leukocyte recruitment, followed later by angiogenic sprouting. Leukocyte influx increases as infection continues. The normal lymphatic vessel network expands first by sprouting and then by enlargement and growth and fusion of newly formed lymphatics. B: In prevention studies, when dexamethasone is given concurrently with infection, blood vessels and lymphatics do not undergo extensive remodeling but remain similar to the pathogen-free state with little or no leukocyte influx. C: In reversal studies, delayed dexamethasone treatment reverses enlargement of blood vessels and growth of angiogenic sprouts almost to pathogen-free values. Leukocyte influx is reduced, as the blood vessels are less remodeled. In contrast to blood vessels, newly formed lymphatics are much more resistant to reversal, except for the disappearance of fine filopodia and formation of some disconnected lymphatic islands.