Heme oxygenase-2 deletion impairs macrophage function: implication in wound healing

Abstract

Heme oxygenase (HO)-2 deficiency impairs wound healing and exacerbates inflammation following injury. We examine the impact of HO-2 deficiency on macrophage function and the contribution of macrophage HO-2 to inflammatory and repair responses to injury. Corneal epithelial debridement was performed in control and macrophage-depleted HO-2(-/-) and wild-type (WT) mice and in bone marrow chimeras. Peritoneal macrophages were collected for determination of phagocytic activity and classically activated macrophage (M1)-alternatively activated macrophage (M2) polarization. Depletion of macrophages delayed corneal healing (13.2%) and increased neutrophil infiltration (54.1%) by day 4 in WT mice, whereas in HO-2(-/-) mice, it did not worsen the already impaired wound healing and exacerbated inflammation. HO-2(-/-) macrophages displayed an altered M1 phenotype with no significant expression of M2 or M2-like activated cells and a 31.3% reduction in phagocytic capacity that was restored by inducing HO-1 activity or supplementing biliverdin. Macrophage depletion had no effect, whereas adoptive transfer of WT bone marrow improved wound healing (34% on day 4) but did not resolve the exaggerated inflammatory response in HO-2(-/-) mice. These findings indicate that HO-2-deficient macrophages are dysfunctional and that macrophage HO-2 is required for proper macrophage function but is insufficient to correct the impaired healing of the HO-2(-/-) cornea, suggesting that corneal epithelial expression of HO-2 is a key to resolution and repair in wound healing.

Keywords: bone marrow transfer; clodronate liposomes; inflammation.

Figure 1.

Effect of macrophage (MØ) depletion on wound healing and neutrophil infiltration. A) Representative images of fluorescein-stained corneas at days 2 and 4 after injury in control and MØ-depleted HO-2^+/+ (WT) and HO-2^−/− mice. B) Wound closure as percent change from day 0. Results are mean ± sem; n = 10–20. *P < 0.05 and **P < 0.01 from control WT mice. C) Neutrophil infiltration into the cornea at day 4 after injury. Left, WT mice; right, HO-2^−/− mice; white bars, control mice receiving PBS liposomes; black bars, MØ-depleted mice receiving clodronate liposomes. Results are mean ± sem; n = 6–10. *P < 0.05 and **P < 0.01 from control (PBS liposome-treated) WT mice.

Figure 2.

Effect of HO-2 deficiency on macrophage phagocytosis. A) Number of Zymosan particles per cell relative to WT control with and without SnCl₂ (1 µM) preincubation. Results are the mean ± sem; n = 2–4. ***P < 0.001. White bars, WT; black bars, HO-2^−/− cells. B) Number of Zymosan particles per cell relative to vehicle control with and without biliverdin (BVD; 10 µM) or Tiron (1 mM) preincubation. Results are the mean ± sem; n = 1–3. ***P < 0.001. White bars, vehicle control cells; black bars, biliverdin-treated cells; gray bars, Tiron-treated cells. C) Number of Zymosan particles per cell relative to control. Results are the mean ± sem; n = 3. *P < 0.05 for HO-2 shRNA-treated (black bar) from control (white bar); ‡P < 0.05 for nontarget shRNA (gray bar). D) Number of Zymosan particles per cell relative to vehicle control with and without SnCl₂ (1 or 5 µM) preincubation. Results are the mean ± sem; n = 2–3. ***P < 0.05 for SnCl₂ 1 µM treated (black bar) from vehicle control (white bar).

Figure 3.

Real-time PCR analysis of mRNA expression of IL-10 (A), TNF (B), IL-1 (C), and IL-6 (D) in PRMs and TG-elicited peritoneal macrophages isolated from WT (white bars) and HO-2^−/− (black bars) mice. Results are presented as mRNA expression relative to WT PRMs and are the mean ± sem; n = 5–10. *P < 0.05, **P < 0.01, and ***P < 0.001 from WT PRMs. Comparison between WT and HO-2^−/− PRMs using unpaired t test: †P < 0.05 and ‡P < 0.001.

Figure 4.

Real-time PCR analysis of mRNA expression of macrophage M1-M2 polarization associated genes. A) CD54, B) CD80, C) iNOS, D) arginase 1, E) CD206, F) CD163, G) Ym1, and H) Fizz1 in PRMs and TG-elicited peritoneal macrophages isolated from WT (white bars) and HO-2^−/− (black bars) mice. Results are presented as mRNA expression relative to WT PRMs and are the mean ± sem; n = 4–10. *P < 0.05, **P < 0.01, and ***P < 0.001 from WT PRMs (i.e., nonelicited macrophages).

Figure 5.

Real-time PCR analysis of mRNA expression of HO-1 (A), Alox15 (B), Alox5 (C), and COX-2 (D) in PRMs and TG-elicited peritoneal macrophages isolated from WT (white bars) and HO-2^−/− (black bars) mice. Results are presented as mRNA expression relative to WT PRMs and are the mean ± sem; n = 5–10; *P < 0.05, **P < 0.01, and ***P < 0.001 from WT PRMs.

Figure 6.

Effect of allogeneic bone marrow transfer on wound healing and neutrophil infiltration. A) Representative images of fluorescein-stained corneas at days 2, 4, and 7 after injury in syngeneic and allogeneic WT and HO-2^−/− mice. B) Wound closure as percent change from day 0. Open squares, WT mice receiving WT bone marrow; closed squares, WT mice receiving HO-2^−/− bone marrow; open circles, HO-2^−/− mice receiving WT bone marrow; closed circles, HO-2^−/− mice receiving HO-2^−/− bone marrow. Results are mean ± sem; n = 5–10. *P < 0.05 comparison of the effect of WT bone marrow on wound healing in WT vs. HO-2^−/− recipients; §P < 0.05 comparison of the effect of HO-2^−/− bone marrow on wound healing in WT vs. HO-2^−/− recipients; and †P < 0.05 comparison between HO-2^−/− recipients of WT vs. HO-2^−/− bone marrow and its effect on wound healing. B) Neutrophil infiltration into the cornea at day 7 after injury. White bars, WT recipients; black bars, HO-2^−/− recipients. Results are mean ± sem; n = 5–10. *P < 0.05 comparison of the effect of WT bone marrow on corneal neutrophil infiltration in WT vs. HO-2^−/− recipients; **P < 0.01 comparison of the effect of HO-2^−/− bone marrow on corneal neutrophil infiltration in WT vs. HO-2^−/− recipient mice.

Figure 7.

Corneas of HO-2–deficient mice maintain an increased macrophage population following injury. A) Representative images of frozen corneal sections stained for CD68 (green). Nuclei are counterstained with DAPI. Corneal sections day 7 after injury from WT mouse receiving WT bone marrow (top left); WT mouse receiving HO-2^−/− bone marrow (top right); WT mouse receiving HO-2^−/− bone marrow (bottom left); and HO-2^−/− receiving HO-2^−/− bone marrow (bottom right). B) Indirect measurement of CD68-positive cells expressed as total number of CD68-positive (green) pixels per square millimeter. Results are the mean ± sem; n = 5. **P < 0.01. Pairwise comparison of the effect of WT bone marrow on corneal macrophage infiltration in WT and HO-2^−/− recipient mice (left) and HO-2^−/− bone marrow in WT vs. HO-2^−/− recipient mice (right).

Figure 8.

Confirmation of chimerism through genotyping of whole blood and immunohistological analysis of corneal HO-2 expression after injury in chimeric mice. A) PCR fragments on 1% agarose gel from 3 WT mice receiving HO-2^−/− bone marrow, followed by 3 HO-2^−/− mice receiving WT bone marrow. Two PCR reactions for each mouse: 1 for the WT (HO-2) fragment followed by the HO-2^−/− (Neo) fragment. B) Representative images of frozen corneal sections stained for HO-2 (red) and CD68 (green). Nuclei are counterstained with DAPI. Left, WT mouse receiving HO-2^−/− bone marrow; right, HO-2^−/− mouse receiving WT bone marrow. HO-2–positive staining in red (top), CD68-positive staining in green (middle), and merged images with DAPI (bottom).