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Heme oxygenase-1 promotes granuloma development and protects against dissemination of mycobacteria.

Regev D, Surolia R, Karki S, Zolak J, Montes-Worboys A, Oliva O, Guroji P, Saini V, Steyn AJ, Agarwal A, Antony VB - Lab. Invest. (2012)

Bottom Line: Inhibition of HO by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor.Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1⁺/⁺ mice.Higher MCP-1 levels were found in bronchoalveolar lavage fluid of M. avium infected HO-1(-/-) mice and CCR2 expression was higher in HO-1⁻/⁻ alveolar macrophages when compared with HO-1⁺/⁺ mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.

ABSTRACT
Non-tuberculous mycobacterial (NTM) infections occur in both immunocompromised and immunocompetent hosts and are an increasingly recognized cause of morbidity and mortality. The hallmark of pulmonary mycobacterial infections is the formation of granuloma in the lung. Our study focuses on the role of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in the regulation of granuloma development and maturation following infection with Mycobacterium avium. We examined the role of HO-1 in regulating monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2), two molecules involved in monocyte-macrophage cell trafficking after infection. We showed that RAW 264.7 mouse monocytes exposed to M. avium expressed HO-1 and MCP-1. Inhibition of HO by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor. HO-1⁻/⁻ mice did not develop organized granuloma in their lungs, had higher lung colony forming unit of M. avium when infected with intratracheal M. avium, and had loose collections of inflammatory cells in the lung parenchyma. Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1⁺/⁺ mice. In HO-1⁻/⁻ mice, mycobacteria were also found in the liver and spleen and showed increased mortality. Peripheral blood monocytes isolated from GFP⁺ mice and given intravenously to HO-1⁺/⁺ mice localized into tight granulomas, while in HO-1⁻/⁻ mice they remained diffusely scattered in areas of parenchymal inflammation. Higher MCP-1 levels were found in bronchoalveolar lavage fluid of M. avium infected HO-1(-/-) mice and CCR2 expression was higher in HO-1⁻/⁻ alveolar macrophages when compared with HO-1⁺/⁺ mice. CCR2 expression localized to granuloma in HO-1⁺/⁺ mice but not in the HO-1⁻/⁻ mice. These findings strongly suggest that HO-1 plays a protective role in the control of M. avium infection.

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M. avium infection induces HO-1 in RAW 264.7 monocyte-macrophages(A, B) Representative light micrograph showing immunocytochemical detection of HO-1 in RAW 264.7. Panel A shows the control cells were incubated in SFM while panel B shows the cells infected with M. avium (50 MOI) for 24 hours. Cells were fixed and stained with anti-HO-1 antibody and visualized by optical microscopy at 400× magnification. The infected cells stained for HO-1 expression (brown) after 24 hours as compared to control cells. Blue color shows counter-stain with hematoxylin. The photomicrograph is the representative of three individual staining. (C) HO-1 relative mRNA expression significantly increases in RAW 264.7 cells after infection with M. avium. Control cells were incubated in SFM and treated cells were incubated with M. avium. For 4, 8 and 24 hours. HO-1 relative gene expression was analyzed by RT-PCR. HO-1 levels significantly increased in cells treated with M. avium as compared to nontreated cells in a time dependent manner (*P < 0.001). Values represent the mean of three different experiments.
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Figure 1: M. avium infection induces HO-1 in RAW 264.7 monocyte-macrophages(A, B) Representative light micrograph showing immunocytochemical detection of HO-1 in RAW 264.7. Panel A shows the control cells were incubated in SFM while panel B shows the cells infected with M. avium (50 MOI) for 24 hours. Cells were fixed and stained with anti-HO-1 antibody and visualized by optical microscopy at 400× magnification. The infected cells stained for HO-1 expression (brown) after 24 hours as compared to control cells. Blue color shows counter-stain with hematoxylin. The photomicrograph is the representative of three individual staining. (C) HO-1 relative mRNA expression significantly increases in RAW 264.7 cells after infection with M. avium. Control cells were incubated in SFM and treated cells were incubated with M. avium. For 4, 8 and 24 hours. HO-1 relative gene expression was analyzed by RT-PCR. HO-1 levels significantly increased in cells treated with M. avium as compared to nontreated cells in a time dependent manner (*P < 0.001). Values represent the mean of three different experiments.

Mentions: To address the role of HO-1 in M. avium infection, we exposed RAW 264.7 cells to M. avium at 50 multiplicity of infection (MOI). One day post infection, the cells were fixed and stained for HO-1. HO-1 was induced following 24 hours infection with M. avium as compared with untreated cells (Figure1 A, B). To substantiate the above observation, we analyzed mRNA levels of HO-1 in RAW 264.7 cells treated with M. avium or untreated as previously described in methods section. Real-time PCR results showed that HO-1 relative gene expression was significantly increased in a time dependent manner in RAW 264.7 cells following M. avium treatment (Figure 1C).


Heme oxygenase-1 promotes granuloma development and protects against dissemination of mycobacteria.

Regev D, Surolia R, Karki S, Zolak J, Montes-Worboys A, Oliva O, Guroji P, Saini V, Steyn AJ, Agarwal A, Antony VB - Lab. Invest. (2012)

M. avium infection induces HO-1 in RAW 264.7 monocyte-macrophages(A, B) Representative light micrograph showing immunocytochemical detection of HO-1 in RAW 264.7. Panel A shows the control cells were incubated in SFM while panel B shows the cells infected with M. avium (50 MOI) for 24 hours. Cells were fixed and stained with anti-HO-1 antibody and visualized by optical microscopy at 400× magnification. The infected cells stained for HO-1 expression (brown) after 24 hours as compared to control cells. Blue color shows counter-stain with hematoxylin. The photomicrograph is the representative of three individual staining. (C) HO-1 relative mRNA expression significantly increases in RAW 264.7 cells after infection with M. avium. Control cells were incubated in SFM and treated cells were incubated with M. avium. For 4, 8 and 24 hours. HO-1 relative gene expression was analyzed by RT-PCR. HO-1 levels significantly increased in cells treated with M. avium as compared to nontreated cells in a time dependent manner (*P < 0.001). Values represent the mean of three different experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017357&req=5

Figure 1: M. avium infection induces HO-1 in RAW 264.7 monocyte-macrophages(A, B) Representative light micrograph showing immunocytochemical detection of HO-1 in RAW 264.7. Panel A shows the control cells were incubated in SFM while panel B shows the cells infected with M. avium (50 MOI) for 24 hours. Cells were fixed and stained with anti-HO-1 antibody and visualized by optical microscopy at 400× magnification. The infected cells stained for HO-1 expression (brown) after 24 hours as compared to control cells. Blue color shows counter-stain with hematoxylin. The photomicrograph is the representative of three individual staining. (C) HO-1 relative mRNA expression significantly increases in RAW 264.7 cells after infection with M. avium. Control cells were incubated in SFM and treated cells were incubated with M. avium. For 4, 8 and 24 hours. HO-1 relative gene expression was analyzed by RT-PCR. HO-1 levels significantly increased in cells treated with M. avium as compared to nontreated cells in a time dependent manner (*P < 0.001). Values represent the mean of three different experiments.
Mentions: To address the role of HO-1 in M. avium infection, we exposed RAW 264.7 cells to M. avium at 50 multiplicity of infection (MOI). One day post infection, the cells were fixed and stained for HO-1. HO-1 was induced following 24 hours infection with M. avium as compared with untreated cells (Figure1 A, B). To substantiate the above observation, we analyzed mRNA levels of HO-1 in RAW 264.7 cells treated with M. avium or untreated as previously described in methods section. Real-time PCR results showed that HO-1 relative gene expression was significantly increased in a time dependent manner in RAW 264.7 cells following M. avium treatment (Figure 1C).

Bottom Line: Inhibition of HO by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor.Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1⁺/⁺ mice.Higher MCP-1 levels were found in bronchoalveolar lavage fluid of M. avium infected HO-1(-/-) mice and CCR2 expression was higher in HO-1⁻/⁻ alveolar macrophages when compared with HO-1⁺/⁺ mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Critical Care and Sleep Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.

ABSTRACT
Non-tuberculous mycobacterial (NTM) infections occur in both immunocompromised and immunocompetent hosts and are an increasingly recognized cause of morbidity and mortality. The hallmark of pulmonary mycobacterial infections is the formation of granuloma in the lung. Our study focuses on the role of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in the regulation of granuloma development and maturation following infection with Mycobacterium avium. We examined the role of HO-1 in regulating monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2), two molecules involved in monocyte-macrophage cell trafficking after infection. We showed that RAW 264.7 mouse monocytes exposed to M. avium expressed HO-1 and MCP-1. Inhibition of HO by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor. HO-1⁻/⁻ mice did not develop organized granuloma in their lungs, had higher lung colony forming unit of M. avium when infected with intratracheal M. avium, and had loose collections of inflammatory cells in the lung parenchyma. Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1⁺/⁺ mice. In HO-1⁻/⁻ mice, mycobacteria were also found in the liver and spleen and showed increased mortality. Peripheral blood monocytes isolated from GFP⁺ mice and given intravenously to HO-1⁺/⁺ mice localized into tight granulomas, while in HO-1⁻/⁻ mice they remained diffusely scattered in areas of parenchymal inflammation. Higher MCP-1 levels were found in bronchoalveolar lavage fluid of M. avium infected HO-1(-/-) mice and CCR2 expression was higher in HO-1⁻/⁻ alveolar macrophages when compared with HO-1⁺/⁺ mice. CCR2 expression localized to granuloma in HO-1⁺/⁺ mice but not in the HO-1⁻/⁻ mice. These findings strongly suggest that HO-1 plays a protective role in the control of M. avium infection.

Show MeSH
Related in: MedlinePlus