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Characterization of Distinct Macrophage Subpopulations during Nitrogen Mustard-Induced Lung Injury and Fibrosis
Alessandro Venosa1, Rama Malaviya1, Hyejeong Choi1
11 Departments of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, and.
Abstract:
Nitrogen mustard (NM) is an alkylating agent known to cause extensive pulmonary injury progressing to fibrosis. This is accompanied by a persistent macrophage inflammatory response. In these studies, we characterized the phenotype of macrophages accumulating in the lung over time following NM exposure. Treatment of rats with NM (0.125 mg/kg, intratracheally) resulted in an increase in CD11b(+) macrophages in histologic sections. These cells consisted of inducible nitric oxide synthase(+) (iNOS) proinflammatory M1 macrophages, and CD68(+), CD163(+), CD206(+), YM-1(+), and arginase-II(+)antiinflammatory M2 macrophages. Although M1 macrophages were prominent 1-3 days after NM, M2 macrophages were most notable at 28 days. At this time, they were enlarged and vacuolated, consistent with a profibrotic phenotype. Flow cytometric analysis of isolated lung macrophages identified three phenotypically distinct subpopulations: mature CD11b(-), CD43(-), and CD68(+) resident macrophages, which decreased in numbers after NM; and two infiltrating (CD11b(+)) macrophage subsets: immature CD43(+) M1 macrophages and mature CD43(-) M2 macrophages, which increased sequentially. Time-related increases in M1 (iNOS, IL-12α, COX-2, TNF-α, matrix metalloproteinase-9, matrix metalloproteinase-10) and M2 (IL-10, pentraxin-2, connective tissue growth factor, ApoE) genes, as well as chemokines/chemokine receptors associated with trafficking of M1 (CCR2, CCR5, CCL2, CCL5) and M2 (CX3CR1, fractalkine) macrophages to sites of injury, were also noted in macrophages isolated from the lung after NM. The appearance of M1 and M2 macrophages in the lung correlated with NM-induced acute injury and the development of fibrosis, suggesting a potential role of these macrophage subpopulations in the pathogenic response to NM.
Insights
Nitrogen mustard exposure causes lung injury and fibrosis by altering macrophage populations. Proinflammatory M1 macrophages appear early, followed by profibrotic M2 macrophages, contributing to the lung
Area of Science:
- Immunology
- Pulmonary Medicine
- Toxicology
Background:
- Nitrogen mustard (NM) is an alkylating agent that induces significant pulmonary injury and fibrosis.
- A persistent macrophage inflammatory response is a hallmark of NM-induced lung damage.
- Understanding the dynamic changes and phenotypes of macrophages is crucial for elucidating NM pathogenesis.
Purpose of the Study:
- To characterize the phenotype and temporal accumulation of macrophages in the lung following NM exposure.
- To differentiate between M1 and M2 macrophage subpopulations and their roles in NM-induced lung injury and fibrosis.
- To investigate the expression of key genes and chemokines associated with macrophage recruitment and function.
Main Methods:
- Rats were intratracheally instilled with Nitrogen mustard (NM).
- Histologic analysis and flow cytometry were used to identify and quantify macrophage populations (M1 and M2).
- Gene expression analysis of M1 and M2 markers, chemokines, and chemokine receptors in isolated lung macrophages was performed.
Main Results:
- NM exposure increased CD11b(+) macrophages, comprising both proinflammatory M1 and anti-inflammatory M2 phenotypes.
- M1 macrophages were prominent early (1-3 days), while M2 macrophages, exhibiting a profibrotic phenotype, increased later (28 days).
- Sequential increases in M1 and M2 macrophages correlated with acute lung injury and subsequent fibrosis development.
Conclusions:
- NM-induced pulmonary injury involves a dynamic shift in macrophage populations, with distinct M1 and M2 phenotypes playing sequential roles.
- The accumulation of profibrotic M2 macrophages is associated with the development of fibrosis after NM exposure.
- These findings suggest that M1 and M2 macrophages are key players in the pathogenic response to Nitrogen mustard.
