Characterization of Distinct Macrophage Subpopulations during Nitrogen Mustard–Induced Lung Injury and Fibrosis

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 1 macrophages in histologic sections. These cells consisted of inducible nitric oxide synthase 1 (iNOS) proinflammatory M1 macrophages, and CD68 1 M1 macrophages and mature CD43 2 M2 macrophages, which increased sequentially. Time-related increases in M1 (iNOS, IL-12a, COX-2, TNF-a, 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 (CX 3 CR1, 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.

Emerging Roles of Apolipoprotein E and Apolipoprotein A-I in the Pathogenesis and Treatment of Lung Disease

“…For example, Apoedeficient mice have increased neutrophilic inflammation and oxidative stress in murine models of ALI induced by acid aspiration, hyperoxia, or pulmonary nanoparticle exposure (25)(26)(27). In addition, hyperoxia and nitrogen mustard exposure increased apoE in the lung, whereas intratracheal administration of apoE to rat lungs prevented hyperoxia-mediated ALI (9,27).…”

Mustard vesicant-induced lung injury: Advances in therapy

“…Similar structural and functional changes are observed in the respiratory tract after NM exposure; these include necrotizing bronchiolitis and thickening of alveolar septa accompanied by decreases in static compliance and end tidal lung volumes within 24 h (Sunil et al, 2011b). This is associated with inflammatory cell accumulation in the airways and lung, and production of reactive oxygen species, reactive nitrogen species, eicosanoids, TNFα, IL-1, IL-6, IL-8, IL-12, IL-13, MCP-1 (CCL-2) and interferon (IFN)-γ, as well as connective tissue growth factor (Anderson et al, 2009; Malaviya et al, 2010, 2012; Mishra et al, 2010; Sawale et al, 2013; Tang and Loke, 2012; Venosa et al, 2015; Wigenstam et al, 2009). Markers of oxidative stress (i.e., malondialdehyde, nitrite-nitrates, reduced glutathione and iNOS), proteolysis (i.e., MMP-9, gelatinase), DNA damage and repair (i.e., PARP-1, phospho-H2A.X), apoptosis (i.e., activated caspase-3 and caspase-9), and autophagy (i.e., LC3B) are also markedly elevated, and there is biochemical evidence of alveolar epithelial damage (i.e., increased bronchoalveolar lavage protein and cell content) (Allon et al, 2009; Calvet et al, 1999b; Das et al, 2003; Guignabert et al, 2005; Malaviya et al, 2010; Sawale et al, 2013; Shohrati et al, 2010; Sunil et al, 2011b; Venosa et al, 2015).…”

“…Airway hyperreactivity to substance P and histamine are noted, consistent with the onset of asthma-like symptoms in victims of SM exposure (Calvet et al, 1994; Emad and Rezaian, 1997; Hefazi et al, 2005). Macrophages remain present in the lung for at least 28 days after acute exposure of rodents to NM (Hoesel et al, 2008; Malaviya et al, 2012; Mukherjee et al, 2009; Venosa et al, 2015). Evidence suggests that these macrophages consist of distinct subpopulations that sequentially accumulate in the lung.…”

“…Evidence suggests that these macrophages consist of distinct subpopulations that sequentially accumulate in the lung. Whereas initially these cells exhibit a proinflammatory/cytotoxic phenotype, contributing to tissue injury, subsequently, during the chronic phase, they become profibrotic playing a role in mustard induced fibrosis (Malaviya et al, 2012; Venosa et al, 2015). The chronic phase of vesicant-induced respiratory injury (> 2-4 weeks in rodents), is characterized by a predominance of enlarged foamy macrophages, lymphocytes and IL-17 + cells, with marked increases in TGF-β, and myofibroblast proliferation, (Malaviya et al, 2015b; Mishra et al, 2012; Venosa et al, 2015).…”

Inflammatory mechanisms of pulmonary injury induced by mustards

“…Exaggerated responses of M2 macrophages are thought to contribute to the development of fibrosis. In an experimental model of NM toxicity, we observed increases in numbers of lung macrophages expressing markers of M2 macrophages including Ym-1, galectin (Gal)-3, and CD68 and expression of antiinflammatory profibrotic genes (i.e., IL-10, connective tissue growth factor) within 3 d suggesting that the process of fibrosis begins early in the pathogenic response (Malaviya et al, 2012; Sunil et al, 2014; Venosa et al, 2015). This is supported by our findings that the appearance of M2 macrophages in the lung correlates with increases in expression of the profibrotic mitogen, TGF-β and fibroplasia at 3 d post NM, and collagen deposits around bronchioles and alveolar septae after 7 d; by 28 d multiple fibrotic foci with mature collagen are evident around the airways, distorting the normal parenchymal structure (Malaviya et al, 2015; Malaviya et al, 2012).…”