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Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

ABSTRACT: Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1? contrib utes to cobalt toxicity in vitro, loss of HIF1? in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1? and HIF2? show unique tissue expression profiles, and HIF2? is known to be the predominant HIF mRNA isoform in the adult lung. Thus, if HIF2? activation by cobalt contributes to pathophysiology of HMLD, we hypothesized that loss of HIF2? in lung epithelium would provide protection from cobalt-induced inflammation. Mice with HIF2?-deficiency in Club and alveolar type II epithelial cells (ATIIs) (HIF2?(?/?)) were exposed to cobalt (60 µg/day) or saline using a subacute occupational exposure model. Bronchoalveolar lavage cellularity, cytokines, qRT-PCR, and histopathology were analyzed. Results show that loss of HIF2? leads to enhanced eosinophilic inflammation and increased goblet cell metaplasia. Additionally, control mice demonstrated a mild recovery from cobalt-induced lung injury compared with HIF2?(?/?) mice, suggesting a role for epithelial HIF2? in repair mechanisms. The expression of important cytokines, such as interleukin (IL)-5 and IL-10, displayed significant differences following cobalt exposure when HIF2?(?/?) and control mice were compared. In summary, our data suggest that although loss of HIF2? does not afford protection from cobalt-induced lung inflammation, epithelial HIF2? signaling does play an important role in modulating the inflammatory and repair response in the lung.

SUBMITTER: Proper SP

PROVIDER: S-EPMC3908723 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

Proper Steven P SP Saini Yogesh Y Greenwood Krista K KK Bramble Lori A LA Downing Nathaniel J NJ Harkema Jack R JR Lapres John J JJ

Toxicological sciences : an official journal of the Society of Toxicology 20131111 2

Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1α contrib utes to cobalt toxicity in vitro, loss of HIF1α in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1α and HIF2α show unique tissue e ...[more]

PMID: 24218148

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Project description:OBJECTIVE:Lung contusion is a major risk factor for the development of acute respiratory distress syndrome. Hypoxia-inducible factor-1? is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension. We set to determine if hypoxia-inducible factor-1? plays a role in the pathogenesis of acute inflammatory response and injury in lung contusion. DESIGN:Nonlethal closed-chest unilateral lung contusion was induced in a hypoxia reporter mouse model and type 2 cell-specific hypoxia-inducible factor-1? conditional knockout mice. The mice were killed at 5-, 24-, 48-, and 72-hour time points, and the extent of systemic and tissue hypoxia was assessed. In addition, injury and inflammation were assessed by measuring bronchoalveolar lavage cells (flow cytometry and cytospin), albumin (permeability injury), and cytokines (inflammation). Isolated type 2 cells from the hypoxia-inducible factor-1? conditional knockout mice were isolated and evaluated for proinflammatory cytokines following lung contusion. Finally, the role of nuclear factor-?B and interleukin-1? as intermediates in this interaction was studied. RESULTS:Lung contusion induced profound global hypoxia rapidly. Increased expression of hypoxia-inducible factor-1? from lung samples was observed as early as 60 minutes, following the insult. The extent of lung injury following lung contusion was significantly reduced in conditional knockout mice at all the time points, when compared with the wild-type littermate mice. Release of proinflammatory cytokines, such as interleukin-1?, interleukin-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant, was significantly lower in conditional knockout mice. These actions are in part mediated through nuclear factor-?B. Hypoxia-inducible factor-1? in lung epithelial cells was shown to regulate interleukin-1? promoter activity. CONCLUSION:Activation of hypoxia-inducible factor-1? in type 2 cell is a major driver of acute inflammation following lung contusion.

Project description:Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air-liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24-48h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.

Project description:Influenza A viruses recruit components of the nuclear import pathway to enter the host cell nucleus and promote viral replication. Here, we analyzed the role of the nuclear import factor importin-?7 in H1N1 influenza virus pulmonary tropism by using various ex vivo imaging techniques (magnetic resonance imaging, confocal laser scanning microscopy, and correlative light-electron microscopy). We infected importin-?7 gene-deficient (?7(-/-)) mice with a recombinant H1N1 influenza virus and compared the in vivo viral kinetics with those in wild-type (WT) mice. In WT mice, influenza virus replication in the bronchial and alveolar epithelium already occurred a few days after infection. Accordingly, extensive mononuclear infiltration and alveolar destruction were present in the lungs of infected WT mice, followed by 100% lethality. Conversely, in ?7(-/-) mice, virus replication was restricted mostly to the bronchial epithelium with marginal alveolar infection, resulting in significantly reduced lung damage and enhanced animal survival. To investigate the host immune response during alveolar virus replication, we studied the role of primary macrophages in virus propagation and clearance. The ability of macrophages to support or clear the virus infection, as well as the host cellular immune responses, did not significantly differ between WT and ?7(-/-) mice. However, cytokine and chemokine responses were generally elevated in WT mice, likely reflective of increased viral replication in the lung. In summary, these data show that a cellular factor, importin-?7, is required for enhanced virus replication in the alveolar epithelium, resulting in elevated cytokine and chemokine levels, extensive mononuclear infiltration, and thus, severe pneumonia and enhanced virulence in mice. Importance: Influenza A viruses are respiratory pathogens that may cause pneumonia in humans. Viral infection and replication in the alveoli of the respiratory tract are believed to be crucial for the development of the acute respiratory distress syndrome associated with fatal outcomes in influenza virus-infected patients. Here, we report the requirement of a cellular factor, importin-?7, for efficient virus replication in the alveolar epithelium of mice. Using complementary ex vivo imaging approaches, we show that influenza virus replication is restricted to the bronchial epithelium, followed by enhanced survival in importin-?7-deficient mice. In contrast, the presence of this gene results in enhanced virus replication in the alveoli, elevated cytokine and chemokine responses, mononuclear infiltration, alveolar destruction, and 100% lethality in wild-type mice. Taken together, our results show that importin-?7 is particularly required for virus replication in the alveolar epithelium in association with severe pneumonia and death in mice.

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Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

Publications

Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

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