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Hypoxia-Inducible Factor (HIF)-1α Promotes Inflammation and Injury Following Aspiration-Induced Lung Injury in Mice

Suresh, Madathilparambil V.*; Balijepalli, Sanjay*; Zhang, Boya*; Singh, Vikas Vikram*; Swamy, Samantha*; Panicker, Sreehari*; Dolgachev, Vladislov A.*; Subramanian, Chitra*; Ramakrishnan, Sadeesh K.; Thomas, Bivin*; Rao, Tejeshwar C.; Delano, Matthew J.*; Machado-Aranda, David*; Shah, Yatrik M.; Raghavendran, Krishnan*

doi: 10.1097/SHK.0000000000001312
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ABSTRACT Acid aspiration-induced lung injury is a common disease in the intensive care unit (ICU) and acute respiratory distress syndrome (ARDS). Hypoxia-inducible factor (HIF)-1α is a major transcription factor responsible for regulating the cellular response to changes in oxygen tension. A clear understanding of the function of HIF-1α in lung inflammatory response is currently lacking. Here, we sought to determine the role of HIF-1α in type 2 alveolar epithelial cells (AEC) in the generation of the acute inflammatory response following gastric aspiration (GA). GA led to profound hypoxia at very early time points following GA. This correlated to a robust increase in HIF-1α, tissue albumin and pro-inflammatory mediators following GA in AECs. The extent of lung injury and the release of pro/anti-inflammatory cytokines were significantly reduced in HIF-1α (−/−) mice. Finally, we report that HIF-1α upregulation of the acute inflammatory response is dependent on NF-κB following GA.

*Department of Surgery, University of Michigan, Ann Arbor, Michigan

Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Department of Cell, Development and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama

Address reprint requests to Krishnan Raghavendran, MD, FACS, Professor of Surgery, 1500 E. Medical center drive, 1B407D, UH, University of Michigan, Ann Arbor, MI 48109. E-mail: kraghave@umich.edu

Received 1 November, 2018

Revised 21 November, 2018

Accepted 18 December, 2018

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Author contributions: Conception and design, MVS and KR performed research, MVS; SB; BZ; VVS; SS; SP; VAD; CS; SKR; BT analysis and interpretation, MVS; MJD; DM-A; YMS and KR drafting the manuscript for important intellectual content, MVS; TCR; and KR.

This work was supported by National Institutes of Health Grants—R01 GM111305-01 (KR).

The authors report no conflicts of interest.

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INTRODUCTION

Aspiration-induced lung injury is a major risk factor for acute respiratory distress syndrome (ARDS). Aspiration pneumonia is the leading cause of pneumonia in the intensive care unit and is one of the leading risk factors for acute lung injury (ALI) and ARDS (1–5). The severity of lung injury following gastric aspiration ranges from a mild, subclinical pneumonitis to progressive respiratory failure with significant morbidity and mortality. Gastric aspiration (GA) also frequently occurs in trauma or ICU patients with an altered state of consciousness such as head trauma, alcohol, or drug-induced alterations in sensorium, or cerebrovascular accidents. Additionally, the initial pneumonitis caused by GA may also be complicated by subsequent bacterial pneumonia (5–9).

The mechanisms underlying the impact of acid aspiration on lung inflammation and infection are poorly understood. Hypoxia is a hallmark of lung injury. The main characteristics of acid aspiration-induced lung injury include increased permeability of the alveolar–capillary interface and interstitial inflammation with edema. It is well understood that acid induced injury is neutrophil mediated, and that chemokines such as macrophage chemoattractant protein-(MCP)-1 induce macrophage aggregation and protective activity (4, 10). While other mediators such as products of the cyclooxygenase system have been well studied, it is unclear whether hypoxia, an early and important physiological response seen in all forms of lung injury, truly contributes to the deterioration of changes following acid aspiration. The cellular response to hypoxia is regulated by a family of transcription factors called the hypoxia-inducible factors (HIFs) (11–13). HIF-1, a nuclear transcription factor, is characterized as the master regulator of cellular oxygen homeostasis. This nuclear transcription factor also regulates the expression of target genes important in angiogenesis, erythropoiesis, energy metabolism, and cell survival (14–16). HIF-1 is composed of two subunits: HIF-1α and HIF-1β. HIF-1 activity is primarily regulated by the abundance of the HIF-1α subunit. Under hypoxic conditions, HIF-1α is stabilized and subsequently translocated to the nucleus where it dimerizes with HIF-1β and activates downstream target genes containing hypoxia-response elements (HRE) within their promoter or enhancer elements (17). Increased expression of HIF-1α leads to pronounced vascular beds with enhanced permeability, indicative of increased downstream expression of Vascular Endothelial Growth Factor (VEGF) (18, 19). There are many factors, such as inflammation and hypoxia that have the potential to stimulate and activate HIF-1α-related pathways. Our laboratory has demonstrated that type II alveolar epithelial cells (AEC), instead of being an innocent bystander in lung injury, primarily direct the inflammatory response to lung contusion (LC), a focal injury to the lungs, specifically via activation of HIF-1α (20). We observed that HIF-1α directly regulates interleukin (IL)-1β, which is a key mediator of the acute inflammatory response after LC (20). Recently, we reported that hypoxic activation of HIF-1α in AEC is critical to the initiation of the inflammatory response after LC (21). In addition, previous studies have demonstrated that HIF-1α acts through a known secondary transcription factor, NF-κB, suggesting a crosstalk and interdependence between NF-κB and HIF-1α signaling (20, 22). Subsequently, we have shown that HIF-1α downregulation of the acute inflammatory response is in part dependent on NF-κBp65 (20).

In the current manuscript we set out to determine the role of HIF-1α in acid aspiration-induced lung injury, and to examine HIF-1α induces epithelial cell injury and cell death. In this study we used C57BL/6 mice, hypoxia reporter mice (oxygen-dependent domain of HIF-1α linked with luciferase [ODD-Luc]), HIF-1α (+/+) mice, and HIF-1α (−/−) triple transgenic conditional knockout mice specific in type II AEC to confirm and characterize the fate of hypoxic AEC in the pathogenesis of acid aspiration-induced lung injury.

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MATERIALS AND METHODS

Animals

Male and female age-matched (6–8 weeks) wild-type (C57BL/6), hypoxia reporter mice with the oxygen-dependent domain of HIF-1α linked with luciferase (ODD-Luc) (Jackson Laboratories, Bar Harbor, Maine), and HIF-1α (triple transgenic conditional knockout specific for type II AEC, SP-C-rtTA_/tg/(tetO)7-CMV-Cretg/tg/HIF-1_flox/flox) mice were used in this study. The triple transgenic mice are capable of respiratory epithelium-specific conditional recombination in the floxed HIF-1α gene upon exposure to doxycycline (20, 21). All procedures performed were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan and complied with state, federal, and National Institutes of Health regulations.

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In Vivo imaging system (IVIS) of ODD-Luc mice

ODD-Luc transgenic mice (uninjured-saline only and injured-acid) were given a single intraperitoneal (i.p.) injection of a mixture of luciferin (50 mg/kg), ketamine (150 mg/kg), and xylazine (12 mg/kg) in sterile water. Twenty minutes later, mice were placed in a light-tight chamber equipped with a charge-coupled device IVIS imaging camera (Xenogen, Alameda, Calif). Photons were collected for a period of 5–20 s, and images were obtained with LIVING IMAGE software (Xenogen) and IGOR image analysis software (Wave Matrics, Lake Oswego, Ore). Upon injection of luciferin, bioluminescent background of normal tissues in the transgenic mice and bioluminescent signals from hypoxia were spontaneously measured noninvasively with an IVIS Spectrum imaging station that has been reported previously (20, 21).

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HIF-1α mice

Triple transgenic mice were created by mating HIF-1_flox/flox and SP-C-rtTA_/tg/(tetO)7-CMV-Cretg/tg transgenic mice. The generated mice, SP-C-rtTA_/tg/(tetO)7-CMV-Cretg/tg/HIF-1_flox/flox, are capable of respiratory epithelium specific conditional recombination in the floxed HIF-1α gene upon exposure to doxycycline (a generous gift from John J. LaPres, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Mich) (14, 23). The HIF-1_flox/flox were originally maintained in a C57BL/6 genetic background, whereas the SP-C-rtTA_/tg/(tetO)7-CMVCretg/tg were generated in an FVB/N genetic background. Aspiration was induced in type II AEC-specific HIF-1α conditional knockout (cKO) age-matched (6–8 weeks) triple transgenic mice (SP-C-rtTA_/tg/(tetO)7-CMV-Cretg/tg/HIF-1_flox/flox), which have been used previously, and control mice (14, 20, 21, 23).

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Doxycycline treatment to achieve recombination in HIF-1α mice

Postnatal recombination was carried out by exposing lactating dams to feed containing doxycycline (625 mg/kg; Harlan Teklad, Madison, Wis) and drinking water (0.8 mg/mL, Sigma Chemical Company, St. Louis, Mo). Triple transgenic mice were then maintained on the same doxycycline-containing food and water till they were 7 weeks of age. Doxycycline treatment was terminated 7 to 10 days before gastric aspiration. These mice will be referred to as HIF-1α (−/−) throughout the paper. Control animals (HIF-1α (+/+) used in the study were triple transgenic [SP-C-rtTA_/tg/(tetO)7-CMVCretg/tg/HIF-1_flox/flox] mice that were maintained on normal food and water ad libitum(20, 21).

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Murine model for gastric aspiration (GA)

The acid component of gastric aspirates is frequently modeled by intratracheal instillation of hydrochloric acid (HCl) in animals. Lung injury in mice following GA of dilute hydrochloric acid (ACID = normal saline (NS) plus HCl, final pH 1.25) is characterized by a bi-phasic response. C57BL/6 mice, ODD-Luc, HIF-1α (+/+), HIF-1α (−/−) mice, 20–25 g (6–8 wk, bred in-house) were used for ACID induction. Mice were anesthetized with 5% isoflurane in oxygen at a rate of 5 L/min. After induction of anesthesia, mice were injected with 30 μL of either normal saline, pH 5.3 (NS, vehicle control) or NS+HCl, pH 1.25 (ACID) via deep oral injection into the trachea. Animals were allowed to recover spontaneously (1, 4, 24).

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Administration of anesthetic, analgesic, and resuscitation

Animals were anesthetized by i.p. injection of ketamine (80–120 mg/kg body weight) and xylazine (5–10 mg/kg body weight). Systemic analgesics were not used due to their effects on the immune system. In the event of severe respiratory distresses beyond 48 h, animals were humanely euthanized (25).

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Lung pressure–volume (P–V) mechanics

Pulmonary respiratory mechanics were measured immediately after blood samples were obtained, and the mice were further exsanguinated by transection of the abdominal inferior vena cava. An 18-gauge metallic cannula was inserted into the trachea through a midline cervical exposure. Animals were then connected to a SCIREQ Flexivent (Montreal, QC, Canada) that allows for simultaneous animal ventilation and data capture. Immediate postmortem ventilation was done with the following parameters: tidal volume 10 mL/kg, respiratory rate 150 breaths/min, and positive end-expiratory pressure 2 cm H2O. With PVrV, a controlled inflation and deflation were performed to measure quasistatic compliance values.

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Albumin concentrations in broncho alveolar lavage (BAL)

Albumin concentrations in the BAL were measured by ELISA using a polyclonal rabbit anti-mouse albumin antibody and HRP-labeled goat anti-rabbit IgG (Bethyl Laboratories Inc, Montgomery, Tex) as described previously (26, 27).

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Determination of cytokine levels in BAL

Soluble concentrations of IL-1β, IL-6, IL-10, CCL6 (MCP-1), CCL12 (MCP-5), MIP-2, TNF α, Myeloperoxidase, and KC in BAL were determined using ELISA. Antibody and recombinant cytokines for these assays were obtained from R&D Systems (Minneapolis, Minn) as described previously (26, 27).

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Histopathology

ODD-Luc and HIF-1α mice lung specimens harvested at the time of death and subsequently fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin. Slides were evaluated by an experienced pathologist and graded for the presence of interstitial neutrophilic infiltrate, intra-alveolar hemorrhage, and pulmonary septal edema as described previously (26, 27).

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Western blot analysis

For NF-kB determination, whole-lung extracts were run on polyacrylamide gels with detailed protocol for sample processing as described previously (20, 27). Polyvinylidene difluoride membranes were probed sequentially with rabbit NFkB-p65 (1:1,000) followed by horseradish peroxidase-conjugated antirabbit IgG antibodies (1:3,000) (Cell Signaling, Danvers, Mass). Western blots were performed with rabbit HIF-1 antibody (Santa Cruz Biotechnology, Calif). Proteins were visualized by chemiluminescence using Super Signal West Pico PLUS Chemiluminescent Substrate (Life Technologies Corporation, Carlsbad, Calif) using a Kodak photo imager (23, 24).

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Preparation and isolation of type 2 alveolar epithelial cells from mice

Crude cell suspensions were prepared from HIF-1α (+/+) and HIF-1α (−/−) injured and uninjured mice after aspiration. Briefly, mice were anesthetized and exsanguinated by opening the peritoneum and clipping the left renal artery. Next, the lungs were perfused with PBS. They were filled with 1.5 mL Dispase via tracheal catheter, and then allowed to collapse naturally. The lungs were immediately covered with crushed ice and incubated for 2 min. Then, they were collected in 2 mL Dispase and incubated for 45 min at room temperature. The lungs were transferred to DMEM with 0.01% DNase I in a 100-mm Petri dish. The digested tissue was carefully teased from the airways with the curved edge of fine-tipped forceps and gently swirled for 5 to 10 min. The resulting suspension was successively filtered through 100 and 35 μm nylon filters, and then through 15 μm nylon mesh. The filtered suspension was centrifuged at 130 × g for 8 min at 4°C and resuspended in the culture media. The cells were incubated with biotinylated anti-CD-32 and biotinylated anti-CD-45 for 30 min at 37°C. Meanwhile, streptavidin-coated magnetic particles were washed completely in a medium with a polypropylene culture tube using a magnetic tube separator. After incubation, the cells were centrifuged (130 × g for 8 min at 4°C), resuspended in 7 mL DMEM, added to the magnetic particles, and incubated with gentle rocking for 30 min at room temperature. At the end of the incubation, the tube was attached to the magnetic tube separator with adhesive tape for 15 min. The cell suspension was aspirated from the bottom of the tube using a narrow-stemmed transfer pipet, centrifuged, and resuspended in culture media and incubated at 37°C. The following day, cultures were washed twice to collect the type 2 cells and the viability of the suspensions was determined by trypan blue exclusion (20, 21).

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TaqMan quantitative polymerase chain reaction

Total RNA was prepared from type 2 cells using RNeasy mini kit, according to the manufacturer's directions (Qiagen, Valencia, Calif). Total RNA was prepared from alveolar macrophages using RNeasy mini kit according to the manufacturer's directions (Qiagen, Germantown, Md). A total of 1 μg of RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, Calif). cDNA was then amplified by real-time quantitative TaqMan PCR using an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, Calif). GAPDH was analyzed as an internal control. TaqMan gene expression reagents or SYBR Green Master PCR mix were used to assay NF-kB, IL-1β, and VEGF-a (Applied Biosystems, Foster City, Calif). Q-PCR data expressed as fold-change in transcript expression and was determined by software developed by Applied Bio systems as described previously (26, 27).

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Statistical methods

Data are expressed as mean ± SEM. Statistical significance was estimated using one-way analysis of variance with GraphPad Prism 6.01 software (GraphPad Software Inc, La Jolla, Calif). Individual inter-group comparisons were analyzed using the two-tailed, unpaired t test with Welch correction. Analyses were run at a significance level of P < 0.05 (20, 26, 27).

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RESULTS

ODD-Luc mice show profound global hypoxia following gastric aspiration (GA)

We recently observed that global hypoxia is a major physiological consequence of lung contusion and is associated with increased nuclear translocation of HIF-1α (20, 21). A mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain fused to luciferase (ODD-Luc) was ubiquitously expressed in all tissues. Hypoxic stress leads to the accumulation of ODD-Luc in the tissues of this mouse model which can be identified by noninvasive bioluminescence measurement. In this study, ODD-Luc mice along with uninjured mice (saline only) were then subjected to IVIS imaging (dorsal and ventral view) to measure the degree of hypoxia 5, 24, 48, and 72 h after GA (n = 3). ODD-Luc mice show profound hypoxia at all-time points after GA (Fig. 1A). The bioluminescent signal is expressed in and displayed as an intensity map. The image display is adjusted to provide optimal contrast and resolution without affecting quantitation based on the maximum photons per second that appeared at 5 h (Fig. 1B). We have recently shown that the degree of luciferase expression in ODD-Luc mice was significantly increased in the lung at 48 h and in the liver at 24 and 48 h after LC (21). In an additional experiment using similar animals, GA was induced and the explanted organs (lungs, liver, kidney, and spleen) were subjected to IVIS. There was profound hypoxia in the lung, liver, kidney, and spleen of ODD-Luc mice (one uninjured and two injured) at 72 h following GA compared with uninjured mice (Fig. 1C). Next, we examined the HIF-1α level following GA. Wild-type (C57BL/6) mice were subjected to GA. The lungs of these injured mice along with uninjured controls were harvested at 0, 1, 5, and 24 h. HIF-1α expression, as determined by Western blot, was initially seen at 1 h after GA, and remained elevated at 24 h. The levels of HIF-1α expression were significantly elevated at all-time points compared with uninjured control (Fig. 1D). To determine the extent of mechanical injury, pressure volume (PV) mechanics were measured at different time intervals following acid administration (C57BL/6). At 1, 5, and 24 h time points, PV measurements indicated that pulmonary compliance was significantly decreased in the acid administered group compared with the uninjured animals (Fig. 1E). These data suggest that acid administration increased cell injury and inflammation.

Fig. 1

Fig. 1

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ODD-Luc mice show increased permeability injury and inflammation following GA

The injury and inflammatory response resulting from GA differ between mice with different genetic backgrounds. We therefore investigated the injury and inflammatory profile of ODD-Luc mice, which are of a FVB background, following acid induced lung injury to assess whether it is similar to that observed in C57BL/6 (WT) mice. After GA was induced in ODD-Luc mice, Broncho alveolar lavage fluid (BAL) was collected at 0,5, 24, 48, and 72 h following GA. ODD-Luc mice BAL was analyzed by ELISA to measure the levels of albumin, an index of permeability injury, and pro-inflammatory cytokines. The levels of albumin were significantly elevated at 24, 48, and 72 h, confirming severe alveolar permeability injury shortly after GA (Fig. 2A). The levels of interleukins (IL-1β, IL-6), monocyte chemotactic proteins (MCP-5), macrophage inflammatory protein (MIP)-2, and keratinocyte chemoattractant (KC) were also measured. These cytokines reflect various attributes of acute inflammation. The levels of IL-1β and IL-6 were significantly elevated at 5 and 24 h following GA (Fig. 2, B and C). Additionally, the levels of MCP-5 were significantly increased at 24 and 48 h (Fig. 2D). The expression of MIP-2 was significantly increased at 24, 48, and 72 h after GA (Fig. 2E). Lastly, there were significant increases in the levels of KC at 5, 24, and 48 h following GA (Fig. 2F).

Fig. 2

Fig. 2

Next, using flow cytometry, we determined the levels of macrophages and neutrophils in BAL fluid collected from ODD-Luc mice at different time intervals following GA. The levels of macrophages were significantly higher in injured mice in comparison with uninjured mice (Fig. 2G). Subsequently, when we examined the apoptosis of macrophages, the ODD-Luc mice show a significant level of macrophage apoptosis at all-time points compared with uninjured mice (Fig. 2H). Neutrophils were significantly higher in injured mice compared with uninjured mice (Fig. 2I). Histological evaluation demonstrated increased lung injury in ODD-Luc mice at all-time points following GA compared with uninjured mice (data not shown). This overall profile of inflammation and injury is consistent with the previously demonstrated research in mice and rodent models, substantiating ODD-Luc mice as a viable model for GA (20, 21).

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Down regulation of HIF-1α in type II AEC results in reduced permeability, lung injury, and inflammation following acid GA

We have determined that HIF-1α plays a key role in the mediation of the acute inflammatory response following LC (20, 21). Our previous studies have also shown that the acute inflammatory response in lung contusion is responsible for deficits in oxygenation, increases in quasistatic pulmonary compliance, and severe permeability injury (24, 26–29). To determine the functional aspect of HIF-1α to the extent of mechanical injury following GA, we used HIF-1α conditional knockout HIF-1α (−/−) mice and the corresponding HIF-1α (+/+) control mice (n = 16). To determine the extent of mechanical injury, pressure volume (PV) mechanics were measured at 5 and 24 h time intervals following acid administration. At both time points, PV measurements indicated that pulmonary compliance and higher inflatable volume at a pressure of V30 (TLC-30) were significantly decreased in the HIF-1α (+/+) group compared with the HIF-1α (−/−) mice (Fig. 3, A and B).

Fig. 3

Fig. 3

Next we examined BAL albumin as a reflection of permeability injury. There was a significant increase in the level of BAL albumin in HIF-1α (+/+) mice at 5, 24, and 48 h following GA compared to HIF-1α (−/−) mice (Fig. 3C). Next, we examined the role of HIF-1α activation on the production of pro/anti-inflammatory mediators following GA by measuring the levels of the interleukins (IL-1β and, IL-6), monocyte chemotactic proteins (MCP-1, MCP-5), chemokines keratinocyte chemoattractant (KC), myeloperoxidase, and TNFα. The levels of IL-1β were significantly lower at 5 and 24 h following GA in the HIF-1α (−/−) mice compared with HIF-1α (+/+) mice (Fig. 3D). The levels of IL-6 in the BAL were significantly higher at 5 h following GA in HIF-1α (+/+) mice (Fig. 3E). The levels of KC were also elevated prominently at 5 and 24 h following GA in HIF-1α (+/+) mice compared with HIF-1α (−/−) mice (Fig. 3F). The levels of MCP1 and MCP-5 were significantly decreased at 5, 24, and 72 h following GA in HIF-1α (−/−) mice compared with HIF-1α (+/+) mice (Fig. 3, G and H). Levels of myeloperoxidase, and TNFα were also elevated prominently at all the time points following aspiration in the HIF-1α (+/+) when compared with HIF-1α (−/−) mice (Fig. 3, I and J).

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HIF-1α (−/−) mice shows reduced inflammation and changes in BAL cellularity

Histological analysis demonstrated increased injury in HIF-1α (+/+) mice at all-time points compared with HIF-1α (−/−) mice following aspiration. Mice for each group were evaluated by an experienced, blinded pathologist, and examined for the presence of interstitial neutrophil infiltrate, and intra alveolar hemorrhage (Fig. 4A). We determined the levels of macrophages and neutrophils in BAL fluid collected from both HIF-1α (+/+) and HIF-1α (−/−) mice at different time intervals using flow cytometry. The number of macrophages was higher at all the time points in HIF-1α (+/+) mice compared with HIF-1α (−/−) mice (Fig. 4B). We have previously reported that neutrophils are mechanistically important in driving the acute inflammatory response following LC (26, 28, 30). At 24, 48, 72 h after GA, there was a significant increase in neutrophil levels in HIF-1α (+/+) mice compared with HIF-1α (−/−) mice (Fig. 4C). Taken together, these data suggest that activation of HIF-1α specifically in type II AEC contributes significantly to lung injury and acute inflammation following GA.

Fig. 4

Fig. 4

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HIF-1α activation in acid aspiration is dependent on NF-κB activation

Nuclear factor-kappa B (NF-κB) is considered the main pro-inflammatory family of transcription factors involved in several relevant medical pathologies. We have recently reported that HIF-1α is a major driver of acute inflammation after LC through type II AEC (20, 21). To determine the precise regulation of NF-κB activation on HIF-1α in type 2 AEC in the injured lung, HIF-1α (+/+) and HIF-1α (−/−) mice were subjected to GA and the lungs of these injured mice and uninjured controls were harvested at 0, 24, and 48 h and assessed for NF-κBp65 activation. As seen in Figure 5A, profound expression of NF-κB was seen in HIF-1α (+/+) mice at 24 and 48 h after GA compared with the HIF-1α (−/−) mice. In a separate experiment, mice were subjected to acid aspiration and type II AECs was isolated from the lungs of injured or uninjured animals. Levels of NF-kB genes were measured by quantitative reverse transcriptase-polymerase chain reaction. In this study the gene expression of NF-kB was found to be significantly elevated in the HIF-1α (+/+) mice following aspiration compared to HIF-1α (−/−) mice (Fig. 5B). Taken together, these results suggest that HIF-1α upregulation of the acute inflammatory response is dependent on NF-κB pathway.

Fig. 5

Fig. 5

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DISCUSSION

Aspiration-induced lung injury is considered an important risk factor for the development of ARDS and subsequent morbidity and mortality (31). This is particularly true in patients with an altered mental status either as a result of traumatic brain injury or consumption of drugs/alcohol (31). Acid-induced airway injury leads to an initial pro-inflammatory environment that most commonly resolves spontaneously in some patients, while it progresses to full blown ARDS in others. The severity of lung injury is related to the amount and acidity of the inoculum. Severe injury often occurs with pH less than 2.5, but can also occur at higher pH (32). The severity of the ALI is further modified by the host response. Inflammation is central to the pathogenesis and outcome of aspiration. Previous studies have reported that the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, increases within 5 h following GA (33). Additionally, it is well reported that acid aspiration peaks early, is neutrophil-mediated and the resolution of acute inflammation is at least in part dependent on macrophage aggregation and phagocytosis by neutrophils (24, 28, 34). The current study shows that upon hypoxic activation, HIF-1α plays a central role in the mediation of increased inflammation with neutrophil accumulation, macrophage apoptosis (predominant cell responsible for resolution of inflammation) and increased permeability injury via its effect on type II AEC during acid aspiration. Additionally, we report that the activation of HIF-1α is mediated through NF-κB.

Hypoxia is the most prominent physiologic feature of lung injury and ultimately leads to activation of HIF-1α. Profound hypoxia was noted on IVIS imaging of ODD-Luc mice at all-time points following GA (Fig. 1). Global hypoxia developed following acid aspiration and was pronounced in the lung, liver, spleen, and kidney. These findings are consistent with our past observation that hypoxia is a very prominent early feature in other models of acute lung injury (20). There are other possible activators of HIF-1α; however, previous studies with other markers of hypoxia such as pimonidazole confirmed that activation of HIF in models of lung injury reflects hypoxia (21).

In GA, reductions in BAL albumin and cytokine levels, as well as less severe histological injury, were observed in ODD mice similar to our previous observations in C57B/L6 mice (4). Additionally, the characteristics of the cellular infiltrate, timing, and nature of the inflammatory profile of these mice were similar to previous observations (4). The cytokines studied reflect the various attributes of acute inflammation leading to early phase reactants (IL-6), neutrophil accumulation (KC, MIP-2), and macrophage aggregation/activation (MCP-1, 5). The same pattern of increased permeability injury and acute inflammation that was seen in the HIF-1α (+/+) mice were significantly reduced with selective knockdown of HIF-1α (HIF−/−) in type II AEC. We have previously confirmed in the setting of LC that HIF-1α inhibition attenuates lung injury and inflammation. Additionally, it has been previously reported that presence of macrophage aggregation and activation constitutes an important mechanism of resolution of inflammation by ingestion of activated neutrophils. Our results suggest that presence of macrophage apoptosis was preferentially higher in the presence of HIF-1α activation, thereby affecting an important mechanism of resolution of acute inflammation. Taken together, the present data further confirms the direct role of HIF-1α, particularly in type II AEC in the initiation and maintenance of acute inflammation following acid aspiration injury.

The data presented in the current manuscript confirms that the transcription and protein expression of NF-κB increase following acid aspiration. HIF-1α is now known to be involved in the innate immune response under hypoxic conditions, and this occurs in part by cross signaling with NF-kB (22). As part of this complex interaction, an extensive crosstalk between the two main molecular players involved, HIF (hypoxia) and NF-kB (inflammation), has been reported (22). The relationship between these two transcriptional regulators is bidirectional. The phosphorylation of IκB (inhibitory κB) and subsequent activation of the NF-κB subunits p50 and p65 (RelA) have been reported to contribute to basal levels of HIF-1α mRNA and protein expression (22, 35).

In conclusion, the data presented in the current manuscript clearly demonstrate that HIF-1α plays a critical role in the regulation of the inflammatory response following acid aspiration-induced lung injury. Specifically, the regulation of type II AEC plays an important role in the propagation of the acute inflammatory response following acid aspiration. Above all, inhibition of HIF-1α is a promising target for therapeutic modulation in patients with aspiration induced lung injury.

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Acknowledgment

The authors thank Dr Jayakrishnan Nandakumar (Assistant Professor of Molecular, Cellular and Developmental Biology, University of Michigan) for his valuable suggestion of the article.

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Keywords:

Aspiration; cytokines; HIF-1α; IVIS; lung injury

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