High-fat Western diet consumption exacerbates silica-induced pulmonary inflammation and fibrosis.

Consumption of a high-fat Western diet (HFWD) contributes to obesity, disrupted adipose endocrine function, and development of metabolic dysfunction (MetDys). Impaired lung function, pulmonary hypertension, and asthma are all associated with MetDys. Over 35% of adults in the U.S. have MetDys, yet interactions between MetDys and hazardous occupational inhalation exposures are largely unknown. Occupational silica-inhalation leads to chronic lung inflammation, progressive fibrosis, and significant respiratory morbidity and mortality. In this study, we aim to determine the potential of HFWD-consumption to alter silica-induced inflammatory responses in the lung. Six-wk old male F344 rats fed a high fat Western diet (HFWD; 45 kcal % fat, sucrose 22.2% by weight) to induce MetDys, or standard rat chow (STD, controls) for 16 wk were subsequently exposed to silica (6 h/d, 5 d/wk, 39 d; Min-U-Sil 5®, 15 mg/m3) or filtered air; animals remained on their assigned diet for the study duration. Indices of lung inflammation and histopathologic assessment of lung tissue were quantified at 0, 4, and 8 wk after cessation of exposure. Combined HFWD+silica exposure increased bronchoalveolar lavage (BAL) total cells, leukocytes, and BAL lactate dehydrogenase compared to STD+silica exposure controls at all timepoints. HFWD+silica exposure increased BAL proinflammatory cytokines at 4 and 8 wk compared to STD+silica exposure. At 8 wk, histopathological analysis confirmed that alveolitis, epithelial cell hypertrophy and hyperplasia, lipoproteinosis, fibrosis, bronchoalveolar lymphoid hyperplasia and granulomas were exacerbated in the HFWD+silica-exposed group compared to STD+silica-exposed controls. Our results suggest an increased susceptibility to silica-induced lung disease caused by HFWD consumption.

Chemokine (C-C Motif) Receptor-Like 2 is not essential for lung injury, lung inflammation, or airway hyperresponsiveness induced by acute exposure to ozone.

Inhalation of ozone (O3), a gaseous air pollutant, causes lung injury, lung inflammation, and airway hyperresponsiveness. Macrophages, mast cells, and neutrophils contribute to one or more of these sequelae induced by O3 Furthermore, each of these aforementioned cells express chemokine (C-C motif) receptor-like 2 (Ccrl2), an atypical chemokine receptor that facilitates leukocyte chemotaxis. Given that Ccrl2 is expressed by cells essential to the development of O3-induced lung pathology and that chemerin, a Ccrl2 ligand, is increased in bronchoalveolar lavage fluid (BALF) by O3, we hypothesized that Ccrl2 contributes to the development of lung injury, lung inflammation, and airway hyperresponsiveness induced by O3 To that end, we measured indices of lung injury (BALF protein, BALF epithelial cells, and bronchiolar epithelial injury), lung inflammation (BALF cytokines and BALF leukocytes), and airway responsiveness to acetyl-ß-methylcholine chloride (respiratory system resistance) in wild-type and mice genetically deficient in Ccrl2 (Ccrl2-deficient mice) 4 and/or 24 hours following cessation of acute exposure to either filtered room air (air) or O3 In air-exposed mice, BALF chemerin was greater in Ccrl2-deficient as compared to wild-type mice. O3 increased BALF chemerin in mice of both genotypes, yet following O3 exposure, BALF chemerin was greater in Ccrl2-deficient as compared to wild-type mice. O3 increased indices of lung injury, lung inflammation, and airway responsiveness. Nevertheless, no indices were different between genotypes following O3 exposure. In conclusion, we demonstrate that Ccrl2 modulates chemerin levels in the epithelial lining fluid of the lungs but does not contribute to the development of O3-induced lung pathology.

Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure.

Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines-including IL-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], TNF receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1ß)-promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1ß, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin; yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indexes of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient compared with wild-type mice, no genotype-related differences in the other indexes existed following O3 exposure. O3 caused AHR to acetyl-ß-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were nonexistent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology.

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2B(f/f)-LysM(Cre)) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14, 17, 21, 25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2B(f/f)-LysM(Cre) mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase-1, markers for AAMs. A 10-fold reduction in IL-6 and a 5-fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH.

Adenosine A2B receptor and hyaluronan modulate pulmonary hypertension associated with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The development of pulmonary hypertension (PH) in patients with COPD is strongly associated with increased mortality. Chronic inflammation and changes to the lung extracellular matrix (ECM) have been implicated in the pathogenesis of COPD, yet the mechanisms that lead to PH secondary to COPD remain unknown. Our experiments using human lung tissue show increased expression levels of the adenosine A2B receptor (ADORA2B) and a heightened deposition of hyaluronan (HA; a component of the ECM) in remodeled vessels of patients with PH associated with COPD. We also demonstrate that the expression of HA synthase 2 correlates with mean pulmonary arterial pressures in patients with COPD, with and without a secondary diagnosis of PH. Using an animal model of airspace enlargement and PH, we show that the blockade of ADORA2B is able to attenuate the development of a PH phenotype that correlates with reduced levels of HA deposition in the vessels and the down-regulation of genes involved in the synthesis of HA.

Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine.

Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-ß-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine.

The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease.

Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A(2B)R) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A(2B)R or treatment with the A(2B)R antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A(2B)R attenuated vascular remodeling and hypertension in our model. Furthermore, direct A(2B)R activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A(2B)R antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.

Pulmonary responses to subacute ozone exposure in obese vs. lean mice.

The purpose of this study was to determine whether obesity affects pulmonary responses following a 3-day ozone exposure. Obese db/db and lean wild-type mice were exposed to ozone (0.3 ppm) for 72 h. In wild-type mice, ozone exposure caused pulmonary injury and inflammation, and these events were associated with reduced pulmonary compliance. In db/db mice, ozone-induced neutrophil recruitment to the lung was reduced and no reduction in compliance was observed. Similar results were obtained in obese Cpe(fat) mice, indicating that loss of leptin signaling in db/db mice does not account for these obesity-related changes. To examine the role of interleukin (IL)-6 in this obesity-related difference in ozone responsiveness, wild-type and IL-6-deficient mice were raised on 10% or 60% fat diets. Compared with 10% fat-fed mice, wild-type 60% fat-fed mice were obese and had reduced neutrophil recruitment following ozone. IL-6 deficiency reduced ozone-induced neutrophil recruitment in 10% fat-fed mice. In contrast, in obese mice, no effect of IL-6 deficiency on neutrophil recruitment was observed. Obesity-related differences in the effect of ozone on compliance were observed in both wild-type and IL-6-deficient mice. Obesity-related differences in serum IL-6 were observed and may account for obesity-related differences in the effect of IL-6 deficiency on neutrophil recruitment. In summary, the neutrophilic inflammation induced by prolonged low level ozone exposure was attenuated in obese mice and appeared to result from an absence of IL-6-dependent neutrophil recruitment in the obese mice.

Allergic airway responses in obese mice.

RATIONALE: Epidemiologic data indicate an increased incidence of asthma in the obese. OBJECTIVES: To determine whether obese mice exhibit augmented pulmonary responses after allergen sensitization and challenge. METHODS: Lean, wild-type (C57BL/6), obese ob/ob, and obese db/db mice were sensitized to ovalbumin (OVA), and then challenged with aerosolized OVA or phosphate-buffered saline (PBS). Changes in total pulmonary resistance (Rl) induced by intravenous methacholine were measured by forced oscillation. Blood was collected, bronchoalveolar lavage (BAL) was performed, and lungs were harvested for measurement of cytokine expression by real-time reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: OVA challenge increased baseline Rl in ob/ob, but not wild-type, mice, and airway responsiveness was greater in ob/ob than wild-type mice, regardless of the challenge. Compared with PBS, OVA challenge caused an increase in the number of BAL fluid (BALF) cells, an increase in lung Th2 cytokine expression, and an increase in serum IgE. Significantly fewer BALF cells were recovered from OVA-challenged ob/ob versus wild-type mice, whereas serum IgE levels were elevated significantly more in ob/ob versus wild-type mice. BALF and lung Th2 cytokine expression was not different in ob/ob versus wild-type mice. Airway responsiveness was greater in db/db versus wild-type mice, regardless of the challenge, and OVA caused airway hyperresponsiveness in db/db but not wild-type mice, despite reduced BALF cells in OVA-challenged db/db versus wild-type mice. CONCLUSIONS: These results demonstrate that obesity enhances OVA-induced changes in pulmonary resistance and serum IgE and that these changes are not the result of increased Th2 type airway inflammation.

Type I interleukin-1 receptor is required for pulmonary responses to subacute ozone exposure in mice.

Interleukin (IL)-1, a proinflammatory cytokine, is expressed in the lung after ozone (O(3)) exposure. IL-1 mediates its effects through the type I IL-1 receptor (IL-1RI), the only signaling receptor for both IL-1alpha and IL-1beta. The purpose of this study was to determine the role of IL-1RI in pulmonary responses to O(3.) To that end, wild-type, C57BL/6 (IL-1RI(+/+)) mice and IL-1RI-deficient (IL-1RI(-/-)) mice were exposed to O(3) either subacutely (0.3 ppm for 72 h) or acutely (2 ppm for 3 h). Subacute O(3) exposure increased bronchoalveolar lavage fluid (BALF) protein, interferon-gamma-inducible protein (IP)-10, soluble tumor necrosis factor receptor 1 (sTNFR1), and neutrophils in IL-1RI(+/+) and IL-1RI(-/-) mice. With the exception of IP-10, all outcome indicators were reduced in IL-1RI(-/-) mice. Furthermore, subacute O(3) exposure increased IL-6 mRNA expression in IL-1RI(+/+), but not IL-1RI(-/-) mice. Acute (2 ppm) O(3) exposure increased BALF protein, IL-6, eotaxin, KC, macrophage inflammatory protein (MIP)-2, IP-10, monocyte chemotactic protein-1, sTNFR1, neutrophils, and epithelial cells in IL-1RI(+/+) and IL-1RI(-/-) mice. For IL-6, eotaxin, MIP-2, and sTNFR1, there were small but significant reductions of these outcome indicators in IL-1RI(-/-) versus IL-1RI(+/+) mice at 6 hours after exposure, but not at other time points, whereas other outcome indicators were unaffected by IL-1RI deficiency. These results suggest that IL-1RI is required for O(3)-induced pulmonary inflammation during subacute O(3) exposure, but plays a more minor role during acute O(3) exposure. In addition, these results suggest that the induction of IL-6 via IL-1RI may be important in mediating the effects of O(3) during subacute exposure.

Pulmonary responses to acute ozone exposure in fasted mice: effect of leptin administration.

Leptin is a satiety hormone that also has proinflammatory effects, including augmentation of ozone-induced pulmonary inflammation. The purpose of this study was to determine whether reductions in endogenous levels of leptin can attenuate pulmonary responses to ozone. To reduce serum leptin, we fasted mice overnight before ozone exposure. Fasting caused a marked reduction in serum leptin to approximately one-sixth the levels observed in fed mice, and continuous infusion of leptin via Alzet micro-osmotic pumps restored serum leptin to, but not above, fed levels. Ozone exposure (2 ppm for 3 h) caused a significant, approximately 40% increase in pulmonary resistance (P < 0.01) and increased airway responsiveness in fasted but not in fed mice. The increased effect of ozone on pulmonary mechanics and airway responsiveness in fasted mice was not observed when leptin was restored via continuous infusion. Ozone exposure caused pulmonary inflammation, as evident by increases in bronchoalveolar lavage cells, protein, and soluble tumor necrosis factor receptors. There was no effect of fasting status on ozone-induced changes in the bronchoalveolar lavage inflammatory profile, and leptin treatment did not alter these responses. Our results indicate that fasting augments ozone-induced changes in pulmonary mechanics and airway responsiveness in mice. These effects of fasting are the result of declines in serum leptin. The mechanistic basis for this protective effect of leptin in fasted mice remains to be determined but is not related to effects on ozone-induced inflammation.

Increased pulmonary responses to acute ozone exposure in obese db/db mice.

Epidemiological studies indicate the incidence of asthma is increased in obese and overweight humans. Responses to ozone (O(3)), an asthma trigger, are increased in obese (ob/ob) mice lacking the satiety hormone leptin. The long form of leptin receptor (Ob-R(b)) is required for satiety; mice lacking this receptor (db/db mice) are also substantially obese. Here, wild-type (WT) and db/db mice were exposed to air or O(3) (2 ppm) for 3 h. Airway responsiveness, measured by the forced oscillation technique, was greater in db/db than WT mice after air exposure. O(3)-induced increases in pulmonary resistance and airway responsiveness were also greater in db/db mice. BALF eotaxin, IL-6, KC, and MIP-2 increased 4 h after O(3) exposure and subsided by 24 h, whereas protein and neutrophils continued to increase through 24 h. For each outcome, the effect of O(3) was significantly greater in db/db than WT mice. Previously published results obtained in ob/ob mice were similar except for O(3)-induced neutrophils and MIP-2, which were not different from WT mice. O(3) also induced pulmonary IL-1beta and TNF-alpha mRNA expression in db/db but not ob/ob mice. Leptin was increased in serum of db/db mice, and pulmonary mRNA expression of short form of leptin receptor (Ob-R(a)) was similar in db/db and WT mice. These data confirm obese mice have innate airway hyperresponsiveness and increased pulmonary responses to O(3). Differences between ob/ob mice, which lack leptin, and db/db mice, which lack Ob-R(b) but not Ob-R(a), suggest leptin, acting through Ob-R(a), can modify some pulmonary responses to O(3).

Role of interleukin-6 in murine airway responses to ozone.

This study sought to examine the role of interleukin-6 (IL-6) in ozone (O(3))-induced airway injury, inflammation, and hyperresponsiveness (AHR). Subacute (72 h) exposure to 0.3 ppm O(3) significantly elevated bronchoalveolar lavage fluid (BALF) protein, neutrophils, and soluble TNF receptors (sTNFR1 and sTNFR2) in wild-type C57BL/6 (IL-6(+/+)) mice; however, all four outcome indicators were significantly reduced in IL-6-deficient (IL-6(-/-)) compared with IL-6(+/+) mice. Acute O(3) exposure (2 ppm for 3 h) increased BALF protein, KC, macrophage inflammatory protein(MIP)-2, eotaxin, sTNFR1, and sTNFR2 in IL-6(+/+) mice. However, MIP-2 and sTNFR2 were not significantly increased following O(3) exposure in IL-6(-/-) mice. Increases in BALF neutrophils induced by O(3) (2 ppm for 3 h) were also significantly reduced in IL-6(-/-) vs. IL-6(+/+) mice. Airway responsiveness to methacholine was measured by whole body plethysmography before and following acute (3 h) or subacute (72 h) exposure to 0.3 ppm O(3). Acute O(3) exposure caused AHR in both groups of mice, but there was no genotype-related difference in the magnitude of O(3)-induced AHR. AHR was absent in mice of either genotype exposed for 72 h. Our results indicate that IL-6 deficiency reduces airway neutrophilia, as well as the levels of BALF sTNFR1 and sTNFR2 following acute high dose and/or subacute low-dose O(3) exposure, but has no effect on O(3)-induced AHR.

Hyperosmolar solution effects in guinea pig airways. III. Studies on the identity of epithelium-derived relaxing factor in isolated perfused trachea using pharmacological agents.

Hyperosmolar challenge of airway epithelium stimulates the release of epithelium-derived relaxing factor (EpDRF), but the identity of EpDRF is not known. We examined the effects of pharmacological agents on relaxant responses of methacholine (3 x 10(-7) M)-contracted guinea pig perfused trachea to mucosal hyperosmolar challenge using D-mannitol. Responses were inhibited by gossypol (5 x 10(-6) M), an agent with diverse actions, by the carbon monoxide (CO) scavenger hemoglobin (10(-6) M), and by the heme oxygenase (HO) inhibitor zinc (II) protoporphyrin IX (10(-4) M). The HO inhibitor chromium (III) mesoporphyrin IX (10(-4) M) was not inhibitory, and the HO activator heme-L-lysinate (3 x 10(-4) M) did not evoke relaxant responses. The CO donor tricarbonyldichlororuthenium (II) dimer (2.2 x 10(-4) M) elicited small relaxation responses. Other agents without an effect on responses included: apyrase, adenosine, 6-anilino-5,8-quinolinequinone (LY83583), proadifen, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), diphenhydramine, glibenclamide, HgCl2, tetrodotoxin, nystatin, alpha-hemolysin, 8-bromoguanosine 3',5'-cyclic monophosphothioate, Rp-isomer, 12-O-tetradecanoylphorbol-13-acetate, cholera toxin, pertussis toxin, thapsigargin, nifedipine, Ca(2+)-free mucosal solution, hydrocortisone, and epidermal growth factor. Cytoskeleton inhibitors, includingerythro-9-(2-hydroxyl-3-nonyl)adenine, colchicine, nocodazole, latrunculin B, and cytochalasins B and D, had no effect on relaxation responses. The results suggest provisionally that a portion of EpDRF activity may be due to CO and that the release of EpDRF does not involve cytoskeletal reorganization.

Hyperosmolar solution effects in guinea pig airways. IV. Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity.

We investigated the in vivo and in vitro effects of lipopolysaccharide (LPS) treatment (4 mg/kg i.p.) on guinea pig airway smooth muscle reactivity and epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Hyperosmolar challenge of the epithelium releases epithelium-derived relaxing factor (EpDRF). Using a two-chamber, whole body plethysmograph 18 h post-treatment, animals treated with LPS were hyporeactive to inhaled MCh aerosol. This could involve an increase in the release and/or actions of EpDRF, because LPS treatment enhanced EpDRF-induced smooth muscle relaxation in vitro in the isolated perfused trachea apparatus. In isolated perfused tracheas the basal transepithelial potential difference (Vt) was increased after LPS treatment. The increase in Vt was inhibited by amiloride and indomethacin. Concentration-response curves for changes in Vt in response to serosally and mucosally applied MCh were biphasic (hyperpolarization, <3 x 10(-7)M; depolarization, >3 x 10(-7)M); MCh was more potent when applied serosally. The hyperpolarization response to MCh, but not the depolarization response, was potentiated after LPS treatment. In both treatment groups, mucosally applied hyperosmolar solution (using added NaCl) depolarized the epithelium; this response was greater in tracheas from LPS-treated animals. The results of this study indicate that airway hyporeactivity in vivo after LPS treatment is accompanied by an increase in the release and/or actions of EpDRF in vitro. These changes may involve LPS-induced bioelectric alterations in the epithelium.