Lung Inflammation In Mouse Model Research Articles

Ratiometric fluorescent probes for capturing endogenous hypochlorous acid in the lungs of mice.

Hypochlorous acid (HClO) is a promising diagnostic marker for inflammation and relevant diseases. Although many probes were previously developed for HClO imaging, the development of organ targeting probes is still lacking. Herein, we designed and synthesized a series of cyanine derivatives as ratiometric fluorescent probes to detect endogenous HClO in the lungs with inflammation. By installing diverse lipid chains and amino groups on cyanine, we identified that ClO1, with one n-octadecane chain and two 2-[[2-(dimethylamino)ethyl]methylamino]-ethyl groups, is a superior probe to target the lungs over other major organs in mice. ClO1 was able to sense both exogenous and endogenous HClO in A549 (human lung epithelial) cells through fluorescence ratiometric imaging. In a lipopolysaccharide (LPS)-induced lung inflammation mouse model, ClO1 effectively captured endogenous HClO in the lungs after intravenous administration. Overall, these cyanine-derived probes merit further development as organ targeting HClO sensors.

Suppressed ubiquitination of Nrf2 by p47phox contributes to Nrf2 activation

Although critical in phagocytosis in innate immunity, reactive oxygen species (ROS) collaterally inflict damage to host phagocytes because they indiscriminate targets. Since Nrf2 increases the expression of anti-oxidant enzymes that nullifies ROS, ROS activating Nrf2 is a critical negative regulatory step for countering the deleterious effects of ROS. Here, we postulate whether, along with ROS activating Nrf2, NADPH oxidase components also participate in direct activation of Nrf2, contributing to protection from ROS. Our results show that the p47phox of the NADPH oxidase, but not p65phox or p40phox, physically binds to Nrf2, activating the Nrf2 function. p47phox binding to Nrf2/Keap1 complex suppresses the ubiquitination of Nrf2, while p47phox becomes ubiquitinated by Keap1. p47phox increases the nuclear translocation of Nrf2 and the expression of Nrf2-dependent genes, whereas genetic ablation of p47phox decreases the expression of those genes. In a lipopolysaccharide-induced acute lung inflammation mouse model, selective expression of p47phox in mouse lungs induces the expression of Nrf2-dependent genes and is sufficient to suppress neutrophilic lung inflammation. Therefore, our findings suggest that p47phox is a novel regulator of Nrf2 function.

Ginsenosides from Korean Red Ginseng ameliorate lung inflammatory responses: inhibition of the MAPKs/NF-κB/c-Fos pathways

BackgroundKorean Red Ginseng (steamed and dried white ginseng, Panax ginseng Meyer) is well known for enhancing vital energy and immune capacity and for inhibiting cancer cell growth. Some clinical studies also demonstrated a therapeutic potential of ginseng extract for treating lung inflammatory disorders. This study was conducted to establish the therapeutic potential of ginseng saponins on the lung inflammatory response. MethodsFrom Korean Red Ginseng, 11 ginsenosides (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, and Rh2) were isolated. Their inhibitory potential and action mechanism were evaluated using a mouse model of lung inflammation, acute lung injury induced by intranasal lipopolysaccharide administration. Their anti-inflammatory activities were also examined in lung epithelial cell line (A549) and alveolar macrophage (MH-S). ResultsAll ginsenosides orally administered at 20 mg/kg showed 11.5–51.6% reduction of total cell numbers in bronchoalveolar lavage fluid (BALF). Among the ginsenosides, Rc, Re, Rg1, and Rh2 exhibited significant inhibitory action by reducing total cell numbers in the BALF by 34.1–51.6% (n = 5). Particularly, Re showed strong and comparable inhibitory potency with that of dexamethasone, as judged by the number of infiltrated cells and histological observations. Re treatment clearly inhibited the activation of mitogen-activated protein kinases, nuclear factor-κB, and the c-Fos component in the lung tissue (n = 3). ConclusionCertain ginsenosides inhibit lung inflammatory responses by interrupting these signaling molecules and they are potential therapeutics for inflammatory lung diseases.

Identification of Lung Inflammation-Related Elevation of Acylamino Acid Releasing Enzyme (APEH) Activity Using an Enzymomics Approach.

Disease-associated alterations of enzymatic functions are potentially useful as disease biomarkers, and here we show that an enzymomics (omics of active enzymes) approach, in which enzymatic activities are screened with panels of substrates, can be an effective way to identify such alterations. In the present study, we used a panel of fluorogenic substrates to search for altered enzyme activities in bronchoalveolar lavage fluid (BALF) from a mouse model of lung inflammation. We found that acylamino acid releasing enzyme (APEH) activity was highly elevated, apparently reflecting the increased population of immune cells in the inflamed lung.

Design and Synthesis of N-Arylphthalimides as Inhibitors of Glucocorticoid-Induced TNF Receptor-Related Protein, Proinflammatory Mediators, and Cytokines in Carrageenan-Induced Lung Inflammation.

N-Arylphthalimides (1-10P) derived from thalidomide by insertion of hydrophobic groups were evaluated for anti-inflammatory activity, and (4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(4-ethoxyphenyl)methylidene]benzohydrazide 6P was identified as a promising anti-inflammatory agent. Further testing confirmed that compared with the control, 6P treatment resulted in a considerable decrease in CD4(+), NF-κB p65(+), TNF-α(+), IL-6(+), GITR(+), and IL-17(+) cell populations and an increase in the Foxp3(+), CD4(+)Foxp3(+), and IκBα(+) populations in whole blood and pleural fluid of a mouse model of lung inflammation. Moreover, treatment with compound 6P decreased the proteins associated with inflammation including TNF-α, IL-6, IL-17, GITR, NF-κB, COX-2, STAT-3, and iNOS and increased the anti-inflammatory mediators such as IL-10 and IL-4. Further, histopathological examination confirmed the potent anti-inflammatory effects of compound 6P. Thus, the N-arylphthalimide derivative 6P acts as a potent anti-inflammatory agent in the carrageenan-induced lung inflammation model, suggesting that this compound may be useful for the treatment of inflammation in a clinical setting.

β-Catenin is required for the differentiation of iNKT2 and iNKT17 cells that augment IL-25-dependent lung inflammation.

BackgroundInvariant Natural Killer T (iNKT) cells have been implicated in lung inflammation in humans and also shown to be a key cell type in inducing allergic lung inflammation in mouse models. iNKT cells differentiate and acquire functional characteristics during development in the thymus. However, the correlation between development of iNKT cells in the thymus and role in lung inflammation remains unknown. In addition, transcriptional control of differentiation of iNKT cells into iNKT cell effector subsets in the thymus during development is also unclear. In this report we show that β-catenin dependent mechanisms direct differentiation of iNKT2 and iNKT17 subsets but not iNKT1 cells.MethodsTo study the role for β-catenin in lung inflammation we utilize mice with conditional deletion and enforced expression of β-catenin in a well-established mouse model for IL-25-dependen lung inflammation.ResultsSpecifically, we demonstrate that conditional deletion of β-catenin permitted development of mature iNKT1 cells while impeding maturation of iNKT2 and 17 cells. A role for β-catenin expression in promoting iNKT2 and iNKT17 subsets was confirmed when we noted that enforced transgenic expression of β-catenin in iNKT cell precursors enhanced the frequency and number of iNKT2 and iNKT17 cells at the cost of iNKT1 cells. This effect of expression of β-catenin in iNKT cell precursors was cell autonomous. Furthermore, iNKT2 cells acquired greater capability to produce type-2 cytokines when β-catenin expression was enhanced.DiscussionThis report shows that β-catenin deficiency resulted in a profound decrease in iNKT2 and iNKT17 subsets of iNKT cells whereas iNKT1 cells developed normally. By contrast, enforced expression of β-catenin promoted the development of iNKT2 and iNKT17 cells. It was important to note that the majority of iNKT cells in the thymus of C57BL/6 mice were iNKT1 cells and enforced expression of β-catenin altered the pattern to iNKT2 and iNKT17 cells suggesting that β-catenin may be a major factor in the distinct pathways that critically direct differentiation of iNKT effector subsets.ConclusionsThus, we demonstrate that β-catenin expression in iNKT cell precursors promotes differentiation toward iNKT2 and iNKT17 effector subsets and supports enhanced capacity to produce type 2 and 17 cytokines which in turn augment lung inflammation in mice.

Activity-Based Probe for N-Acylethanolamine Acid Amidase.

N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme.

Role of microRNAs in allergic asthma: present and future.

MicroRNAs (miRNAs) modulate gene transcription in response to environmental stressors and other stimuli. A role for miRNAs in inflammation and immunity has been demonstrated and further evidence suggests that miRNAs also play a role in allergic asthma. Studies investigating the differential expression of miRNAs in biological fluids between asthma patients and controls have been published, as have their role in immune cell subsets. Further development of miRNAs in therapy has been addressed. miRNA-146a has been implicated in autoimmunity and allergic inflammation and miRNA-155 in the development of atopy. Targeting of miRNA-1 and miRNA-145 has been used to inhibit lung inflammation in mouse models of asthma. Although these recent findings need to be confirmed, miRNAs may prove to be useful as potential biomarkers of disease. However, their use as therapeutic targets in the lung remains unclear. There may be a potential role for using circulating miRNAs as biomarkers of disease status or response to therapy. The use of miRNAs as asthma therapy remains to be determined.

OX40 blockade inhibits house dust mite driven allergic lung inflammation in mice and in vitro allergic responses in humans.

The costimulatory receptor OX40 is expressed on activated T cells and regulates T-cell responses. Here, we show the efficacy and mechanism of action of an OX40 blocking antibody using the chronic house dust mite (HDM) mouse model of lung inflammation and in vitro HDM stimulation of cells from HDM allergic human donors. We have demonstrated that OX40 blockade leads to a reduction in the number of eosinophils and neutrophils in the lavage fluid and lung tissue of HDM sensitized mice. This was accompanied by a decrease in activated and memory CD4(+) T cells in the lungs and further analysis revealed that both the Th2 and Th17 populations were inhibited. Improved lung function and decreased HDM-specific antibody responses were also noted. Significantly, efficacy was observed even when anti-OX40 treatment was delayed until after inflammation was established. OX40 blockade also inhibited the release of the Th2 cytokines IL-5 and IL-13 from cells isolated from HDM allergic human donors. Altogether, our data provide evidence of a role of the OX40/OX40L pathway in ongoing allergic lung inflammation and support clinical studies of a blocking OX40 antibody in Th2 high severe asthma patients.

Inhalation of glycopyrronium inhibits cigarette smoke-induced acute lung inflammation in a murine model of COPD

Glycopyrronium bromide (GB) is a muscarinic receptor antagonist that has been used as a long-acting bronchodilator in chronic obstructive pulmonary disease (COPD) patients. The aim of this study was to investigate the anti-inflammatory activity of inhaled GB in a cigarette smoke-induced acute lung inflammation mouse model. We found that aerosol pre-treatment with GB suppresses the accumulation of neutrophils and macrophages in the bronchoalveolar lavage fluid (BALF) in cigarette smoke (CS)-exposed mice. GB at doses of 300 and 600 μg/ml significantly inhibited the CS-induced increases in the mRNA and protein expression levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1 and transforming growth factor (TGF)-β1 in lung tissues and the BALF. Moreover, GB at a dose of 600 μg/ml significantly inhibited the CS-induced changes in glutathione (GSH) and myeloperoxidase (MPO) activities in the BALF, decreased the CS-induced expression of matrix metalloproteinases (MMP)-9, and increased the CS-induced expression of tissue inhibitor of metalloproteinases (TIMP)-1, as determined through the immunohistochemical staining of lung tissue. Our results demonstrate the beneficial effects of inhaled GB on the inflammatory reaction in COPD.

A comprehensive analysis of oxidative stress in the ozone-induced lung inflammation mouse model

Ozone is an oxidizing environmental pollutant that contributes significantly to respiratory health. Exposure to increased levels of ozone has been associated with worsening of symptoms of patients with asthma and COPD (chronic obstructive pulmonary disease). In the present study, we investigated the acute and chronic effects of ozone exposure-induced oxidative stress-related inflammation mechanics in mouse lung. In particular, we investigated the oxidative stress-induced effects on HDAC2 (histone deacetylase 2) modification and activation of the Nrf2 (nuclear factor erythroid-related factor 2) and HIF-1α (hypoxia-inducible factor-1α) signalling pathways. Male C57BL/6 mice were exposed to ozone (3 p.p.m.) for 3 h a day, twice a week for a period of 1, 3 or 6 weeks. Control mice were exposed to normal air. After the last exposure, mice were killed for BAL (bronchoalveolar lavage) fluid and lung tissue collection. BAL total cell counts were elevated at all of the time points studied. This was associated with increased levels of chemokines and cytokines in all ozone-exposed groups, indicating the presence of a persistent inflammatory environment in the lung. Increased inflammation and Lm (mean linear intercept) scores were observed in chronic exposed mice, indicating emphysematous changes were present in lungs of chronic exposed mice. The antioxidative stress response was active (indicated by increased Nrf2 activity and protein) after 1 week of ozone exposure, but this ability was lost after 3 and 6 weeks of ozone exposure. The transcription factor HIF-1α was elevated in 3- and 6-week ozone-exposed mice and this was associated with increased gene expression levels of several HIF-1α target genes including Hdac2 (histone deacetylase 2), Vegf (vascular endothelial growth factor), Keap1 (kelch-like ECH-associated protein 1) and Mif (macrophage migration inhibitory factor). HDAC2 protein was found to be phosphorylated and carbonylated in nuclear and cytoplasm fractions, respectively, and was associated with a decrease in DNA-binding activity and protein expression of HDAC2. Decreased HDAC2 activity, most likely a direct result of protein modification, in combination with the loss of the antioxidative stress response and activation of the HIF-1α pathway, contribute to the inflammatory response and emphysema observed in ozone-exposed mice.

PXS-4681A, a Potent and Selective Mechanism-Based Inhibitor of SSAO/VAP-1 with Anti-Inflammatory Effects In Vivo

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Abstract 6: Co-targeting of Thrombomodulin and the Endothelial Protein C Receptor Protects the Pulmonary Vasculature in a Mouse Model of Acute Endothelial Injury

Background Recombinant activated protein C is protective in animal models of sepsis, stroke, and focal thrombosis, but has a poor side effect profile. Thrombomodulin (TM) and the Endothelial Protein C Receptor (EPCR) are endothelial membrane proteins, which partner to bind thrombin and activate protein C at sites of inflammation, ischemia, and thrombosis. Both proteins can be lost from the endothelial surface in a variety of disease states. We have shown previously that targeted delivery of TM to the Platelet Endothelial Cell Adhesion Molecule (PECAM-1) on the endothelial surface is protective in mouse models of lung inflammation and ischemia. Unlike endogenous TM, however, recombinant TM targeted to PECAM-1 does not effectively partner with endogenous EPCR. Objective We hypothesized that co-delivery of TM and EPCR would enhance in vitro activity and therapeutic efficacy in vivo. Methods TM and EPCR were fused to single chain fragments of two anti-PECAM antibodies, which are known to demonstrate collaborative binding enhancement. TM and EPCR fusion proteins were tested on PECAM-expressing cells and mouse endothelial cells. Fusion proteins were injected intravenously in C57BL/6 mice prior to intratracheal injection of endotoxin. Expression of VCAM-1 was measured using qPCR and MIP-2 was quantified in bronchoalveolar lavage (BAL) fluid. Endothelial barrier dysfunction was quantified by measuring transendothelial leak of 125I-labeled albumin. Results TM and EPCR fusion proteins target PECAM-1 on endothelial cells and demonstrate collaborative binding. Independent of binding effects, the EPCR fusion protein results in a 50% increase in the activation of protein C by cell-bound TM, an effect blocked by anti-EPCR antibody. Consistent with in vitro results, the combination of TM and EPCR fusion proteins enhances protection in endotoxin-induced lung injury, reducing VCAM expression, BAL MIP-2, and transendothelial leak of albumin (p < 0.001), as compared to TM fusion protein alone. Conclusion Co-targeting of TM and EPCR to PECAM-1 enhances activation of protein C and represents a novel and promising therapeutic strategy for the treatment of diseases involving acute endothelial injury.

CD8α⁺β⁻ and CD8α⁺β⁺ plasmacytoid dendritic cells induce Foxp3⁺ regulatory T cells and prevent the induction of airway hyper-reactivity.

Dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self-antigens. Here, we demonstrate for the first time that mouse plasmacytoid DCs (pDCs) can be segregated into three distinct populations, exhibiting phenotypic and functional differences, according to their surface expression of CD8α or CD8β as CD8α−β−, CD8α+β− or CD8α+β+. In a mouse model of lung inflammation, adoptive transfer of CD8α+β− or CD8α+β+ pDCs prevents the development of airway hyperreactivity. The tolerogenic features of these subsets are associated with increased production of retinoic acid, which leads to the enhanced induction of Foxp3+ regulatory T cells compared to CD8α−β− pDCs. Our data thus identify subsets of pDCs with potent tolerogenic functions that may contribute to the maintenance of tolerance in mucosal sites such as the lungs.

Early detection of lung inflammation: Exploiting T1‐effects of iron oxide particles using UTE MRI

At high magnetic fields diagnostic proton MRI of the lung is problematic, because of fast T2* relaxation. The application of superparamagnetic contrast agents and the exploitation of the corresponding T2* effect is inefficient with conventional MRI methods, which limits the early detection of lung diseases. However, a simple theoretical treatment shows that in the lung, by the use of ultra-short echo time sequences, T2* effects can be neglected while T(1) shortening effects can be used for signal detection. In our study, we have applied a theoretically and experimentally optimized 3D ultra-short echo time sequence to lung phantoms and to a mouse model of lung inflammation, which was induced by systemic bacterial infection. Following the systemic application of very small superparamagnetic iron oxide nanoparticles, a significant signal increase in the lung of infected animals was detected already at 24 h postinfection, compared to control mice (17%, P < 0.001). Iron accumulation in the lung parenchyma as consequence of the host immune response was histologically confirmed. By conventional T2*- and T(2)-weighted imaging, neither structural changes nor formation of substantial edema were observed.

Integrin β3 is not critical for neutrophil recruitment in a mouse model of pneumococcal pneumonia

Streptococcus pneumoniae is one of the most common causes of bacterial pneumonias in humans. Neutrophil migration into lungs infected with S. pneumoniae is central to the host defense but the mechanisms of neutrophil recruitment, as mediated by S. pneumoniae, into lungs are incompletely understood. Therefore, we have assessed the role of integrin αvβ3 by evaluating its subunit β3 in a mouse model of lung inflammation induced by S. pneumonia. Integrin subunit β3 knockout (β3(-/-)) and wild-type (WT) mice were intratracheally instilled with either S. pneumoniae or saline. Other groups of WT mice were treated intraperitoneally with 25μg or 50μg of antibody against integrin β3 or with isotype-matched antibody at 1h before instillation of S. pneumoniae. Mice were killed 24h after infection. Flow cytometry confirmed the absence or presence of integrin subunit β3 on peripheral blood neutrophils in β3(-/-) or WT mice, respectively. Neutrophil numbers in bronchoalveolar lavage (BAL) from infected β3(-/-) and WT mice showed no differences. Neutrophil numbers in BAL of infected WT mice treated with β3 antibody were lower compared with those without antibody but similar to those of mice administered isotype-matched antibody. Many neutrophils were present in the perivascular spaces of the lungs in β3(-/-) mice. Lungs from infected β3(-/-) mice had negligible mitogen-activated protein kinase expression compared with those of infected WT mice. Thus, integrin β3 or its heterodimer αvβ3 is not critical for neutrophil migration into lungs infected with S. pneumoniae.

Inflammation provoked byMycoplasma pneumoniaeextract: implications for combination treatment with clarithromycin and dexamethasone

Recently, combination treatment with a macrolide and a steroid for Mycoplasma pneumoniae (Mp) pneumonia has been reported to be effective. Thus, the effect of this combination on a mouse model of lung inflammation associated with Mp extract (the LIMEX mouse) was studied. Interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were induced in Mp extract-treated RAW264.7 cells, and this induction was inhibited by dexamethasone, parthenolide, SB203580 or LY294002. This suggested that Mp extract activates nuclear factor κB-, p38- and PI-3K-linked pro-inflammatory signals. The LIMEX mice were then either treated with or without clarithromycin and/or dexamethasone. Clarithromycin administration enhanced the production of IL-6, TNF-α, macrophage inflammatory protein-1α, monocyte chemotactic protein-1 and RANTES, while their production was perfectly suppressed by the combination of clarithromycin and dexamethasone. IL-17, IL-23, keratinocyte-derived chemokine (KC) and interferon-γ levels were not affected by clarithromycin treatment, but they were significantly suppressed by the combination of dexamethasone and clarithromycin. Collectively, some components of Mp extract provoked an inflammatory reaction in the RAW 264.7 cell line and LIMEX mice. Whereas the lung reaction in LIMEX mice was further exacerbated by clarithromycin treatment, it was resolved by the combinational treatment with clarithromycin and dexamethasone.

Pharmacological characterisation of anti-inflammatory compounds in acute and chronic mouse models of cigarette smoke-induced inflammation

BackgroundCandidate compounds being developed to treat chronic obstructive pulmonary disease are typically assessed using either acute or chronic mouse smoking models; however, in both systems compounds have almost always been administered prophylactically. Our aim was to determine whether the prophylactic effects of reference anti-inflammatory compounds in acute mouse smoking models reflected their therapeutic effects in (more clinically relevant) chronic systems.MethodsTo do this, we started by examining the type of inflammatory cell infiltrate which occurred after acute (3 days) or chronic (12 weeks) cigarette smoke exposure (CSE) using female, C57BL/6 mice (n = 7-10). To compare the effects of anti-inflammatory compounds in these models, mice were exposed to either 3 days of CSE concomitant with compound dosing or 14 weeks of CSE with dosing beginning after week 12. Budesonide (1 mg kg-1; i.n., q.d.), roflumilast (3 mg kg-1; p.o., q.d.) and fluvastatin (2 mg kg-1; p.o., b.i.d.) were dosed 1 h before (and 5 h after for fluvastatin) CSE. These dose levels were selected because they have previously been shown to be efficacious in mouse models of lung inflammation. Bronchoalveolar lavage fluid (BALF) leukocyte number was the primary endpoint in both models as this is also a primary endpoint in early clinical studies.ResultsTo start, we confirmed that the inflammatory phenotypes were different after acute (3 days) versus chronic (12 weeks) CSE. The inflammation in the acute systems was predominantly neutrophilic, while in the more chronic CSE systems BALF neutrophils (PMNs), macrophage and lymphocyte numbers were all increased (p < 0.05). In the acute model, both roflumilast and fluvastatin reduced BALF PMNs (p < 0.01) after 3 days of CSE, while budesonide had no effect on BALF PMNs. In the chronic model, therapeutically administered fluvastatin reduced the numbers of PMNs and macrophages in the BALF (p ≤ 0.05), while budesonide had no effect on PMN or macrophage numbers, but did reduce BALF lymphocytes (p < 0.01). Roflumilast's inhibitory effects on inflammatory cell infiltrate were not statistically significant.ConclusionsThese results demonstrate that the acute, prophylactic systems can be used to identify compounds with therapeutic potential, but may not predict a compound's efficacy in chronic smoke exposure models.

Surfactant protein-A reduces translocation of mediators from the lung into the circulation

ABSTRACTThe objective of this study was to characterize a mouse model of lung inflammation and determine the effect of surfactant protein A (SP-A, or sftpa) on the transfer of inflammatory mediators from these injured lungs into the systemic circulation. Lung inflammation was induced in either sftpa-deficient (−/−) or wild-type (+/+) spontaneously breathing, adult mice via intranasal lipopolysaccharide (LPS). Four hours later, lungs were isolated, perfused, and mechanically ventilated for 2 hours. Perfusate was collected for analysis over the duration of ventilation and lung lavage was obtained in groups of animals immediately before and after mechanical ventilation (MV). Lavage analysis showed an increase in interleukin-6 (IL6) and tumor necrosis factor-alpha (TNFα) 4 hours after LPS, with a further increase in IL6 following MV. LPS and MV also caused an increase in total cell and neutrophil numbers as well as total protein in the lavage compared to controls. Perfusate analysis revealed a significant increase in IL6 and TNFα after LPS and MV, with significantly greater levels of these mediators in sftpa (−/−) versus (+/+) mice. The authors conclude that LPS followed by MV resulted in lung inflammation and injury, and that SP-A significantly influenced inflammatory mediator release from these inflamed lungs into the perfusate.

IL-17A–Producing γδ T and Th17 Lymphocytes Mediate Lung Inflammation but Not Fibrosis in Experimental Silicosis

IL-17-producing T lymphocytes play a crucial role in inflammation, but their possible implication in fibrosis remains to be explored. In this study, we examined the involvement of these cells in a mouse model of lung inflammation and fibrosis induced by silica particles. Upregulation of IL-17A was associated with the development of experimental silicosis, but this response was markedly reduced in athymic, gammadelta T cell-deficient or CD4(+) T cell-depleted mice. In addition, gammadelta T lymphocytes and CD4(+) T cells, but not macrophages, neutrophils, NK cells or CD8 T cells, purified from the lungs of silicotic mice markedly expressed IL-17A. Depletion of alveolar macrophages or neutralization of IL-23 reduced upregulation of IL-17A in the lung of silicotic mice. IL-17R-deficient animals (IL-17R(-/-)) or IL-17A Ab neutralization, but not IL-22(-/-) mice, developed reduced neutrophil influx and injury during the early lung response to silica. However, chronic inflammation, fibrosis, and TGF-beta expression induced by silica were not attenuated in the absence of IL-17R or -22 or after IL-17A Ab blockade. In conclusion, a rapid lung recruitment of IL-17A-producing T cells, mediated by macrophage-derived IL-23, is associated with experimental silicosis in mice. Although the acute alveolitis induced by silica is IL-17A dependent, this cytokine appears dispensable for the development of the late inflammatory and fibrotic lung responses to silica.