Lung Volumes in a Mouse Model of Pulmonary Allergic Inflammation.

PURPOSE: Air trapping, often attested in humans by elevated residual volume (RV) and ratio of RV on total lung capacity (RV/TLC), is frequently observed in asthma. Confirming these alterations in experimental asthma would be important for translational purposes. Herein, lung volumes were investigated in a mouse model of pulmonary allergic inflammation. METHODS: Eight- to 10-week-old male C57BL/6 and BALB/c mice were exposed once daily to intranasal house dust mite (HDM) for 10 consecutive days. All readouts were measured 24 h after the last exposure. Lung volumes were assessed with the flexiVent using a new automated method consisting of degassing the lungs followed by a full-range pressure-volume maneuver. The weight and the volume of the lungs were also measured ex vivo and a lobe was further processed for histological analyses. RESULTS: HDM exposure led to tissue infiltration with inflammatory cells, goblet cell hyperplasia, thickening of the airway epithelium, and elevated ex vivo lung weight and volume. It also decreased TLC and vital capacity but without affecting RV and RV/TLC. These observations were similar between the two mouse strains. CONCLUSION: Alterations of lung volumes in a murine model of pulmonary allergic inflammation are inconsistent with observations made in human asthma. These discrepancies reflect the different means whereby lung volumes are measured between species. The invasive method used herein enables RV to be measured more precisely and without the confounding effect of air trapping, suggesting that changes in RV and RV/TLC using this method in mice should be interpreted differently than in humans.

More airway smooth muscle in males versus females in a mouse model of asthma: A blessing in disguise?

NEW FINDINGS: What is the central question of this study? The lung response to inhaled methacholine is reputed to be greater in male than in female mice. The underpinnings of this sex disparity are ill defined. What is the main finding and its importance? We demonstrated that male airways exhibit a greater content of airway smooth muscle than female airways. We also found that, although a more muscular airway tree in males might contribute to their greater responsiveness to inhaled methacholine than females, it might also curb the heterogeneity in small airway narrowing. ABSTRACT: Mouse models are helpful in unveiling the mechanisms underlying sex disparities in asthma. In comparison to their female counterparts, male mice are hyperresponsive to inhaled methacholine, a cardinal feature of asthma that contributes to its symptoms. The physiological details and the structural underpinnings of this hyperresponsiveness in males are currently unknown. Herein, BALB/c mice were exposed intranasally to either saline or house dust mite once daily for 10 consecutive days to induce experimental asthma. Twenty-four hours after the last exposure, respiratory mechanics were measured at baseline and after a single dose of inhaled methacholine that was adjusted to trigger the same degree of bronchoconstriction in both sexes (it was twice as high in females). Bronchoalveolar lavages were then collected, and the lungs were processed for histology. House dust mite increased the number of inflammatory cells in bronchoalveolar lavages to the same extent in both sexes (asthma, P = 0.0005; sex, P = 0.96). The methacholine response was also markedly increased by asthma in both sexes (e.g., P = 0.0002 for asthma on the methacholine-induced bronchoconstriction). However, for a well-matched bronchoconstriction between sexes, the increase in hysteresivity, an indicator of airway narrowing heterogeneity, was attenuated in males for both control and asthmatic mice (sex, P = 0.002). The content of airway smooth muscle was not affected by asthma but was greater in males (asthma, P = 0.31; sex, P < 0.0001). These results provide further insights regarding an important sex disparity in mouse models of asthma. The increased amount of airway smooth muscle in males might contribute functionally to their greater methacholine response and, possibly, to their decreased propensity for airway narrowing heterogeneity.

Force adaptation through the intravenous route in naïve mice.

Aim of the study: Force adaptation is a process whereby the contractile capacity of the airway smooth muscle increases during a sustained contraction (aka tone). Tone also increases the response to a nebulized challenge with methacholine in vivo, presumably through force adaptation. Yet, due to its patchy pattern of deposition, nebulized methacholine often spurs small airway narrowing heterogeneity and closure, two important enhancers of the methacholine response. This raises the possibility that the potentiating effect of tone on the methacholine response is not due to force adaptation but by furthering heterogeneity and closure. Herein, methacholine was delivered homogenously through the intravenous (i.v.) route. Materials and Methods: Female and male BALB/c mice were subjected to one of two i.v. methacholine challenges, each of the same cumulative dose but starting by a 20-min period either with or without tone induced by serial i.v. boluses. Changes in respiratory mechanics were monitored throughout by oscillometry, and the response after the final dose was compared between the two challenges to assess the effect of tone. Results: For the elastance of the respiratory system (Ers), tone potentiated the methacholine response by 64 and 405% in females (37.4 ± 10.7 vs. 61.5 ± 15.1 cmH2O/mL; p = 0.01) and males (33.0 ± 14.3 vs. 166.7 ± 60.6 cmH2O/mL; p = 0.0004), respectively. For the resistance of the respiratory system (Rrs), tone potentiated the methacholine response by 129 and 225% in females (9.7 ± 3.5 vs. 22.2 ± 4.3 cmH2O·s/mL; p = 0.0003) and males (10.7 ± 3.1 vs. 34.7 ± 7.9 cmH2O·s/mL; p < 0.0001), respectively. Conclusions: As previously reported with nebulized challenges, tone increases the response to i.v. methacholine in both sexes; albeit sexual dimorphisms were obvious regarding the relative resistive versus elastic nature of this potentiation. This represents further support that tone increases the lung response to methacholine through force adaptation.

The cumulative effect of methacholine on large and small airways when deep inspirations are avoided.

BACKGROUND AND OBJECTIVE: The effect of serial incremental concentrations of methacholine is only slightly cumulative when assessed by spirometry. This limited cumulative effect may be attributed to the bronchodilator effect of deep inspirations that are required between concentrations to measure lung function. Using oscillometry, the response to methacholine can be measured without deep inspirations. Conveniently, oscillometry can also dissociate the contribution of large versus small airways. Herein, oscillometry was used to assess the cumulative effect of methacholine in the absence of deep inspirations on large and small airways. METHODS: Healthy and asthmatic volunteers underwent a multiple-concentration methacholine challenge on visit 1 and a single-concentration challenge on visit 2 using the highest concentration of visit 1. The maximal response was compared between visits to assess the cumulative effect of methacholine. The lung volume was also measured after the final concentration to assess hyperinflation. RESULTS: In both healthy and asthmatic subjects, increases in resistance at 19 Hz (Rrs19 ), reflecting large airway narrowing, did not differ between the multiple- and the single-concentration challenge. However, increases in resistance at 5 Hz (Rrs5 ) minus Rrs19 , reflecting small airway narrowing, were 117 and 270% greater in the multiple- than the single-concentration challenge in healthy (p = 0.006) and asthmatic (p < 0.0001) subjects, respectively. Hyperinflation occurred with both challenges and was greater in the multiple- than the single-concentration challenge in both groups. CONCLUSION: Without deep inspirations, the effect of methacholine is cumulative on small airways but not on large airways. Lung hyperinflation and derecruitment may partially explain these different responses.

Sensitive physiological readouts to evaluate countermeasures for lipopolysaccharide-induced lung alterations in mice.

Despite decades of research, studies investigating the physiological alterations caused by an acute bout of inflammation induced by exposing the lung to lipopolysaccharide have yielded inconsistent results. This can be attributed to small effects and/or a lack of fitted physiological testing. Herein, a comprehensive investigation of lung mechanics was conducted on 270 male C57BL/6 mice at 24, 48, or 96 h after an intranasal exposure to saline or lipopolysaccharide at either 1 or 3 mg/kg (30 mice per group). Traditional techniques that probe the lung using small-amplitude perturbations (i.e., oscillometry) were used, together with less conventional and new techniques that probe the lung using maneuvers of large amplitudes. The latter include a partial and a full-range pressure-volume maneuvers to measure quasi-static elastance, compliance, total lung volume, vital capacity, and residual volume. The results demonstrate that lung mechanics assessed by oscillometry was only slightly affected by lipopolysaccharide, confirming previous findings. In contradistinction, lipopolysaccharide markedly altered mechanics when the lung was probed with maneuvers of large amplitudes. With the dose of 3 mg/kg at the peak of inflammation (48 h postexposure), lipopolysaccharide increased quasi-static elastance by 26.7% (P < 0.0001) and decreased compliance by 34.5% (P < 0.0001). It also decreased lung volumes, including total lung capacity, vital capacity, and residual volume by 33.3%, 30.5%, and 43.3%, respectively (all P < 0.0001). These newly reported physiological alterations represent sensitive outcomes to efficiently evaluate countermeasures (e.g., drugs) in the context of several lung diseases.

Effects of airway smooth muscle contraction and inflammation on lung tissue compliance.

There are renewed interests in using the parameter K of Salazar-Knowles' equation to assess lung tissue compliance. K either decreases or increases when the lung's parenchyma stiffens or loosens, respectively. However, whether K is affected by other common features of respiratory diseases, such as inflammation and airway smooth muscle (ASM) contraction, is unknown. Herein, male C57BL/6 mice were treated intranasally with either saline or lipopolysaccharide (LPS) at 1 mg/kg to induce pulmonary inflammation. They were then subjected to either a multiple or a single-dose challenge with methacholine to activate ASM to different degrees. A quasi-static pressure-driven partial pressure-volume (P-V) maneuver was performed before and after methacholine. The Salazar-Knowles' equation was then fitted to the deflation limb of the P-V loop to obtain K, as well as the parameter A, an estimate of lung volume (inspiratory capacity). The fitted curve was also used to derive the quasi-static elastance (Est) at 5 cmH2O. The results demonstrate that LPS and both methacholine challenges increased Est. LPS also decreased A, but did not affect K. In contradistinction, methacholine decreased both A and K in the multiple-dose challenge, whereas it decreased K but not A in the single-dose challenge. These results suggest that LPS increases Est by reducing the open lung volume (A) and without affecting tissue compliance (K), whereas methacholine increases Est by decreasing tissue compliance with or without affecting lung volume. We conclude that lung tissue compliance, assessed using the parameter K of Salazar-Knowles' equation, is insensitive to inflammation but sensitive to ASM contraction.

Additive effects of orchiectomy and intermittent hypoxia on lung mechanics and inflammation in C57BL/6J male mice.

NEW FINDINGS: What is the central question of this study? Does endogenous testosterone modulate the consequences of intermittent hypoxia (IH) in the lungs of male mice? What is the main finding and its importance? Orchiectomized mice exposed to IH develop a pattern that is similar to emphysema or obstructive lung disease with elevated lung volumes, low pulmonary elastance during a methacholine challenge test and high counts of lymphocytes in bronchoalveolar lavages. Since low testosterone levels and other respiratory diseases are common in sleep apnoea, there is a clear clinical relevance to these results. ABSTRACT: We tested the hypothesis that low testosterone levels modulate the pulmonary responses to intermittent hypoxia (IH; used as a model of sleep apnoea (SA)) in male mice. We used intact (SHAM) or orchiectomized (ORX) mice exposed to IH for 14 days (12 h/day, 10 cycles/h, 6% oxygen) or to normoxia (Nx). We first measured ventilation and metabolic rates in freely behaving mice (whole-body plethysmography) and then respiratory mechanics in tracheotomized mice (flexiVent). We assessed the respiratory system resistance and elastance (Ers ), Newtonian resistance (resistance of the large airways), tissue damping and tissue elastance (H) under baseline conditions and during a methacholine challenge test. We also measured the quasi-static compliance and inspiratory capacity with partial pressure-volume loops. Finally, inflammatory cells were counted in the broncho-alveolar lavage (BAL) and we measured lung volume by water displacement. ORX-IH mice had higher tidal volume, inspiratory capacity and lung volume compared to the other groups, but showed signs of low efficiency of O2 exchange rate relative to minute ventilation. During the methacholine challenge, orchiectomy decreased the values of most mechanical parameters and IH reduced Ers and H leading to very low values in ORX-IH mice. Finally, the total number of cells and the number of lymphocytes in BAL were both increased by IH in ORX mice. Since reduced lung elasticity, low O2 extraction, increased lung volumes and inflammation are signs of emphysematous lung disease, we conclude that testosterone might prevent lung emphysema during IH exposures.

Double-chamber plethysmography versus oscillometry to detect baseline airflow obstruction in a model of asthma in two mouse strains.

AIM OF THE STUDY: The current gold standard to assess respiratory mechanics in mice is oscillometry, a technique from which several readouts of the respiratory system can be deduced, such as resistance and elastance. However, these readouts are often not altered in mouse models of asthma. This is in stark contrast with humans, where asthma is generally associated with alterations when assessed by either oscillometry or other techniques. In the present study, we have used double-chamber plethysmography (DCP) to evaluate the breathing pattern and the degree of airflow obstruction in a mouse model of asthma. MATERIALS AND METHODS: Female C57BL/6 and BALB/c mice were studied at day 1 using DCP, as well as at day 11 using both DCP and oscillometry following a once-daily exposure to either house-dust mite (HDM) or saline for 10 consecutive days. RESULTS: All DCP readouts used to describe either the breathing pattern (e.g., tidal volume and breathing frequency) or the degree of airflow obstruction (e.g., specific airway resistance) were different between mouse strains at day 1. Most of these strain differences persisted at day 11. Most oscillometric readouts (e.g., respiratory system resistance and elastance) were also different between strains. Changes caused by HDM were obvious with DCP, including decreases in tidal volume, minute ventilation, inspiratory time and mid-tidal expiratory flow and an increase in specific airway resistance. HDM also caused some strain specific alterations in breathing pattern, including increases in expiratory time and end inspiratory pause, which were only observed in C57BL/6 mice. Oscillometry also detected a small but significant increase in tissue elastance in HDM versus saline-exposed mice. CONCLUSIONS: DCP successfully identified differences between C57BL/6 and BALB/c mice, as well as alterations in mice from both strains exposed to HDM. We conclude that, depending on the study purpose, DCP may sometimes outweigh oscillometry.

Airway smooth muscle tone increases actin filamentogenesis and contractile capacity.

Force adaptation of airway smooth muscle (ASM) is a process whereby the presence of tone (i.e., a sustained contraction) increases the contractile capacity. For example, tone has been shown to increase airway responsiveness in both healthy mice and humans. The goal of the present study is to elucidate the underlying molecular mechanisms. The maximal force generated by mouse tracheas was measured in response to 10-4 M of methacholine following a 30-min period with or without tone elicited by the EC30 of methacholine. To confirm the occurrence of force adaptation at the cellular level, traction force generated by cultured human ASM cells was also measured following a similar protocol. Different pharmacological inhibitors were used to investigate the role of Rho-associated coiled-coil containing protein kinase (ROCK), protein kinase C (PKC), myosin light chain kinase (MLCK), and actin polymerization in force adaptation. The phosphorylation level of the regulatory light chain (RLC) of myosin, the amount of actin filaments, and the activation level of the actin-severing protein cofilin were also quantified. Although ROCK, PKC, MLCK, and RLC phosphorylation was not implicated, force adaptation was prevented by inhibiting actin polymerization. Interestingly, the presence of tone blocked the activation of cofilin in addition to increasing the amount of actin filaments to a maximal level. We conclude that actin filamentogenesis induced by tone, resulting from both actin polymerization and the prevention of cofilin-mediated actin cleavage, is the main molecular mechanism underlying force adaptation.

Airway smooth muscle adapting in dynamic conditions is refractory to the bronchodilator effect of a deep inspiration.

Airway smooth muscle (ASM) is continuously strained during breathing at tidal volume. Whether this tidal strain influences the magnitude of the bronchodilator response to a deep inspiration (DI) is not clearly defined. The present in vitro study examines the effect of tidal strain on the bronchodilator effect of DIs. ASM strips from sheep tracheas were mounted in organ baths and then subjected to stretches (30% strain), simulating DIs at varying time intervals. In between simulated DIs, the strips were either held at a fixed length (isometric) or oscillated continuously by 6% (length oscillations) to simulate tidal strain. The contractile state of the strips was also controlled by adding either methacholine or isoproterenol to activate or relax ASM, respectively. Although the time-dependent gain in force caused by methacholine was attenuated by length oscillations, part of the acquired force in the oscillating condition was preserved postsimulated DIs, which was not the case in the isometric condition. Consequently, the bronchodilator effect of simulated DIs (i.e., the decline in force postsimulated versus presimulated DIs) was attenuated in oscillating versus isometric conditions. These findings suggest that an ASM operating in a dynamic environment acquired adaptations that make it refractory to the decline in contractility inflicted by a larger strain simulating a DI.

CD34 regulates the skeletal muscle response to hypoxia.

Chronic obstructive pulmonary disease (COPD) can sometimes be associated with skeletal muscle atrophy. Hypoxemic episodes, which occur during disease exacerbation and daily physical activity, are frequent in COPD patients. However, the link between hypoxemia and muscle atrophy remains unclear, along with mechanisms of muscle hypoxic stress response. Myogenic progenitors (MPs) and fibro/adipogenic progenitors (FAPs) express CD34 and participate to muscle mass maintenance. Although there is evidence linking CD34 expression and muscle repair, the link between CD34 expression, muscle wasting and the hypoxic stress observed in COPD has never been studied. Using a 2-day model of exposure to hypoxic conditions, we investigated the impact of hypoxia on skeletal muscle wasting and function, and elucidated the importance of CD34 expression in that response. A 2-day exposure to hypoxic conditions induces muscle atrophy, which was significantly worse in Cd34-/- mice compared to wild type (WT). Moreover, the lack of CD34 expression negatively impacts the maximal strength of the extensor digitorum longus muscle in response to hypoxia. Following exposure to hypoxic conditions, FAPs (which support MPs differentiation and myogenesis) are significantly lower in Cd34-/- mice compared to WT animals while the expression of myogenic regulatory factors and degradation factors (Atrogin) are similar. CD34 expression is important in the maintenance of muscle mass and function in response to hypoxic stress. These results highlight a new potential role for CD34 in muscle mass maintenance in hypoxic stress such as observed in COPD.

CD34 Differentially Regulates Contractile and Noncontractile Elements of Airway Reactivity.

Airway hyperresponsiveness (AHR), a major hallmark of asthma, results from alterations of contractile and noncontractile elements of airway reactivity. CD34 is a sialomucin that is expressed on various cells involved in asthma, such as eosinophils and airway smooth muscle precursors, highlighting its potential influence in AHR. To study the role of CD34 in regulating the contractile and noncontractile elements of AHR, AHR was induced by chronic exposure to house dust mite (HDM) antigen. To assess the role of CD34 on the contractile elements of AHR, airway reactivity and airway smooth muscle contractility in response to methacholine were measured. To assess CD34's role in regulating the noncontractile elements of AHR, a chimeric mouse model was used to determine the impact of CD34 expression on inflammatory versus microenvironmental cells in AHR development. Extracellular matrix production, mucus production, and mast cell degranulation were also measured. Whereas wild-type mice developed AHR in response to HDM, a loss of airway reactivity was observed in Cd34-/- mice 24 hours after the last exposure to HDM compared with naive controls. This was reversed when airway reactivity was measured 1 week after the last HDM exposure. Additionally, mast cell degranulation and mucus production were altered in the absence of CD34 expression. Importantly, simultaneous expression of CD34 on cells originating from the hematopoietic compartment and the microenvironment was needed for expression of this phenotype. These results provide evidence that CD34 is required for AHR and airway reactivity maintenance in the early days after an inflammatory episode in asthma.

Impact of immunization against OxLDL on the pulmonary response to cigarette smoke exposure in mice.

BACKGROUND: Cigarette smoke exposure can affect pulmonary lipid homeostasis and cause a progressive increase in pulmonary antibodies against oxidized low-density lipoproteins (OxLDL). Similarly, increased anti-OxLDL antibodies are observed in atherosclerosis, a pathology also tightly associated with smoking and lipid homeostasis disruption. Several immunization strategies against oxidized lipid species to help with their clearance have been shown to reduce the formation of atherosclerotic lesions. Since oxidized lipids are generated during cigarette smoke exposure, we investigated the impact of a prophylactic immunization protocol against OxLDL on the pulmonary effects of cigarette smoke exposure in mice. METHODS: Mice were immunized systemically with a mixture of human OxLDL (antigen source) and AddaVax (adjuvant) or PBS alone prior to the initiation of acute (2 week) or sub-chronic (8 weeks) cigarette smoke exposure protocols. Anti-OxLDL antibodies were measured in the bronchoalveolar lavage (BAL) fluid and serum by direct ELISA. Pulmonary impacts of cigarette smoke exposure and OxLDL immunization were assessed by measuring BAL inflammatory cells, lung functions, and changes in lung structure and gene levels of matrix/matrix-related genes. RESULTS: Immunization to OxLDL led to a marked increase in circulating and pulmonary antibodies against OxLDL that persisted during cigarette smoke exposure. OxLDL immunization did not exacerbate or reduce the inflammatory response following acute or sub-chronic exposure to cigarette smoke. OxLDL immunization alone had effects similar to cigarette smoke exposure on lung functions but OxLDL immunization and cigarette smoke exposure had no additive effects on these parameters. No obvious changes in lung histology, airspace or levels of matrix and matrix-related genes were caused by OxLDL immunization compared to vehicle treatment. CONCLUSIONS: Overall, this study shows for the first time that a prophylactic immunization protocol against OxLDL can potentially have detrimental effects lung functions, without having additive effects over cigarette smoke exposure. This work sheds light on a complex dynamic between anti-OxLDL antibodies and the pulmonary response to cigarette smoke exposure.

The underlying physiological mechanisms whereby anticholinergics alleviate asthma.

The mechanisms whereby anticholinergics improve asthma outcomes, such as lung function, symptoms, and rate of exacerbation, can be numerous. The most obvious is by affecting the contraction of airway smooth muscle (ASM). The acetylcholine released from the cholinergic nerves is the most important bronchoconstrictor that sets the baseline degree of contractile activation of ASM in healthy individuals. Although the degree of ASM's contractile activation can also be fine-tuned by a plethora of other bronchoconstrictors and bronchodilators in asthma, blocking the ceaseless effect of acetylcholine on ASM by anticholinergics reduces, at any given moment, the overall degree of contractile activation. Because the relationships that exist between the degree of contractile activation, ASM force, ASM shortening, airway narrowing, airflow resistance, and respiratory resistance are not linear, small decreases in the contractile activation of ASM can be greatly amplified and thus translate into important benefits to a patient's well-being. Plus, many inflammatory and remodeling features that are often found in asthmatic lungs synergize with the contractile activation of ASM to increase respiratory resistance. This review recalls that the proven effectiveness of anticholinergics in the treatment of asthma could be merely attributed to a small reduction in the contractile activation of ASM.

Airway smooth muscle tone increases airway responsiveness in healthy young adults.

Force adaptation, a process whereby sustained spasmogenic activation (viz., tone) of airway smooth muscle (ASM) increases its contractile capacity, has been reported in isolated ASM tissues in vitro, as well as in mice in vivo. The objective of the present study was to assess the effect of tone on airway responsiveness in humans. Ten healthy volunteers underwent methacholine challenge on two occasions. One challenge consisted of six serial doses of saline followed by a single high dose of methacholine. The other consisted of six low doses of methacholine 5 min apart followed by a higher dose. The cumulative dose was identical for both challenges. After both methacholine challenges, subjects took a deep inspiration (DI) to total lung capacity as another way to probe ASM mechanics. Responses to methacholine and the DI were measured using a multifrequency forced oscillation technique. Compared with a single high dose, the challenge preceded by tone led to an elevated response measured by respiratory system resistance (Rrs) and reactance at 5 Hz. However, there was no difference in the increase in Rrs at 19 Hz, suggesting a predominant effect on smaller airways. Increased tone also reduced the efficacy of DI, measured by an attenuated maximal dilation during the DI and an increased renarrowing post-DI. We conclude that ASM tone increases small airway responsiveness to inhaled methacholine and reduces the effectiveness of DI in healthy humans. This suggests that force adaptation may contribute to airway hyperresponsiveness and the reduced bronchodilatory effect of DI in asthma.

Acute effects of nicotine-free and flavour-free electronic cigarette use on lung functions in healthy and asthmatic individuals.

We designed a crossover and placebo-controlled trial to investigate the impact of a 1-h acute vaping session of nicotine-free and flavour-free e-liquid on the pulmonary functions and respiratory mechanics of healthy and asthmatic individuals. This study shows that a 1-h vaping session of a high-grade and contaminant-free mixture of propylene glycol and glycerol using a commercially available electronic cigarette performed in a controlled environment does not significantly impact pulmonary functions, respiratory mechanics or symptoms in healthy or asthmatic subjects.

FTY720 promotes pulmonary fibrosis when administered during the remodelling phase following a bleomycin-induced lung injury.

Fibrosis complicates numerous pathologies including interstitial lung diseases. Sphingosine analogs such as FTY720 can alleviate lung injury-induced fibrosis in murine models. Contradictorily, FTY720 also promotes in vitro processes normally leading to fibrosis and high doses in vivo foster lung fibrosis by enhancing vascular leakage into the lung. The goal of this study was to determine the effect of low doses of FTY720 on lung fibrosis triggered by an acute injury in mice. We first defined the time-boundaries delimiting the inflammatory and remodelling phases of an injury elicited by bleomycin based on neutrophil counts, total lung capacity and lung stiffness. Thereafter, FTY720 (0.1 mg/kg) was delivered during either the inflammatory or the remodelling phases of bleomycin-induced injury. While FTY720 decreased fibrosis by 60% and lung stiffness by 28% when administered during the inflammatory phase, it increased fibrosis (2.1-fold) and lung stiffness (1.7-fold) when administered during the remodelling phase. FTY720-induced worsening of fibrosis was associated with an increased expression of connective tissue growth factor, but not with vascular leakage into the lung. Thus, the timing of FTY720 delivery following a bleomycin-induced lung injury determines pro-vs anti-fibrotic outcomes.

Lung CD200 Receptor Activation Abrogates Airway Hyperresponsiveness in Experimental Asthma.

In allergic asthma, homeostatic pathways are dysregulated, which leads to an immune response toward normally innocuous antigens. The CD200-CD200 receptor pathway is a central regulator of inflammation, and CD200 expression was recently found to be down-regulated in circulating leukocytes of patients with asthma. Given the antiinflammatory properties of CD200, we investigated whether local delivery of recombinant CD200 (rCD200) could reinstate lung homeostasis in an experimental model of asthma. Brown Norway rats were sensitized with ovalbumin (OVA) and alum. rCD200 was intratracheally administered 24 hours before OVA challenge, and airway responsiveness to methacholine was measured 24 hours after the allergen challenge. Inflammation was also assessed by measuring cell recruitment and cytokine levels in bronchoalveolar lavages, as well as lung and draining lymph node accumulation of dendritic cells (DCs) and T cells. In sensitized rats, rCD200 abolished airway hyperresponsiveness, whereas the sham treatment had no effect. In addition, rCD200 strongly reduced OVA-induced lung accumulation of myeloid DCs, CD4(+) T cells, and T helper type 2 cells. This was associated with a strong reduction of OVA-induced IL-13 level and with an increase of IL-10 in supernatants of bronchoalveolar lavages. Lung eosinophilia and draining lymph node accumulation of myeloid DCs and T cells were not affected by rCD200. Overall, these data reveal that rCD200 can inhibit airway hyperresponsiveness in a model of asthma by a multistep mechanism associated with local alterations of the T cell response and the cytokine milieu.

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma.

BACKGROUND: In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus. METHODS: BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes. RESULTS: AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4(+) T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs. CONCLUSION: Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.