Analysis Of Bronchoalveolar Lavage Cells Research Articles

Lung injury and oxidative stress induced by inhaled chlorine in mice is associated with proinflammatory activation of macrophages and altered bioenergetics

Chlorine (Cl2) gas is a highly toxic and oxidizing irritant that causes life-threatening lung injuries. Herein, we investigated the impact of Cl2-induced injury and oxidative stress on lung macrophage phenotype and function. Spontaneously breathing male C57BL/6J mice were exposed to air or Cl2 (300 ppm, 25 min) in a whole-body exposure chamber. Bronchoalveolar lavage (BAL) fluid and cells, and lung tissue were collected 24 h later and analyzed for markers of injury, oxidative stress and macrophage activation. Exposure of mice to Cl2 resulted in increases in numbers of BAL cells and levels of IgM, total protein, and fibrinogen, indicating alveolar epithelial barrier dysfunction and inflammation. BAL levels of inflammatory proteins including surfactant protein (SP)-D, soluble receptor for glycation end product (sRAGE) and matrix metalloproteinase (MMP)-9 were also increased. Cl2 inhalation resulted in upregulation of phospho-histone H2A.X, a marker of double-strand DNA breaks in the bronchiolar epithelium and alveolar cells; oxidative stress proteins, heme oxygenase (HO)-1 and catalase were also upregulated. Flow cytometric analysis of BAL cells revealed increases in proinflammatory macrophages following Cl2 exposure, whereas numbers of resident and antiinflammatory macrophages were not altered. This was associated with increases in numbers of macrophages expressing cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), markers of proinflammatory activation, with no effect on mannose receptor (MR) or Ym-1 expression, markers of antiinflammatory activation. Metabolic analysis of lung cells showed increases in glycolytic activity following Cl2 exposure in line with proinflammatory macrophage activation. Mechanistic understanding of Cl2-induced injury will be useful in the identification of efficacious countermeasures for mitigating morbidity and mortality of this highly toxic gas.

Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences.

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in BAL were CD14+CD16+, which contrasts the majority of CD14+CD16- classical monocytes in PBMC. Percentages of CD16-expressing BAL macrophages were reduced in BAL from horses with sEA compared to healthy horses. While PBMC lymphocytes predominantly contain CD4+ T cells, B cells and few CD8+ T cells, BAL lymphocytes comprised mainly CD8+ T cells, fewer CD4+ T cells and hardly any B cells. These lymphocyte subsets’ distributions were similar between all groups. After PMA/ionomycin stimulation in vitro, lymphocyte activation (CD154 and T helper cell cytokine expression) was analyzed in BAL cells of 26 of the horses and group differences were observed (p=0.01–0.11). Compared to healthy horses’ BAL, CD154+ lymphocytes from horses with mEA, and CD4+IL-17A+ lymphocytes from horses with sEA were increased in frequency. Activated CD4+ T helper cells were more frequent in asthmatics’ (mEA, sEA) compared to healthy horses’ PBMC lymphocytes. In summary, FC analysis of BAL cells identified increased polymorphonuclear cells frequencies in sEA as established, while macrophage percentages were mildly reduced, and lymphocyte populations remained unaffected by EA. Cytokine production differences of BAL lymphocytes from horses with sEA compared to healthy horses’ cells point towards a functional difference, namely increased local type 3 responses in sEA.

Modeling Asthma in Mice Using Common Aeroallergens.

Aeroallergens are common inducers of asthma in humans and are widely used in experimental research to generate animal models of this disease. In this chapter, we describe four mouse models of aeroallergen-induced asthma. These models differ in type and number of allergens used, route and duration of allergen exposure, and utilization of an adjuvant, representing different mechanistic variants of asthma. In addition, we describe several basic methods that are commonly used in mechanistic studies of asthma in mice. These methods include tracheotomy and bronchoalveolar lavage, cytospin and morphologic analysis of bronchoalveolar lavage cells, and lung harvest and digestion for generation of single-cell suspension.

Differential gene expression and Ingenuity Pathway Analysis of bronchoalveolar lavage cells from horses with mild/moderate neutrophilic or mastocytic inflammation on BAL cytology

Mild to moderate equine asthma syndrome (mEAS) affects horses of all ages and breeds. To date, the etiology and pathophysiology of mEAS are active areas of research, and it remains incompletely understood whether mEAS horses with different immune cell ‘signatures’ on BAL cytology represent different phenotypes, distinct pathobiological mechanisms (endotypes), varied environmental conditions, disease severity, genetic predispositions, or all of the above. In this descriptive study, we compared gene expression data from BAL cells isolated from horses with normal BALF cytology (n = 5), to those isolated from horses with mild/moderate neutrophilic inflammation (n = 5), or mild/moderate mastocytic inflammation (n = 5). BAL cell protein lysates were analyzed for cytokine/chemokine levels using Multiplex Bead Immunoassay, and for select proteins using immunoblot. The transcriptome, determined by RNA-seq and analyzed with DEseq2, contained 20, 63, and 102 significantly differentially expressed genes in horses with normal vs. neutrophilic, normal vs. mastocytic, and neutrophilic vs. mastocytic BALF cytology, respectively. Pathway analyses revealed that BAL-isolated cells from horses with neutrophilic vs. normal cytology showed enrichment in inflammation pathways, and horses with mastocytic vs. normal cytology showed enrichment in pathways involved in fibrosis and allergic reaction. BAL cells from horses with mastocytic mEAS, compared to neutrophilic mEAS, showed enrichment in pathways involved in alteration of tissue structures. Cytokine analysis determined that IL-1β was significantly different in the lysates from horses with neutrophilic inflammation compared to those with normal or mastocytic BAL cytology. Immunoblot revealed significant difference in the relative level of MMP2 in horses with neutrophilic vs. mastocytic mEAS. Upregulation of mRNA transcripts involved in the IL-1 family cytokine signaling axis (IL1a, IL1b, and IL1R2) in neutrophilic mEAS, as well as KIT mRNA in mastocytic mEAS, are novel, potentially clinically relevant, findings of this study. These findings further inform our understanding of inflammatory cell subtypes in mEAS.

Uncovering Salient Cellular Heterogeneity in Human Lung Transplantation Using Single-Cell RNA Sequencing

Lung transplant (LT) recipients experience a low survival rate compared with other solid organ transplant recipients mainly due to development of chronic lung allograft dysfunction (CLAD). Acute rejection (AR) is a major risk factor for CLAD. We have applied single-cell RNA sequencing (scRNAseq) to allograft-derived cells during quiescence, AR, and CLAD, in order to determine key cellular elements in dysfunctional lung allografts. Fresh bronchoalveolar lavage (BAL) cells from one LT patient (4792 cells) with stable lung function and one with AR (1791) as well as enzymatically and mechanically dissociated CLAD lung tissue (9797 cells) were subjected to barcoding and library construction using 10X Genomics 3' expression V2 chemistry. Library constructs were then sent for scRNAseq. Data were analysed using the Seurat package in R. scRNAseq analysis of BAL cells demonstrated at least three distinct subsets of macrophages including a cluster with a clear inflammatory phenotype that was highly present in AR (cluster 5, Fig 1A). Another macrophage subset with a regenerative phenotype was represented mostly in the stable patient sample (cluster 11, Fig 1A). The AR sample also contained a distinct cytotoxic T cell cluster (cluster 14, Fig 1A), absent in the stable patient sample (Fig 1A, B, and C). Comparison of CLAD lung tissue to the AR BAL sample revealed co-clustering of cytotoxic T cells (cluster 1, Fig1D), suggesting further expansion of these cells as allograft dysfunction progresses. In addition, unique cell subsets of T, B, and NK cells were seen in CLAD lung tissue but not in BAL (Fig 1D, E, and F). Using scRNAseq, we offer insights into the cellular composition of stable and AR BAL and created a map of tissue-resident cells in a CLAD lung. Further application of this powerful tool is underway and will deepen our understanding of the cellular complexity of LT immunobiology and its role in allograft injury and dysfunction.

Absence of Angiotensin ll type 2 (AT2) Receptors Increases Airway Inflammation and Responsiveness in an Allergic Mouse Model of Asthma

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: Ang II receptor type‐1 and type‐2 (AT1 and AT2). This lab has previously found that AT2 receptor agonist plays a significant role in reducing airway inflammation and reactivity (FASEB J April 2018 32: 829.6). For the current study, we characterized the role of AT2 receptors in airway hyperreactivity and inflammation using AT2 receptor knockout (AT2KO; Hein L, Barsh et. al. Nature. 1995;377:744–747) and corresponding wild‐type (WT) mice that were divided into two experimental groups each: control (CON) and allergen sensitized‐challenged (SEN). Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in AT2KO SEN mice compared to WT SEN (324.7±66% in AT2KO SEN vs 166±56.3%, p<0.05). Total cell count analysis showed increased number of cells in AT2KO SEN (3.3±0.2 × 106 vs. 1.4±5 × 106 in WT SEN, p<0.001). Differential BAL cell analysis showed increased eosinophils (73±8% in AT2KO SEN vs. 50.11±2.84% in WT SEN, p<0.05). Macrophages were lower in AT2KO SEN than WT SEN (15±4% vs. 40±3%; p<0.001). These data indicate that absence of AT2 receptors increases airway inflammation and responsiveness in our model of asthma.Support or Funding InformationInstitutional Research Grant Long Island UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of omega-3 fatty acid supplementation on resolvin (RvE1)-mediated suppression of inflammation in a mouse model of asthma

Objective: ResolvinE1 (RvE1), an endogenous lipid mediator derived from omega 3 fatty acids contributes to resolution of allergic inflammatory responses. We investigated effects of RvE1 (R) and omega 3 fatty acids (O) on airway reactivity and inflammation using allergic mice.Methods: Mice were divided into control (nonasthmatic; CON) and allergen sensitized-challenged (asthmatic; SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2 μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. RvE1 was administered i.p. postallergen challenge, while omega 3 fatty acids (fish oil) were administered via oral gavage once daily (days 1–13). Whole body plethysmography and bronchoalveolar lavage (BAL) studies were performed on day 14.Results: RvE1 attenuated airway responsiveness to methacholine (48 mg/ml) in treated asthmatic mice vs. nontreated (150 ± 27.88% in SEN vs. 54 ± 7.52% in SEN + R, p < .05). No difference was observed with omega-3 supplementation (115 ± 19.28% in SEN + O) or treatment with both RvE1 and omega 3 fatty acids (39 ± 12.37% in SEN + R + O vs. 54 ± 7.52% in SEN + R). Differential BAL cell analysis showed that RvE1 decreased eosinophils and neutrophils in SEN mice (p < .005) while no difference was observed with omega-3 fatty acids. SEN + R + O group had similar results as RvE1 treated mice, suggesting that only RvE1 attenuated inflammation.Conclusions: RvE1 attenuated airway responsiveness and inflammation in asthmatic mice. Omega-3 fatty acids, although a precursor for RvE1 formation, had no additive effects on RvE1 decreases in airway inflammation and airway reactivity. Our data suggests that omega-3 supplementation has little effect on airway inflammation and reactivity in our model of asthma.

Protective Role of Surfactant Protein-D Against Lung Injury and Oxidative Stress Induced by Nitrogen Mustard.

Nitrogen mustard (NM) is a vesicant known to cause acute pulmonary injury which progresses to fibrosis. Macrophages contribute to both of these pathologies. Surfactant protein (SP)-D is a pulmonary collectin that suppresses lung macrophage activity. Herein, we analyzed the effects of loss of SP-D on NM-induced macrophage activation and lung toxicity. Wild-type (WT) and SP-D-/- mice were treated intratracheally with PBS or NM (0.08 mg/kg). Bronchoalveolar lavage (BAL) fluid and tissue were collected 14 days later. In WT mice, NM caused an increase in total SP-D levels in BAL; multiple lower molecular weight forms of SP-D were also identified, consistent with lung injury and oxidative stress. Flow cytometric analysis of BAL cells from NM treated WT mice revealed the presence of proinflammatory and anti-inflammatory macrophages. Whereas loss of SP-D had no effect on numbers of these cells, their activation state, as measured by proinflammatory (iNOS, MMP-9), and anti-inflammatory (MR-1, Ym-1) protein expression, was amplified. Loss of SP-D also exacerbated NM-induced oxidative stress and alveolar epithelial injury, as reflected by increases in heme oxygenase-1 expression, and BAL cell and protein content. This was correlated with alterations in pulmonary mechanics. In NM-treated SP-D-/-, but not WT mice, there was evidence of edema, epithelial hypertrophy and hyperplasia, bronchiectasis, and fibrosis, as well as increases in BAL phospholipid content. These data demonstrate that activated lung macrophages play a role in NM-induced lung injury and oxidative stress. Elucidating mechanisms regulating macrophage activity may be important in developing therapeutics to treat mustard-induced lung injury.

Longitudinal Mass Cytometric Profiling of BAL Cells in Lung Transplant Recipients

We have previously demonstrated the feasibility of phenotyping bronchoalveolar lavage (BAL) cells in lung transplant recipients (LTRs) using mass cytometry (MC). MC allows multidimensional analysis of BAL cells without autofluorescence. We are using MC to examine longitudinal changes in BAL cells in LTRs, with the goal of identifying novel determinants of acute rejection and chronic lung allograft dysfunction (CLAD).

Limonene‐induced Activation of A2A Adenosine Receptors Reduces Airway Inflammation and Reactivity in a Mouse Model of Asthma

Animal models of asthma have shown that limonene (terpene in citrus fruits) reduces inflammation and airway reactivity. However, the mechanism of these effects is unknown. We hypothesized that limonene activates A2A adenosine receptors (A2AAR) to produce its effects in asthma, after comparing Glide scores for limonene (−5.19) and A2AAR agonist CGS 21680 (−8.43). We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) as well NECA (nonselective adenosine analog) in A2AAR knock‐out (A2AKO) and wild‐type (WT) mice. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20 μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13 (n=6 mice per group). Limonene was administered as an inhalation prior to allergen challenges in one group of allergic mice for both WT and KO (SEN+LIM). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Airway responsiveness to MCh (48 mg/ml) in WT SEN group was significantly lowered with limonene treatment (420±92.57% in SEN vs. 56.23±25% in SEN+LIM). However, limonene did not have an effect in A2AKO (553±85% in SEN vs. 588±34% in SEN+LIM). NECA‐induced increased airway responsiveness was attenuated in limonene‐treated WT SEN mice (1002±161 in SEN vs. 250±226%, p&lt;0.05) while no effect was observed in A2AKO groups (541±44% in SEN vs. 528±147% in SEN+LIM). Differential BAL cell analysis showed that limonene reduced levels of eosinophils in WT SEN (70.66±2.6% in SEN vs. 28.66±1.45% in SEN+LIM, p&lt;0.05) while no difference was observed in A2AKO (76.33±1.35% in SEN vs 79±0.69% in SEN+LIM). These data suggest that limonene‐induced reduction in airway inflammation and airway reactivity occurs via activation of A2AAR.Support or Funding InformationLong Island University start‐up funding (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Omega‐3 Fatty Acid Supplementation on Resolvin (RvE1)‐mediated Suppression of Inflammation in a Mouse Model of Asthma

ResolvinE1(RvE1), an endogenous lipid mediator derived from eicosapentaenoic acid (omega 3 fatty acids), contributes to the resolution of an allergic inflammatory response. We investigated the effects of RvE1 and Omega 3 fatty acids in airway reactivity and inflammation using an asthmatic murine model. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. RvE1 was administered intraperitoneally post‐allergen challenge while fish oil was administered via oral gavage once daily (day 1 through 13). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. RvE1attenuated airway responsiveness to methacholine (MCh; 48mg/ml) in treated mice (150±27.88% in SEN vs. 54±7.52% in SEN+RvE1, p&lt;0.05). However, no difference was observed with either omega‐3 supplementation alone (115±19.28%in SEN+omega‐3) or additive effect of omega‐3 on RvE1 treatment (39±12.37% in SEN+RvE1+omega‐3 vs. 54±7.52% in SEN+RvE1). Differential BAL cell analysis showed that post‐treatment with RVE1 decreased eosinophils (40 ± 2.7% in SEN vs.10.2 ± 1.4% in SEN+RvE1, p&lt;0.005). Omega‐3 treated SEN showed no difference (45 ± 3%) while SEN+RvE1+omega‐3 group had similar results as RvE1 treated mice (10.2 ± 1.4% in SEN+RvE1 vs.10.6 ± 1.4% in SEN+RvE1+omega‐3), suggesting that RvE1 only attenuated the eosinophilia. Our data suggests that omega‐3 supplementation has little effect on airway inflammation and reactivity. Further, omega‐3 fatty acid, although a precursor for resolvin formation, did not have additive effects on resolvin‐mediated decreases in airway inflammation and airway reactivity.Support or Funding InformationLong Island University start‐up funds (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin ll Type 2 (AT2) Receptors: Novel Target in Asthma

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: AT1 and AT2. This lab has previously found that AT1 antagonist losartan reduces airway inflammation and reactivity. The AT2 receptors counterbalance the effects of AT1 and are known to be anti‐inflammatory. However, it is not known if AT2 receptors have any effects in asthma. We studied the effects of AT2 agonist novokinin and antagonist PD 12339 administered in vivo in a mouse model of asthma. Mice were divided into control (non‐allergic; CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in allergic mice compared to control (563.71±40% in SEN vs. 208.79±20.84% in CON, p&lt;0.05). However, novokinin (i.p. 0.3mg/kg) treated SEN mice had significantly attenuated MCh‐indcued airway responsiveness (563.71±40% in SEN vs. 252±11% in SEN+Novokinin, p&lt;0.001). The AT2 antagonist PD 12339 (i.p.5mg/kg) significantly increased MCh airway responsiveness (563.71±40% in SEN vs. 757±30% in SEN+PD 12239, p&lt;0.05). Differential BAL cell analysis showed novokinin also decreased eosinophils (221±21 in SEN vs. 122±18 in SEN+PD 12239, p&lt;0.001), while PD 12239 had no effect on eosinophils (245± 16 cells in SEN+PD 12339). Our data suggests that activation of AT2 receptors may attenuate airway reactivity and inflammation in our model of asthma.Support or Funding InformationLong Island University start‐up funds (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long-Term Persistence of Donor Alveolar Macrophages in Human Lung Transplant Recipients That Influences Donor-Specific Immune Responses.

Steady-state alveolar macrophages (AMs) are long-lived lung-resident macrophages with sentinel function. Evidence suggests that AM precursors originate during embryogenesis and populate lungs without replenishment by circulating leukocytes. However, their presence and persistence are unclear following human lung transplantation (LTx). Our goal was to examine donor AM longevity and evaluate whether AMs of recipient origin seed the transplanted lungs. Origin of AMs was accessed using donor-recipient HLA mismatches. We demonstrate that 94-100% of AMs present in bronchoalveolar lavage (BAL) were donor derived and, importantly, AMs of recipient origin were not detected. Further, analysis of BAL cells up to 3.5years post-LTx revealed that the majority of AMs (>87%) was donor derived. Elicitation of de novo donor-specific antibody (DSA) is a major post-LTx complication and a risk factor for development of chronic rejection. The donor AMs responded to anti-HLA framework antibody (Ab) with secretion of inflammatory cytokines. Further, in an experimental murine model, we demonstrate that adoptive transfer of allogeneic AMs stimulated humoral and cellular immune responses to alloantigen and lung-associated self-antigens and led to bronchiolar obstruction. Therefore, donor-derived AMs play an essential role in the DSA-induced inflammatory cascade leading to obliterative airway disease of the transplanted lungs.

Genetic Deletion of β-Arrestin-2 and the Mitigation of Established Airway Hyperresponsiveness in a Murine Asthma Model

β-Arrestin-2 (βarr2) is a ubiquitously expressed cytosolic protein that terminates G protein-coupled receptor signaling and transduces G protein-independent signaling. We previously showed that mice lacking βarr2 do not develop an asthma phenotype when sensitized to, and challenged with, allergens. The current study evaluates if an established asthma phenotype can be mitigated by deletion of βarr2 using an inducible Cre recombinase. We sensitized and challenged mice to ovalbumin (OVA) and demonstrated that on Day (d) 24 the allergic asthma phenotype was apparent in uninduced βarr2 and wild-type (WT) mice. In a second group of OVA-treated mice, tamoxifen was injected on d24 to d28 to activate Cre recombinase, and OVA aerosol challenge was continued through d44. The asthma phenotype was assessed using lung mechanics measurements, bronchoalveolar lavage cell analysis, and histological assessment of mucin and airway inflammation. Compared with their respective saline-treated controls, OVA-treated WT mice and mice expressing the inducible Cre recombinase displayed a significant asthma phenotype at d45. Whereas tamoxifen treatment had no significant effect on the asthma phenotype in WT mice, it inhibited βarr2 expression and caused a significant reduction in airway hyper-responsiveness (AHR) in Cre-inducible mice. These findings suggest that βarr2 is actively required for perpetuation of the AHR component of the allergic asthma phenotype. Our finding that βarr2 participates in the perpetuation of AHR in an asthma model means that targeting βarr2 may provide immediate and potentially long-term relief from daily asthma symptoms due to AHR irrespective of inflammation.

CD34 Is Required for Dendritic Cell Trafficking and Pathology in Murine Hypersensitivity Pneumonitis

Although recent work has shown that CD34 plays an important role in the trafficking of inflammatory cells during Th2-biased inflammatory responses, its role in Th1/Th17-biased disease as well as dendritic cell (DC) trafficking is unknown. We used CD34-deficient mice (Cd34(-/-)) to investigate the role of CD34 in the Th1/Th17-biased lung inflammatory disease, hypersensitivity pneumonitis (HP). HP was induced in wild-type (wt) and Cd34(-/-) mice by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Lung inflammation was assessed by histology and analysis of bronchoalveolar lavage cells. Primary and secondary immune responses were evaluated by cytokine recall responses of pulmonary inflammatory cells as well as draining lymph node cells. Cd34(-/-) mice were highly resistant to the development of HP and exhibited an inflammatory pattern more reflective of a primary response to S. rectivirgula rather than the chronic lymphocytosis that is typical of this disease. Cytokine recall responses from Cd34(-/-) lymph node cells were dampened and consistent with a failure of antigen-loaded Cd34(-/-) DCs to deliver antigen and prime T cells in the draining lymph nodes. In agreement with this interpretation, adoptive transfer of wt DCs into Cd34(-/-) mice was sufficient to restore normal sensitivity to HP. CD34 was found to be expressed by wt DCs, and Cd34(-/-) DCs exhibited an impaired ability to chemotax toward a subset of chemokines in vitro. Finally, expression of human CD34 in Cd34(-/-) mice restored normal susceptibility to HP. We conclude that CD34 is expressed by mucosal DCs and plays an important role in their trafficking through the lung and to the lymph nodes. Our data also suggest that CD34 may play a selective role in the efficient migration of these cells to a subset of chemokines.

Noninvasive Quantitative Tomography of the Therapeutic Response to Dexamethasone in Ovalbumin-Induced Murine Asthma

Animal models of pulmonary inflammation are critical for understanding the pathophysiology of asthma and for developing new therapies. Current conventional assessments in mouse models of asthma and chronic obstructive pulmonary disease rely on invasive measures of pulmonary function and terminal characterization of cells infiltrating into the lung. The ability to noninvasively visualize and quantify the underlying biological processes in mouse pulmonary models in vivo would provide a significant advance in characterizing disease processes and the effects of therapeutics. We report the utility of near-infrared imaging agents, in combination with fluorescence molecular tomography (FMT) imaging, for the noninvasive quantitative imaging of mouse lung inflammation in an ovalbumin (OVA)-induced chronic asthma model. BALB/c mice were intraperitoneally sensitized with OVA-Alum (aluminum hydroxide) at days 0 and 14, followed by daily intranasal challenge with OVA in phosphate-buffered saline from days 21 to 24. Dexamethasone and control therapies were given intraperitoneally 4 h before each intranasal inhalation of OVA from days 21 to 24. Twenty-four hours before imaging, the mice were injected intravenously with 5 nmol of the cathepsin-activatable fluorescent agent, ProSense 680. Quantification by FMT revealed in vivo cysteine protease activity within the lung associated with the inflammatory eosinophilia, which decreased in response to dexamethasone treatment. Results were correlated with in vitro laboratory tests (bronchoalveolar lavage cell analysis and immunohistochemistry) and revealed good correlation between these measures and quantification of ProSense 680 activation. We have demonstrated the ability of FMT to noninvasively visualize and quantify inflammation in the lung and monitor therapeutic efficacy in vivo.

Repeated exposure to low-dose diesel exhaust after allergen challenge exaggerates asthmatic responses in mice

Background In conjunction with allergens, diesel exhaust particles act as an adjuvant to enhance IgE responses, inducing expression of cytokines/chemokines and adhesion molecules, and increasing airway hyper-responsiveness (AHR). As most studies were designed to expose animals to diesel exhaust throughout the periods of both sensitization and allergen challenge, it remains unclear whether diesel exhaust (DE) exposure exaggerates airway responses in asthmatic animals. Objective To study effects of exposure to low-dose DE on AHR and allergic airway inflammation in asthmatic mice. Methods BALB/c mice were sensitized by intraperitoneal injection of ovalbumin and challenged by intranasal administration with ovalbumin. They were exposed to low-dose DE for 7 h/day, 5 days/week, for up to 12 weeks. AHR to methacholine was evaluated by whole-body plethysmography as well as bronchoalveolar lavage cell analysis and cytokine gene expression in lungs. Results Repeated exposure of asthmatic mice to low-dose DE resulted in increased AHR and gene expression of several pro-asthmatic cytokines/chemokines, but these effects rapidly subsided with continued exposure to DE. Conclusion Repeated exposure to low-dose DE after ovalbumin challenge exaggerates allergic responses in mice, but effects are not prolonged with continuous DE exposure.

Early exposure to a nonhygienic environment alters pulmonary immunity and allergic responses

The hygiene hypothesis suggests that early life exposure to a nonhygienic environment that contains endotoxin reduces the risk of developing allergic diseases. The mechanisms underlying the hygiene hypothesis are unclear and may involve subtle immune system interactions that occur during maturation. Experimental objectives of this study were to use a novel animal model to test the hygiene hypothesis and to characterize early life immune system responses to a nonhygienic environment. Mice were reared in corn dust, a grain-processing byproduct with a high-endotoxin content and microbial products or in a low-endotoxin environment. The influence of early or later life exposure to corn dust on a subsequent allergen stimulus (ovalbumin) was assessed by bronchoalveolar lavage (BAL) cell analysis, lung histology, serum IgE, and BAL cytokine measurements. The influence of the corn dust environment on the developing pulmonary immune system was assessed by BAL cell analysis and immunostaining of lung tissue. The corn dust environment contained significantly more endotoxin (P < 0.001), and the dust exposures attenuated the cellular inflammatory response to ovalbumin in the adult mouse (P < 0.01) but did not reduce serum IgE levels or alter baseline BAL fluid proinflammatory cytokine levels. The corn dust environment did not induce significant neutrophilia in lavage fluid but significantly increased the number of antigen-presenting cells in alveolar walls early in life by approximately 37%. In conclusion, exposure to a nonhygienic environment did not induce significant airway neutrophilia, yet altered the population of immunologically active cells in the lung and reduced subsequent allergic inflammation.

Imatinib as a Novel Antifibrotic Agent in Bleomycin-induced Pulmonary Fibrosis in Mice

Imatinib mesylate is a potent and specific tyrosine kinase inhibitor against c-ABL, BCR-ABL, and c-KIT, and has been demonstrated to be highly active in chronic myeloid leukemia and gastrointestinal stromal tumors. We examined the antifibrotic effects of imatinib using a bleomycin-induced lung fibrosis model in mice because imatinib also inhibits tyrosine kinase of platelet-derived growth factor receptors (PDGFRs). Imatinib inhibited the growth of primary murine lung fibroblasts and the autophosphorylation of PDGFR-beta induced by PDGF. Administration of imatinib significantly prevented bleomycin-induced pulmonary fibrosis in mice, partly by reducing the number of mesenchymal cells incorporating bromodeoxyuridine. Analysis of bronchoalveolar lavage cells demonstrated that imatinib did not suppress early inflammation on Days 7 and 14 caused by bleomycin. These results suggest that imatinib has the potential to prevent pulmonary fibrosis by inhibiting the proliferation of mesenchymal cells, and that imatinib might be useful for the treatment of pulmonary fibrosis in humans.

Influence of lipopolysaccharide exposure on airway function and allergic responses in developing mice.

Exposure to endotoxin has been associated with an exacerbation of asthmatic responses in humans and animal models. However, recent evidence suggests that microbial exposure in early life may protect from the development of asthma and atopy. In this study, we sought to evaluate the effects of lipopolysaccaride (LPS) on airway function in developing mice. In addition, we evaluated the influence of LPS on subsequent allergen sensitization and challenge. Under light anesthesia, 2-3-week-old Balb/c mice received a single intranasal instillation of LPS or sterile physiologic saline. Measurements of airway function were obtained in unrestrained animals, using whole-body plethysmography. Airway responsiveness was expressed in terms of % enhanced pause (Penh) increase from baseline to aerosolized methacholine (Mch). In additional studies, we assessed the functional and cellular responses to ovalbumin sensitization and challenge following prior exposure to LPS. We found that exposure to LPS induced transient airway hyperresponsiveness to Mch. These functional changes were associated with the recruitment of neutrophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid. Airway responsiveness after allergen sensitization and challenge was decreased by prior exposure to LPS. The analysis of BAL cells and cytokines (interferon-gamma and interleukin-4) did not reveal alterations in the overall Th1/Th2 balance. Our findings suggest that LPS leads to airway hyperresponsiveness in developing mice, and may protect against the development of allergen-driven airway dysfunction.