Margaret Solon

Airway Epithelial miRNA Expression Is Altered in Asthma

RATIONALE Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. OBJECTIVES To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. METHODS We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. MEASUREMENTS AND MAIN RESULTS Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. CONCLUSIONS Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).

Chitotriosidase is the primary active chitinase in the human lung and is modulated by genotype and smoking habit

BACKGROUND Chitinolytic enzymes play important roles in the pathophysiology of allergic airway responses in mouse models of asthma. Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1) have chitinolytic activity, but relatively little is known about their expression in human asthma. OBJECTIVE We sought to determine the expression and activity of AMCase and CHIT1 in healthy subjects, subjects with asthma, and habitual smokers, taking account of the null 24-bp duplication in the CHIT1 gene. METHODS We measured chitinase activity in bronchoalveolar lavage (BAL) fluid at multiple pHs by using a synthetic chitin substrate. We also determined AMCase and CHIT1 gene expression in epithelial brushings and BAL fluid macrophages by means of real time RT-PCR. Paired DNA samples were genotyped for the CHIT1 duplication. RESULTS In all subgroups the pH profile of chitinase activity in BAL fluid matched that of CHIT1, but not AMCase, and chitinase activity was absent in subjects genetically deficient in active CHIT1. Although AMCase protein was detectable in lavage fluid, AMCase transcripts in macrophages were consistent with an isoform lacking enzymatic activity. Median chitinase activity in BAL fluid tended to be lower than normal in asthmatic subjects but was increased 7-fold in habitual smokers, where CHIT1 gene expression in macrophages was increased. CONCLUSIONS Chitinase activity in the lung is the result of CHIT1 activity. Although AMCase protein is detectable in the lung, our data indicate that it is inactive. Chitinase activity is not increased in subjects with asthma and in fact tends to be decreased. The high levels of chitinase activity in habitual smokers result from upregulation of CHIT1 gene expression, especially in macrophages.

A protective role for periostin and TGF-β in IgE-mediated allergy and airway hyperresponsiveness

The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up‐regulated in the airway epithelium of asthmatics and to increase active TGF‐β. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown.

The H antigen at epithelial surfaces is associated with susceptibility to asthma exacerbation.

RATIONALE Acute asthma exacerbations, precipitated by viral infections, are a significant cause of morbidity, but not all patients with asthma are equally susceptible. OBJECTIVES To explore susceptibility factors for asthma exacerbations, we considered a role for histoblood group antigens because they are implicated in mechanisms of gastrointestinal viral infection, specifically the O-secretor mucin glycan phenotype. We investigated if this phenotype is associated with susceptibility to asthma exacerbation. METHODS We performed two consecutive case-control studies in subjects with asthma who were either prone or resistant to asthma exacerbations. Exacerbation-prone cases had frequent use of prednisone for an asthma exacerbation and frequent asthma-related healthcare utilization, whereas exacerbation-resistant control subjects had rarely reported asthma exacerbations. The frequency of different mucin glycan phenotypes, defined by the presence or absence of H (O), A, B, or AB antigens, was compared in cases and control subjects. MEASUREMENTS AND MAIN RESULTS In an initial study consisting of 49 subjects with asthma (23 cases and 26 control subjects), we found that having the O-secretor phenotype was associated with a 5.8-fold increase in the odds of being a case (95% confidence interval, 1.7-21.0; P = 0.006). In a replication study consisting of 204 subjects with asthma (101 cases and 103 control subjects), we found that having the O-secretor phenotype was associated with a 2.3-fold increased odds of being a case (95% confidence interval, 1.2-4.4; P = 0.02). CONCLUSIONS The O-secretor mucin glycan phenotype is associated with susceptibility to asthma exacerbation. Clinical trial registered at www.clinicaltrials.gov (NCT00201266).

DAP12 Is Required for Macrophage Recruitment to the Lung in Response to Cigarette Smoke and Chemotaxis toward CCL2

DAP12 is an adapter protein that associates with several receptors in macrophages. Little is known about the biological role of DAP12 in alveolar macrophages. In genome-wide profiling, we previously found that two DAP12-associated receptors, myeloid DAP12-associated lectin-1 and triggering receptor expressed on myeloid cells 2 (TREM2), were highly induced in alveolar macrophages from habitual smokers. Here, we found that transcript levels for these receptors in alveolar macrophages increased with packs per day of cigarettes smoked and expression of TREM2 protein was increased in lung macrophages of former smokers with emphysema compared with that in controls. In vitro, cigarette smoke directly induced expression of myeloid DAP12-associated lectin-1 and TREM2 and activation of DAP12 signaling in mouse macrophages. To determine whether DAP12 plays a role in cigarette smoke-induced pulmonary inflammation, we exposed wild-type and DAP12-deficient mice to chronic cigarette smoke and found significant reduction in recruitment of alveolar macrophages in DAP12-deficient mice. Because cigarette smoking induces the macrophage chemoattractant CCL2, we tested the chemotactic ability of DAP12-deficient macrophages and found abrogation of chemotaxis toward CCL2 in vitro. Airway administration of CCL2 also resulted in a significant reduction of macrophage recruitment to the lungs of DAP12-deficient mice compared with that in controls. DAP12 was also required for normal macrophage migration in a “scratch” assay. Reconstitution studies revealed that phosphorylation of the DAP12 ITAM was required for normal migration in vitro and association with TREM2 was sufficient for normal migration. These findings indicate that DAP12, possibly through association with TREM2, contributes to alveolar macrophage chemotaxis and recruitment to the lung and may mediate macrophage accumulation in lung diseases such as emphysema.

Intelectin-1 Is a Prominent Protein Constituent of Pathologic Mucus Associated with Eosinophilic Airway Inflammation in Asthma

To the Editor: Intelectin-1 (ITLN-1) is an epithelial cell protein that is up-regulated in asthma (1). ITLN-1 is a pleotropic adipokine (also known as omentin-1) with roles in the gut ranging from host defense against pathogenic bacteria to promotion of insulin-stimulated glucose uptake (2–4). The host defense roles of ITLN-1 may result from its ability to bind structures expressed by microorganisms in a carbohydrate-dependent manner (5). ITLN-1 is also a binding partner for lactoferrin (6), and ITLN-1 may cooperate with lactoferrin in host defense (6, 7). Little is known about the function of ITLN-1 in human asthma. One possibility is that it participates in pathways of inflammation downstream of IL-13 (1). Indeed, studies in a mouse model of asthma suggest that ITLN-1 mediates IL-13–induced monocyte chemotactic protein-1 and -3 production in epithelial cells (8). Another possibility is that ITLN-1 is a component of airway mucus and contributes to pathologic mucus gel formation in disease. Supporting this possibility are studies in the gastrointestinal tract showing that ITLN-1 is a goblet cell protein that is secreted with mucus into the intestinal lumen (9). In addition, other studies in the intestine have suggested mucin–intelectin interactions that could alter the biophysical properties of mucus (10). Some of the results of these studies have been previously reported in the form of an abstract (11) Because mucus pathology causes mucus plugging and airway occlusion (12, 13), especially in fatal asthma (14), we set out to determine if ITLN-1 is a component of pathologic mucus in acute asthma. We first immunostained lung tissue sections from cases of fatal asthma and found prominent ITLN-1 immunostaining in the pathologic mucus plugs that occlude the airways (Figures 1A–1C). The cellular source of the ITLN-1 appears to be goblet cells (Figure 1C). We next measured ITLN-1 protein in sputum from 11 patients with acute severe asthma and two control groups (35 subjects with chronic stable asthma and 11 healthy control subjects) (Table 1). We found that ITLN-1 protein levels in the subgroup of patients with asthma in exacerbation were significantly higher than in stable asthma and in healthy control subjects (Figure 1D). We also noted that the increase in ITLN-1 in acute asthma was driven by the subgroup with increased sputum eosinophils (>2%) (Figure 1E), a finding that is consistent with ITLN-1’s regulation by IL-13 in airway epithelial cells (1). ITLN-1 up-regulation is thus a feature of “Th2-high” asthma, and the known pathologic characteristics of this disease endotype can be extended to include high ITLN-1 protein concentrations in mucus forming during disease exacerbations. Figure 1. Intelectin-1 (ITLN-1) protein in airway biospecimens and binding of ITLN-1 to airway mucins and lactoferrin. Sections of lung tissue from lungs of patients with fatal asthma were stained with an anti–ITLN-1 antibody or peptide blocking control. ... Table 1: Subject Characteristics The prominent immunostaining for ITLN-1 in mucus plugs in fatal asthma and the high concentrations of ITLN-1 in sputum in acute severe asthma prompted us to explore if ITLN-1 can bind to human airway mucins. ITLN-1 is a lectin with known specificity for galactosyl structures, especially the galactofuranosyl sugars expressed by microorganisms (5). To determine if ITLN-1 binds to human airway mucin glycans, we developed a plate-based binding assay using high-molecular-weight mucin preparations that we purified from induced sputum samples from subjects with chronic stable asthma (“mucin study”; Table 1). Specifically, we used biotinylated recombinant ITLN-1 to probe mucin coated on microtiter plates (see online supplement). Biotinylated jacalin, a tetrameric plant seed lectin with specificity for galactose, was used as a positive control. Although jacalin showed binding to mucin, ITLN-1 did not (Figure 1F). It is possible that ITLN-1 cannot recognize the pyranosyl forms of galactose in human mucin, but another possibility is that mucin glycans prevent binding through steric hindrance. It could also be that the plate assay is suboptimal for measuring ITLN-1 binding to mucin because other proteins or cofactors involved in an ITLN-1–mucin interaction in vivo are not represented in vitro. Because ITLN-1 has been characterized as the lactoferrin receptor (6, 7), we considered if ITLN-1 interacts with lactoferrin in airway mucus in acute asthma. We found that lactoferrin levels in sputum from patients with acute severe asthma are significantly higher than in control samples (Figure 1G). Notably, the concentration of lactoferrin ranged from 500 to 1,000 μg/ml in some of these sputum samples, a 1,000-fold higher concentration than ITLN-1. This large amount of lactoferrin in asthmatic mucus may bind and concentrate ITLN-1 in mucus. To examine the binding of ITLN-1 to lactoferrin, we used a plate-binding assay similar to the one we used for mucin-ITLN-1 binding. In this way, we found that biotinylated ITLN-1 binds avidly to immobilized lactoferrin (Figure 1H). This binding was inhibited by heparin, suggesting a charge-based interaction between ITLN-1 and lactoferrin’s basic N-terminal region (15), and increased by methyl galactofuranoside. Galactofuranoside is found in many microbial polysaccharides and is recognized as a preferred glycan ligand for ITLN-1 (7, 16). Our data suggest that ITLN-1 binding to galactofuranosyl residues on microorganisms might improve its ability to bind lactoferrin and target it to regions of high microorganism burden. We conclude that ITLN-1 is a prominent protein component of pathologic mucus in fatal asthma and in acute severe asthma, especially in the context of eosinophilic airway inflammation. The binding of ITLN-1 to lactoferrin is increased by galactofuranoside providing a mechanism by which ITLN-1 can cooperate with lactoferrin to defend against microbes.

Alveolar macrophage recruitment and activation by chronic second hand smoke exposure in Mice

Approximately 15% of cases of COPD occur in non-smokers. Among the potential risk factors for COPD in non-smokers is second-hand smoke (SHS) exposure. However, the Surgeon General reported in 2006 that the evidence linking second hand smoke and COPD is insufficient to infer a causal relationship, largely because current evidence does not establish a biological link. The goal of this study was to determine whether SHS exposure can induce alveolar macrophage recruitment and expression of activation markers that we have previously demonstrated in human smokers and in mouse models of emphysema. To achieve these goals, we studied mice exposed to an ambient mixture of predominantly [89%] sidestream smoke at increasing doses over 3 months. We found that second hand smoke exposure induced a dose-dependent increase in alveolar macrophage recruitment (mean ± sd; 224,511 ± 52,330 vs 166,152 ± 47,989 macrophages/ml of bronchoalveolar lavage in smoke-exposed vs air-exposed controls at 3 months, p = 0.003). We also found increased expression of several markers of alveolar macrophage activation (PLA2g7, dkfzp434l142, Trem-2, and pirin, all p < 0.01 at 3 months) and increased lavage levels of two inflammatory mediators associated with COPD (CCL2 [MCP-1], 58 ± 12 vs. 43 ± 22 pg/ml, p = 0.03; and TNFα, 138 ± 43 vs 88 ± 78 pg/ml, p = 0.04 at 3 months). These findings indicate that second smoke exposure can cause macrophage recruitment and activation, providing a biological link between second-hand smoke exposure and the development of inflammatory processes linked to COPD.

Depletion of major pathogenic cells in asthma by targeting CRTh2

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.