Matthew J. Edwards

Expression and Cellular Provenance of Thymic Stromal Lymphopoietin and Chemokines in Patients with Severe Asthma and Chronic Obstructive Pulmonary Disease

Asthma and chronic obstructive pulmonary disease (COPD) are associated with Th2 and Th1 differentiated T cells. The cytokine thymic stromal lymphopoietin (TSLP) promotes differentiation of Th2 T cells and secretion of chemokines which preferentially attract them. We hypothesized that there is distinct airways expression of TSLP and chemokines which preferentially attract Th1- and Th2-type T cells, and influx of T cells bearing their receptors in asthma and COPD. In situ hybridization, immunohistochemistry, and ELISA were used to examine the expression and cellular provenance of TSLP, Th2-attracting (TARC/CCL17, MDC/CCL22, I-309/CCL1), and Th1-attracting (IP-10/CXCL10, I-TAC/CXCL11) chemokines in the bronchial mucosa and bronchoalveolar lavage fluid of subjects with moderate/severe asthma, COPD, and controls. Cells expressing mRNA encoding TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in severe asthma and COPD as compared with non-smoker controls (p < 0.02). This pattern was reflected in bronchoalveolar lavage fluid protein concentrations. Expression of the same chemokines was also increased in ex- and current smokers. The cellular sources of TSLP and chemokines were strikingly similar in severe asthma and COPD. The numbers of total bronchial mucosal T cells expressing the chemokine receptors CCR4, CCR8, and CXCR3 did not significantly differ in asthma, COPD, and controls. Both asthma and COPD are associated with elevated bronchial mucosal expression of TSLP and the same Th1- and Th2-attracting chemokines. Increased expression of these chemokines is not, however, associated with selective accumulation of T cells bearing their receptors.

IL-33–Dependent Type 2 Inflammation during Rhinovirus-induced Asthma Exacerbations In Vivo

RATIONALE Rhinoviruses are the major cause of asthma exacerbations; however, its underlying mechanisms are poorly understood. We hypothesized that the epithelial cell-derived cytokine IL-33 plays a central role in exacerbation pathogenesis through augmentation of type 2 inflammation. OBJECTIVES To assess whether rhinovirus induces a type 2 inflammatory response in asthma in vivo and to define a role for IL-33 in this pathway. METHODS We used a human experimental model of rhinovirus infection and novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in the asthmatic and healthy airways during a rhinovirus infection. Additionally, we cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants of rhinovirus-infected bronchial epithelial cells (BECs) to assess type 2 cytokine production in the presence or absence of IL-33 receptor blockade. MEASUREMENTS AND MAIN RESULTS IL-4, IL-5, IL-13, and IL-33 are all induced by rhinovirus in the asthmatic airway in vivo and relate to exacerbation severity. Further, induction of IL-33 correlates with viral load and IL-5 and IL-13 levels. Rhinovirus infection of human primary BECs induced IL-33, and culture of human T cells and ILC2s with supernatants of rhinovirus-infected BECs strongly induced type 2 cytokines. This induction was entirely dependent on IL-33. CONCLUSIONS IL-33 and type 2 cytokines are induced during a rhinovirus-induced asthma exacerbation in vivo. Virus-induced IL-33 and IL-33-responsive T cells and ILC2s are key mechanistic links between viral infection and exacerbation of asthma. IL-33 inhibition is a novel therapeutic approach for asthma exacerbations.

Rhinovirus-induced IL-25 in asthma exacerbation drives type 2 immunity and allergic pulmonary inflammation.

IL-25 critically links rhinovirus infection and allergic asthma exacerbations. IL-25 Horns in on Asthma Attacks The common cold isn’t so common in people with asthma. Rhinoviruses—the main causes of the common cold—can make asthma attacks worse. Now, Beale et al. report that one way this happens is because rhinoviruses can induce interleukin-25 (IL-25) in lung epithelial cells. They found that IL-25 is more highly expressed in people with asthma than in healthy controls. In a mouse model of allergic asthma, rhinovirus infection induced IL-25 production, and blocking the IL-25 receptor could reduce rhinovirus-induced symptom exacerbation. These data suggest that blocking IL-25 is a promising therapeutic strategy in asthmatics, something to consider as the cold season approaches. Rhinoviruses (RVs), which are the most common cause of virally induced asthma exacerbations, account for much of the burden of asthma in terms of morbidity, mortality, and associated cost. Interleukin-25 (IL-25) activates type 2–driven inflammation and is therefore potentially important in virally induced asthma exacerbations. To investigate this, we examined whether RV-induced IL-25 could contribute to asthma exacerbations. RV-infected cultured asthmatic bronchial epithelial cells exhibited a heightened intrinsic capacity for IL-25 expression, which correlated with donor atopic status. In vivo human IL-25 expression was greater in asthmatics at baseline and during experimental RV infection. In addition, in mice, RV infection induced IL-25 expression and augmented allergen-induced IL-25. Blockade of the IL-25 receptor reduced many RV-induced exacerbation-specific responses including type 2 cytokine expression, mucus production, and recruitment of eosinophils, neutrophils, basophils, and T and non-T type 2 cells. Therefore, asthmatic epithelial cells have an increased intrinsic capacity for expression of a pro–type 2 cytokine in response to a viral infection, and IL-25 is a key mediator of RV-induced exacerbations of pulmonary inflammation.

The Sphingosine 1-Phosphate Receptor Agonist FTY720 Differentially Affects the Sequestration of CD4+/CD25+ T-Regulatory Cells and Enhances Their Functional Activity

The sphingosine 1-phosphate (S1P) receptor agonist FTY720 is well known for its immunomodulatory activity, sequestering lymphocytes from blood and spleen into secondary lymphoid organs and thereby preventing their migration to sites of inflammation. Because inflammation is critically dependent on a balance between Ag-specific Th/effector cells and T-regulatory cells, we investigated the effect of FTY720 on T-regulatory cell trafficking and functional activity. An increased number of CD4+/CD25+ T cells was found in blood and spleens of FTY720-treated mice, and transfer of these cells resulted in a significantly more pronounced accumulation in spleens but not lymph nodes after treatment, suggesting that this compound differentially affects the homing properties of T-regulatory cells compared with other T cell subsets. Indeed, CD4+/CD25+ T cells express lower levels of S1P1 and S1P4 receptors and demonstrate a reduced chemotactic response to S1P. Moreover, analysis of the functional response of FTY720-treated CD4+/CD25+ T cells revealed an increased suppressive activity in an in vitro Ag-specific proliferation assay. This correlated with enhanced function in vivo, with T-regulatory cells obtained from FTY720-treated mice being able to suppress OVA-induced airway inflammation. Thus, FTY720 differentially affects the sequestration of T-regulatory cells and importantly, increases the functional activity of T-regulatory cells, suggesting that it may have disease-modifying potential in inflammatory disorders.

Notch2 Is Required for Inflammatory Cytokine-Driven Goblet Cell Metaplasia in the Lung

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.

A novel method for assessing unchallenged levels of mediators in nasal epithelial lining fluid

To the Editor: There is a need to develop noninvasive methods to sample epithelial lining fluid (ELF) from the respiratory system. The nasal mucosa is easily accessible, and it has long been recognized that there is a strong functional and immunologic relationship between the nose and bronchi. It is possible to obtain samples of ELF by means of nasal lavage, which has been used to measure inflammatory protein secretion after nasal allergen challenge. However, nasal allergen challenge exaggerates natural exposures, and the unknown dilution factor from nasal lavage is a significant confounder and might dilute the mediators to less than the detection limit of the assay. Therefore there is a need to measure mediators in the undisturbed ELF. We propose a method for sampling nasal ELF using a synthetic absorptive matrix (SAM), separating the fluid by means of centrifugation, and analyzing the sample with a multiplex quantitative protein assay for a panel of inflammatory mediators. The advantage of this technique is that it analyzes undiluted ELF at the tissue interface without need for stimulation of the mucosa. We conducted a case-control study of children with symptomatic allergic rhinitis and healthy control subjects from the Copenhagen Prospective Study on Asthma in Childhood birth cohort to assess whether unchallenged levels of nasal mediators could be detected in ELF collected with SAM and whether such levels associated with symptoms of allergic rhinitis. The study was conducted August to September 2008 and was approved by the Copenhagen Ethics Committee. Case status was determined based on allergic rhinitis with troublesome symptoms in the previous 24 hours, and control

Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors

Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.

Treatment with Anti–Gremlin 1 Antibody Ameliorates Chronic Hypoxia/SU5416–Induced Pulmonary Arterial Hypertension in Mice

The expression of the bone morphogenetic protein antagonist, Gremlin 1, was recently shown to be increased in the lungs of pulmonary arterial hypertension patients, and in response to hypoxia. Gremlin 1 released from the vascular endothelium may inhibit endogenous bone morphogenetic protein signaling and contribute to the development of pulmonary arterial hypertension. Here, we investigate the impact of Gremlin 1 inhibition in disease after exposure to chronic hypoxia/SU5416 in mice. We investigated the effects of an anti-Gremlin 1 monoclonal antibody in the chronic hypoxia/SU5416 murine model of pulmonary arterial hypertension. Chronic hypoxic/SU5416 exposure of mice induced upregulation of Gremlin 1 mRNA in lung and right ventricle tissue compared with normoxic controls. Prophylactic treatment with an anti-Gremlin 1 neutralizing mAb reduced the hypoxic/SU5416-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy. Importantly, therapeutic treatment with an anti-Gremlin 1 antibody also reduced pulmonary vascular remodeling and right ventricular hypertrophy indicating a role for Gremlin 1 in the progression of the disease. We conclude that Gremlin 1 plays a role in the development and progression of pulmonary arterial hypertension in the murine hypoxia/SU5416 model, and that Gremlin 1 is a potential therapeutic target for pulmonary arterial hypertension.

Therapy directed against thymic stromal lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine and has been shown to be one of the factors released by epithelial cells following allergen contact with an important role instructing dendritic cells (DCs) to induce a T-helper type 2 (Th2) response. These TSLP-DC stimulate CD4(+) T cells to induce a proallergic cytokine profile, suggesting TSLP plays a crucial role in the initiation of the allergic cascade. Recently, evidence has also accumulated that TSLP could play a role in enhancement of the effector stages of the allergic response, with TSLP in synergy with IL-1 and tumor necrosis factor (TNF)-alpha shown to amplify cytokine secretion from mast cells. Also, the clinical relevance of TSLP has been demonstrated by both high levels of TSLP in skin biopsies from lesional atopic dermatitis patients and also increased expression of TSLP in asthmatic airway epithelial cells which correlated with reduced lung function. These studies suggest a critical role for TSLP as a driving factor in the emerging concept of tissue-specific control of immunity with TSLP secretion at the epithelial-DC interface acting as an initial factor in the proallergic cascade.

A role for the transcription factor RelB in IFN-α production and in IFN-α-stimulated cross-priming

Chimeric mice generated with bone marrow from RelB‐deficient (–/–), RelB‐heterozygous (+/–) and wild‐type (+/+) mice were used to determine how total or partial absence of the transcription factor RelB in haematopoietic cells affects the immune response generated after lymphocytic choriomeningitis virus (LCMV) infection. In RelB–/– chimeras, early virus replication was enhanced and LCMV clearance was impaired. Although plasmacytoid dendritic cell numbers were similar, serum interferon (IFN)‐α levels in RelB–/– and RelB+/– chimeras were markedly lower than in RelB+/+ chimeras during early LCMV infection. Further, both RelB–/– and RelB+/– chimeras mounted a lower‐magnitude LCMV‐specific CD8+ T cell response than their RelB+/+ counterparts, although the LCMV‐specific CD8+ T cells present were differentiated into functional cytotoxic cells. In LCMV‐infected RelB–/– mice, induction of cross‐priming to an independently injected soluble protein, which depends on the IFN‐α/β made during the viral infection, was also impaired. Notably, provision of exogenous IFN‐α did not restore the ability of RelB–/– mice to cross‐prime. In summary, these results show that the RelB/NF‐κB pathway is required for optimal IFN‐α production after LCMV infection and suggest a crucial role for RelB in IFN‐α‐stimulated cross‐priming of CD8+ T cell responses.