Magdalena M. Gorska

Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33

BACKGROUNDnAsthma in a mouse model spontaneously resolves after cessation of allergen exposure. We developed a mouse model in which asthma features persisted for 6 months after cessation of allergen exposure.nnnOBJECTIVEnWe sought to elucidate factors contributing to the persistence of asthma.nnnMETHODSnWe used a combination of immunologic, genetic, microarray, and pharmacologic approaches to dissect the mechanism of asthma persistence.nnnRESULTSnElimination of T cells though antibody-mediated depletion or lethal irradiation and transplantation of recombination-activating gene (Rag1)(-/-) bone marrow in mice with chronic asthma resulted in resolution of airway inflammation but not airway hyperreactivity or remodeling. Elimination of T cells and type 2 innate lymphoid cells (ILC2s) through lethal irradiation and transplantation of Rag2(-/-)γc(-/-) bone marrow or blockade of IL-33 resulted in resolution of airway inflammation and hyperreactivity. Persistence of asthma required multiple interconnected feedback and feed-forward circuits between ILC2s and epithelial cells. Epithelial IL-33 induced ILC2s, a rich source of IL-13. The latter directly induced epithelial IL-33, establishing a positive feedback circuit. IL-33 autoinduced, generating another feedback circuit. IL-13 upregulated IL-33 receptors and facilitated IL-33 autoinduction, thus establishing a feed-forward circuit. Elimination of any component of these circuits resulted in resolution of chronic asthma. In agreement with the foregoing, IL-33 and ILC2 levels were increased in the airways of asthmatic patients. IL-33 levels correlated with disease severity.nnnCONCLUSIONSnWe present a critical network of feedback and feed-forward interactions between epithelial cells and ILC2s involved in maintaining chronic asthma. Although T cells contributed to the severity of chronic asthma, they were redundant in maintaining airway hyperreactivity and remodeling.

Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma

BACKGROUNDnTH2 cells can further differentiate into dual-positive TH2/TH17 cells. The presence of dual-positive TH2/TH17 cells in the airways and their effect on asthma severity are unknown.nnnOBJECTIVEnWe sought to study dual-positive TH2/TH17 cells in bronchoalveolar lavage (BAL) fluid from asthmatic patients, examine their response to glucocorticoids, and define their relevance for disease severity.nnnMETHODSnBronchoscopy and lavage were performed in 52 asthmatic patients and 25 disease control subjects. TH2 and TH2/TH17 cells were analyzed by using multicolor flow cytometry and confocal immunofluorescence microscopy. Cytokines were assayed by means of ELISA.nnnRESULTSnDual-positive TH2/TH17 cells were present at a higher frequency in BAL fluid from asthmatic patients compared with numbers seen in disease control subjects. High-level IL-4 production was typically accompanied by high-level IL-17 production and coexpression of GATA3 and retinoic acid receptor-related orphan receptor γt. Increased presence of TH2/TH17 cells was associated with increased IL-17 production in lavage fluid. TH2/TH17 cell counts and IL-17 production correlated with PC20 for methacholine, eosinophil counts, and FEV1. TH2/TH17 cells, unlike TH2 cells, were resistant to dexamethasone-induced cell death. They expressed higher levels of mitogen-activated protein-extracellular signal-regulated kinase kinase 1, a molecule that induces glucocorticoid resistance. On the basis of the dominance of BAL fluid TH2 or TH2/TH17 cells, we identified 3 subgroups of asthma: TH2(predominant), TH2/TH17(predominant), and TH2/TH17(low). The TH2/TH17(predominant) subgroup manifested the most severe form of asthma, whereas the TH2/TH17(low) subgroup had the mildest asthma.nnnCONCLUSIONnAsthma is associated with a higher frequency of dual-positive TH2/TH17 cells in BAL fluid. The TH2/TH17(predominant) subgroup of asthmatic patients manifested glucocorticoid resistance in vitro. They also had the greatest airway obstruction and hyperreactivity compared with the TH2(predominant) and TH2/TH17(low) subgroups.

Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic airways

BACKGROUNDnMany airway cells manifest signs of chronic activation in asthma. The mechanism of this chronic activation is unknown.nnnOBJECTIVESnWe sought to study the activation of the mitogen-activated protein kinase (MAPK) signaling pathway in airway cells.nnnMETHODSnEndobronchial biopsy specimens from patients with severe and mild asthma (n = 17 in each group) and healthy control subjects (n = 15) were analyzed for the phosphorylated MAPKs extracellular signal-regulated kinase (ERK) 1/2, p38, and Jun N-terminal kinase (JNK) and their downstream effectors by means of immunofluorescence staining. Airway epithelial activation of ERK1/2 and p38 was studied by using Western blotting. Epithelial function was studied by means of real-time PCR, ELISA, and the thymidine incorporation assay.nnnRESULTSnWe detected strong phospho-ERK1/2 staining in airway epithelium and smooth muscle cells in biopsy specimens from asthmatic patients. Fluorescent areas per image, as well as mean fluorescence intensity, were significantly (P < .0001) different among the 3 study groups (patients with severe asthma, patients with mild asthma, and healthy control subjects). Patients with severe asthma also demonstrated strong phospho-p38 staining, mostly in epithelial cells, which was significantly different from that in patients with mild asthma (P = .0001) and healthy control subjects (P = .02). Phospho-JNK primarily stained airway smooth muscle cells. Healthy subjects showed the highest intensity of phospho-JNK staining compared with that seen in patients with severe (P = .004) and mild asthma (P = .003). Inhibition of ERK1/2 and p38 in primary airway epithelial cells blocked their proliferation and expression of select, but not all, chemokines.nnnCONCLUSIONSnSignificant phosphorylation of ERK1/2 and p38 and their correlation with disease severity suggests that the foregoing signaling pathways play an important role in asthma. The ERK1/2 and p38 pathways regulate epithelial cell secretory function and proliferation.

Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma

BACKGROUNDnExisting asthma models develop tolerance when chronically exposed to the same allergen.nnnOBJECTIVEnWe sought to establish a chronic model that sustains features of asthma long after discontinuation of allergen exposure.nnnMETHODSnWe immunized and exposed mice to a combination of single, double, or triple allergens (dust mite, ragweed, and Aspergillus species) intranasally for 8 weeks. Airway hyperreactivity (AHR) and morphologic features of asthma were studied 3 weeks after allergen exposure. Signaling effects of the allergens were studied on dendritic cells.nnnRESULTSnSensitization and repeated exposure to a single allergen induced tolerance. Sensitization to double and especially triple allergens broke through tolerance and established AHR, eosinophilic inflammation, mast cell and smooth muscle hyperplasia, mucus production, and airway remodeling that persisted at least 3 weeks after allergen exposure. Mucosal exposure to triple allergens in the absence of an adjuvant was sufficient to induce chronic airway inflammation. Anti-IL-5 and anti-IL-13 antibodies inhibited inflammation and AHR in the acute asthma model but not in the chronic triple-allergen model. Multiple allergens produce a synergy in p38 mitogen-activated protein kinase signaling and maturation of dendritic cells, which provides heightened T-cell costimulation at a level that cannot be achieved with a single allergen.nnnCONCLUSIONSnSensitivity to multiple allergens leads to chronic asthma in mice. Multiple allergens synergize in dendritic cell signaling and T-cell stimulation that allows escape from the single allergen-associated tolerance development.

A mouse model links asthma susceptibility to prenatal exposure to diesel exhaust

BACKGROUNDnMost asthma begins in the first years of life. This early onset cannot be attributed merely to genetic factors because the prevalence of asthma is increasing. Epidemiologic studies have indicated roles for prenatal and early childhood exposures, including exposure to diesel exhaust. However, little is known about the mechanisms. This is largely due to a paucity of animal models.nnnOBJECTIVEnWe aimed to develop a mouse model of asthma susceptibility through prenatal exposure to diesel exhaust.nnnMETHODSnPregnant C57BL/6 female mice were given repeated intranasal applications of diesel exhaust particles (DEPs) or PBS. Offspring underwent suboptimal immunization and challenge with ovalbumin (OVA) or received PBS. Pups were examined for features of asthma; lung and liver tissues were analyzed for transcription of DEP-regulated genes.nnnRESULTSnOffspring of mice exposed to DEPs were hypersensitive to OVA, as indicated by airway inflammation and hyperresponsiveness, increased serum OVA-specific IgE levels, and increased pulmonary and systemic TH2 and TH17 cytokine levels. These cytokines were primarily produced by natural killer (NK) cells. Antibody-mediated depletion of NK cells prevented airway inflammation. Asthma susceptibility was associated with increased transcription of genes known to be specifically regulated by the aryl hydrocarbon receptor and oxidative stress. Features of asthma were either marginal or absent in OVA-treated pups of PBS-exposed mice.nnnCONCLUSIONnWe created a mouse model that linked maternal exposure to DEPs with asthma susceptibility in offspring. Development of asthma was dependent on NK cells and associated with increased transcription from aryl hydrocarbon receptor- and oxidative stress-regulated genes.

IL-2 and IL-4 Stimulate MEK1 Expression and Contribute to T Cell Resistance against Suppression by TGF-β and IL-10 in Asthma

The T cell-driven airway inflammation in chronic asthma is uninhibited and sustained. We examined the resistance of T cells from asthmatic patients against suppression by TGF-β, IL-10 and glucocorticoids and explored its signaling mechanism. CD4+CD25− T cells from allergic asthmatic subjects demonstrated increased TCR-stimulated proliferation as compared with healthy and chronic obstructive pulmonary disease controls. This proliferation was resistant to inhibition by TGF-β, IL-10, and dexamethasone and to anergy induction. CD4 T cells from asthmatic patients, but not chronic obstructive pulmonary disease, allergic rhinitis, and healthy subjects, showed increased expression of MEK1, heightened phosphorylation of ERK1/2, and increased levels of c-Fos. IL-2 and IL-4 stimulated the expression of MEK1 and c-Fos and induced T cell resistance. The inhibition of MEK1 reversed, whereas induced expression of c-Fos and JunB promoted T cell resistance against TGF-β– and IL-10–mediated suppression. We have uncovered an IL-2– and IL-4–driven MEK1 induction mechanism that results in heightened ERK1/2 activation in asthmatic T cells and make them resistant to certain inhibitory mechanisms.

Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin

Background: Type 2 innate lymphoid cells (ILC2s) represent an important type 2 immune cell. Glucocorticoid regulation of human ILC2s is largely unknown. Objective: We sought to assess steroid resistance of human blood and airway ILC2s from asthmatic patients and to examine its mechanism of induction. Methods: We studied human blood and lung ILC2s from asthmatic patients and control subjects using flow cytometry and ELISA. Results: Dexamethasone inhibited (P = .04) chemoattractant receptor–homologous molecule expressed on TH2 lymphocytes and type 2 cytokine expression by blood ILC2s stimulated with IL‐25 and IL‐33. However, it did not do so when ILC2s were stimulated with IL‐7 and thymic stromal lymphopoietin (TSLP), 2 ligands of IL‐7 receptor &agr;. Unlike blood ILC2s, bronchoalveolar lavage (BAL) fluid ILC2s from asthmatic patients were resistant to dexamethasone. BAL fluid from asthmatic patients had increased TSLP but not IL‐7 levels. BAL fluid TSLP levels correlated (r = 0.74) with steroid resistance of ILC2s. TSLP was synergistically induced in epithelial cells by IL‐13 and human rhinovirus. Mechanistically, dexamethasone upregulated ILC2 expression of IL‐7 receptor &agr;, which augmented and sustained signal transducer and activator of transcription (STAT) 5 signaling by TSLP. TSLP induced mitogen‐activated protein kinase kinase (MEK), c‐Fos, inhibitor of DNA binding 3, phosphorylated signal transducer and activator of transcription (pSTAT) 3, and pSTAT5, molecules linked to steroid resistance. Dexamethasone inhibited c‐Fos, inhibitor of DNA binding 3, and pSTAT3 but not pSTAT5 and MEK. The MEK inhibitor trametinib, the Janus kinase–STAT inhibitor tofacitinib, and the STAT5 inhibitor pimozide reversed steroid resistance of BAL ILC2s. Conclusions: Dexamethasone inhibited type 2 cytokine production by blood ILC2s. IL‐7 and TSLP abrogated this inhibition and induced steroid resistance of ILC2s in a MEK‐ and STAT5‐dependent manner. BAL fluid ILC2s from asthmatic patients with increased TSLP levels were steroid resistant, which was reversed by clinically available inhibitors of MEK and STAT5.

ERK1 is important for Th2 differentiation and development of experimental asthma

The ERK1/2 signaling pathway regulates a variety of T‐cell functions. We observed dynamic changes in the expression of ERK1/2 during T‐helper cell differentiation. Specifically, the expression of ERK1/2 was decreased and increased by IL‐12 and IL‐4, respectively. To address this subject further, we examined the specific role of ERK1 in Th2 differentiation and development of experimental asthma using ERK1‐/‐ mice. ERK1‐/‐ mice were unable to mount airway inflammation and hyperreactivity in two different models of asthma, acute and chronic. ERK1‐/‐mice had reduced expression of Th2 cytokines IL‐4 and IL‐5 but not IL17A or IFN‐γ. They had reduced levels of allergen‐specific IgE and blood eosinophils. T cells from immunized ERK1‐/‐ mice manifested reduced proliferation in response to the sensitizing allergen. ERK1‐/‐ T cells had reduced and short‐lived expression of JunB following TCR stimulation, which likely contributed to their impaired Th2 differentiation. Immunized ERK1‐/‐ mice showed reduced numbers of CD44 high CD4 T cells in the spleen. In vitro studies demonstrated that Th2 but not Th1 cells from ERK1 ‐/‐ mice had reduced numbers of CD44 high cells. Finally, CD4 T cells form ERK1 ‐/‐ mice expressed higher levels of BIM under growth factor‐deprived conditions and reduced Mcl‐1 on stimulation. As a result, the survival of CD4 T cells, especially CD44high Th2 cells, was much reduced in ERK1‐/‐ mice. We conclude that ERK1 plays a nonredundant role in Th2 differentiation and development of experimental asthma. ERK1 controls Th2 differentiation and survival through its effect on JunB and BIM, respectively.—Goplen, N., Karim, Z., Guo, L., Zhuang, Y., Huang, H., Gorska, M. M., Gelfand, E., Pagés, G., Pouysségur, J., Alam, R. ERK1 is important for Th2 differentiation and development of experimental asthma. FASEB J. 26, 1934‐1945 (2012). www.fasebj.org

Mechanism of TH2/TH17-predominant and neutrophilic TH2/TH17-low subtypes of asthma.

Background: The mechanism of TH2/TH17‐predominant and TH2/TH17‐low asthma is unknown. Objective: We sought to study the immune mechanism of TH2/TH17‐predominant and TH2/TH17‐low asthma. Methods: In a previously reported cohort of 60 asthmatic patients, 16 patients were immunophenotyped with TH2/TH17‐predominant asthma and 22 patients with TH2/TH17‐low asthma. We examined bronchoalveolar lavage (BAL) fluid leukocytes, cytokines, mediators, and epithelial cell function for these asthma subgroups. Results: Patients with TH2/TH17‐predominant asthma had increased IL‐1&bgr;, IL‐6, IL‐23, C3a, and serum amyloid A levels in BAL fluid, and these correlated with IL‐1&bgr; and C3a levels. TH2/TH17 cells expressed higher levels of the IL‐1 receptor and phospho‐p38 mitogen‐activated protein kinase. Anakinra, an IL‐1 receptor antagonist protein, inhibited BAL TH2/TH17 cell counts. TH2/TH17‐low asthma had 2 distinct subgroups: neutrophilic asthma (45%) and pauci‐inflammatory asthma (55%). This contrasted with patients with TH2/TH17‐predominant and TH2‐predominant asthma, which included neutrophilic asthma in 6% and 0% of patients, respectively. BAL fluid neutrophils strongly correlated with BAL fluid myeloperoxidase, IL‐8, IL‐1&agr;, IL‐6, granulocyte colony‐stimulating factor, and GM‐CSF levels. Sixty percent of the patients with neutrophilic asthma had a pathogenic microorganism in BAL culture, which suggested a subclinical infection. Conclusion: We uncovered a critical role for the IL‐1&bgr; pathway in patients with TH2/TH17‐predminant asthma. A subgroup of patients with TH2/TH17‐low asthma had neutrophilic asthma and increased BAL fluid IL‐1&agr;, IL‐6, IL‐8, granulocyte colony‐stimulating factor, and GM‐CSF levels. IL‐1&agr; was directly involved in IL‐8 production and likely contributed to neutrophilic asthma. Sixty percent of neutrophilic patients had a subclinical infection.

UNC119 inhibits dynamin and dynamin-dependent endocytic processes

Unc119 is an adapter signaling molecule, which regulates activation of tyrosine kinases in T cells, eosinophils and fibroblasts. It plays an important role in the photoreceptor synapses of the retina. Recently, we have shown that it inhibits bacterial uptake through macropinocytosis. In this paper we demonstrate a role for Unc119 in clathrin- and caveolae-based endocytosis as well as macropinocytosis. Depletion of Unc119 in fibroblasts increases, whereas overexpression inhibits uptake of transferrin, FM4-64, albumin, viruses, and ligand-coated beads. Physiological stimuli that upregulate the expression of Unc119 also inhibits endocytosis. Unc119 has the opposite effect on cholera toxin B uptake, which represents a clathrin- and dynamin-independent endocytic process. Unc119 interacts with dynamin, a key effector molecule of many endocytic processes. More importantly, Unc119 inhibits the GTPase activity of dynamin. Binding of Unc119 to dynamin decreases the association with its binding partner amphiphysin, a known regulator of dynamin activation. Thus, Unc119 regulates various endocytic pathways through dynamin and sets a threshold point for vesicular trafficking.