Sean Lund

Lung type 2 innate lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production.

BACKGROUND Cysteinyl leukotrienes (CysLTs) contribute to asthma pathogenesis, in part through cysteinyl leukotriene receptor 1 (CysLT1R). Recently discovered lineage-negative type 2 innate lymphoid cells (ILC2s) potently produce IL-5 and IL-13. OBJECTIVES We hypothesized that lung ILC2s might be activated by leukotrienes through CysLT1R. METHODS ILC2s (Thy1.2(+) lineage-negative lymphocytes) and CysLT1R were detected in the lungs of wild-type, signal transducer and activator of transcription 6-deficient (STAT6(-/-)), and recombination-activating gene 2-deficient (RAG2(-/-)) mice by means of flow cytometry. T(H)2 cytokine levels were measured in purified lung ILC2s stimulated with leukotriene D₄ (LTD₄) in the presence or absence of the CysLT1R antagonist montelukast. Calcium influx was measured by using Fluo-4 intensity. Intranasal leukotriene C₄, D₄, and E₄ were administered to naive mice, and levels of ILC2 IL-5 production were determined. Finally, LTD₄ was coadministered with Alternaria species repetitively to RAG2(-/-) mice (with ILC2s) and IL-7 receptor-deficient mice (lack ILC2s), and total ILC2 numbers, proliferation (Ki-67(+)), and bronchoalveolar lavage fluid eosinophil numbers were measured. RESULTS CysLT1R was expressed on lung ILC2s from wild-type, RAG2(-/-), and STAT6(-/-) naive and Alternaria species-challenged mice. In vitro LTD₄ induced ILC2s to rapidly generate high levels of IL-5 and IL-13 within 6 hours of stimulation. Interestingly, LTD4, but not IL-33, induced high levels of IL-4 by ILC2s. LTD₄ administered in vivo rapidly induced ILC2 IL-5 production that was significantly reduced by montelukast before treatment. Finally, LTD₄ potentiated Alternaria species-induced eosinophilia, as well as ILC2 accumulation and proliferation. CONCLUSIONS We present novel data that CysLT1R is expressed on ILC2s and LTD₄ potently induces CysLT1R-dependent ILC2 production of IL-4, IL-5, and IL-13. Additionally, LTD₄ potentiates Alternaria species-induced eosinophilia and ILC2 proliferation and accumulation.

Increased ILC2s in the eosinophilic nasal polyp endotype are associated with corticosteroid responsiveness

Group 2 innate lymphoid cells (ILC2s) have recently been identified in human nasal polyps, but whether numbers of ILC2s differ by polyp endotype or are influenced by corticosteroid use is unknown. Here, we show that eosinophilic nasal polyps contained double the number of ILC2s vs. non-eosinophilic polyps. Polyp ILC2s were also reduced by 50% in patients treated with systemic corticosteroids. Further, using a fungal allergen challenge mouse model, we detected greatly reduced Th2 cytokine-producing and Ki-67+ proliferating lung ILC2s in mice receiving dexamethasone. Finally, ILC2 Annexin V staining revealed extensive apoptosis after corticosteroid treatment in vivo and in vitro. Thus, ILC2s are elevated in the eosinophilic nasal polyp endotype and systemic corticosteroid treatment correlated with reduced polyp ILC2s. Finally, allergen-challenged mice showed reduced ILC2s and increased ILC2 apoptosis after corticosteroid treatment suggesting that ILC2 may be responsive to corticosteroids in eosinophilic respiratory disease.

Allergen challenge in allergic rhinitis rapidly induces increased peripheral blood type 2 innate lymphoid cells that express CD84.

Type 2 innate lymphoid cells (ILC2) produce high levels of Th2 cytokines. Our study demonstrates that cat allergen challenge in allergic rhinitis subjects rapidly induces increased peripheral blood ILC2.

Type 2 Innate Lymphoid Cells in Allergic Disease.

Type II innate lymphoid cells (ILC2) are a novel population of lineage-negative cells that produce high levels of Th2 cytokines IL-5 and IL-13. ILC2 are found in human respiratory and gastrointestinal tissue as well as in skin. Studies from mouse models of asthma and atopic dermatitis suggest a role for ILC2 in promoting allergic inflammation. The epithelial cytokines IL-25, IL-33, and TSLP, as well as the lipid mediator leukotriene D4, have been shown to potently activate ILC2 under specific conditions and supporting the notion that many separate pathways in allergic disease may result in stimulation of ILC2. Ongoing investigations are required to better characterize the relative contribution of ILC2 in allergic inflammation as well as mechanisms by which other cell types including conventional T cells regulate ILC2 survival, proliferation, and cytokine production. Importantly, therapeutic strategies to target ILC2 may reduce allergic inflammation in afflicted individuals. This review summarizes the development, surface marker profile, cytokine production, and upstream regulation of ILC2, and focuses on the role of ILC2 in common allergic diseases.

Innate Type 2 Response to Alternaria Extract Enhances Ryegrass-Induced Lung Inflammation

Background: Exposure to the fungal allergen Alternaria alternata as well as ryegrass pollen has been implicated in severe asthma symptoms during thunderstorms. We have previously shown that Alternaria extract induces innate type 2 lung inflammation in mice. We hypothesized that the innate eosinophilic response to Alternaria extract may enhance lung inflammation induced by ryegrass. Methods: Mice were sensitized to ryegrass allergen and administered a single challenge with A. alternata extract before or after final ryegrass challenges. Levels of eosinophils, neutrophils, Th2 cells, innate lymphoid cells (ILC2), interleukin (IL)-5 and IL-13 in bronchoalveolar lavage (BAL) as well as inflammation and mucus were assessed. Results: Mice receiving ryegrass sensitization and challenge developed an eosinophilic lung response. A single challenge with Alternaria extract given 3 days before or 3 days after ryegrass challenges resulted in increased eosinophils, peribronchial inflammation and mucus production in the airways compared with ryegrass-only challenges. Type 2 ILC2 and Th2 cell recruitment to the airways was increased after Alternaria extract exposure in ryegrass-challenged mice. Innate immune challenges with Alternaria extract induced BAL eosinophilia, Th2 cell recruitment as well as ILC2 expansion and proliferation. Conclusions: A single exposure to Alternaria extract in ryegrass-sensitized and -challenged mice enhances the type 2 lung inflammatory response, including airway eosinophilia, peribronchial infiltrate, and mucus production, possibly through Th2 cell recruitment and ILC2 expansion. If translated to humans, exposure to both grass pollen and Alternaria may be a potential cause of thunderstorm-related asthma.

Leukotriene C4 Potentiates IL-33–Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R−/− mice, but not CysLT2R−/− mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R−/− mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata. Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-β signaling

Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.

Virulence Role of the GlcNAc Side Chain of the Lancefield Cell Wall Carbohydrate Antigen in Non-M1-Serotype Group A Streptococcus

ABSTRACT Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses. IMPORTANCE The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model. The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model.

Impaired induction of allergic lung inflammation by Alternaria alternata mutant MAPK homologue Fus3

ABSTRACT The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.