Publications by authors named "Donald N Cook"

64 Publications

Adipocyte P2Y14 receptors play a key role in regulating whole-body glucose and lipid homeostasis.

JCI Insight 2021 May 24;6(10). Epub 2021 May 24.

Molecular Recognition Section and.

Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.
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http://dx.doi.org/10.1172/jci.insight.146577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262345PMC
May 2021

A new wrinkle for skin dendritic cell migration.

Blood 2021 May;137(20):2716-2717

National Institute of Environmental Health Sciences.

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http://dx.doi.org/10.1182/blood.2020010619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138550PMC
May 2021

Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes.

Front Immunol 2021 28;12:661290. Epub 2021 Apr 28.

Department of Immunology, Duke University Medical Center, Durham, NC, United States.

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14CD24 macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14CD24 mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.
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http://dx.doi.org/10.3389/fimmu.2021.661290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113646PMC
April 2021

Glucocorticoids and Androgens Protect From Gastric Metaplasia by Suppressing Group 2 Innate Lymphoid Cell Activation.

Gastroenterology 2021 May 7. Epub 2021 May 7.

Molecular Endocrinology Group, Signal Transduction Laboratory, North Carolina. Electronic address:

Background & Aims: The immune compartment is critical for maintaining tissue homeostasis. A weak immune response increases susceptibility to infection, but immune hyperactivation causes tissue damage, and chronic inflammation may lead to cancer development. In the stomach, inflammation damages the gastric glands and drives the development of potentially preneoplastic metaplasia. Glucocorticoids are potent anti-inflammatory steroid hormones that are required to suppress gastric inflammation and metaplasia. However, these hormones function differently in males and females. Here, we investigate the impact of sex on the regulation of gastric inflammation.

Methods: Endogenous glucocorticoids and male sex hormones were removed from mice using adrenalectomy and castration, respectively. Mice were treated with 5α-dihydrotestosterone (DHT) to test the effects of androgens on regulating gastric inflammation. Single-cell RNA sequencing of gastric leukocytes was used to identify the leukocyte populations that were the direct targets of androgen signaling. Type 2 innate lymphoid cells (ILC2s) were depleted by treatment with CD90.2 antibodies.

Results: We show that adrenalectomized female mice develop spontaneous gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) but that the stomachs of adrenalectomized male mice remain quantitatively normal. Simultaneous depletion of glucocorticoids and sex hormones abolished the male-protective effects and triggered spontaneous pathogenic gastric inflammation and SPEM. Treatment of female mice with DHT prevented gastric inflammation and SPEM development when administered concurrent with adrenalectomy and also reversed the pathology when administered after disease onset. Single-cell RNAseq of gastric leukocytes revealed that ILC2s expressed abundant levels of both the glucocorticoid receptor (Gr) and androgen receptor (Ar). We demonstrated that DHT treatment potently suppressed the expression of the proinflammatory cytokines Il13 and Csf2 by ILC2s. Moreover, ILC2 depletion protected the stomach from SPEM development.

Conclusions: Here, we report a novel mechanism by which glucocorticoids and androgens exert overlapping effects to regulate gastric inflammation. Androgen signaling within ILC2s prevents their pathogenic activation by suppressing the transcription of proinflammatory cytokines. This work revealed a critical role for sex hormones in regulating gastric inflammation and metaplasia.
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http://dx.doi.org/10.1053/j.gastro.2021.04.075DOI Listing
May 2021

UDP-glucose and P2Y14 receptor amplify allergen-induced airway eosinophilia.

J Clin Invest 2021 Apr;131(7)

Immunity, Inflammation and Disease Laboratory.

Airway eosinophilia is a hallmark of allergic asthma and is associated with mucus production, airway hyperresponsiveness, and shortness of breath. Although glucocorticoids are widely used to treat asthma, their prolonged use is associated with several side effects. Furthermore, many individuals with eosinophilic asthma are resistant to glucocorticoid treatment, and they have an unmet need for novel therapies. Here, we show that UDP-glucose (UDP-G), a nucleotide sugar, is selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. Mice lacking P2Y14R, the receptor for UDP-G, had decreased airway eosinophilia and airway hyperresponsiveness compared with wild-type mice in a protease-mediated model of asthma. P2Y14R was dispensable for allergic sensitization and for the production of type 2 cytokines in the lung after challenge. However, UDP-G increased chemokinesis in eosinophils and enhanced their response to the eosinophil chemoattractant, CCL24. In turn, eosinophils triggered the release of UDP-G into the airway, thereby amplifying eosinophilic recruitment. This positive feedback loop was sensitive to therapeutic intervention, as a small molecule antagonist of P2Y14R inhibited airway eosinophilia. These findings thus reveal a pathway that can be therapeutically targeted to treat asthma exacerbations and glucocorticoid-resistant forms of this disease.
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http://dx.doi.org/10.1172/JCI140709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011887PMC
April 2021

Structure-Activity Relationship of Heterocyclic P2Y Receptor Antagonists: Removal of the Zwitterionic Character with Piperidine Bioisosteres.

J Med Chem 2021 04 31;64(8):5099-5122. Epub 2021 Mar 31.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

A known zwitterionic, heterocyclic P2YR antagonist was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2YR affinity. Piperidine amide , 3-aminopropynyl , and 5-(hydroxymethyl)isoxazol-3-yl) congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing (; MRS4654; IC, 15 nM) and less active phenylamide-containing () scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2YR antagonists for in vivo application.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00164DOI Listing
April 2021

Scavenger Receptor BI Attenuates IL-17A-Dependent Neutrophilic Inflammation in Asthma.

Am J Respir Cell Mol Biol 2021 06;64(6):698-708

Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina.

Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI) and SR-BI-deficient (SR-BI) mice were sensitized (Days 0 and 7) and then challenged (Days 14, 15, and 16) with a house dust mite (HDM) preparation administered through oropharyngeal aspiration. Airway inflammation and cytokine production were quantified on Day 17. When compared with SR-BI mice, the HDM-challenged SR-BI mice had increased neutrophils and pulmonary IL-17A production in BAL fluid. This augmented IL-17A production in SR-BI mice originated from a non-T-cell source that included neutrophils and alveolar macrophages. Given that SR-BI regulates adrenal steroid hormone production, we tested whether the changes in SR-BI mice were glucocorticoid dependent. Indeed, SR-BI mice were adrenally insufficient during the HDM challenge, and corticosterone replacement decreased pulmonary neutrophilia and IL-17A production in SR-BI mice. Taken together, these data indicate that SR-BI dampens pulmonary neutrophilic inflammation and IL-17A production in allergic asthma at least in part by maintaining adrenal function.
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http://dx.doi.org/10.1165/rcmb.2020-0007OCDOI Listing
June 2021

Regulation of Immune Responses by Nonhematopoietic Cells in Asthma.

J Immunol 2021 01;206(2):292-301

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

Nonhematopoietic cells are emerging as important contributors to many inflammatory diseases, including allergic asthma. Recent advances have led to a deeper understanding of how these cells interact with traditional immune cells, thereby modulating their activities in both homeostasis and disease. In addition to their well-established roles in gas exchange and barrier function, lung epithelial cells express an armament of innate sensors that can be triggered by various inhaled environmental agents, leading to the production of proinflammatory molecules. Advances in cell lineage tracing and single-cell RNA sequencing have expanded our knowledge of rare, but immunologically important nonhematopoietic cell populations. In parallel with these advances, novel reverse genetic approaches are revealing how individual genes in different lung-resident nonhematopoietic cell populations contribute to the initiation and maintenance of asthma. This knowledge is already revealing new pathways that can be selectively targeted to treat distinct forms of asthma.
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http://dx.doi.org/10.4049/jimmunol.2000885DOI Listing
January 2021

Exploration of Alternative Scaffolds for P2Y Receptor Antagonists Containing a Biaryl Core.

J Med Chem 2020 09 6;63(17):9563-9589. Epub 2020 Aug 6.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

Various heteroaryl and bicyclo-aliphatic analogues of zwitterionic biaryl P2Y receptor (P2YR) antagonists were synthesized, and affinity was measured in P2YR-expressing Chinese hamster ovary cells by flow cytometry. Given this series' low water solubility, various polyethylene glycol derivatives of the distally binding piperidin-4-yl moiety of moderate affinity were synthesized. Rotation of previously identified 1,2,3-triazole attached to the central -benzoic acid core () provided moderate affinity but not indole and benzimidazole substitution of the aryl-triazole. The corresponding P2YR region is predicted by homology modeling as a deep, sterically limited hydrophobic pocket, with the outward pointing piperidine moiety being the most flexible. Bicyclic-substituted piperidine ring derivatives of naphthalene antagonist , e.g., quinuclidine (MRS4608, IC ≈ 20 nM at hP2YR/mP2YR), or of triazole , preserved affinity. Potent antagonists , , , and (10 mg/kg) protected in an ovalbumin/ mouse asthma model, and PEG conjugate reduced chronic pain. Thus, we expanded P2YR antagonist structure-activity relationship, introducing diverse physical-chemical properties.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040948PMC
September 2020

Role of Environmental Adjuvants in Asthma Development.

Authors:
Donald N Cook

Curr Allergy Asthma Rep 2020 06 16;20(9):42. Epub 2020 Jun 16.

Immunogenetics Group, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Dr., Research Triangle Park, Durham, NC, 27709, USA.

Purpose Of Review: This review summarizes recent progress in our understanding how environmental adjuvants promote the development of asthma.

Recent Findings: Asthma is a heterogeneous set of lung pathologies with overlapping features. Human studies and animal models suggest that exposure to different environmental adjuvants activate distinct immune pathways, which in turn give rise to distinct forms, or endotypes, of allergic asthma. Depending on their concentrations, inhaled TLR ligands can activate either type 2 inflammation, or Th17 differentiation, along with regulatory responses that function to attenuate inflammation. By contrast, a different category of environmental adjuvants, proteases, activate distinct immune pathways and prime predominantly type 2 immune responses. Asthma is not a single disease, but rather a group of pathologies with overlapping features. Different endotypes of asthma likely arise from perturbations of distinct immunologic pathways during allergic sensitization.
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http://dx.doi.org/10.1007/s11882-020-00935-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344337PMC
June 2020

Cholesterol 25-hydroxylase promotes efferocytosis and resolution of lung inflammation.

JCI Insight 2020 06 4;5(11). Epub 2020 Jun 4.

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, North Carolina, USA.

Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.
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http://dx.doi.org/10.1172/jci.insight.137189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308063PMC
June 2020

Epithelial membrane protein 2 governs transepithelial migration of neutrophils into the airspace.

J Clin Invest 2020 01;130(1):157-170

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.

Whether respiratory epithelial cells regulate the final transit of extravasated neutrophils into the inflamed airspace or are a passive barrier is poorly understood. Alveolar epithelial type 1 (AT1) cells, best known for solute transport and gas exchange, have few established immune roles. Epithelial membrane protein 2 (EMP2), a tetraspan protein that promotes recruitment of integrins to lipid rafts, is highly expressed in AT1 cells but has no known function in lung biology. Here, we show that Emp2-/- mice exhibit reduced neutrophil influx into the airspace after a wide range of inhaled exposures. During bacterial pneumonia, Emp2-/- mice had attenuated neutrophilic lung injury and improved survival. Bone marrow chimeras, intravital neutrophil labeling, and in vitro assays suggested that defective transepithelial migration of neutrophils into the alveolar lumen occurs in Emp2-/- lungs. Emp2-/- AT1 cells had dysregulated surface display of multiple adhesion molecules, associated with reduced raft abundance. Epithelial raft abundance was dependent upon putative cholesterol-binding motifs in EMP2, whereas EMP2 supported adhesion molecule display and neutrophil transmigration through suppression of caveolins. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular neutrophil traffic into the alveolus.
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http://dx.doi.org/10.1172/JCI127144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934223PMC
January 2020

(Inverse) Agonists of Retinoic Acid-Related Orphan Receptor γ: Regulation of Immune Responses, Inflammation, and Autoimmune Disease.

Annu Rev Pharmacol Toxicol 2020 01 6;60:371-390. Epub 2019 Aug 6.

Immunogenetics Section, Immunity, Inflammation, and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

Retinoic acid-related orphan receptor γt (RORγt) functions as a ligand-dependent transcription factor that regulates multiple proinflammatory genes and plays a critical role in several inflammatory and autoimmune diseases. Various endogenous and synthetic RORγ (inverse) agonists have been identified that regulate RORγ transcriptional activity, including many cholesterol intermediates and oxysterols. Changes in cholesterol biosynthesis and metabolism can therefore have a significant impact on the generation of oxysterol RORγ ligands and, consequently, can control RORγt activity and inflammation. These observations contribute to a growing literature that connects cholesterol metabolism to the regulation of immune responses and autoimmune disease. Loss of RORγ function in knockout mice and in mice treated with RORγ inverse agonists results in reduced production of proinflammatory cytokines, such as IL-17A/F, and increased resistance to autoimmune disease in several experimental rodent models. Thus, RORγt inverse agonists might provide an attractive therapeutic approach to treat a variety of autoimmune diseases.
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http://dx.doi.org/10.1146/annurev-pharmtox-010919-023711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952538PMC
January 2020

Therapeutic suppression of pulmonary neutrophilia and allergic airway hyperresponsiveness by a RORγt inverse agonist.

JCI Insight 2019 06 11;5. Epub 2019 Jun 11.

Immunity, Inflammation, and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA.

Airway neutrophilia occurs in approximately 50% of patients with asthma and is associated with particularly severe disease. Unfortunately, this form of asthma is usually refractory to corticosteroid treatment, and there is an unmet need for new therapies. Pulmonary neutrophilic inflammation is associated with Th17 cells, whose differentiation is controlled by the nuclear receptor, RORγt. Here, we tested whether VTP-938, a selective inverse agonist of this receptor, can reduce disease parameters in animal models of neutrophilic asthma. When administered prior to allergic sensitization through the airway, the RORγt inverse agonist blunted allergen-specific Th17 cell development in lung-draining lymph nodes and attenuated allergen-induced production of IL-17. VTP-938 also reduced pulmonary production of IL-17 and airway neutrophilia when given during the allergen challenge of the model. Finally, in an environmentally relevant model of allergic responses to house dust extracts, VTP-938 suppressed production of IL-17 and neutrophilic inflammation, and also markedly diminished airway hyperresponsiveness. Together, these findings suggest that orally available inverse agonists of RORγt might provide an effective therapy to treat glucocorticoid-resistant neutrophilic asthma.
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http://dx.doi.org/10.1172/jci.insight.125528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675583PMC
June 2019

Endogenous glucocorticoids prevent gastric metaplasia by suppressing spontaneous inflammation.

J Clin Invest 2019 03 18;129(3):1345-1358. Epub 2019 Feb 18.

Molecular Endocrinology Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.

In the stomach, chronic inflammation causes metaplasia and creates a favorable environment for the evolution of gastric cancer. Glucocorticoids are steroid hormones that repress proinflammatory stimuli, but their role in the stomach is unknown. In this study, we show that endogenous glucocorticoids are required to maintain gastric homeostasis. Removal of circulating glucocorticoids in mice by adrenalectomy resulted in the rapid onset of spontaneous gastric inflammation, oxyntic atrophy, and spasmolytic polypeptide-expressing metaplasia (SPEM), a putative precursor of gastric cancer. SPEM and oxyntic atrophy occurred independently of lymphocytes. However, depletion of monocytes and macrophages by clodronate treatment or inhibition of gastric monocyte infiltration using the Cx3cr1 knockout mouse model prevented SPEM development. Our results highlight the requirement for endogenous glucocorticoid signaling within the stomach to prevent spontaneous gastric inflammation and metaplasia, and suggest that glucocorticoid deficiency may lead to gastric cancer development.
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http://dx.doi.org/10.1172/JCI123233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391099PMC
March 2019

Imaging Precision-Cut Lung Slices to Visualize Leukocyte Localization and Trafficking.

Methods Mol Biol 2018 ;1799:237-246

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.

Pulmonary dendritic cells (DCs) are potent antigen-presenting cells that can activate both naïve and memory/effector T cells. However, very little is known of how movements and localization of DCs contribute to these events. To study this, we have developed new procedures that combine precision-cut lung slices with cell staining using fluorescently tagged antibodies to detect individual cell types. In this chapter, we describe these methods in detail and show how they can be used to study the localization of not only DCs but also other leukocytes of interest, as well as structural cells within the lung.
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http://dx.doi.org/10.1007/978-1-4939-7896-0_18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773260PMC
February 2019

Isolation and Purification of Epithelial and Endothelial Cells from Mouse Lung.

Methods Mol Biol 2018 ;1799:59-69

Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.

It is well established that responses to inhaled environmental agents are controlled by the coordinated actions by multiple immune cell types, including macrophages, dendritic cells, and lymphocytes. Recent evidence indicates that some structural cells can also contribute to the initiation and propagation of immune responses. For example, airway epithelial cells can promote eosinophilic inflammation in response to allergen inhalation. Much remains to be learned, however, regarding how each of these cell types interact with the others, and how these interactions shape immune responses to inhaled agents. Such studies have been hampered by the lack of reliable methods to isolate multiple and distinct populations of cells from the same tissue sample. Consequently, investigators have had to choose between using different protocols to isolate different populations of cells from different animals and accept that for some populations, cell yields can be very low. To overcome these difficulties, we have developed a convenient and practical method to isolate and purify subpopulations of epithelial and endothelial cells from mouse lung. Here, we describe these methods in detail.
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http://dx.doi.org/10.1007/978-1-4939-7896-0_6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779162PMC
February 2019

Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide.

Am J Physiol Lung Cell Mol Physiol 2018 08 19;315(2):L202-L211. Epub 2018 Apr 19.

Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina , Chapel Hill, North Carolina.

Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AMs) in the steady state but is dramatically upregulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AMs with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AMs was dependent upon the myeloid differentiation primary response 88 signaling pathway and the transcription factor NF-κB. In addition to upregulating display of NRP2 on the cell membrane, inhaled LPS also triggered AMs to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine (C-C motif) ligand 2 ( Ccl2) in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AMs regulates LPS-induced inflammatory cell recruitment to the airways and reveal a novel role for NRP2 during innate immune responses in the lungs.
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http://dx.doi.org/10.1152/ajplung.00067.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139664PMC
August 2018

Pathogenic T17 inflammation is sustained in the lungs by conventional dendritic cells and Toll-like receptor 4 signaling.

J Allergy Clin Immunol 2018 10 14;142(4):1229-1242.e6. Epub 2017 Nov 14.

Immunogenetics Group, Immunity, Inflammation and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC. Electronic address:

Background: Mechanisms that elicit mucosal T17 cell responses have been described, yet how these cells are sustained in chronically inflamed tissues remains unclear.

Objective: We sought to understand whether maintenance of lung T17 inflammation requires environmental agents in addition to antigen and to identify the lung antigen-presenting cell (APC) types that sustain the self-renewal of T17 cells.

Methods: Animals were exposed repeatedly to aspiration of ovalbumin alone or together with environmental adjuvants, including common house dust extract (HDE), to test their role in maintaining lung inflammation. Alternatively, antigen-specific effector/memory T17 cells, generated in culture with CD4 T cells from Il17a fate-mapping mice, were adoptively transferred to assess their persistence in genetically modified animals lacking distinct lung APC subsets or cell-specific Toll-like receptor (TLR) 4 signaling. T17 cells were also cocultured with lung APC subsets to determine which of these could revive their expansion and activation.

Results: T17 cells and the consequent neutrophilic inflammation were poorly sustained by inhaled antigen alone but were augmented by inhalation of antigen together with HDE. This was associated with weight loss and changes in lung physiology consistent with interstitial lung disease. The effect of HDE required TLR4 signaling predominantly in lung hematopoietic cells, including CD11c cells. CD103 and CD11b conventional dendritic cells interacted directly with T17 cells in situ and revived the clonal expansion of T17 cells both ex vivo and in vivo, whereas lung macrophages and B cells could not.

Conclusion: T17-dependent inflammation in the lungs can be sustained by persistent TLR4-mediated activation of lung conventional dendritic cells.
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http://dx.doi.org/10.1016/j.jaci.2017.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951733PMC
October 2018

Reversing SKI-SMAD4-mediated suppression is essential for T17 cell differentiation.

Nature 2017 11 25;551(7678):105-109. Epub 2017 Oct 25.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA.

T helper 17 (T17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in T17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables T17 cell differentiation remains elusive. Here we reveal that TGFβ enables T17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into T17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and T17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and T17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and T17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable T17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls T17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating T17-related diseases.
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http://dx.doi.org/10.1038/nature24283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743442PMC
November 2017

Early Endometriosis in Females Is Directed by Immune-Mediated Estrogen Receptor α and IL-6 Cross-Talk.

Endocrinology 2018 01;159(1):103-118

Receptor Biology Group, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina.

Endometriosis is a gynecological disease that negatively affects the health of 1 in 10 women. Although more information is known about late stage disease, the early initiation of endometriosis and lesion development is poorly understood. Herein, we use a uterine tissue transfer mouse model of endometriosis to examine early disease development and its dependence on estradiol (E2) and estrogen receptor (ER) α within 72 hours of disease initiation. Using wild-type and ERα knockout mice as hosts or donors, we find substantial infiltration of neutrophils and macrophages into the peritoneal cavity. Examining cell infiltration, lesion gene expression, and peritoneal fluid, we find that E2/ERα plays a minor role in early lesion development. Immune-mediated signaling predominates E2-mediated signaling, but 48 hours after the initiation of disease, a blunted interleukin (IL)-6-mediated response is found in developing lesions lacking ERα. Our data provide evidence that the early initiation of endometriosis is predominantly dependent on the immune system, whereas E2/ERα/IL-6-mediated cross-talk plays a partial role. These findings suggest there are two phases of endometriosis-an immune-dependent phase and a hormone-dependent phase, and that targeting the innate immune system could prevent lesion attachment in this susceptible population of women.
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http://dx.doi.org/10.1210/en.2017-00562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761597PMC
January 2018

TNF is required for TLR ligand-mediated but not protease-mediated allergic airway inflammation.

J Clin Invest 2017 Sep 31;127(9):3313-3326. Epub 2017 Jul 31.

Immunity, Inflammation and Disease Laboratory.

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.
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http://dx.doi.org/10.1172/JCI90890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669552PMC
September 2017

NIAID, NIEHS, NHLBI, and MCAN Workshop Report: The indoor environment and childhood asthma-implications for home environmental intervention in asthma prevention and management.

J Allergy Clin Immunol 2017 Oct 10;140(4):933-949. Epub 2017 May 10.

Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC.

Environmental exposures have been recognized as critical in the initiation and exacerbation of asthma, one of the most common chronic childhood diseases. The National Institute of Allergy and Infectious Diseases; National Institute of Environmental Health Sciences; National Heart, Lung, and Blood Institute; and Merck Childhood Asthma Network sponsored a joint workshop to discuss the current state of science with respect to the indoor environment and its effects on the development and morbidity of childhood asthma. The workshop included US and international experts with backgrounds in allergy/allergens, immunology, asthma, environmental health, environmental exposures and pollutants, epidemiology, public health, and bioinformatics. Workshop participants provided new insights into the biologic properties of indoor exposures, indoor exposure assessment, and exposure reduction techniques. This informed a primary focus of the workshop: to critically review trials and research relevant to the prevention or control of asthma through environmental intervention. The participants identified important limitations and gaps in scientific methodologies and knowledge and proposed and prioritized areas for future research. The group reviewed socioeconomic and structural challenges to changing environmental exposure and offered recommendations for creative study design to overcome these challenges in trials to improve asthma management. The recommendations of this workshop can serve as guidance for future research in the study of the indoor environment and on environmental interventions as they pertain to the prevention and management of asthma and airway allergies.
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http://dx.doi.org/10.1016/j.jaci.2017.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632590PMC
October 2017

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells.

J Vis Exp 2017 04 5(122). Epub 2017 Apr 5.

Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH;

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.
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http://dx.doi.org/10.3791/55465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512285PMC
April 2017

Distinct functions of CXCR4, CCR2, and CX3CR1 direct dendritic cell precursors from the bone marrow to the lung.

J Leukoc Biol 2017 05 1;101(5):1143-1153. Epub 2017 Feb 1.

Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.

Precursors of dendritic cells (pre-DCs) arise in the bone marrow (BM), egress to the blood, and finally migrate to peripheral tissue, where they differentiate to conventional dendritic cells (cDCs). Upon their activation, antigen-bearing cDCs migrate from peripheral tissue to regional lymph nodes (LNs) in a manner dependent on the chemokine receptor, CCR7. To maintain immune homeostasis, these departing cDCs must be replenished by new cDCs that develop from pre-DCs, but the molecular signals that direct pre-DC trafficking from the BM to the blood and peripheral tissues remain poorly understood. In the present study, we found that pre-DCs express the chemokine receptors CXCR4, CCR2, and CX3CR1, and that each of these receptors has a distinct role in pre-DC trafficking. Flow cytometric analysis of pre-DCs lacking CXCR4 revealed that this receptor is required for the retention of pre-DCs in the BM. Analyses of mice lacking CCR2 or CX3CR1, or both, revealed that they promote pre-DC migration to the lung at steady state. CCR2, but not CX3CR1, was required for pre-DC migration to the inflamed lung. Thus, these multiple chemokine receptors cooperate in a step-wise fashion to coordinate the trafficking of pre-DCs from the BM to the circulation and peripheral tissues.
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http://dx.doi.org/10.1189/jlb.1A0616-285RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380375PMC
May 2017

Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism.

Nucl Receptor Res 2015;2. Epub 2015 Dec 16.

Cell Biology Section, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs). We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated.
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http://dx.doi.org/10.11131/2015/101185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750502PMC
December 2015

Inhaled house dust programs pulmonary dendritic cells to promote type 2 T-cell responses by an indirect mechanism.

Am J Physiol Lung Cell Mol Physiol 2015 Nov 18;309(10):L1208-18. Epub 2015 Sep 18.

Immunity, Inflammation and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina;

The induction of allergen-specific T helper 2 (Th2) cells by lung dendritic cells (DCs) is a critical step in allergic asthma development. Airway delivery of purified allergens or microbial products can promote Th2 priming by lung DCs, but how environmentally relevant quantities and combinations of these factors affect lung DC function is unclear. Here, we investigated the ability of house dust extract (HDE), which contains a mixture of environmental adjuvants, to prime Th2 responses against an innocuous inhaled antigen. Inhalational exposure to HDE conditioned lung conventional DCs, but not monocyte-derived DCs, to induce antigen-specific Th2 differentiation. Conditioning of DCs by HDE was independent of Toll-like receptor 4 signaling, indicating that environmental endotoxin is dispensable for programming DCs to induce Th2 responses. DCs directly treated with HDE underwent maturation but were poor stimulators of Th2 differentiation. In contrast, DCs treated with bronchoalveolar lavage fluid (BALF) from HDE-exposed mice induced robust Th2 differentiation. DC conditioning by BALF was independent of the proallergic cytokines IL-25, IL-33, and thymic stromal lymphopoietin. BALF treatment of DCs resulted in upregulation of CD80 but low expression of CD40, CD86, and IL-12p40, which was associated with Th2 induction. These findings support a model whereby environmental adjuvants in house dust indirectly program DCs to prime Th2 responses by triggering the release of endogenous soluble factor(s) by airway cells. Identifying these factors could lead to novel therapeutic targets for allergic asthma.
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http://dx.doi.org/10.1152/ajplung.00256.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652150PMC
November 2015

Complement receptor C5aR1/CD88 and dipeptidyl peptidase-4/CD26 define distinct hematopoietic lineages of dendritic cells.

J Immunol 2015 Apr 13;194(8):3808-19. Epub 2015 Mar 13.

Immunity, Inflammation and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709;

Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103(+) DCs arise from FLT3-dependent DC precursors (preDCs), whereas CD11b(hi) DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11b(hi) DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of fractionated lung DCs from C57BL/6 mice and found that monocyte-derived DCs (moDCs), including CD11b(hi)Ly-6C(lo) tissue-resident and CD11b(hi)Ly-6C(hi) inflammatory moDCs, express the complement 5a receptor 1/CD88, whereas preDC-derived conventional DCs (cDCs), including CD103(+) and CD11b(hi) cDCs, express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine, and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that most blood-derived lymph node-resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, cDCs, but not moDCs, stimulated naive T cell proliferation. We anticipate that the use of Abs against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses.
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http://dx.doi.org/10.4049/jimmunol.1402195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390500PMC
April 2015

Epigenetic control of Ccr7 expression in distinct lineages of lung dendritic cells.

J Immunol 2014 Nov 8;193(10):4904-13. Epub 2014 Oct 8.

Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; and

Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.
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http://dx.doi.org/10.4049/jimmunol.1401104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225164PMC
November 2014

Modulation of distinct asthmatic phenotypes in mice by dose-dependent inhalation of microbial products.

Environ Health Perspect 2014 Jan 29;122(1):34-42. Epub 2013 Oct 29.

Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.

Background: Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2-associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood.

Objective: We studied the nature and longevity of asthma-like responses triggered by inhalation of allergen together with environmentally relevant doses of inhaled lipopolysaccharide (LPS).

Methods: Ovalbumin (OVA) was instilled into the airways of mice together with a wide range of LPS doses. Following a single OVA challenge, or multiple challenges, animals were assessed for pulmonary cytokine production, airway inflammation, and airway hyperresponsiveness (AHR).

Results: Mice instilled with OVA together with very low doses (≤10⁻³ μg) of LPS displayed modest amounts of Th2 cytokines, with associated airway eosinophilia and AHR after a single challenge, and these responses were sustained after multiple OVA challenges. When the higher but still environmentally relevant dose of 10⁻¹ μg LPS was used, mice initially displayed similar Th2 responses, as well as Th17-associated neutrophilia. After multiple OVA challenges, however, the 10⁻¹ μg LPS animals also accumulated large numbers of allergen-specific T regulatory (Treg) cells with high levels of inducible co-stimulatory molecule (ICOS). As a result, asthma-like features in these mice were shorter-lived than in mice sensitized using lower doses of LPS.

Conclusions: The nature and longevity of Th2, Th17, and Treg immune responses to inhaled allergen are dependent on the quantity of LPS inhaled at the time of allergic sensitization. These findings might account in part for the heterogeneity of inflammatory infiltrates seen in lungs of asthmatics.
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http://dx.doi.org/10.1289/ehp.1307280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3888577PMC
January 2014
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