Michelle M. Epstein

Do Mouse Models of Allergic Asthma Mimic Clinical Disease

Experimental mouse models of allergic asthma established almost 10 years ago offered new opportunities to study disease pathogenesis and to develop new therapeutics. These models focused on the factors governing the allergic immune response, on modeling clinical behavior of allergic asthma, and led to insights into pulmonary pathophysiology. Although mouse models rarely completely reproduce all the features of human disease, after sensitization and respiratory tract challenges with antigen, wild-type mice develop a clinical syndrome that closely resembles allergic asthma, characterized by eosinophilic lung inflammation, airway hyperresponsiveness (AHR), increased IgE, mucus hypersecretion, and eventually, airway remodeling. There are, however, differences between mouse and human physiology that threaten to limit the value of mouse models. Three examples of such differences relate to both clinical manifestations of disease and underlying pathogenesis. First, in contrast to patients who have increased methacholine-induced AHR even when they are symptom-free, mice exhibit only transient methacholine-induced AHR following allergen exposure. Second, chronic allergen exposure in patients leads to chronic allergic asthma, whereas repeated exposures in sensitized mice causes suppression of disease. Third, IgE and mast cells, in humans, mediate early- and late-phase allergic responses, though both are unnecessary for the generation of allergic asthma in mice. Taken together, these observations suggest that mouse models of allergic asthma are not exact replicas of human disease and thus, question the validity of these models. However, observations from mouse models of allergic asthma support many existing paradigms, although some novel discoveries in mice have yet to be verified in patients. This review presents an overview of the clinical aspects of disease in mouse models of allergic asthma emphasizing (1) the factors influencing the pathophysiological responses during the initiation and perpetuation of disease, (2) the utility of mouse models for studying clinical manifestations of disease, and (3) the applicability of mouse models for testing new treatments for allergic asthma.

Treatment of allergic asthma: Modulation of Th2 cells and their responses

Atopic asthma is a chronic inflammatory pulmonary disease characterised by recurrent episodes of wheezy, laboured breathing with an underlying Th2 cell-mediated inflammatory response in the airways. It is currently treated and, more or less, controlled depending on severity, with bronchodilators e.g. long-acting beta agonists and long-acting muscarinic antagonists or anti-inflammatory drugs such as corticosteroids (inhaled or oral), leukotriene modifiers, theophyline and anti-IgE therapy. Unfortunately, none of these treatments are curative and some asthmatic patients do not respond to intense anti-inflammatory therapies. Additionally, the use of long-term oral steroids has many undesired side effects. For this reason, novel and more effective drugs are needed. In this review, we focus on the CD4+ Th2 cells and their products as targets for the development of new drugs to add to the current armamentarium as adjuncts or as potential stand-alone treatments for allergic asthma. We argue that in early disease, the reduction or elimination of allergen-specific Th2 cells will reduce the consequences of repeated allergic inflammatory responses such as lung remodelling without causing generalised immunosuppression.

Long-Lived Th2 Memory in Experimental Allergic Asthma

Although life-long immunity against pathogens is beneficial, immunological memory responses directed against allergens are potentially harmful. Because there is a paucity of information about Th2 memory cells in allergic disease, we established a model of allergic asthma in BALB/c mice to explore the generation and maintenance of Th2 memory. We induced disease without the use of adjuvants, thus avoiding Ag depots, and found that unlike allergic asthma in mice immunized with adjuvant, immunizing with soluble and aerosol OVA resulted in pathological lung lesions resembling human disease. To test memory responses we allowed mice with acute disease to recover and then re-exposed them to aerosol OVA a second time. Over 400 days later these mice developed OVA-dependent eosinophilic lung inflammation, airway hyperresponsiveness, mucus hypersecretion, and IgE. Over 1 year after recuperating from acute disease, mice had persistent lymphocytic lung infiltrates, Ag-specific production of IL-4 and IL-5 from spleen and lung cells in vitro, and elevated IgG1. Moreover, when recuperated mice were briefly aerosol challenged, we detected early expression of Th2 cytokine RNA in lungs. Taken together, these data demonstrate the presence of long-lived Th2 memory cells in spleen and lungs involved in the generation of allergic asthma upon Ag re-exposure.

Conditional Deletion of Histone Deacetylase 1 in T Cells Leads to Enhanced Airway Inflammation and Increased Th2 Cytokine Production

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4+ T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection

Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

Fate of Transgenic DNA from Orally Administered Bt MON810 Maize and Effects on Immune Response and Growth in Pigs

We assessed the effect of short-term feeding of genetically modified (GM: Bt MON810) maize on immune responses and growth in weanling pigs and determined the fate of the transgenic DNA and protein in-vivo. Pigs were fed a diet containing 38.9% GM or non-GM isogenic parent line maize for 31 days. We observed that IL-12 and IFNγ production from mitogenic stimulated peripheral blood mononuclear cells decreased (P<0.10) following 31 days of GM maize exposure. While Cry1Ab-specific IgG and IgA were not detected in the plasma of GM maize-fed pigs, the detection of the cry1Ab gene and protein was limited to the gastrointestinal digesta and was not found in the kidneys, liver, spleen, muscle, heart or blood. Feeding GM maize to weanling pigs had no effect on growth performance or body weight. IL-6 and IL-4 production from isolated splenocytes were increased (P<0.05) in response to feeding GM maize while the proportion of CD4+ T cells in the spleen decreased. In the ileum, the proportion of B cells and macrophages decreased while the proportion of CD4+ T cells increased in GM maize-fed pigs. IL-8 and IL-4 production from isolated intraepithelial and lamina propria lymphocytes were also increased (P<0.05) in response to feeding GM maize. In conclusion, there was no evidence of cry1Ab gene or protein translocation to the organs and blood of weaning pigs. The growth of pigs was not affected by feeding GM maize. Alterations in immune responses were detected; however, their biologic relevance is questionable.

Dendritic polyglycerolsulfate near infrared fluorescent (NIRF) dye conjugate for non-invasively monitoring of inflammation in an allergic asthma mouse model.

Background Non-invasive in vivo imaging strategies are of high demand for longitudinal monitoring of inflammation during disease progression. In this study we present an imaging approach using near infrared fluorescence (NIRF) imaging in combination with a polyanionic macromolecular conjugate as a dedicated probe, known to target L- and P-selectin and C3/C5 complement factors. Methodology/Principal Findings We investigated the suitability of dendritic polyglycerol sulfates (dPGS), conjugated with a hydrophilic version of the indocyanine green label with 6 sulfonate groups (6S-ICG) to monitor sites of inflammation using an experimental mouse model of allergic asthma. Accumulation of the NIRF-conjugated dPGS (dPGS-NIRF) in the inflamed lungs was analyzed in and ex vivo in comparison with the free NIRF dye using optical imaging. Commercially available smart probes activated by matrix metalloproteinases (MMP) and cathepsins were used as a comparative control. The fluorescence intensity ratio between lung areas of asthmatic and healthy mice was four times higher for the dPGS in comparison to the free dye in vivo at four hrs post intravenous administration. No significant difference in fluorescence intensity between healthy and asthmatic mice was observed 24 hrs post injection for dPGS-NIRF. At this time point ex-vivo scans of asthmatic mice confirmed that the fluorescence within the lungs was reduced to approximately 30% of the intensity observed at 4 hrs post injection. Conclusions/Significance Compared with smart-probes resulting in a high fluorescence level at 24 hrs post injection optical imaging with dPGS-NIRF conjugates is characterized by fast uptake of the probe at inflammatory sites and represents a novel approach to monitor lung inflammation as demonstrated in mice with allergic asthma.

Ephrin-A1 Suppresses Th2 Cell Activation and Provides a Regulatory Link to Lung Epithelial Cells

Gene expression screening showed decreased ephrin-A1 expression in CD4+ T cells of asthma patients. Ephrin-A1 is the ligand of the Eph receptor family of tyrosine kinases, forming the largest family of receptor tyrosine kinases. Their immune regulatory properties are largely unknown. This study demonstrates significantly reduced ephrin-A1 expression in T cells of asthma patients using real time-PCR. Immunohistological analyses revealed strong ephrin-A1 expression in lung tissue and low expression in cortical areas of lymph nodes. It is absent in T cell/B cell areas of the spleen. Colocalization of ephrin-A1 and its receptors was found only in the lung, but not in lymphoid tissues. In vitro activation of T cells reduced ephrin-A1 at mRNA and protein levels. T cell proliferation, activation-induced, and IL-2-dependent cell death were inhibited by cross-linking ephrin-A1, and not by engagement of Eph receptors. However, anti-EphA1 receptor slightly enhances Ag-specific and polyclonal proliferation of PBMC cultures. Furthermore, activation-induced CD25 up-regulation was diminished by ephrin-A1 engagement. Ephrin-A1 engagement reduced IL-2 expression by 82% and IL-4 reduced it by 69%; the IFN-γ expression remained unaffected. These results demonstrate that ephrin-A1 suppresses T cell activation and Th2 cytokine expression, while preventing activation-induced cell death. The reduced ephrin-A1 expression in asthma patients may reflect the increased frequency of activated T cells in peripheral blood. That the natural ligands of ephrin-A1 are most abundantly expressed in the lung may be relevant for Th2 cell regulation in asthma and Th2 cell generation by mucosal allergens.

A comparative approach linking molecular dynamics of altered peptide ligands and MHC with in vivo immune responses.

Background The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood. Results We analyzed the immunological consequences of the interaction of MHC class II (I-Au) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4+ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation.

Repeated Aerosol Allergen Exposure Suppresses Inflammation in B-Cell-Deficient Mice with Established Allergic Asthma

Background: Repeated allergen administration is a well-established therapeutic strategy for desensitizing patients with allergic disease. Similarly, repeated inhalation of antigen by mice with established allergen-induced asthma suppresses allergic inflammation. The mechanisms underlying antigen-dependent suppression of allergic immune responses remain unknown. In previous studies, we found that repeated aerosol antigen challenges in sensitized mice reduced eosinophils while increasing plasma cells and antibody in the lungs. We sought to test whether plasma cells and antibody played a role in suppression of allergic disease. Methods: We primed wild-type and B-cell-deficient (µMT) mice with 25 µg ovalbumin (OVA) precipitated in alum on days 0 and 5, nebulized weekly with 1% OVA, 1 h, twice daily, for up to 6 weeks, and assessed lung inflammation, mucus hypersecretion, and IgE/IgG1. Results: Kinetic studies revealed that initial aerosol exposure induced high numbers of eosinophils, lymphocytes, and macrophages within lung infiltrates and increased mucus production in wild-type mice. After 3–4 weeks of antigen exposure, eosinophils diminished while lymphocytes, plasma cells, and macrophages and mucus hypersecretion increased. However, by 6 weeks, lung inflammation and mucus hypersecretion were dramatically reduced. In contrast, repeated aerosol challenges maintained OVA-specific IgG1 and IgE production. Repeated aerosol antigen challenges in µMT mice resulted in reduced lung inflammation and mucus hypersecretion and the development of smooth muscle hypertrophy of the pulmonary microvasculature. Conclusions: B cells and antibody do not appear to play a role in antigen-dependent suppression of allergic responses in mice.