A Novel Animal Model of Emphysema Induced by Anti-Elastin Autoimmunity.

Loss of immune tolerance to self-antigens can promote chronic inflammation and disrupt the normal function of multiple organs, including the lungs. Degradation of elastin, a highly insoluble protein and a significant component of the lung structural matrix, generates proinflammatory molecules. Elastin fragments (EFs) have been detected in the serum of smokers with emphysema, and elastin-specific T cells have also been detected in the peripheral blood of smokers with emphysema. However, an animal model that could recapitulate T cell-specific autoimmune responses by initiating and sustaining inflammation in the lungs is lacking. In this study, we report an animal model of autoimmune emphysema mediated by the loss of tolerance to elastin. Mice immunized with a combination of human EFs plus rat EFs but not mouse EFs showed increased infiltration of innate and adaptive immune cells to the lungs and developed emphysema. We cloned and expanded mouse elastin-specific CD4+ T cells from the lung and spleen of immunized mice. Finally, we identified TCR sequences from the autoreactive T cell clones, suggesting possible pathogenic TCRs that can cause loss of immune tolerance against elastin. This new autoimmune model of emphysema provides a useful tool to examine the immunological factors that promote loss of immune tolerance to self.

Selective cleavage of fibrinogen by diverse proteinases initiates innate allergic and antifungal immunity through CD11b.

Proteinases are essential drivers of allergic airway disease and innate antifungal immunity in part through their ability cleave the clotting factor fibrinogen (FBG) into fibrinogen cleavage products (FCPs) that signal through Toll-like receptor 4 (TLR4). However, the mechanism by which FCPs engage TLR4 remains unknown. Here, we show that the proteinases from Aspergillus melleus (PAM) and other allergenic organisms rapidly hydrolyze FBG to yield relatively few FCPs that drive distinct antifungal mechanisms through TLR4. Functional FCPs, termed cryptokines, were characterized by rapid loss of the FBG α chain with substantial preservation of the ß and γ chains, including a γ chain sequence (Fibγ390-396) that binds the integrin Mac-1 (CD11b/CD18). PAM-derived cryptokines could be generated from multiple FBG domains, and the ability of cryptokines to induce fungistasis in vitro and innate allergic airway disease in vivo strongly depended on both Mac-1 and the Mac-1-binding domain of FBG (Fibγ390-396). Our findings illustrate the essential concept of proteinase-activated immune responses and for the first time link Mac-1, cryptokines, and TLR4 to innate antifungal immunity and allergic airway disease.

Small molecule targeting of the STAT5/6 Src homology 2 (SH2) domains to inhibit allergic airway disease.

Asthma is a chronic inflammatory disease of the lungs and airways and one of the most burdensome of all chronic maladies. Previous studies have established that expression of experimental and human asthma requires the IL-4/IL-13/IL-4 receptor α (IL-4Rα) signaling pathway, which activates the transcription factor STAT6. However, no small molecules targeting this important pathway are currently in clinical development. To this end, using a preclinical asthma model, we sought to develop and test a small-molecule inhibitor of the Src homology 2 domains in mouse and human STAT6. We previously developed multiple peptidomimetic compounds on the basis of blocking the docking site of STAT6 to IL-4Rα and phosphorylation of Tyr641 in STAT6. Here, we expanded the scope of our initial in vitro structure-activity relationship studies to include central and C-terminal analogs of these peptides to develop a lead compound, PM-43I. Conducting initial dose range, toxicity, and pharmacokinetic experiments with PM-43I, we found that it potently inhibits both STAT5- and STAT6-dependent allergic airway disease in mice. Moreover, PM-43I reversed preexisting allergic airway disease in mice with a minimum ED50 of 0.25 µg/kg. Of note, PM-43I was efficiently cleared through the kidneys with no long-term toxicity. We conclude that PM-43I represents the first of a class of small molecules that may be suitable for further clinical development against asthma.

Fibrinogen cleavage products and Toll-like receptor 4 promote the generation of programmed cell death 1 ligand 2-positive dendritic cells in allergic asthma.

BACKGROUND: Inhaled protease allergens preferentially trigger TH2-mediated inflammation in allergic asthma. The role of dendritic cells (DCs) on induction of TH2 cell responses in allergic asthma has been well documented; however, the mechanism by which protease allergens induce TH2-favorable DCs in the airway remains unclear. OBJECTIVE: We sought to determine a subset of DCs responsible for TH2 cell responses in allergic asthma and the mechanism by which protease allergens induce the DC subset in the airway. METHODS: Mice were challenged intranasally with protease allergens or fibrinogen cleavage products (FCPs) to induce allergic airway inflammation. DCs isolated from mediastinal lymph nodes were analyzed for surface phenotype and T-cell stimulatory function. Anti-Thy1.2 and Mas-TRECK mice were used to deplete innate lymphoid cells and mast cells, respectively. Adoptive cell transfer, bone marrow DC culture, anti-IL-13, and Toll-like receptor (TLR) 4-deficient mice were used for further mechanistic studies. RESULTS: Protease allergens induced a remarkable accumulation of TH2-favorable programmed cell death 1 ligand 2 (PD-L2)+ DCs in mediastinal lymph nodes, which was significantly abolished in mice depleted of mast cells and, to a lesser extent, innate lymphoid cells. Mechanistically, FCPs generated by protease allergens triggered IL-13 production from wild-type mast cells but not from TLR4-deficient mast cells, which resulted in an increase in the number of PD-L2+ DCs. Intranasal administration of FCPs induced an increase in numbers of PD-L2+ DCs in the airway, which was significantly abolished in TLR4- and mast cell-deficient mice. Injection of IL-13 restored the PD-L2+ DC population in mice lacking mast cells. CONCLUSION: Our findings unveil the "protease-FCP-TLR4-mast cell-IL-13" axis as a molecular mechanism for generation of TH2-favorable PD-L2+ DCs in allergic asthma and suggest that targeting the PD-L2+ DC pathway might be effective in suppressing allergic T-cell responses in the airway.

Advances and Evolving Concepts in Allergic Asthma.

Allergic asthma is a heterogeneous disorder that defies a unanimously acceptable definition, but is generally recognized through its highly characteristic clinical expression of dyspnea and cough accompanied by clinical data that document reversible or exaggerated airway constriction and obstruction. The generally rising prevalence of asthma in highly industrialized societies despite significant therapeutic advances suggests that the fundamental cause(s) of asthma remain poorly understood. Detailed analyses of both the indoor (built) and outdoor environments continue to support the concept that not only inhaled particulates, especially carbon-based particulate pollution, pollens, and fungal elements, but also many noxious gases and chemicals, especially biologically derived byproducts such as proteinases, are essential to asthma pathogenesis. Phthalates, another common class of chemical pollutant found in the built environment, are emerging as potentially important mediators or attenuators of asthma. Other biological products such as endotoxin have also been confirmed to be protective in both the indoor and outdoor contexts. Proasthmatic factors are believed to activate, and in some instances initiate, pathologic inflammatory cascades through complex interactions with pattern recognition receptors (PRRs) expressed on many cell types, but especially airway epithelial cells. PRRs initiate the release of proallergic cytokines such as interleukin (IL)-33, IL-25, and others that coordinate activation of innate lymphoid cells type 2 (ILC2), T helper type 2 cells, and immunoglobulin E-secreting B cells that together promote additional inflammation and the major airway remodeling events (airway hyperresponsiveness, mucus hypersecretion) that promote airway obstruction. Proteinases, with airway fungi and viruses being potentially important sources, are emerging as critically important initiators of these inflammatory cascades in part through their effects on clotting factors such as fibrinogen. Recent clinical trials have demonstrated that targeting inflammatory pathways orchestrated through IL-4, IL-5, IL-13, and the prostaglandin receptor CRTH2 is potentially highly effective in adult asthma.

Microglia and amyloid precursor protein coordinate control of transient Candida cerebritis with memory deficits.

Bloodborne infections with Candida albicans are an increasingly recognized complication of modern medicine. Here, we present a mouse model of low-grade candidemia to determine the effect of disseminated infection on cerebral function and relevant immune determinants. We show that intravenous injection of 25,000 C. albicans cells causes a highly localized cerebritis marked by the accumulation of activated microglial and astroglial cells around yeast aggregates, forming fungal-induced glial granulomas. Amyloid precursor protein accumulates within the periphery of these granulomas, while cleaved amyloid beta (Aß) peptides accumulate around the yeast cells. CNS-localized C. albicans further activate the transcription factor NF-κB and induce production of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF), and Aß peptides enhance both phagocytic and antifungal activity from BV-2 cells. Mice infected with C. albicans display mild memory impairment that resolves with fungal clearance. Our results warrant additional studies to understand the effect of chronic cerebritis on cognitive and immune function.

Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine.

Electronic nicotine delivery systems (ENDS) or e-cigarettes have emerged as a popular recreational tool among adolescents and adults. Although the use of ENDS is often promoted as a safer alternative to conventional cigarettes, few comprehensive studies have assessed the long-term effects of vaporized nicotine and its associated solvents, propylene glycol (PG) and vegetable glycerin (VG). Here, we show that compared with smoke exposure, mice receiving ENDS vapor for 4 months failed to develop pulmonary inflammation or emphysema. However, ENDS exposure, independent of nicotine, altered lung lipid homeostasis in alveolar macrophages and epithelial cells. Comprehensive lipidomic and structural analyses of the lungs revealed aberrant phospholipids in alveolar macrophages and increased surfactant-associated phospholipids in the airway. In addition to ENDS-induced lipid deposition, chronic ENDS vapor exposure downregulated innate immunity against viral pathogens in resident macrophages. Moreover, independent of nicotine, ENDS-exposed mice infected with influenza demonstrated enhanced lung inflammation and tissue damage. Together, our findings reveal that chronic e-cigarette vapor aberrantly alters the physiology of lung epithelial cells and resident immune cells and promotes poor response to infectious challenge. Notably, alterations in lipid homeostasis and immune impairment are independent of nicotine, thereby warranting more extensive investigations of the vehicle solvents used in e-cigarettes.

A Fungal Protease Model to Interrogate Allergic Lung Immunity.

Allergic airway diseases (asthma and chronic rhinosinusitis) are among the most common of all human diseases in heavily industrialized societies. Animal models of asthma have provided remarkable insight into allergic disease pathogenesis and will continue to drive the discovery of new therapeutic insights. We provide in this chapter a detailed protocol for inducing allergic immunity in the lungs of mice using a purified fungal protease and include related protocols for assessing immune endpoints.

Fungi in Mucoobstructive Airway Diseases.

Asthma, chronic rhinosinusitis, and related incurable allergic afflictions of the upper and lower airways are medically important because of their association with the disabling symptom of dyspnea and, at least for asthma, the potential to cause fatal asphyxiation. Extensive research over the past two decades has uncovered both the physiological basis of airway obstruction in asthma and key governing molecular pathways. Exaggerated airway constriction in response to diverse provocative stimuli, termed airway hyperresponsiveness, is mediated through the cytokines interleukin 4 (IL-4) and IL-13 and the transcription factor signal transducer and activator of transcription 6 (STAT6). Overproduction of mucus has long been known to be an essential second component of airway obstruction and is also mediated in part through the IL-4/IL-13/STAT6 pathway. In this review, we discuss a second major signaling pathway which underlies mucus production that is mediated through proteinase-cleaved fibrinogen signaling through Toll-like receptor 4. Unexpectedly, our analysis of human sputum and paranasal sinus fluid indicates that in most cases of severe allergic airway disease, a unique type of airway fungal infection, termed airway mycosis, is pathogenically linked to these conditions. We further discuss how fungal and endogenous proteinases mediate the fibrinogenolysis that is essential to both Toll-like receptor 4 signaling and fibrin deposition that, together with mucus, contribute to airway obstruction.

MicroRNA-22 controls interferon alpha production and erythroid maturation in response to infectious stress in mice.

MicroRNA-22 (miR-22) is a highly conserved microRNA that can regulate cell proliferation, oncogenesis, and cell maturation, especially during stress. In hematopoietic stem cells (HSCs), miR-22 has been reported to be involved in the regulation of key self-renewal factors, including Tet2. Recent work demonstrates that miR-22 also participates in regulation of the interferon (IFN) response, and expression profiling studies suggest that it is variably expressed at different stages in erythroid differentiation. We thus hypothesized that miR-22 regulates maturation of erythroid progenitors during stress hematopoiesis through its interaction with IFN. We compared the blood and bone marrow of wild-type (WT) and miR-22-deficient mice at baseline and upon infectious challenge with systemic lymphochoriomeningitis (LCMV) virus. miR-22-deficient mice maintained platelet counts better than WT mice during infection, but they showed significantly reduced red blood cells and hemoglobin. Analysis of bone marrow progenitors demonstrated better overall survival and improved HSC homeostasis in infected miR-22-null mice compared with WT, which was attributable to a blunted IFN response to LCMV challenge in the miR-22-null mice. We found that miR-22 was expressed exclusively in stage II erythroid precursors and downregulated upon infection in WT mice. Our results indicate that miR-22 promotes the IFN response to viral infection and that it functions at baseline as a brake to slow erythroid differentiation and maintain adequate erythroid potential. Impaired regulation of erythrogenesis in the absence of miR-22 can lead to anemia during infection.

Allergen-encoded signals that control allergic responses.

PURPOSE OF REVIEW: The purpose is to review the important recent advances made in how innate immune cells, microbes, and the environment contribute to the expression of allergic disease, emphasizing the allergen-related signals that drive allergic responses. RECENT FINDINGS: The last few years have seen crucial advances in how innate immune cells such as innate lymphoid cells group 2 and airway epithelial cells and related molecular pathways through organismal proteinases and innate immune cytokines, such as thymic stromal lymphopoietin, IL-25, and IL-33 contribute to allergy and asthma. Simultaneously with these advances, important progress has been made in our understanding of how the environment, and especially pathogenic organisms, such as bacteria, viruses, helminths, and especially fungi derived from the natural and built environments, either promote or inhibit allergic inflammation and disease. Of specific interest are how lipopolysaccharide mediates its antiallergic effect through the ubiquitin modifying factor A20 and the antiallergic activity of both helminths and protozoa. SUMMARY: Innate immune cells and molecular pathways, often activated by allergen-derived proteinases acting on airway epithelium and macrophages as well as additional unknown factors, are essential to the expression of allergic inflammation and disease. These findings suggest numerous future research opportunities and new opportunities for therapeutic intervention in allergic disease.