In vivo Hypoxia and a Fungal Alcohol Dehydrogenase Influence the Pathogenesis of Invasive Pulmonary Aspergillosis

Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and 1H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses

Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils

Surface expression of FcεRI on BP eosinophils.

<p>Interaction of FcεRIα with IgE or FcεRIβ was evaluated using the proximity ligation assay on non-permeabilized preparations of circulating granulocytes or skin cryosections from BP patients or controls. Eosinophils were identified by their unique nuclear morphology (bi-lobed nucleus stained with DAPI) using high resolution confocal microscopy. Interaction of FcεRIα/IgE on peripheral blood and tissue eosinophils from BP patient (A, B) or controls (C, D). Insets are enlarged to show nuclear morphology. Scale bar = 25 uM. Interaction of FcεRIα/FcεRIβ on eosinophils in lesional biopsies (E–H). Panel H is an image of the same BP sample depicted in panel G, captured at higher magnification for resolution of nuclear morphology. Scale bar = 50 uM.</p

Disease activity and autoantibody profiles in patients with BP.

<p>Subjects (n = 48) were enrolled before receiving any immunosuppressive treatment for BP. Since patients were enrolled over several years, disease activity was scored with a BP Index (A) or the more comprehensive Bullous Disease Area Index (BPDAI) criteria (B). A strong correlation (Spearman’s r = 0.7193, p<0.0001) was observed between these two scoring systems (C). Sera were collected from untreated BP patients (BP sera; BPS), patients evaluated for other autoimmune skin diseases (other autoimmune sera; OAS), or age- and gender-matched controls (normal human sera; NHS) and evaluated for BP180 IgG, BP230 IgG, total IgE and BP180 IgE by ELISA. Each point represents the average of duplicate samples from an individual patient with the N per group indicated. Mann-Whitney U-test, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Evaluation of IgE receptor expression on circulating eosinophils from BP patients.

<p>Peripheral granulocytes were stained with fluorescently tagged antibodies specific for human CD203c, CD49d, CD16, and FcεRI-α, FcεRIβ, CD23 or IgE for flow cytometric analysis. Eosinophils were identified by gating on the CD16⁻/CD49d⁺/CD203c⁻ population. Eosinophils from healthy controls (A), active BP patients (B), or basophils (CD16⁻/CD49d⁺/CD203c⁺) from active BP patients (C) were evaluated. The degree of specific staining is indicated by the open histogram compared to appropriate isotype control shown by the shaded histogram. Staining is representative of 10 BP patients and 11 age- and gender-matched controls.</p

Patient characteristics and mRNA expression of FcεRI α, β, and γ receptor chains^a.

a<p>mRNA expression was evaluated by RT-PCR after immunomagnetic purification of eosinophils.</p>b<p>reported as IU/ml, normal range ≤100.</p>c<p>expressed as Index Units, positive test ≥19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p>e<p>Not done.</p><p>Patient characteristics and mRNA expression of FcεRI α, β, and γ receptor chains^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107725#nt110" target="_blank">a</a>}.</p

Correlation^a between antibody levels, eosinophil counts, and disease severity in untreated BP patients.

a<p>determined using Spearman’s rank correlation coefficient (r) and p = *<0.05, **<0.01, ***0.001.</p>b<p>reported as IU/ml, normal range ≤100.</p>c<p>expressed as Index Units, positive test ≥19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p><p>Correlation^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107725#nt101" target="_blank">a</a>} between antibody levels, eosinophil counts, and disease severity in untreated BP patients.</p

Samples evaluated for FcεRI expression using the proximity ligation assay.

a<p>eosinophils were enriched from peripheral blood using density centrifugation and adhered to glass slides.</p>b<p>expressed as IU of IgE, normal range ≤100 IU.</p>c<p>ELISA Index Value, normal range ≤19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p>e<p>not done.</p><p>Samples evaluated for FcεRI expression using the proximity ligation assay.</p