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. 2021 Aug 27;7(9):698.
doi: 10.3390/jof7090698.

Chronic Occupational Mold Exposure Drives Expansion of Aspergillus-Reactive Type 1 and Type 2 T-Helper Cell Responses

Chris D Lauruschkat  1 , Sonja Etter  1 , Elisabeth Schnack  2 , Frank Ebel  2 , Sascha Schäuble  3 , Lukas Page  1 , Dana Rümens  1 , Mariola Dragan  4 , Nicolas Schlegel  4 , Gianni Panagiotou  3   5 , Olaf Kniemeyer  6 , Axel A Brakhage  6 , Hermann Einsele  1 , Sebastian Wurster  7 , Juergen Loeffler  1
Affiliations

Chronic Occupational Mold Exposure Drives Expansion of Aspergillus-Reactive Type 1 and Type 2 T-Helper Cell Responses

Chris D Lauruschkat et al. J Fungi (Basel). .

Abstract

Occupational mold exposure can lead to Aspergillus-associated allergic diseases including asthma and hypersensitivity pneumonitis. Elevated IL-17 levels or disbalanced T-helper (Th) cell expansion were previously linked to Aspergillus-associated allergic diseases, whereas alterations to the Th cell repertoire in healthy occupationally exposed subjects are scarcely studied. Therefore, we employed functional immunoassays to compare Th cell responses to A. fumigatus antigens in organic farmers, a cohort frequently exposed to environmental molds, and non-occupationally exposed controls. Organic farmers harbored significantly higher A. fumigatus-specific Th-cell frequencies than controls, with comparable expansion of Th1- and Th2-cell frequencies but only slightly elevated Th17-cell frequencies. Accordingly, Aspergillus antigen-induced Th1 and Th2 cytokine levels were strongly elevated, whereas induction of IL-17A was minimal. Additionally, increased levels of some innate immune cell-derived cytokines were found in samples from organic farmers. Antigen-induced cytokine release combined with Aspergillus-specific Th-cell frequencies resulted in high classification accuracy between organic farmers and controls. Aspf22, CatB, and CipC elicited the strongest differences in Th1 and Th2 responses between the two cohorts, suggesting these antigens as potential candidates for future bio-effect monitoring approaches. Overall, we found that occupationally exposed agricultural workers display a largely balanced co-expansion of Th1 and Th2 immunity with only minor changes in Th17 responses.

Keywords: Aspergillus; adaptive immunity; biomarker; cytokines; hypersensitivity; immunoassay; inflammation; mold exposure.

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Conflict of interest statement

The authors have no conflict of interest related to this study.

Figures

Figure 1
Figure 1
Subjects with occupational mold exposure harbor increased frequencies of A. fumigatus-specific T-helper (Th) cells. PBMCs from 10 organic farmers (red) and 10 non-occupationally exposed control subjects (green) were stimulated with A. fumigatus mycelial lysate (AfuLy). Frequencies and phenotypes of CD154+ Th cells (CD4+ cells) were determined by flow cytometry. (a) Total background-adjusted AfuLy-specific Th cell frequencies. (b) Mean percentages of effector/memory phenotypes (inner pie) and Th cell lineages (outer ring) among CD4+CD154+ AfuLy-specific Th cells in organic farmers and controls. Tcm = central memory Th cells, Tem = effector memory Th cells, Teff = effector Th cells (§ 1% in organic farmers, 2% in controls). (c,d) Frequencies of AfuLy-reactive CD154+ effector/memory Th cell populations (c) and Th1/Th2/Th17 cells (d) among CD4+ Th cells in organic farmers and controls. (a,c,d) Background-adjusted individual values, medians (black bars), and inter-quartile ranges (colored boxes) are shown. Two-sided Mann–Whitney-U-test. Asterisks indicate significant differences (p < 0.05).
Figure 2
Figure 2
Organic farmers show enhanced Th1, Th2, and innate immune cell-derived cytokine responses to A. fumigatus antigens. Whole-blood samples from 10 organic farmers (red) and 10 non-occupationally exposed controls (green) were stimulated with A. fumigatus mycelial lysate (AfuLy), Aspf4, or Aspf9/Crf1. Cytokine concentrations in plasma supernatants were quantified using a Luminex assay. Individual and median (black bars) background-adjusted cytokine concentrations of INF-γ (a), IL-4 (b), IL-5 (c), IL-13 (d), IL-17 (e), IL-10 (f), IL-6 (g), IL-21 (h), MIP-1α (i), and GM-CSF (j) are shown. Colored boxes represent inter-quartile ranges. Two-sided Mann–Whitney-U-test with Benjamini–Hochberg correction for an FDR of 0.2. No comparison reached FDR-corrected significance between organic farmers and controls (p < 0.05, FDR < 0.2). Black circles indicate a trend toward significance (p < 0.05, FDR > 0.2).
Figure 3
Figure 3
Machine learning corroborates a selection of highly correlated T-cellular and APC-derived cytokine responses with robust discriminatory power between occupationally exposed subjects and controls. (a) Heatmap of Spearman’s rank correlation coefficients of individual cytokine concentrations elicited by A. fumigatus mycelial lysate (AfuLy, Luminex assay results). FDR-corrected significance of Spearman coefficients is indicated by asterisks. ** p < 0.01, *** p < 0.001. (b) Random forest analysis to determine the classification accuracy of combinations of up to three cytokine responses to AfuLy along with flow cytometrically determined CD154+ AfuLy-reactive T-helper cell frequencies. The top 20 combinations with the highest classification accuracy between organic farmers and controls are shown. Only cytokine markers passing the pre-filtering step (Figure S4) based on their relative induction in the two cohorts (median-to-median ratio > 2.0) and p-value (p < 0.2) were considered for both panels.
Figure 4
Figure 4
PBMCs from organic farmers display enhanced Th1 and Th2 responses to selected A. fumigatus antigens compared with non-occupationally exposed controls. PBMCs from 10 organic farmers (red) and 10 non-occupationally exposed control subjects (green) were stimulated with A. fumigatus mycelial lysate (AfuLy) or proteins. Numbers of IFN-γ (a), IL-5 (b), and IL-17 (c) producing cells (spot forming cells, SFCs) per million PBMCs were determined by ELISPOT. Individual background-adjusted SFC counts, medians (black bars), and inter-quartile ranges (colored boxes) are shown. Two-sided Mann–Whitney-U-test with Benjamini–Hochberg correction for an FDR of 0.2. Asterisks indicate significant differences between organic farmers and controls (p < 0.05, FDR < 0.2). Black circles indicate a trend toward significance (p < 0.05, FDR > 0.2).
Figure 5
Figure 5
ELISPOT reveals a cluster of proteinaceous A. fumigatus antigens that elicit enhanced Th1 and Th2 signature cytokine response in subjects with occupational mold exposure. Volcano plot combining log2-transformed ratios of median SFCs counts for each antigen/cytokine pair in organic farmers and controls with the corresponding p-values (Mann–Whitney-U-test). Raw data are derived from Figure 4. Antigen/cytokine pairs are classified as insignificant (“+” symbols, median-to-median ratio <2 and/or p > 0.2), potentially significant (circles, median-to-median ratio > 2 and 0.05 < p < 0.2), or significant (diamonds, median-to-median ratio >2 and p < 0.05). Antigen/cytokine pairs with medians of 0 SFCs in both cohorts are not displayed.
Figure 6
Figure 6
Chronic occupational mold exposure moderately correlates with a Th2-polarized immune phenotype. (a) Heatmap of Spearman’s rank correlation coefficients between the duration of service in organic farming and individual Th1/Th2 ratios (flow cytometry, FC) or ratios of Th1 (IFN-γ) and Th2 (IL-4, IL5, and IL-13) signature cytokine release detectable by WB-based Luminex analysis and ELISPOT after stimulation with A. fumigatus antigens. * p < 0.05. (b) Individual log2-transformed ratios of IFN-γ and IL-5 spot forming cell counts (SFC) upon stimulation with Aspf8 and CatB (x-value) compared with the subjects’ duration of service in organic farming (y-value). Spearman’s rank correlation coefficients (ρ) and their two-sided p-values are given.
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