Asthma-related environmental fungus, Alternaria, activates dendritic cells and produces potent Th2 adjuvant activity

Abstract

Asthma is thought to result from dysregulated Th2-like airway inflammatory responses to the environment. Although the etiology of asthma is not fully understood in humans, clinical and epidemiological evidence suggest a potential link between exposure to environmental fungi, such as Alternaria, and development and/or exacerbation of asthma. The goal of this project was to investigate the mechanisms of airway Th2 responses by using Alternaria as a clinically relevant model for environmental exposure. Airway exposure of naive animals to an experimental Ag, OVA, or a common allergen, short ragweed pollen, induced no or minimal immune responses to these Ags. In contrast, mice developed strong Th2-like immune responses when they were exposed to these Ags in the presence of Alternaria extract. Extracts of other fungi, such as Aspergillus and Candida, showed similar Th2 adjuvant effects, albeit not as potently. Alternaria stimulated bone marrow-derived dendritic cells (DCs) to express MHC class II and costimulatory molecules, including OX40 ligand, in vitro. Importantly, Alternaria inhibited IL-12 production by activated DCs, and DCs exposed to Alternaria enhanced Th2 polarization of CD4(+) T cells. Furthermore, adoptive airway transfer of DCs, which had been pulsed with OVA in the presence of Alternaria, showed that the recipient mice had enhanced IgE Ab production and Th2-like airway responses to OVA. Thus, the asthma-related environmental fungus Alternaria produces potent Th2-like adjuvant effects in the airways. Such immunogenic properties of certain environmental fungi may explain their strong relationships with human asthma and allergic diseases.

FIGURE 1

Fungi promote Th2-type airway sensitization in a mouse airway sensitization and challenge model. (A) A schematic representation of a mouse airway sensitization and challenge model. On days 0 and 7, BALB/c mice were intranasally exposed to 100 μg OVA with or without 50 μg fungal extract. On day 14, plasma was collected for analysis of OVA-specific IgE, IgG1 and IgG2a. On days 21, 22, and 23, mice were challenged by intranasal administration of 100 μg OVA. On day 24, BAL fluids, lungs, mediastinal lymph node (MLN), and spleen were collected for analyses of inflammation and cytokine responses. (B) Mice were intranasally exposed to OVA alone or OVA with fungal extracts (Alt; Alternaria, Asp; Aspergillus, Can; Candida). The day 14 levels of OVA-specific Abs, including IgE, IgG1, and IgG2a in the plasma, were measured by ELISA. On day 24, BAL fluids and lungs were collected and analyzed for the (C) number of eosinophils and the (D) levels of IL-13 in lung homogenates, respectively. Results are the mean ± SEM (n=6-9 in each group). * p<0.05, ** p<0.01 compared to mice previously exposed to OVA alone.

FIGURE 2

Recall response of draining lymph node cells and airway reactivity in mice exposed to OVA and Alternaria extract. (A) Mice were intranasally exposed to OVA plus Alternaria extract and challenged intranasally with OVA as in Figure 1A. On day 24, mediastinal lymph node (MLN) cells were harvested and cultured with medium alone or with 100 μg/ml OVA or BSA for 4 days. The levels of IL-5 and IL-13 in the supernatants were measured by ELISA. Results are the mean ± SD of four separate wells from one of two experiments, which showed similar results. (B) Mice were intranasally exposed to OVA alone, Alternaria extract alone or OVA plus Alternaria extract as shown in Figure 1A, and then challenged with OVA on days 42, 43, and 44. Airway reactivity to methacholine was examined on day 45. Results are a percentage of baseline (before PBS or methacholine challenge) and presented as mean ± SEM (n=5 mice in each group). * p<0.05, # p<0.05 compared to mice exposed to OVA alone or Alternaria extract alone, respectively.

FIGURE 3

Alternaria promotes Th2-type airway sensitization to short ragweed pollen (SRW). On days 0 and 7, mice were intranasally exposed to SRW extract alone (SRW) or SRW extract plus Alternaria extract (SRW+Alt). (A) On day 14, plasma was collected, and the levels of SRW-specific Abs were measured by ELISA. On days 21, 22, and 23, mice were then challenged intranasally with SRW alone. (B) On day 24, BAL fluids were analyzed for airway eosinophilia and IL-13 levels. Results are the mean ± SEM (n=7-8 in each group). * p<0.05, ** p<0.01 compared to mice exposed to SRW alone.

FIGURE 4

Alternaria enhances expression of MHC class II and co-stimulatory molecules by bone marrow (BM)-derived DCs. BM-derived DCs were incubated with medium alone (Med), 100 μg/ml Alternaria extract (Alt), or 1 μg/ml LPS for 24 h (MHC II, CD40, CD80 and CD86) or 48 h (OX40L). The expression of MHC class II and costimulatory molecules was analyzed by FACS. (A) Representative histograms show expression on DCs incubated with medium alone or Alternaria extract. (B) Mean expression levels are shown from 4 independent experiments (Mean ± SEM). * p<0.05, ** p<0.01 compared to medium alone. MFI; mean fluorescence intensity.

FIGURE 5

Cytokine production by DCs stimulated with Alternaria extract, LPS, or both. (A) BM-derived DCs were incubated with medium alone, 100 μg/ml Alternaria extract (Alt), or 1 μg/ml LPS for indicated times. IL-6 and IL-12p70 levels in the supernatants were measured by ELISA. Results are mean ± SEM (n=3-4). (B) BM-derived DCs were incubated with medium alone, 100 μg/ml Alternaria extract (Alt), 1 μg/ml LPS, or a combination of Alt and LPS for 24 h. IL-12p70 and TNF-α levels in the supernatants were measured by ELISA. Results are mean ± SEM (n=3).

FIGURE 6

DCs stimulated with Alternaria extract promote production of Th2 cytokines by allogenic CD4+ T cells. BM-derived DCs from BALB/c mice were incubated with medium alone (control DCs) or 75 μg/ml Alternaria extract (Alt-stimulated DCs) for 24 h and washed. (A) Allogenic CD4+ T cells from B6 mice were cultured with these DCs for 6 days, and supernatants analyzed for cytokines by ELISA. Results show mean ± SEM of 3 independent experiments. * p<0.05, ** p<0.01 compared to control DCs. (B) On day 4 of co-culture, cells were restimulated with anti-CD3/CD28 for 24 hours. Intracellular cytokines were stained and analyzed by FACS by gating on CD4+ T cells, as described in the Materials and Methods. Representative data are shown from 3 reproducible experiments. Numbers indicate percentages of IL-4- and IFN-γ-positive cells.

FIGURE 7

DCs stimulated with Alternaria and LPS induce distinctive cytokines from Ag-specific CD4+ T cells. BM-derived DCs from B6 mice were pulsed with 100 μg/ml OVA in medium alone (OVA), or in the presence of 75 μg/ml Alternaria extract (OVA+Alt) or 1 μg/ml LPS (OVA+LPS) for 24 hours and washed extensively. CD4+ T cells from OTII mice were incubated with these OVA-pulsed DCs. On day 6, supernatants were analyzed for IL-4, IL-13, and IFN-γ and, on day 10, for IL-5 by ELISA. Results show mean ± SEM from 3 independent experiments. * p<0.05, ** p<0.01 compared to DCs pulsed with OVA alone. # p<0.05 compared to DCs pulsed with OVA in the presence of Alternaria.

FIGURE 8

Intranasal transfer of DCs pulsed with OVA in the presence of Alternaria ex vivo enhances Th2-type humoral immunity and airway inflammation to OVA in vivo. BM-derived DCs were pulsed in vitro with medium alone, 1 mg/ml OVA, or OVA in the presence of 75 μg/ml Alternaria extract for 18 hours. DCs were then washed extensively and transferred intranasally (0.5 × 10⁶ cells) into naive mice. (A) On days 0 and 7, DCs were transferred; on day 14, plasma was collected, and the levels of OVA-specific IgE, IgG1, and IgG2a Abs were measured by ELISA. (B) DCs were transferred on day 0, and on days 14, 15, and 16, mice were challenged by intranasal administration of OVA. On day 17, BAL fluids were analyzed for the number of eosinophils. Results are the mean ± SEM (n=7-10 in each group). * p<0.05, ** p<0.01 compared to DCs pulsed with OVA alone.