Surface availability of beta-glucans is critical determinant of host immune response to Cladosporium cladosporioides

Abstract

Background: It is well accepted that mold exposure is a major contributor to the development of asthma, and beta-glucans are often used as a surrogate for mold exposure in the environment. Beta-glucans are an important component of mold spores and are recognized by the immune system by their receptor, Dectin-1. Cladosporium cladosporioides spores have a high beta-glucan content, but the beta-glucans are not available on the surface of live spores.

Objective: We sought to determine whether altering the exposure of beta-glucans in C cladosporioides through heat killing could alter the immune response through binding to Dectin-1.

Methods: In a murine model of mold-induced asthma, mice were repeatedly exposed to either live or heat-killed C cladosporioides and the phenotype was determined by the measurement of airway hyperresponsiveness, airway inflammation, and cytokine production. Pro-inflammatory cytokines from dendritic cells were measured by using quantitative PCR and ELISA.

Results: Live C cladosporioides induced robust airway hyperresponsiveness, eosinophilia, and a predominately TH2 response, while heat-killed C cladosporioides induced a strong TH17 response and neutrophilic inflammation, but very mild airway hyperresponsiveness. Heat killing of C cladosporioides spores effectively exposed beta-glucans on the surface of the spores and increased binding to Dectin-1. In the absence of Dectin-1, heat-killed spores induced a predominantly TH2 response analogous to live spores. Furthermore, the production of TH17-skewing IL-6, IL-23, and TNF-α by dendritic cells in response to heat-killed C cladosporioides was dependent on Dectin-1.

Conclusions: The host immune response to C cladosporioides is dependent on the surface availability of beta-glucans rather than the total beta-glucan content.

FIG E1.

A, Live cells gating with live dead stain (Invitrogen). B, Gating of lymphocytes in the live population based on forward scatter (FSC) and side scatter (SSC). C, CD4 gating from the lymphocyte population.

FIG E2.

C clad or C clad HK spores were stained with an AF488 dye (green) or not stained and then incubated with anti-beta glucan and a secondary antibody (red). C clad HK, Heat-killed C cladosporioides.

FIG 1.

Heat killing of C cladosporioides prevents the development of murine asthma. A, BALB/c mice were exposed i.t. to 10⁶ live or heat-killed C cladosporioides (C clad or C clad HK) spores 3 times a week for 3 weeks and 48 hours after the last challenge, AHR was measured by flexiVent. B, BALF was analyzed for total and differential counts. C, Total serum IgE. Data are representative of 3 independent experiments and are expressed as mean and SEM. n = 4 to 6 mice per group. Eos, Eosinophils; HK, heat killed; i.t., intratracheally; Lymp, lymphocytes; Mac, macrophages; Neut, neutrophils. *P < .05, **P < .01, and ***P < .001 as compared with saline. #P < .05 and ###P < .001 as compared with C clad HK.

FIG 2.

Induction of a T_H17 response by heat-killed C cladosporioides (C clad HK). Contour plots (A) and quantification of intracellular cytokine staining for IL-13 or IL-17A in CD4+ lung cells (B). C, Levels of IL-17A and IL-4 in BALF. D, Relative levels of mRNA for IL-17A and IL-4 in whole lung. Data are representative of 3 independent experiments and are expressed as mean and SEM. n = 4 to 6 mice per group. HPRT, Hypoxanthine phosphoribosyltransferase. *P < .05, **P < .01, and ***P < .001 as compared with saline. #P < .05, ##P < .01, and ###P < .001 as compared with C clad HK.

FIG 3.

Heat-killed C cladosporioides have increased surface exposure of beta-glucans and binding to Dectin-1. A, Immunofluorescence images of beta-glucan staining taken at ×1000 for live and heat-killed C cladosporioides spores. Scale bar is 10 μm. B, Quantification of beta-glucan fluorescence intensity per spore. C, Number of spores bound to recombinant Dectin-1. Data are representative of 2 independent experiments and are expressed as mean and SEM. n = 3 samples per group. C clad HK, Heat-killed C cladosporioides. *P < .05.

FIG 4.

Heat-killed C cladosporioides prevents murine asthma in a Dectin-1–dependent manner. A, Wild-type C57BI/6 or Dectin-1^−/− mice were exposed to heat-killed C cladosporioides spores, and AHR was assessed 48 hours later. B, Total and differential counts of BALF. Data are representative from 2 independent experiments and are expressed as mean and SEM. n = 4 to 6 mice per group. C clad HK, Heat-killed C cladosporioides; Eos, eosinophils; KO, knockout; Lymp, lymphocytes; Mac, macrophages; Neut, neutrophils; WT, wild type. **P < .01 and ***P < .001 as compared with saline. ##P < .01 and ###P < .001 as compared with wild-type mice exposed to C cladosporioides.

FIG 5.

In the absence of Dectin-1, heat-killed C cladosporioides induces a T_H2 response. Contour plots (A) and quantification of intracellular cytokine staining for IL-13 or IL-17A in CD4+ lung cells (B). C, Levels of IL-17A in BALF and mRNA in whole lung. D, Levels of IL-4 in BALF and mRNA in whole lung. Data are representative from 2 independent experiments and are expressed as mean and SEM. n = 4 to 6 mice per group. C clad HK, Heat-killed C cladosporioides; KO, knockout; WT, wild type. **P < .01 and ***P < .001 as compared with saline. #P < .05, ##P < .01, and ###P < .001 as compared with wild-type mice exposed to Ccladosporioides.

FIG 6.

Expression of T_H17-skewing cytokines in the lungs is dependent on Dectin-1. A and C, Expression of IL-6 in the lungs of mice exposed to live (Fig 6, A) or heat-killed (Fig 6, C) C cladosporioides. B and D, Expression of TNF-α in the lungs of mice exposed to live (Fig 6, B) or heat-killed (Fig 6, D) C cladosporioides. Data are representative from 2 independent experiments and are expressed as mean and SEM. n = 4 to 6 mice per group. C clad HK, Heat-killed C cladosporioides; HPRT, hypoxanthine phosphoribosyltransferase; KO, knockout; WT, wild type. *P < .05, **P < .01, and ***P < .001 as compared with saline. #P < .05 and ###P < .001 as compared with Dectin-1^−/− mice exposed to C cladosporioides.

FIG 7.

Proinflammatory cytokine production in BMDCs is dependent on Dectin-1. Expression of IL-6 (A), TNF-α (B), and IL-23 (C) in BMDCs. Level of IL-6 (D) and TNF-α (E) in culture supernatants of BMDCs. Data are representative of 3 independent experiments and are expressed as mean and SEM. n = 4 samples per group. C clad HK, Heat-killed C cladosporioides; HPRT, hypoxanthine phosphoribosyltransferase; KO, knockout; WT, wild type. *P < .05, **P < .01, and ***P < .001 as compared with saline. ##P < 0.01 and ###P < .001 as compared with Dectin-1^−/− BMDCs exposed to C cladosporioides.