Distinct CD4+-T-cell responses to live and heat-inactivated Aspergillus fumigatus conidia

Abstract

Aspergillus fumigatus is an important fungal pathogen that causes invasive pulmonary disease in immunocompromised hosts. Respiratory exposure to A. fumigatus spores also causes allergic bronchopulmonary aspergillosis, a Th2 CD4+-T-cell-mediated disease that accompanies asthma. The microbial factors that influence the differentiation of A. fumigatus-specific CD4+ T lymphocytes into Th1 versus Th2 cells remain incompletely defined. We therefore examined CD4+-T-cell responses of immunologically intact mice to intratracheal challenge with live or heat-inactivated A. fumigatus spores. Live but not heat-inactivated fungal spores resulted in recruitment of gamma interferon (IFN-gamma)-producing, fungus-specific CD4+ T cells to lung airways, achieving A. fumigatus-specific frequencies exceeding 5% of total CD4+ T cells. While heat-inactivated spores did not induce detectable levels of IFN-gamma-producing, A. fumigatus-specific CD4+ T cells in the airways, they did prime CD4+ T-cell responses in draining lymph nodes that produced greater amounts of interleukin 4 (IL-4) and IL-13 than T cells responding to live conidia. While immunization with live fungal spores induced antibody responses, we found a marked decrease in isotype-switched, A. fumigatus-specific antibodies in sera of mice following immunization with heat-inactivated spores. Our studies demonstrate that robust Th1 T-cell and humoral responses are restricted to challenge with fungal spores that have the potential to germinate and cause invasive infection. How the adaptive immune system distinguishes between metabolically active and inactive fungal spores remains an important question.

FIG. 1.

Similar CD4⁺-T-cell recruitment and proliferation in the MLN of mice challenged with live or heat-inactivated conidia. (A and B) Representative confocal images of resting, live A. fumigatus conidia (A) or of heat-inactivated spores (B). Bars, 5 μm. (C and D) Bone marrow-derived macrophages cultured with live (C) or heat-inactivated (D) A. fumigatus conidia. Bars, 10 μm. (E) Live or HIC homogenates were analyzed by 10% SDS-PAGE and immunoblotting with anti-A. fumigatus polyclonal antibodies. (F) Mice were infected with 10⁸ live A. fumigatus spores or with a similar dose of heat-inactivated conidia, and the numbers of CD4⁺ T lymphocytes recruited to the MLN were analyzed at different times postchallenge. Each symbol represents one mouse. Horizontal black lines indicate the mean value per group. Results are representative of two separate experiments. (G) T-cell proliferation by total MLN cells obtained from mice challenged with heat-inactivated conidia (black bars) or with a similar dose of live spores (gray bars) at various times postchallenge. Each bar represents the average response of three to four mice per group per time point. Results are representative of two individual experiments. (H) CD4⁺ T cells were purified from the MLN of mice that had been challenged 10 days earlier and restimulated in vitro with APCs pulsed with hyphal fragments. Results are representative of four individual experiments with five mice per group.

FIG. 2.

CD4⁺ T cells specific for various A. fumigatus antigens are activated after a single pulmonary challenge with live A. fumigatus conidia. (A) (Left) Molecular weights (MW, in thousands) and functions of A. fumigatus allergens. (Right) SDS-PAGE of the A. fumigatus allergens expressed and purified in E. coli. The recombinant proteins depicted in the gel are larger than their native counterparts due to the addition of N-terminal periplasmic targeting sequences and a C-terminal histidine tag. (B) Purified CD4⁺ T cells from the MLN of mice infected 10 days earlier (gray bars) or from the lymph nodes of naïve mice (black bars) were stimulated with the indicated A. fumigatus antigens or with live conidia. Each bar represents the average proliferation of triplicate wells. Results are representative of three individual experiments. (C) CD4⁺ T cells were purified from the MLN of mice challenged 10 days earlier with heat-inactivated conidia or with live A. fumigatus spores. Purified CD4⁺ T cells were stimulated with APCs pulsed with Asp f 2 or Asp f 3. Representative results of two individual experiments are shown. *, P < 0.05; **, P < 0.01.

FIG. 3.

CD4⁺ T cells primed with live A. fumigatus spores display a predominant Th1 cytokine profile, while Th2 cytokines predominate in the profile of A. fumigatus-specific CD4⁺ T cells primed by inactivated fungus. (A) CD4⁺ T cells were purified from the MLN of mice challenged 7 days earlier with live or heat-inactivated A. fumigatus spores. Purified CD4⁺ T cells were stimulated with APCs pulsed with hyphal fragments. Culture supernatants were collected 72 h post-culture initiation and assayed for the presence of cytokines by ELISA. Results shown are representative of two individual experiments. **, P < 0.01. (B and C) Representative images of lung sections stained with GMS. (B) Immunocompetent B6 mouse infected with live A. fumigatus spores 7 days earlier. (C) Neutrophil-depleted B6 mouse 3 days after infection with live A. fumigatus conidia.

FIG. 4.

A. fumigatus-specific CD4⁺ T cells are recruited to the airways of mice challenged with heat-inactivated fungus but are unable to produce IFN-γ. (A) Numbers of CD4⁺ T cells in the BALF of mice challenged with live or heat-inactivated A. fumigatus spores at various times postinfection. Results are representative of two separate experiments. (B and C) Mice were challenged with live or inactivated fungus and BALF collected 7 days later. BALF total cells were pooled from five mice per group and stimulated with either hypha-pulsed APCs, APCs alone, or phorbol myristate acetate for 5 h prior to ICCS analysis. Plots depicted are from gated CD4⁺ T cells. Results shown are representative of three individual experiments. (B) IFN-γ production; (C) TNF-α production. (D) Cytokines present in the BALF of mice challenged 7 days earlier with HIC or live fungus. Samples were analyzed by ELISA. Results shown are representative of two individual experiments.

FIG. 5.

Absence of A. fumigatus-specific IgG antibodies in the sera of mice challenged with inactivated fungus. B6 mice were challenged with an intratracheal dose of heat-inactivated conidia (black triangles) or a similar dose of live A. fumigatus spores (gray triangles), and hypha-specific (A), Asp f 2-specific (B), and Asp f 3-specific (C) IgG serum antibody titers were determined. The responses depicted here are the values measured at day 22 and are representative of measurements at other time points (day 14 and day 30 postinfection). Values shown are the average responses of four mice per group.