Caspofungin Increases Fungal Chitin and Eosinophil and γδ T Cell-Dependent Pathology in Invasive Aspergillosis

Abstract

The polysaccharide-rich fungal cell wall provides pathogen-specific targets for antifungal therapy and distinct molecular patterns that stimulate protective or detrimental host immunity. The echinocandin antifungal caspofungin inhibits synthesis of cell wall β-1,3-glucan and is used for prophylactic therapy in immune-suppressed individuals. However, breakthrough infections with fungal pathogen Aspergillus fumigatus are associated with caspofungin prophylaxis. In this study, we report in vitro and in vivo increases in fungal surface chitin in A. fumigatus induced by caspofungin that was associated with airway eosinophil recruitment in neutropenic mice with invasive pulmonary aspergillosis (IA). More importantly, caspofungin treatment of mice with IA resulted in a pattern of increased fungal burden and severity of disease that was reversed in eosinophil-deficient mice. Additionally, the eosinophil granule proteins major basic protein and eosinophil peroxidase were more frequently detected in the bronchoalveolar lavage fluid of lung transplant patients diagnosed with IA that received caspofungin therapy when compared with azole-treated patients. Eosinophil recruitment and inhibition of fungal clearance in caspofungin-treated mice with IA required RAG1 expression and γδ T cells. These results identify an eosinophil-mediated mechanism for paradoxical caspofungin activity and support the future investigation of the potential of eosinophil or fungal chitin-targeted inhibition in the treatment of IA.

FIGURE 1

Caspofungin increases surface chitin exposure in A. fumigatus in vitro and in the lungs of mice with IA. (A and B) Af293 conidia were cultured and germinated (4h at 37°C) in the presence of caspofungin or control conditions, then fixed prior to WGA-APC staining. (A) Representative histogram overlay from 3 experiments. (B) Summary of median fluorescence intensity of WGA staining from 3 experiments (n=3/group). (C) Infection timeline. BALB/c mice were depleted of neutrophils and infected with 5x10⁶ Af293 conidia, with a subset of mice treated with i.p. caspofungin daily until harvest at 72h post-infection. (D) Representative fungal morphology in GMS-stained control lung and caspofungin-treated mice. Red arrows highlight swollen or burst hyphal tips, blue arrows highlight short, thickened hyphae. Panels are representative of sections from 3 mice/group. (E) Representative immune fluorescence staining of d3 frozen lung sections from caspofungin treated or untreated mice with IA, stained for CCR3+ cells (green) and calcofluor white (red) to identify fungal chitin (n=3/group). Scale bars (D, E) are equivalent to 20 μm. ****p<0.0001.

FIGURE 2

Airway eosinophil accumulation is increased with caspofungin treatment in mice with IA. A, B, and C, BALB/c mice were neutrophil-depleted, infected, and treated or untreated with caspofungin as described for Fig. 1 (timeline in Fig. 1C). BALF was analyzed for eosinophil recruitment as described in Materials and Methods. D and E, analysis of BALF from caspofungin-treated mice with IA that constititively express lung AMCase (SPAM+) compared to transgene-negative littermates (SPAM-). A, representative flow plots depicting gating of BALF CD45^hiLy6G⁻SiglecF⁺CD11c⁻ eosinophils (Eos) and CD45^hiLy6G⁻SiglecF⁺CD11c⁺ alveolar macrophages (AM). B and D, total cells. C and E, frequency (left) and total number (right) of eosinophils. Data shown are a summary of 2–3 experiments. *p<0.05. **p<0.01.

FIGURE 3

Increased detection of eosinophil granule proteins in aspergillosis patients treated with caspofungin. Major basic protein (A) and eosinophil peroxidase (B) in the BALF of lung transplant patients with or without aspergillosis quantified by ELISA. Patients were further subdivided by antifungal therapy: those that received azoles (Infected) or those that received caspofungin alone or in combination with azoles (Inf+Caspo). Statistical analysis was performed as described in Materials and Methods.

FIGURE 4

Increased disease severity and lack of fungal clearance in caspofungin-treated mice is eosinophil-dependent. Wild-type BALB/c or eosinophil-deficient (ΔdblGATA1) mice were infected and treated or left untreated with caspofungin as described for Fig. 1. (A) Survival. (B) Disease Score. (C) Fungal DNA (burden) (15–30 mice/group, summary of 3–6 experiments). (A–C) n=15–30 mice/group, data are a summary of 3–6 experiments. (D) Change in fungal DNA burden with caspofungin treatment calculated from results shown in Fig. 4A. (E) Fungal burden as measured by quantification of GMS staining of histological sections (n=3–4 mice/group). (F) Representative GMS staining of histological sections from wild-type (top) or eosinophil−/− mice (bottom). Scale bar is equivalent to 100 μm. *p<0.05. **p<0.01. ****p<0.0001.

FIGURE 5

Disease severity, fungal burden, and eosinophil recruitment are not increased in caspofungin-treated RAG1−/− mice. Mice deficient in RAG1 (BALB/c background) were infected with A. fumigatus as described for Fig. 1. (A) Disease severity and (B) Fungal burden (n=8). (C) Frequency (left) and total BALF eosinophils (right). Data shown are a summary of two experiments.

FIGURE 6

Modulation of CD3+TCRδ+ cells in caspofungin-treated mice with IA. Neutropenic mice were infected and harvested at 48 hours post-infection, with cell suspensions derived from lung homogenates analyzed by flow cytometry for expression of γδ T cell markers. A) Representative dot plots from two experiments. B) Frequency (left) and total numbers (right) of lung CD3+TCRδ+ cells. C) Representative histogram from two experiments depicting GFP fluorescence of CD3+TCRδ+ cells from IL4-GFP-reporter mice (4get). *p<0.05.

FIGURE 7

Caspofungin-mediated eosinophil recruitment and pathology require γδ T cells. γδ T cell-deficient mice were infected with Af293, monitored and harvested as described for Fig. 1. (A) Survival (n=9–10, summary of two experiments). (B) Display of fungal burden determined by PCR quantification of fungal DNA (7–8mice/group, summary of two experiments). (C) Representative GMS staining of lung sections from γδ T cell-deficient mice with the indicated treatment. Scale bar is equivalent to 100 μM. X. Inset, bottom left of right panel, determination of fungal burden by quantification of GMS staining in treated and untreated mice. (D, E) BALF cell populations as determined by flow cytometry. (E) Frequency (left) and total number (right) of airway eosinophils in the indicated experimental groups. Data shown are a summary of two experiments. *p<0.05. **p<0.01. ***p<0.001.

FIGURE 8

Effects of caspofungin treatment and requirement for γδ T cells in lung expression of immunomodulatory genes in mice with IA. Neutropenic C57BL/6 (B6) wild-type or γδ T cell-deficient mice were infected with A. fumigatus, treated or untreated with caspofungin, and harvested for qRT-PCR analysis of the indicated genes in lung homogenate extracts at 48 hours post-infection. A) Wild-type B6 mice treated or untreated with caspofungin. B) Wild-type or TCRδ−/− mice infected and treated with caspofungin. *p<0.05.