Aspergillus fumigatus Infection in Humans With STAT3-Deficiency Is Associated With Defective Interferon-Gamma and Th17 Responses

Abstract

In humans, loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3) gene is frequently associated with susceptibility to bacterial as well as fungal infections including aspergillosis, although its pathogenesis remains largely unknown. In the present study, we investigated the immune responses obtained after stimulation with Aspergillus fumigatus in STAT3-deficient patients. A. fumigatus conidial killing efficiencies of both monocytes and neutrophils isolated from whole blood samples of STAT3-deficient patients were not different compared to those of healthy controls. After stimulation with A. fumigatus conidia, lower concentrations of adaptive cytokines (IFN-γ, IL-17 and IL-22) were secreted by peripheral blood mononuclear cells from STAT3-deficient patients compared to those from healthy controls. Moreover, the frequency of IFN-γ and IL-17 producing CD4+ T cells was lower in STAT3-deficient patients vs. healthy controls. Among the STAT3-deficient patients, those with aspergillosis showed further lower secretion of IFN-γ upon stimulation of their PBMCs with A. fumigatus conidia compared to the patients without aspergillosis. Together, our study indicated that STAT3-deficiency leads to a defective adaptive immune response against A. fumigatus infection, particularly with a lower IFN-γ and IL-17 responses in those with aspergillosis, suggesting potential therapeutic benefit of recombinant IFN-γ in STAT3-deficient patients with aspergillosis.

Keywords: Aspergillus; IgE; IgG; aspergillosis; autosomal dominant hyper-IgE syndrome (AD-HIES); innate/adaptive immunity; loss-of-function mutation; signal transducer and activator of transcription 3 (STAT3).

Figure 1

Total IgE (A), specific IgE (B), and IgG (C) against A. fumigatus. Comparison of sera from STAT3 deficient-patients (STAT3^+/−, representing heterozygous mutation) without aspergillosis (w/o asp; n = 21), with ongoing aspergillosis (asp ongoing; n = 8) or with prior aspergillosis (asp prior; n = 3), with healthy controls (n = 20), CPA (n = 10), and ABPA (n = 11) patients, and patients receiving substitutive intravenous immunoglobulin (IVIG; n = 5). OD: optic density; *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (the mean values are presented in the figures).

Figure 2

A. fumigatus conidial phagocytosis and killing by monocytes and neutrophils. (A) Phagocytosis of FITC-labeled conidia (parental strain) by CD14⁺ monocytes from STAT3-deficient patient (STAT3^+/−) and controls. Extracellular conidia are labeled with CFW (in blue) whereas intracellular conidia are pre-FITC-labeled (in green). (B) Phagocytosis of Δku80 conidia by CD14⁺ monocytes from four STAT3-deficient patients and six healthy controls. (C) Killing of Δku80 conidia by CD14⁺ monocytes from four each of STAT3-deficient patients and healthy controls, and evaluated by colony forming unit (CFU) counting. (D) Killing of Δku80 conidia by neutrophils at different conidia: neutrophils ratios from six each of STAT3-deficient patients and healthy controls.

Figure 3

Analysis of adaptive immune response after stimulating PBMCs with conidia for 5-days. 2 × 10⁵ conidia were co-incubated with 2 × 10⁵ PBMC from STAT3-deficient patients (STAT3^+/−) and healthy controls. Secretion of IFN-γ, IL-17, IL-22, IL-10, IL-5 were analyzed by ELISA and expressed in pg/mL, while, percent CD4+ T cells expressing IFN-γ, IL-17, IL-4 was analyzed by FACS; ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

Analysis of the cytokine production by PBMCs isolated from STAT3-deficient patients with or without aspergillosis upon interaction with A. fumigatus. 2 × 10⁵ conidia were co-incubated with 2 × 10⁵ PBMC during 1 (innate response) or 5 days (adaptive response). (A) TNF-α, IL-1β, and IL-6 secreted by PBMCs isolated from STAT3-deficient patients (STAT3^+/−) with aspergillosis (STAT3 asp) (n = 6), without aspergillosis (STAT3 w/o asp) (n = 6) and healthy controls upon one-day interaction with A. fumigatus conidia; cytokines were analyzed by ELISA. (B) IFN-γ and IL-17 secreted upon stimulation of PBMCs isolated from STAT3-asp, STAT3-w/o-asp and healthy controls with A. fumigatus conidia for 5-days; ELISA (secreted) and FACS (intracellular) were performed. (C) Analysis of IFN-γ, IL-17 and IL-5 secretion in one STAT3-deficient patient with ongoing ABPA compared to STAT3-deficient patient with aspergillosis (n = 5); *p < 0.05, ** p < 0.01, and ***p < 0.001.