Genetic Variation in PFKFB3 Impairs Antifungal Immunometabolic Responses and Predisposes to Invasive Pulmonary Aspergillosis

Abstract

Activation of immune cells in response to fungal infection involves the reprogramming of their cellular metabolism to support antimicrobial effector functions. Although metabolic pathways such as glycolysis are known to represent critical regulatory nodes in antifungal immunity, it remains undetermined whether these are differentially regulated at the interindividual level. In this study, we identify a key role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the immunometabolic responses to Aspergillus fumigatus. A genetic association study performed in 439 recipients of allogeneic hematopoietic stem cell transplantation (HSCT) and corresponding donors revealed that the donor, but not recipient, rs646564 variant in the PFKFB3 gene increased the risk of invasive pulmonary aspergillosis (IPA) after transplantation. The risk genotype impaired the expression of PFKFB3 by human macrophages in response to fungal infection, which was correlated with a defective activation of glycolysis and the ensuing antifungal effector functions. In patients with IPA, the risk genotype was associated with lower concentrations of cytokines in the bronchoalveolar lavage fluid samples. Collectively, these findings demonstrate the important contribution of genetic variation in PFKFB3 to the risk of IPA in patients undergoing HSCT and support its inclusion in prognostic tools to predict the risk of fungal infection in this clinical setting. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. Activation of glycolysis is essential for innate immune cells to mount effective antifungal responses. In this study, we report the contribution of genetic variation in the key glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) to the risk of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation. The PFKFB3 genotype associated with increased risk of infection was correlated with an impairment of the antifungal effector functions of macrophages in vitro and in patients with IPA. This work highlights the clinical relevance of genetic variation in PFKFB3 to the risk of IPA and supports its integration in risk stratification and preemptive measures for patients at high risk of IPA.

Keywords: Aspergillus; PFKFB3; antifungal immunity; immunometabolism; invasive pulmonary aspergillosis; macrophage; single nucleotide polymorphism; stem cell transplantation.

FIG 1

The PFKFB3 locus influences the production of cytokines by PBMCs. (A) Levels (log₂) of TNF and IL-6 according to rs674430 genotypes, and (B) IFN-γ and IL-22 according to rs646564 genotypes detected after stimulation of PBMCs from the 500FG cohort with A. fumigatus for 24 h or 7 days, respectively. Data are expressed as mean values ± standard errors of the means (SEM). Overall P values were determined using a linear regression model with age and gender as covariates.

FIG 2

Genetic variation in PFKFB3 influences the risk of IPA. Cumulative incidence of IPA in 439 eligible HSCT recipients from the IFIGEN cohort according to the recipient or donor PFKFB3 genotypes at rs646564 (A) and rs674430 (B). Data were censored at 24 months, and relapse and death were competing events. P values are for Gray’s test.

FIG 3

The rs646564 SNP in PFKFB3 inhibits the activation of glycolysis in macrophages. (A and B) Expression of PFKFB3 in macrophages infected with A. fumigatus for 2 h according to different rs646564 genotypes (A) or following treatment with 3PO (representative of three independent experiments) (B). The pixel density of the PFKFB3 was normalized to β-actin. (C and D) Glucose consumption (C) and lactate secretion (D) by macrophages left untreated or infected with A. fumigatus for 24 h, according to different rs646564 genotypes or following treatment with 3PO. Data are expressed as mean values ± SEM. Ctrl, control.

FIG 4

Antifungal effector mechanisms of macrophages are impaired by the rs646564 SNP in PFKFB3. (A) Production of IL-1β, TNF, IL-6, and IL-10 by macrophages infected with A. fumigatus for 24 h according to different rs646564 genotypes or following treatment with 3PO. (B) Conidiacidal activity of macrophages according to different rs646564 genotypes or following treatment with 3PO. (C) Production of cytosolic ROS (left) and mitochondrial ROS (right) by macrophages infected with A. fumigatus for 4 h according to different rs646564 genotypes or following treatment with 3PO. Data are expressed as mean fluorescence intensity (MFI). DHE, dihydroethidium. (D) Phagocytosis of macrophages according to different rs646564 genotypes or following treatment with 3PO. Data are expressed as mean values ± SEM.

FIG 5

PFKFB3 regulates cytokine production in IPA. (A and B) Levels of IL-1β, TNF, and IL-6 (A) and IFN-γ, IL-17, and IL-22 (B) in BAL fluid samples from patients diagnosed with IPA (n = 16 for GG; n = 7 for TT). Data are expressed as mean values ± SEM.