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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.

SUBMITTER: Goncalves SM

PROVIDER: S-EPMC8263003 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:β2-integrins are heterodimeric surface receptors that are expressed specifically by leukocytes and consist of a variable α (CD11a-d) and a common β-subunit (CD18). Functional impairment of CD18, which causes leukocyte adhesion deficiency type-1 results in an immunocompromised state characterized by severe infections, such as invasive pulmonary aspergillosis (IPA). The underlying immune defects have largely been attributed to an impaired migratory and phagocytic activity of polymorphonuclear granulocytes (PMN). However, the exact contribution of β2-integrins for PMN functions in-vivo has not been elucidated yet, since the mouse models available so far display a constitutive CD18 knockout (CD18-/- or CD18hypo). To determine the PMN-specific role of β2-integrins for innate effector functions and pathogen control, we generated a mouse line with a Ly6G-specific knockdown of the common β-subunit (CD18Ly6G cKO). We characterized CD18Ly6G cKO mice in-vitro to confirm the PMN-specific knockdown of β2-integrins. Next, we investigated the clinical course of IPA in A. fumigatus infected CD18Ly6G cKO mice with regard to the fungal burden, pulmonary inflammation and PMN response towards A. fumigatus. Our results revealed that the β2-integrin knockdown was restricted to PMN and that CD18Ly6G cKO mice showed an aggravated course of IPA. In accordance, we observed a higher fungal burden and lower levels of proinflammatory innate cytokines, such as TNF-α, in lungs of IPA-infected CD18Ly6G cKO mice. Bronchoalveolar lavage revealed higher levels of CXCL1, a stronger PMN-infiltration, but concomitantly elevated apoptosis of PMN in lungs of CD18Ly6G cKO mice. Ex-vivo analysis further unveiled a strong impairment of PMN effector function, as reflected by an attenuated phagocytic activity, and a diminished generation of reactive oxygen species (ROS) and neutrophil-extracellular traps (NET) in CD18-deficient PMN. Overall, our study demonstrates that β2-integrins are required specifically for PMN effector functions and contribute to the clearance of A. fumigatus by infiltrating PMN, and the establishment of an inflammatory microenvironment in infected lungs.

Project description:Animal model systems are a critical component of the process of discovery and development of new antifungal agents for treatment and prevention of invasive aspergillosis. The persistently neutropenic rabbit model of invasive pulmonary aspergillosis (IPA) has been a highly predictive system in identifying new antifungal agents for treatment and prevention of this frequently lethal infection. Since its initial development, the persistently neutropenic rabbit model of IPA has established a strong preclinical foundation for dosages, drug disposition, pharmacokinetics, safety, tolerability, and efficacy for deoxycholate amphotericin B, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, caspofungin, micafungin, anidulafungin, voriconazole, posaconazole, isavuconazole, and ibrexafungerp in treatment of patients with invasive aspergillosis. The findings of combination therapy with a mould-active triazole and an echinocandin in this rabbit model also predicted the outcome of the clinical trial for voriconazole plus anidulafungin for treatment of IPA. The plasma pharmacokinetic parameters and tissue disposition for most antifungal agents approximate those of humans in persistently neutropenic rabbits. Safety, particularly nephrotoxicity, has also been highly predictive in the rabbit model, as exemplified by the differential glomerular filtration rates observed in animals treated with deoxycholate amphotericin B, liposomal amphotericin B, amphotericin B lipid complex, and amphotericin B colloidal dispersion. A panel of validated outcome variables measures therapeutic outcome in the rabbit model: residual fungal burden, markers of organism-mediated pulmonary injury (lung weights and infarct scores), survival, and serum biomarkers. In selected antifungal studies, thoracic computerized tomography (CT) is also used with diagnostic imaging algorithms to measure therapeutic response of pulmonary infiltrates, which exhibit characteristic radiographic patterns, including nodules and halo signs. Further strengthening the predictive properties of the model, therapeutic response to successfully developed antifungal agents for treatment of IPA has been demonstrated over the past two decades by biomarkers of serum galactomannan and (1→3)-β-D-glucan with patterns of resolution, that closely mirror those documented responses in patients with IPA. The decision to move from laboratory to clinical trials should be predicated upon a portfolio of complementary and mutually validating preclinical laboratory animal models studies. Other model systems, including those in mice, rats, and guinea pigs, are also valuable tools in developing clinical protocols. Meticulous preclinical investigation of a candidate antifungal compound in a robust series of complementary laboratory animal models will optimize study design, de-risk clinical trials, and ensure tangible benefit to our most vulnerable immunocompromised patients with invasive aspergillosis.

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

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