Dexamethasone Promotes Aspergillus fumigatus Growth in Macrophages by Triggering M2 Repolarization via Targeting PKM2

Abstract

Since long-term corticosteroid treatment is associated with emerging opportunistic fungal infections causing high morbidity and mortality in immune-suppressed individuals, here we characterized the impact of dexamethasone (Dex) treatment on Aspergillus fumigatus-related immune modulation. We found by high content screening and flow cytometric analyses that during monocyte-to-macrophage differentiation, as little as 0.1 µg/mL Dex resulted in a shift in macrophage polarization from M1 to M2-like macrophages. This macrophage repolarization mediated via Dex was characterized by significant upregulation of the M2 marker CD163 and downmodulation of M1 markers CD40 and CD86 as well as changes in phenotypic properties and adherence. These Dex-mediated phenotypic alterations were furthermore associated with a metabolic switch in macrophages orchestrated via PKM2. Such treated macrophages lost their ability to prevent Aspergillus fumigatus germination, which was correlated with accelerated fungal growth, destruction of macrophages, and induction of an anti-inflammatory cytokine profile. Taken together, repolarization of macrophages following corticosteroid treatment and concomitant switch to an anti-inflammatory phenotype might play a prominent role in triggering invasive aspergillosis (IA) due to suppression of innate immunological responses necessary to combat extensive fungal outgrowth.

Dexamethasone Creates a Suppressive Microenvironment and Promotes Aspergillus fumigatus Invasion in a Human 3D Epithelial/Immune Respiratory Model

“…Dexamethasone powder (Sigma, St. Louis, MO, USA #D4902) was diluted in absolute Ethanol (Carl Roth, Karlsruhe, Germany #D65.1) to a final stock concentration of 20 mg/mL as previously described [15]. It was then stored at −20 • C until needed.…”

“…Due to the limited availability of pulmonary macrophages [19], donor monocytes were derived from normal healthy donor whole blood buffy coats using anti-human CD14 Magnetic Particles (BD ImagTM, Franklin Lakes, NJ #557769, USA) and cell separation magnets (BD ImagTM, Franklin Lakes, NJ #552311, USA). Purity was confirmed using flow cytometry as previously described [15]. Polarization was accomplished using 50 ng/mL rhGM-CSF (#572905, Biolegend, San Diego, CA, USA) for 7 days to obtain M1 (GM-MDMs, corresponds to GM-CSF-generated monocyte-derived macrophages [MDMs])-like macrophages, respectively [15].…”

“…Purity was confirmed using flow cytometry as previously described [15]. Polarization was accomplished using 50 ng/mL rhGM-CSF (#572905, Biolegend, San Diego, CA, USA) for 7 days to obtain M1 (GM-MDMs, corresponds to GM-CSF-generated monocyte-derived macrophages [MDMs])-like macrophages, respectively [15].…”

“…The dsRed-expressing isolate of A. fumigatus strain number AF293 was utilized in this study. The organism was grown on Aspergillus Complete Media (ACM) for 5 days at 35 • C as previously described [15,18]. Conidia were then harvested and suspensions filtered using a sterile 40 um pore size filter (BD Falcon, Franklin Lakes, NJ #352340, USA).…”

“…These diseases include invasive Aspergillosis (IA), an extremely serious condition with a mortality rate up to 95% in untreated cases and around 40% in treated ones. Through Glucocorticoid Receptor-mediated signaling, steroids regulate cellular functions, metabolism, differentiation, survival, proliferation, and general immune responses [14,15]. These endogenous substances are cholesterol-derived hormones produced by the adrenal gland [16], but synthetic versions have been developed and are commonly applied whenever potent immune regulation is required [17].…”