Introduction Invasive aspergillosis in lung transplant recipients on immunosuppression is associated with high morbidity and mortality. The calcineurin inhibitor tacrolimus (FK506) inhibits the calcineurin-NFAT axis, which impairs the innate response to fungal infection.1 Dendritic cells (DC’s) play a pivotal role in signalling to the adaptive immune system in infection – immature DC’s phagocytose antigen, leading to maturation into DC’s capable of stimulating T-cells. We investigated the effect of FK506 on DC function in invasive aspergillosis by assessing phenotypic maturation of DC’s in response to Aspergillus fumigatus (AF) infection.
Methods Healthy volunteer PBMC’s negatively isolated by Ficoll® gradient were differentiated into DC’s with GM-CSF and IL-4. Day 5 cells were matured with IFN-γ. Day 7 cells were treated with FK506 and/or inoculated with swollen conidia of A.fumigatus (MOI 1:1). Cells were then stained with PE-bound anti-CD83 (a late maturation marker) and PerCP-Cy5.5-bound anti-CD-209 (a DC-specific marker) and analysed by the ImageStream® imaging flow cytometer. Statistical analysis was performed with Graphpad Prism v6.0, using unpaired t-tests with Welch correction.
Results 5000 cells/condition were analysed. DC’s were subsetted by gating for CD-209 positivity. FK506 was not toxic to cells (similar cell viability between groups).
We demonstrated up-regulation of CD83 (measured by mean fluorescent intensity) with IFN-γ stimulation of DC’s (12534 ± 799.3 vs. 26228 ± 1462, p < 0.0001; mean fluorescent units +/-SEM), AF infection of unstimulated DC’s (12534 ± 799.3 vs. 29888 ± 1393, p < 0.0001) and for AF infection of IFN-γ-stimulated DC’s (26228 ± 1462 vs. 36778 ± 1356, p < 0.0001).
CD83 mean fluorescent intensity was reduced with FK506 treatment of IFN-γ-stimulated DC’s (26228 ± 1462, vs. 20219 ± 846.0, p = 0.0004), AF-infected unstimulated DC’s (29888 ± 1393 vs. 24289 ± 1253, p = 0.0028), and AF-infected, IFN-γ-stimulated DC’s (36778 ± 1356 vs. 30159 ± 1279, p = 0.0004), but unchanged for unstimulated, un-infected DC’s (12534 ± 799.3, vs. 11942 ± 762.5, p = 0.5921).
Conclusion Both A.fumigatus infection and IFN-γ stimulation promote phenotypic maturation of DC’s in vitro, and treatment with FK506 inhibits maturation in this context. This suggests an inhibitory effect of FK506 on innate antigen presentation to T-cells and may impair the adaptive immune response to invasive aspergillosis in lung transplants recipients.
Reference 1 Herbst S, Shah A, Mazon Moya M, et al. EMBO Mol Med. 2015;7(3):240–58