Background Lung transplant recipients on calcineurin inhibitor immunosuppression have increased susceptibility to, and increased mortality from, invasive aspergillosis. Tacrolimus (FK506) diminishes the innate immune response to Aspergillus fumigatus infection partly by inhibition of the calcineurin-NFAT axis. We investigated the effects of FK506 on transcriptional regulation in dendritic cells (DC’s), and assessed interferon-gamma as a treatment, with a combination of RNA-Seq and histone modification ChIP-seq.
Methods Healthy volunteer monocytes were negatively isolated from gradient-centrifugation-selected PBMC’s and differentiated into DC’s with GM-CSF and IL-4. DC’s were treated with FK506, interferon-gamma and/or inoculated with swollen conidia of A.fumigatus (MOI 1:1). For RNA-Seq, extracted mRNA was poly-A purified and reverse-transcribed to ds-DNA, and for ChIP-seq, DNA was cross-linked, sonicated, then immunoprecipitated with antibodies against histone marks H3k4me1 and H3k27ac. Resultant DNA was PCR-amplified to generate libraries for next generation sequencing on the Illumina HiSeq 2500. Computational sequencing analysis pipelines used open-source C++ and R-based packages (Bowtie, Kallisto, edgeR and MACS).
Results A.fumigatus infection in DC’s elicited upregulation of genes belonging to two key groups of early-phase response transcription factors – the early growth response family (EGR1 – log fold-change 4.90, FDR p-value = 0.0003) and the nuclear receptor family (NR4A2 – logFC 6.96, p = 1.56 × 10–6). FK506 treatment ablated significant differential expression of these genes whilst subsequent interferon-gamma treatment restored their upregulation (EGR1 – logFC 4.43, p = 0.00093; NR4A2 – logFC 5.56, p = 0.00034).
Active gene enhancers regions were identified by presence of significant peaks of H3k4me1 and H3k27ac antibody binding. Motif analysis of enhancers within regulatory domains around differentially-expressed genes identified enrichment of core binding motifs of NFAT (p = 7.8 × 10-9) and FOXF2 (p = 8.6 × 10–10) transcription factors, which was lost after FK506 treatment.
Conclusions Transcriptome analysis has revealed the key genes involved in early dendritic cell responses to A.fumigatus infection, and their ablation by FK-506 treatment suggests a deleterious genome-level effect of calcineurin inhibitors in this context. Furthermore, interferon-gamma treatment restores a more favourable transcriptomic response to infection in FK506-treated DC’s. The condition-dependent differential enrichment of enhancer motifs suggests a role for both suspected (NFAT) and previously unidentified (FOXF2) transcription factors in the DC response to A.fumigatus infection.