Intracellular Cryptococcus neoformans Disrupts the Transcriptome Profile of M1- and M2-Polarized Host Macrophages

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, we used an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-?B (NF-?B) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, we identified common gene expression changes that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes. Overall design: Naïve macrophage (naïve, M0, overall control), M1-polarized mock-infected (M1 control), M1-polarized pathogen-infected, M2-polarized mock-infected (M2 control), M1-polarized pathogen-infected were compared in a pairwise manner. Samples were used in triplicate, but analyses supported removal of 1 replicate for all treatments except M2-polarized pathogen-infected