Modulation of Macrophage Antifungal Activity by the Transcriptional Coregulator, CITED1

Abstract

The fungal pathogen Cryptococcus neoformans (Cn) causes 220,000 cryptococcosis cases and 181,000 deaths annually, mostly in immunocompromised people. Alveolar macrophages serve as the first line of defense against Cn, which typically enters the body as inhaled propagules and is critical to the outcome of the infection. The ability of macrophages to eliminate Cn is influenced by their polarization state, a set of transitory phenotypes characterized by the altered expression of >1000 genes, with macrophages polarized to the proinflammatory M1 state by IFNγ-stimulation exhibiting the highest fungicidal activity. RNA sequencing-based transcriptome profiling was employed to assess if Cn impacts the expression of M1-associated genes in RAW264.7 macrophages. Intracellular infection partially reverted the gene expression profile to a naïve (M0)-like state. To ascertain the mechanism underlying this gene expression, transcriptional regulators were identified among the differentially expressed genes (DEGs) in mock vs. Cn-infected cells. Amongst these, CITED1 exhibited the largest fold increase in expression. CITED1 encodes a member of the CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail (CITED) family of transcriptional co-regulators. Since CITED2 inhibits M1 polarization by preventing STAT1 from recruiting the histone acetyltransferase, CBP/p300, to gene cis-regulatory sites, we hypothesized that CITED1 would have similar effects. This was tested using a loss- and gain-of-function approach coupled with RNAseq. Surprisingly, ectopic CITED1 expression increased the expression of multiple interferon-stimulated genes (ISGs), including Ccl2, ifit1, Isg15, and Oas2, and this was reversed In Cited1 null cells. These findings imply an antagonistic relationship between CITED proteins in controlling macrophage inflammatory activity. Additionally, it was found that Cited1 activity is regulated at multiple levels post-IFNγ stimulation. Cited1 transcription was found to be STAT1-dependent, and IFNγ increased CITED1 phosphorylation and nuclear accumulation. Collectively, these data demonstrate that CITED1 enhances the transcriptional response to IFNγ stimulation and acts as a positive regulator of macrophage proinflammatory function.