Modulation of Macrophage Antifungal Activity by the Transcriptional Co-regulator CITED1 - ABSTRACT
Cryptococcus neoformans (Cn) is a ubiquitous pathogenic yeast found in urban environments that infects most
people within the first few years of life. Infection typically occurs through the inhalation of Cn propagules, and in
immunocompetent individuals these are efficiently ingested and killed by alveolar macrophages. However, if
Cn avoids destruction, it utilizes macrophages as a growth niche and disseminates from the lungs, resulting in
fungal meningitis, which is lethal if not treated (~82% mortality rate), causing ~181,000 deaths annually. The
emergence of drug-resistant strains and the toxic side-effects of existing antifungal agents necessitates the
development of new strategies to combat Cn infections. To facilitate this, an improved understanding of how
Cn avoids destruction by macrophages is necessary. Therefore, the overarching goal of our research
program is to understand how Cn infection impacts macrophage fungicidal activity and identify host
factors that enhance it. Macrophage anticryptococcal activity is increased by M1 polarization, which is
stimulated by IFNγ exposure. This triggers the altered expression of >1000 genes, largely directed by STAT1,
including Nos2, which encodes iNOS, an enzyme required to produce antimicrobial reactive nitrogen species.
During the prior funding period, we showed that while intracellular Cn infection partially reversed gene
expression changes associated with M1 polarization, iNOS levels were increased. This was accompanied by
expression of CITED1, which we showed to function as a co-activator of STAT1-regulated genes. As a step
towards achieving our long-term goal, we will investigate how CITED1 enhances the IFNγ response and
affects the polarization and fungicidal activity of Cn-infected macrophages. Specifically, we will: Determine
how CITED1 impacts the M1 transcriptome and outcome of intracellular Cn infection. We will (A) identify
CITED1-regulated genes using a combination of transcriptome and chromatin profiling and (B) Determine the
effect of CITED1 on the M1 phenotype by measuring its impact on proinflammatory cytokine secretion and the
iNOS-dependent anticryptococcal activity of these cells. 2) Determine how CITED1 modulates IFNγ-
stimulated gene (ISG) expression. As our preliminary data indicates a positive feedback relationship
between STAT1 and CITED1, we will (A) investigate the mechanism by which CITED1 increases the
expression of STAT1-regulated ISGs by measuring the effect of CITED1 on CBP:STAT1 chromatin complexes
and (B) determine how STAT1-regulates Cited1 transcription using reporter and CUT&RUN-based assays.
Additionally, we will investigate how IFNγ stimulates the nuclear translocation of CITED1 proteins using a live
cell imaging approach. Collectively, these studies will provide new insights into how intracellular Cn-stimulated
expression of CITED1 alters ISG expression in host macrophages and how this might influence their
phenotype and fungicidal activity.
