The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondii

Abstract

The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."

Fig 1. High-throughput screening identifies genes that restore function to the STAT1 pathway in Toxoplasma-infected cells.

(A) Schematic showing workflow of STAT1 screen in Toxoplasma-infected cells. (B) Results of screen from experimental replicates, presented as fold change relative to infected cells transfected with an empty cDNA control vector. Uninfected cells transfected with empty cDNA controls are indicated in red, and infected cells transfected with MGC cDNA clones are in black. cDNAs that enhanced STAT1 activity ≥2.5-fold in both experiments are shown in the box in the upper right and inset (uninfected controls removed for clarity). (C) Protein domain family (Pfam)-based classification of nine validated cDNA hits that enhanced STAT1 activity ≥2.5-fold. (D) Protein–protein interaction network based on human orthologs of these hits (black) and STAT1 (red). Green indicates a network neighbor of STAT1. Data for panel B can be found in file S1 Data.

Fig 2. STAT1 enhancers selectively potentiate the STAT1 pathway.

Heatmap of showing row Z-scores based on log2 raw luminescent values for enhancers (right) tested on various signaling pathway reporters (top), either unstimulated (-) or stimulated with the cytokines shown on bottom (+). Values are averaged from three experiments. Color scheme legend is shown.

Fig 3. Ectopic expression of TLX potentiates a subset of IFN-γ-inducible transcripts.

(A) Heatmap and hierarchical clustering analysis of 341 genes differentially regulated (≥1.5-fold, FDR ≤ 5%) plus or minus IFN-γ stimulation of cells expressing either control vector or TLX. Row Z-scores of the mean expression data for duplicate arrays are shown. (B) Bubble plot showing enriched GO terms for each of the three clusters indicated in panel A. Bubble size indicates the number of genes associated with each term, while color intensity indicates fold enrichment. (C) Heatmap showing selected genes associated with central nervous system (CNS) development and function (from cluster 3) and immune function (from cluster 2).

Fig 4. TLX depletion in astrocyte cells impairs a subset of IFN-γ-inducible targets.

(A) Quantitative reverse transcription PCR (RT-qPCR) measuring TLX expression in U251 cells following treatment with either control siRNA or siRNA targeting TLX. (B) Heatmap and hierarchical clustering analysis of 1,418 genes differentially regulated (≥1.5-fold, FDR ≤ 5%) plus or minus IFN-γ stimulation of cells treated with either control siRNA or siRNA targeting TLX. Row Z-scores of the mean expression data for duplicate arrays are shown. (C) Bubble plot showing enriched GO terms for each of the three clusters indicated in panel A. Bubble size indicates number of genes associated with each term. Bubble color indicates whether genes associated with each term were up-regulated (red) or down-regulated (blue), while color intensity indicates fold enrichment. (D–E) Heatmaps showing (D) TLX-dependent genes associated with CNS development and function (from cluster 3) and (E) TLX-dependent, IFN-γ-regulated immune genes (from cluster 1). Asterisks in panel D indicate genes previously shown to be TLX dependent in the CNS [47].

Fig 5. TLX potentiation of STAT1 targets requires both the DNA binding domain and the ligand binding domain.

(A) STAT1 reporter activity in unstimulated or IFN-γ-stimulated U2OS cells transduced with wild-type TLX (WT) or TLX truncation mutants consisting of the DNA binding domain only (∆DBD) or the ligand binding domain only (∆LBD). (B) RT-qPCR for CXCL9 and CXCL10 expression in U2OS cells transfected with WT TLX or truncation constructs. (C) RT-qPCRfor CXCL10 and OAS2 expression in unstimulated or IFN-γ-treated U251 astrocytes pretreated with famprofazone or DMSO. Mean and standard deviation is shown for three biological replicates. * = p ≤ 0.01. Experiment was repeated three times with similar results. Data for panels A, B, and C can be found in file S1 Data.

Fig 6. TLX expression enhances occupancy of pSTAT1 on the CXCL9 and CXCL10 promoters.

ChIP with rabbit monoclonal antibody to phosphorylated STAT1 (Y701) or control rabbit IgG. U2OS cells expressing either control vector or TLX were either left untreated or stimulated with IFN-γ for 2 h. The experiment was repeated twice with similar results. Data can be found in file S1 Data.

Fig 7. TLX is induced during Toxoplasma CNS infection and is required for parasite control.

(A–D) Immunohistochemical staining of TLX in formalin-fixed paraffin-embedded sections from Toxoplasma-infected mouse brain. Sections were stained with anti-TLX (A and C) or secondary antibody as a control (B and D). (A) TLX positive cell (arrow) adjacent to Toxoplasma cyst. (C) Large cells with neuronal morphology are TLX positive. (E) Representative plots showing flow cytometric detection of intracellular interleukin-12 (IL-12) p40 in CD11c positive splenocytes from Mx1-cre TLX^f/f and TLX^f/f controls pretreated with Poly(I:C) prior to infection with values shown for the individual animal. (F) Graph of data from panel E for four-to-six mice per group. (G–H) Immunohistochemical detection of parasites in formalin-fixed paraffin embedded section. Free parasites are indicated by arrows. (I) qPCR-based measurement of parasite DNA in brain. Experiments were repeated two-to-three times with similar results. Data for panel F and I can be found in file S1 Data.