Transcriptional Profiling of Monocytes Deficient in Nuclear Orphan Receptors NR4A2 and NR4A3 Reveals Distinct Signalling Roles Related to Antigen Presentation and Viral Response

Abstract

The nuclear receptor sub-family 4 group A (NR4A) family are early response genes that encode proteins that are activated in several tissues/cells in response to a variety of stressors. The NR4A family comprises NR4A1, NR4A2 and NR4A3 of which NR4A2 and NR4A3 are under researched and less understood, particularly in the context of immune cells. NR4A expression is associated with multiple diseases e.g. arthritis and atherosclerosis and the development of NR4A-targetting molecules as therapeutics is a current focus in this research field. Here, we use a combination of RNA-sequencing coupled with strategic bioinformatic analysis to investigate the down-stream effects of NR4A2 and NR4A3 in monocytes and dissect their common and distinct signalling roles. Our data reveals that NR4A2 and NR4A3 depletion has a robust and broad-reaching effect on transcription in both the unstimulated state and in the presence of LPS. Interestingly, many of the genes affected were present in both the unstimulated and stimulated states revealing a previously unappreciated role for the NR4As in unstimulated cells. Strategic clustering and bioinformatic analysis identified both distinct and common transcriptional roles for NR4A2 and NR4A3 in monocytes. NR4A2 notably was linked by both bioinformatic clustering analysis and transcription factor interactome analysis to pathways associated with antigen presentation and regulation of MHC genes. NR4A3 in contrast was more closely linked to pathways associated with viral response. Functional studies further support our data analysis pointing towards preferential/selective roles for NR4A2 in the regulation of antigen processing with common roles for NR4A2 and NR4A3 evident with respect to cell migration. Taken together this study provides novel mechanistic insights into the role of the enigmatic nuclear receptors NR4A2 and NR4A3 in monocytes.

Keywords: NR4A; NR4A2; NR4A3; cell signalling; monocytes; nuclear orphan receptor; nuclear receptor; transcriptomics.

Figure 1

Knockdown of NR4A2 has a distinct transcriptional profile in the basal state. Principal component analysis (PCA) plot of transduced cells based on RNA-seq data between shNT and shNR4A2 cells (A). Volcano plot of significant DEGs (p<0.05) upregulated (log2fc>/=1) (green) or downregulated (log2fc</=-1) (red) by knockdown of NR4A2 in THP-1 cells based on RNA-seq data (B). Top gene ontology (GO) terms associated with knockdown of NR4A2 in THP-1 cells based on RNA-seq data (C). Summary table of significant DEGs upregulated (green) and downregulated (red) by knockdown of NR4A2 in THP-1 cells based on RNA-seq data (D).

Figure 2

Knockdown of NR4A2 has a distinct transcriptional profile in the presence of LPS. Principal component analysis (PCA) plot of transduced cells based on RNA-seq data between shNT and shNR4A2 cells in the presence of LPS (A). Volcano plot of significant DEGs (p<0.05) upregulated (log2fc>/=1) (green) or downregulated (log2fc</=-1) (red) by knockdown of NR4A2 in THP-1 cells in the presence of LPS based on RNA-seq data (B). Top gene ontology (GO) terms associated with knockdown of NR4A2 in THP-1 cells in the presence of LPS based on RNA-seq data (C). Summary table of significant DEGs upregulated (green) and downregulated (red) by knockdown of NR4A2 in THP-1 cells in the presence of LPS based on RNA-seq data (D).

Figure 3

Knockdown of NR4A3 has a distinct transcriptional profile in the basal state. Principal component analysis (PCA) plot of transduced cells based on RNA-seq data between shNT and shNR4A3 cells (A). Volcano plot of significant DEGs (p<0.05) upregulated (log2fc>/=1) (green) or downregulated (log2fc</=-1) (red) by knockdown of NR4A3 in THP-1 cells based on RNA-seq data (B). Top gene ontology (GO) terms associated with knockdown of NR4A3 in THP-1 cells based on RNA-seq data (C). Summary table of significant DEGs upregulated (green) and downregulated (red) by knockdown of NR4A3 in THP-1 cells based on RNA-seq data (D).

Figure 4

Knockdown of NR4A3 has a distinct transcriptional profile in the presence of LPS. Principal component analysis (PCA) plot of transduced cells based on RNA-seq data between shNT and shNR4A3 in the presence of LPS cells (A). Volcano plot of significant DEGs (p<0.05) upregulated (log2fc>/=1) (green) or downregulated (log2fc</=-1) (red) by knockdown of NR4A3 in THP-1 cells in the presence of LPS based on RNA-seq data (B). Top gene ontology (GO) terms associated with knockdown of NR4A3 in THP-1 cells in the presence of LPS based on RNA-seq data (C). Summary table of significant DEGs upregulated (green) and downregulated (red) by knockdown of NR4A3 in THP-1 cells in the presence of LPS based on RNA-seq data (D).

Figure 5

Analysis of gene clusters regulated by NR4A2 alone, NR4A3 alone and by both NR4A2 and NR4A3. Venn diagram outlining clusters of genes regulated by NR4A2 alone (cluster 1), NR4A2 and NR4A3 (cluster 2) and NR4A3 alone (cluster 3) from RNA-seq analysis using a threshold of p-adj </=0.05 for statistical significance (A). Table describing the top 3 most enriched GO terms in genes associated with clusters 1-3 (top panel) in addition to the statistically significantly expressed genes in those clusters associated with the GO terms (bottom panel) (B).

Figure 6

Knockdown of NR4A2 but not NR4A3 increases bead association, while knockdown of either NR4A2 or NR4A3 increases cell migration. Knockdown of NR4A2 selectively promotes antigen presentation while cell migration is comparably enhanced by loss of NR4A2 or NR4A3. Fluorescent bead association assay in which shNT THP-1 cells, NR4A2 and NR4A3 depleted cells were treated +/-1μg/ml LPS for 2hrs followed by addition of 1 μm fluorescent beads source for a further 2hrs. Fluorescence was measured at 535/575 nm. Results expressed as fold over control (FOC) (unstimulated shNT-THP-1) for n= 4 independent experiments (A). Cell migration assay in which shNT-THP-1 cells were exposed for 2hrs to conditioned media from shNT-THP1, NR4A2 and NR4A3 depleted cells +/- 100ng/ml LPS (22hrs). Results expressed as migrated cells for n=4 independent experiments (B). Cell migration assay in which shNT-THP-1 cells were exposed for 2hrs to conditioned media from shNT-THP1, NR4A2 and NR4A3 depleted cells + 100ng/ml LPS (22hrs). Prior to migration assay conditioned media was treated +/- an anti-MCP1 neutralising antibody (2ng/ml) for approximately 1hr. Results expressed as migrated cells for n=3 independent experiments (C). Note data from non-antibody treated media is also included as part of (B) Representative images (10X magnification) of migrated cells +/- LPS, +/- anti-MCP-1 fixed to transwell inserts shown in (D). Statistical analysis (A, B & C) was performed using 2-way ANOVA (assuming sphericity) followed by Sidak’s multiple comparisons test (A&B). **p</= 0.01; ns, p > 0.05.

Figure 7

NR4A expression and depletion in THP-1 monocytes. RAW TPM values (mean +/- SEM) extracted from RNA-seq for NR4A1 (A) NR4A2 (B) and NR4A3 (C) in shNT-THP1 cells in the control (unstimulated state) and following LPS treatment (2.5μg/ml for 2hrs) all shown on common axis. RAW TPM values extracted from RNA-seq for NR4A1 (D) NR4A2 (E) and NR4A3 (F) in shNT-THP1 cells, shNR4A2-THP1 cells and shNR4A3-THP1 cells in the control (unstimulated state) and following LPS treatment (2.5μg/ml for 2hrs). N=3 independent experiments. Adjusted p-values (Padj) for specific comparisons are contained in the tables below the TPM data. Statistically significant Padj are denoted in bold font. Comparisons between shNR4A2 cells and control are denoted in the red boxes, comparisons between the shNR4A3 cells and control are denoted in the blue boxes. Statistically significant comparisons are further signposted using bars on the TPM graphs.

Figure 8

Individual knockdown of NR4A2 and NR4A3 alters the expression of distinct and overlapping genes. RAW TPM values (mean +/- SEM) extracted from RNA-seq for POU4F2 (A) HUNK (B) NCAM1 (C) CACNA2D1 (D) MCF2 (E) and IL-6 (F) in shNT-THP1 cells, shNR4A2-THP1 cells and shNR4A3-THP1 cells in the control (unstimulated state) and following LPS treatment (2.5μg/ml for 2hrs). N=3 independent experiments. Adjusted p-values (Padj) for specific comparisons are contained in the tables below the TPM data. Statistically significant Padj are denoted in bold font. Comparisons between shNR4A2 cells and control are denoted in the red boxes, comparisons between the shNR4A3 cells and control are denoted in the blue boxes. Statistically significant comparisons are further signposted using bars on the TPM graphs.

Figure 9

Transcription factor enrichment analysis of genes in NR4A2 and NR4A3 depleted cells. Table depicting the top 10 results ranked by z-score from Metacore transcription factor enrichment analysis performed on NR4A2 (red) and NR4A3 (blue) depleted cells. Network object name refers to the enriched transcription factor network, p-value refers to the statistical significance of the observation and z-score refers to the distance from the expected mean.

Figure 10

Knockdown of NR4A2 alters MHC Class1 gene expression. RAW TPM values (mean +/- SEM) extracted from RNA-seq for genes associated with MHC-Class I (green rectangles) and MHC-Class II (purple rectangles). Data shown for HLA-B (A) HLA-C (B) HLA-F (C) HLA-DRA (D) HLA-DRB1 (E) and HLA-DPB1 (F) in shNT-THP1 cells, shNR4A2-THP1 cells and shNR4A3-THP1 cells in the control (unstimulated state) and following LPS treatment (2.5μg/ml for 2hrs). N=3 independent experiments. Adjusted p-values (Padj) for specific comparisons are contained in the tables below the TPM data. Statistically significant Padj are denoted in bold font. Comparisons between shNR4A2 cells and control are denoted in the red boxes, comparisons between the shNR4A3 cells and control are denoted in the blue boxes. Statistically significant comparisons are further signposted using bars on the TPM graphs.