RNA-sequencing Identifies Novel Pathways in Sarcoidosis Monocytes

Abstract

Sarcoidosis is a complex systemic granulomatous disorder of unknown etiology. Genome-wide association studies have not been able to explain a causative role for nucleotide variation in its pathogenesis. The goal of the present study was to identify the gene expression profile and the cellular pathways altered in sarcoidosis monocytes via RNA-sequencing. Peripheral blood monocytes play a role in sarcoidosis inflammation. Therefore, we determined and compared the transcriptional signature of monocytes from peripheral blood from sarcoidosis patients and healthy controls via RNA-sequencing. We found 2,446 differentially expressed (DE) genes between sarcoidosis and healthy control monocytes. Analysis of these DE genes showed enrichment for ribosome, phagocytosis, lysosome, proteasome, oxidative phosphorylation and metabolic pathways. RNA-sequencing identified upregulation of genes involved in phagocytosis and lysosomal pathway in sarcoidosis monocytes, whereas genes involved in proteasome degradation and ribosomal pathways were downregulated. Further studies are needed to investigate the role of specific genes involved in the identified pathways and their possible interaction leading to sarcoidosis pathology.

Figure 1

Differential gene expression and pathways between control and sarcoidosis monocytes. (a) Scatter plot of the entire gene expression data (healthy versus sarcoidosis monocytes) analyzed by DEseq2 analysis tool, where the log₂-fold change (>0.5) of each gene is plotted against the total number of counts recorded for that gene. DE genes (FDR < 5%) are highlighted in red. (b) Significant pathways modulated in sarcoidosis monocytes as compared to healthy controls. Pathway analysis was done on the DE genes (log₂-fold change > 0.6 and FDR < 5%) using iPathwayGuide analysis tool that uses two types of evidence: the over-representation on the horizontal axis (pORA) and the perturbation on the vertical axis (pAcc). Significant pathways (FDR < 5%) are shown in red, whereas non-significant are in black. The size of the circle is proportional to the number of genes in that pathway.

Figure 2

Heat maps of DE genes of three significant pathways in sarcoidosis versus healthy monocytes. (a) Heatmap of the 143 genes involved in metabolic pathways between two groups. (b) Heatmaps of 19 genes involved in phagocytosis and (c) Heatmaps of 9 genes involved in proteasome (log₂-fold change > 0.5 & FDR < 5%). Dendrograms according to means identifying genes levels in all three pathways show two distinct clusters. Red shade represents high expression and blue shade represents low expression.

Figure 3

Downregulation of genes involved in ribosomal pathway in sarcoidosis monocytes. Graphic illustration of pathway analysis of DE (log₂-fold change with FDR < 0.05) genes related to ribosome in sarcoidosis monocytes. The pathway diagram is overlaid with the computed perturbation of each gene. The perturbation accounts both for the gene’s measured fold changes and for accumulated perturbation propagated from any upstream genes (accumulation). The color intensity corresponds to the level of upregulation (red) or downregulation (blue) of the DE genes in sarcoidosis monocytes versus healthy monocytes. Note: A number of genes encoding large and small ribosomal subunits were downregulated.

Figure 4

Upregulation of genes involved in lysosomal pathway in sarcoidosis monocytes. Graphic illustration of pathway analysis of DE (log₂-fold change with FDR < 0.05) genes related to lysosome in sarcoidosis monocytes. The pathway diagram is overlaid with the computed perturbation of each gene. The perturbation accounts both for the gene’s measured fold changes and for accumulated perturbation propagated from any upstream genes (accumulation). The color intensity corresponds to the level of upregulation (red) or downregulation (blue) of the DE genes in sarcoidosis monocytes versus healthy monocytes. Note: Various genes involved in lysosomal membrane proteins and acid hydrolases were upregulated except LAPTM4B gene which was downregulated.

Figure 5

Proteasome in sarcoidosis monocytes. Graphic illustration of pathway analysis of DE (log₂-fold change with FDR < 0.05) genes related to proteasome and immunoproteasome in sarcoidosis monocytes. The pathway diagram is overlaid with the computed perturbation of each gene. The perturbation accounts both for the gene’s measured fold changes and for accumulated perturbation propagated from any upstream genes (accumulation). Note: Several genes involved in regulatory and core particles and immunoproteasomes were downregulated (blue) in sarcoidosis monocytes.

Figure 6

Validation of RNA-seq data by qRT-PCR. Total RNA was extracted from the monocytes from independent sets of 10 sarcoidosis patients and 10 healthy controls. Isolated RNAs were and reverse-transcribed using the Reverse Transcription System. The primers targeted (a) ATP6AP1, (b) CYBB, (c) LAMP2, (d) SERPIN1A, (e) RPSA and (f) RPL10A to amplify cDNA using iQSYBR Green Supermix. Relative mRNA levels were calculated by normalizing to β-actin. Box plots represent the normalized expression level of each gene of monocytes of healthy controls and sarcoidosis monocytes. Data were analyzed using the paired, two-tailed Student’s t test and the results were expressed as fold change. *Represents a p value < 0.05 and **signifies a p < 0.001.