Interleukin 27, Similar to Interferons, Modulates Gene Expression of Tripartite Motif (TRIM) Family Members and Interferes with Mayaro Virus Replication in Human Macrophages

Abstract

Background: The Tripartite motif (TRIM) family includes more than 80 distinct human genes. Their function has been implicated in regulating important cellular processes, including intracellular signaling, transcription, autophagy, and innate immunity. During viral infections, macrophages are key components of innate immunity that produce interferons (IFNs) and IL27. We recently published that IL27 and IFNs induce transcriptional changes in various genes, including those involved in JAK-STAT signaling. Furthermore, IL27 and IFNs share proinflammatory and antiviral pathways in monocyte-derived macrophages (MDMs), resulting in both common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs) encoding antiviral proteins. Interestingly, many TRIM proteins have been recognized as ISGs in recent years. Although it is already very well described that TRIM expression is induced by IFNs, it is not fully understood whether TRIM genes are induced in macrophages by IL27. Therefore, in this study, we examined the effect of stimulation with IL27 and type I, II, and III IFNs on the mRNA expression profiles of TRIM genes in MDMs.

Methods: We used bulk RNA-seq to examine the TRIM expression profile of MDMs treated with IFNs or IL27. Initially, we characterized the expression patterns of different TRIM subfamilies using a heatmap. Subsequently, a volcano plot was employed to identify commonly differentially expressed TRIM genes. Additionally, we conducted gene ontology analysis with ClueGO to explore the biological processes of the regulated TRIMs, created a gene-gene interaction network using GeneMANIA, and examined protein-protein interactions with the STRING database. Finally, RNA-seq data was validated using RT-qPCR. Furthermore, the effect of IL27 on Mayaro virus replication was also evaluated.

Results: We found that IL27, similar to IFNs, upregulates several TRIM genes' expression in human macrophages. Specifically, we identified three common TRIM genes (TRIM19, 21, and 22) induced by IL27 and all types of human IFNs. Additionally, we performed the first report of transcriptional regulation of TRIM19, 21, 22, and 69 genes in response to IL27. The TRIMs involved a broad range of biological processes, including defense response to viruses, viral life cycle regulation, and negative regulation of viral processes. In addition, we observed a decrease in Mayaro virus replication in MDMs previously treated with IL27.

Conclusions: Our results show that IL27, like IFNs, modulates the transcriptional expression of different TRIM-family members involved in the induction of innate immunity and an antiviral response. In addition, the functional analysis demonstrated that, like IFN, IL27 reduced Mayaro virus replication in MDMs. This implies that IL27 and IFNs share many similarities at a functional level. Moreover, identifying distinct TRIM groups and their differential expressions in response to IL27 provides new insights into the regulatory mechanisms underlying the antiviral response in human macrophages.

Keywords: RNA-seq; interferons; interleukin 27; macrophages; tripartite motif.

Figure 1

Study workflow.

Figure 2

TRIMs expression of MDMs treated with IFNα, IFNε, IFNγ, IFNλ, and IL27. The following RNA-seqs were reanalyzed: GSE262963 (GEO)) for MDMs treated or not with IL27; GSE158434 (GEO) for MDMs treated or not with IFNα, IFNε, IFNγ, and IFNλ. (A) Heat map of TRIM expression in MDMs treated with IFNα, IFNε, IFNγ, IFNλ, and IL27. The heat map was obtained from R software (version 4.2.0) [64], where each column represents a condition (treatment to MDM) with a color: IFNα (brown), IFNγ (turquoise), IFNλ (pink), and IL27 (blue-violet). The heat map shows rows with Log₂FC values for 72 selected TRIMs (including subfamilies) indicating up-regulation (green) or down-regulation (red) of their expression. MA plots of differentially expressed genes (DEGs) of TRIMs. Each MA plot shows DEGs of TRIMs in IFNε-treated MDMs (B), DEGs of TRIMs in IFNα-treated MDMs (C), DEGs of TRIMs in IFNλ-treated MDMs (D), DEGs of TRIMs in IFNγ-treated MDMs (E), and DEGs of TRIMs in IL27-treated MDMs (F). (G) Gene-gene association network DEGs of TRIM expression in MDMs treated or not with IFNα, IFNε, IFNγ, IFNλ, and IL27. The network was obtained from Cytoscape. Genes are shown as an ellipse, grouped according to the treatment(s) in MDMs. The node colors represent the up-regulation (green) or down-regulation (red) of TRIM expression based on the mean (Log₂FC).

Figure 3

DEGs expression of IFNs or IL27-dependent or independent TRIM in MDMs. The following RNA-seqs were reanalyzed: GSE262963 (GEO) for MDMs treated or not with IL27; GSE158434 (GEO) for MDMs treated or not with IFNα, IFNε, IFNγ, IFNλ. Gene Ontology (GO) analysis was achieved in Cytoscape using the plugin ClueGO. Each GO diagram shows the common biological processes implicated in IFNs or IL27-dependent TRIMs (A) and the common biological processes implicated in IFNs or IL27-independent TRIMs (B). The number of genes regulated (the gene count) is shown for each biological process. Each GO network was realized using Cytoscape, which displays each TRIM involved in IFN or IL27-dependent biological processes (C) and the TRIM involved in IFN or IL27-independent biological processes (D). Each biological process is represented by an edge of a specific color.

Figure 4

The gene-gene interaction network of TRIM significantly up-regulated the transcripts of MDMs from the transcriptomic analysis. Each network of significantly up-regulated TRIM genes, such as TRIM21 (A), TRIM22 (B), TRIM25 (C), TRIM69 (D), TRIM19/PML (E), and TRIM31 (F), was obtained from GeneMANIA. The functions of the genes are represented by colored sections in the circle nodes, with the corresponding functions indicated in the legend labeled “Functions” using a color code. Meanwhile, the types of interactions between genes are represented by the color of the lines, and the legend labeled “Networks” provides a color code for understanding the different types of interactions. Additionally, the size of each node corresponds to the strength of the interaction.

Figure 5

The gene-gene interaction network of TRIM significantly down-regulated the transcripts of MDMs from the transcriptomic analysis. Each network of significantly down-regulated TRIM genes, such as TRIM58 (A), TRIM24 (B), and TRIM32 (C), was obtained from GeneMANIA. The functions of the genes are represented by colored sections in the circle nodes, with the corresponding functions indicated in the legend labeled “Functions” using a color code. Meanwhile, the types of interactions between genes are represented by the color of the lines, and the legend labeled “Networks” provides a color code for understanding the different types of interactions. Additionally, the size of each node corresponds to the strength of the interaction.

Figure 6

Protein-protein interaction network of TRIM-regulated transcripts of MDMs from the transcriptomic analysis. The STRING output shows 20 interactors for each analysis based on the selected TRIM and its confidence threshold. TRIM21 with 0.7 of confidence (A), TRIM22 with 0.4 of confidence (B), TRIM25 with 0.7 of confidence (C), TRIM19 with 0.7 of confidence (D), TRIM31 with 0.7 of confidence (E), TRIM24 with 0.7 of confidence (F), and TRIM32 with 0.7 of confidence (G). Nodes are proteins; the edges represent protein-protein interactions contributing to a shared function. The full donut represents the functional enrichment analysis.

Figure 7

Expression of common TRIMs among treatments with IFNs or IL27 in MDMs. (A) TRIM expression in MDMs treated or not with IFNα, IFNε, IFNγ, IFNλ, and IL27. The following RNA-seq were reanalyzed: GSE262963 (GEO) for MDMs treated or not with IL27; GSE158434 (GEO) for MDMs treated or not with IFNα, IFNε, IFNγ, and IFNλ. Logarithm of fold change (Log₂FC) ratios for TRIM21, TRIM22, TRIM25, TRIM69, TRIM19, and TRIM31 in Treatment/Control. n = 3. Data are represented as mean ± SEM. (B) TRIM expression in MDMs treated or not with IFNβ, IFNγ, IFNλ1, and IL27. Cell lysates were obtained at 24 h post-treatment, and RT-qPCR was performed. Logarithm of fold change (Log₂FC) ratios for TRIM21, TRIM22, TRIM25, TRIM69, TRIM19, and TRIM31 in Treatment/Control. n = 4, without technical triplicates. Data are represented as mean ± SEM. A Log₂FC of 0.6 and −0.6 were considered as up-regulation or down-regulation of gene expression, respectively (dotted lines). Linear regression analysis of Log₂FC ratios between mRNA level transcriptomes and mRNA level validation was performed for each TRIM (C). The R square is shown.

Figure 8

MAYV replication in human macrophages pre-treated with IL27 and expression of TRIM19, 21, 22, and 69 and infected with MAYV. Human macrophages were pre-treated with IL27 (1, 5, 10, 25 ng/mL) or IFNβ (25 ng/mL) for 6 h, followed by infection with MAYV at a MOI of 0.5. Culture supernatants and cells were collected at 24 h.p.i. Viral titration was determined by plaque assay on Vero cells using culture supernatants, while cell lysates underwent RT-qPCR analysis. MAYV replication in MDMs treated with IL27 (A). Percentage inhibition of MAYV replication and EC50 of IL27 in MDMs (B). The logarithm of fold change (Log₂FC) ratios for TRIM19 (C), TRIM21 (D), TRIM22 (E), and TRIM69 (F) relative to the control are shown. The study included three replicates (n = 3, without technical triplicates), and the data are presented as mean ± SEM. A Log₂FC of 0.6 and −0.6 indicated up-regulation and down-regulation of gene expression, respectively (dotted lines). Asterisks refer to statistically significant differences between control and treatments as: ** p < 0.01, *** p < 0.001.