The Importance of mRNA stabilisation for expression of Th17 phenotype

The epigenetic regulation of immunity and its role in inflammatory disease has become a growing focus of research over the last few years.  Much of this work has investigated chromatin modification and its impact on the accessibility of transcription factor binding sites.  An additional epigenetic mechanism for regulating cell phenotype is the chemical modification of RNA transcripts. These modifications include the 2′O-methylation of nucleotides (position 2 on the ribose ring) and the methylation of adenine (at the amino group, position 6; termed m⁶A) and cytosine bases (at position 5; termed m⁵C).  These modifications may modulate the half-life of transcripts and enhance translation.

There are a range of enzymes that perform RNA modification, including the methyltransferase-like (METTL) enzymes such as METTL3, which has been shown to have a role in controlling regulatory T cell [Tong et al. doi: 10.1038/cr.2018.7] and T follicular helper cell phenotypes [Yao et al. doi: 10.1038/s41467-021-21594-6]; METTL enzymes are generally associated with m⁶A.  Another enzyme with RNA methyltransferase activity is the NOP2/sun protein family member 2, Nsun2, which is associated with m⁵C RNA modification [Frye & Watt. doi: 10.1016/j.cub.2006.04.027].  Increased m⁵C methylation has been shown in CD4⁺ T cells from individuals with systemic lupus erythematosus [Lu et al. DOI: 10.1186/s10020-023-00643-4]; in addition, methylation of the ll17a mRNA by Nsun2 can enhance its translation in rat T cells [Wang et al. doi: 10.1016/j.bbrc.2017.09.069].

The role of Nsun2 in the expression of the Th17 cell phenotype and its impact on intestinal inflammation was the subject of a recently published study by scientists from the Chinese Academy of Sciences in Beijing [Yang et al.  doi: 10.1038/s41467-023-36595-w].  This work was centred on the production of an Nsun2 knockout (Nsun2 ^-/-) mouse, using CRISPR/cas9 methodology to delete a 56bp section from the middle of exon 2.  In wild-type mice (C57BL/6J), Nsun2 was expressed primarily in lymphoid tissues.  Differentiated T cells also showed increased expression relative to naïve T cells, with some phenotypes showing higher expression, particularly Th17 cells.  In contrast, expression of Nsun2 protein could not be demonstrated in lymphoid tissues from Nsun2 ^-/- mice.  Overall, loss of Nsun2 did not affect the relative proportions of myeloid cells, B cells or T cells (both CD4⁺ T helper cells and CD8⁺ cytotoxic T cells) in leukocyte populations isolated from both spleen and peripheral blood; however, the proportion of IL-17-expressing cells in each leukocyte population was significantly reduced, as was the potential of naïve CD4⁺ T cells for in vitro differentiation into Th17 cells.

Following on from their initial observations of the Nsun2 ^-/- mouse, Yang and colleagues investigated the relationship between Nsun2 and RORγt, the main transcription factor determining Th17 cell phenotype.  Using immunoprecipitation-mass spectrometry, it was shown that (apart from itself) the protein to show the strongest direct interaction with RORγt was Nsun2.  This interaction was demonstrated visually within the nuclei of in vitro differentiated Th17 cells using a proximity ligation assay, which relies on co-localisation of the two proteins in order to generate a fluorescent signal (based on the detection of an oligonucleotide produced by the amplification of hybridised and ligated antibody-linked primers sequences).   Employing an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq), it was found that in Nsun2 ^-/- Th17 cells, the absence of Nsun2 did not affect the ability of RORγt to bind to transcription start sites, nor did it affect the ability of RORγt to initiate transcription, with no change in the frequency of chromatin-associated RNAs.  Immunoprecipitation assays also confirmed that both RORγt and Nsun2 had matching chromatin biding characteristics, as using either protein as the target in the assay enriched for Il17a and Il17f chromatin-associated transcripts to a similar extent.

RNAseq was used to show that 430 genes were down-regulated in Nsun2^-/- cells (relative to wild-type).  Approximately 40 % of these genes demonstrated Nsun2 binding and show m⁵C and in turn, 40 % of these genes were also RORγt target genes, including Il17a and Il17f.  Using Il17a as a reference gene, it was shown that the phenotype of Th17 cells could be rescued by retroviral transfection of Nsun2 and that the rescue was dependent on the protein’s methyl transferase activity, as transfection of Nsun2 with a C to A single point mutation at base 321 was unable to rescue the phenotype.   The authors were able to demonstrate that Il17a mRNA in Th17 cells from wild-type mice was more stable than the same mRNA in Th17 cells from Nsun2^-/- mice, with a half-life of approximately 2 hours, as compared to less than 30 minutes for the knockout cells.  By making a RNA reporter minigene based on the mRNA of the Il17a gene, it was shown that m⁵C modification of cytosine 424 within the reporter could increase the reporter’s stability when transfected into Th17 cells, in agreement with the previous findings of Wang and colleagues [doi: 10.1016/j.bbrc.2017.09.069] who had demonstrated that modification of the equivalent cytosine residue in the rat Il17a mRNA also enhanced its half-life.

The importance of Nsun2 for Th17-driven inflammation was demonstrated using the DSS-induced colitis model of inflammatory bowel disease.  Using this model, it was shown that Nsun2^-/- mice had reduced susceptibility to disease, showing less body weight loss and reduced pathological changes in the large bowel, as compared to wild-type mice.  This reduction in disease was associated with a decrease in the proportion of Th17 cells, but not other CD4⁺ T cell populations, within the spleen and mesenteric lymph nodes of the Nsun2^-/- mice.  The reduced potential for colitis development associated with the absence of Nsun2 was negated by the administration of exogenous IL-17.  In addition to the results from the DSS model, the adoptive transfer of naïve T cells from Nsun2^-/- mice to Rag1^-/- knockout mice failed to induce colitis and was associated with a reduction in Th17 cells, in contrast to the transfer of naïve T cells. RNAseq analysis was used to define all the major cell populations involved in the development of DSS-induced colitis.  Specific changes in gene expression were observed in wild-type mice within the different cell types, in response to DSS treatment.  Many of these changes were ameliorated in the absence of Nsun2 expression and were specific to cells expressing IL-17 receptors.

Overall, the authors demonstrated that regulation of RNA stability can influence cell phenotype and the development of inflammatory disease.  This raises the possibility that RNA modifying enzymes, like Nsun2, which are recruited by transcription factors to modulate the execution of their phenotypic programme, could be useful therapeutic targets.

Epistem Services

In a similar manner to the techniques described here, at Epistem there is a routine deployment of multi-modal assessment studies of a wide variety of tissues and cells utilising a range of expertise to delineate cellular compartmentalisation, form and function.

To aid this assessment, Laser Capture Microscopy (LCM) is a powerful tool used at Epistem to select individual cells from a population in a tissue for input into downstream histochemical profiling or transcriptomic analysis via single cell RNA-Seq (scRNA-Seq) allowing for precise categorisation of the cell state with a comparably high resolution to Slide-seq.

This high resolution is especially important given the complexity created by the heterogeneity of population cells, where subpopulations in a pool are a primary target but could be missed. The subpopulations can be identified and spatially assigned to aid understanding of the driving virulence force behind disease or tumour progression.