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The magnitude of IFN-g responses is fine-tuned by DNA architecture and the non-coding transcript of Ifng-as1

ABSTRACT: IFN-g, critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA Deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired IFN-g response and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-g production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this lncRNA and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.

ORGANISM(S): Mus musculus

PROVIDER: GSE132531 | GEO | 2019/07/19

REPOSITORIES: GEO

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Project description:Intracellular parasites reprogram the host functions for their survival and reproduction. Conversely, the infected host attempts to defend the microbial insult. The extent and relevance of parasite-mediated host response in vivo remains poorly studied. We utilized Eimeria falciformis, an obligate intracellular parasite completing its entire life cycle in the mouse intestinal epithelium, to identify and validate the host determinants of the parasite infection. The most prominent mouse genes induced during the onset of asexual (24 hrs) and sexual (144 hrs) parasite cycle include IFNg-regulated factors, e.g., immunity-related GTPases IRGA6/B6/D/M2/M3, guanylate-binding proteins GBP2/3/5/8, chemokines CxCL9-11 and several enzymes of the kynurenine pathway including indoleamine 2,3-dioxygenase 1 (IDO1). These results indicated a multifarious innate defense (tryptophan catabolism, IRG, GBP, chemokines signaling) mounted by epithelial cells, and a consequential adaptive immune response (chemokines-cytokines signaling, lymphocyte recruitment). A notable increase in the inflammation- and immunity-associated transcripts correlated with the severity of infection and influx of B-cells, T-cells and macrophages to the parasitized tissue. Indeed, parasite growth was enhanced in the animals inhibited for CxCr3, a major chemokine receptor on immune cells. Interestingly, despite a prominent induction, the mouse IRGB6 failed to recognize and disrupt the parasitophorous vacuole in the parasite cultures, implying an immune evasion by E. falciformis. Likewise, the oocyst output was impaired in IFNg-R-/- and IDO1-/- mice, which signifies a subversion of IFNg-signaling by the parasite to promote its growth. In brief, the Eimeria-rodent model shows contrasting roles of IFNg-signaling for parasite development, identifies a retinue of potential host determinants, and epitomizes its efficacy for in vivo parasite-host interaction studies. Microarray experiments were performed as dual-color hybridizations on Agilent mouse whole genome catalog 44K arrays. To compensate for dye-specific effects, a dye-reversal color-swap was applied.

Project description:Background & Aims: Inflammatory bowel diseases (IBD) are clinically manifested as ulcerative colitis (UC) and Crohn’s Disease (CD). Novel high throughput technologies revealed new categories of genes, including the long non-coding RNAs (lncRNAs), which have been involved in the pathogenesis of different human diseases; however the role and function of lncRNAs in the UC pathogenesis has not been evaluated. Methods: The gene expression patterns for both non-coding (lncRNAs) and coding (mRNA) transcripts were profiled in UC (n=8) and control (n=7) patients by ArrayStar assays. The differentially expressed genes were used to develop lncRNA signatures in UC samples. Ingenuity Software Analysis (IPA) program was used to identify the up-stream regulators of IFNG-AS1. Jurkat T cells were activated by PMA/ionomycin and IFNG and TNF protein levels were assessed by ELISA assay. Two anti-sense molecules were designed and used to block IFNG-AS1 expression levels. Colonic tissues from TNBS-treated and IL10-/- mice were used to extract RNA and examine INFG-AS1 expression by real-time PCR. Results: A unique set of lncRNAs was found to be differentially expressed between UC (n=15) and control samples (n=16). Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, p-value: 7.07E-06). Bioinformatic analyses showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine, interferon-gamma (IFNG). Using the TNBS and IL10-/- mouse models of colitis, we found increased colonic expression of this lncRNA during active colitis. Utilizing the Jurkat T cell model system, we explored the mechanisms of action and found that IFNG-AS1 can positively regulate IFNG expression. Conclusions: Novel lncRNA signatures were identified to differentiate UC patients with active disease, in remission and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and was found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taken together, our study revealed novel lncRNA signatures deregulated in ulcerative colitis and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of non-coding RNA mechanisms on regulation of IBD-related inflammatory responses.

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The magnitude of IFN-g responses is fine-tuned by DNA architecture and the non-coding transcript of Ifng-as1

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