Endogenous retroelements promote tolerance to dietary antigens

Keywords

• Oral Tolerance

• Retroelements

• Type I Interferon

• Gut Immunity

Main Findings

Oral tolerance, or systemic unresponsiveness to food antigens first encountered within the gastrointestinal tract, protects the body from inappropriate allergic reactions to dietary antigens. This tolerogenic environment is primarily shaped by the induction of dietary antigen-specific regulatory T cells (Tregs), a process driven by secreted factors such as TGF-β and retinoic acid produced by conventional dendritic cells (cDCs). Rivera et al. aim to uncover the upstream mediators that regulate this induction, focusing particularly on the role of retroelements in establishing oral tolerance. Retroelements are mobile genetic elements that integrate into the genome via a reverse-transcribed RNA intermediate. Emerging evidence suggests they play critical roles in maintaining homeostasis and regulating immunity. In a recent preprint (not peer-reviewed), Rivera et al. report that these elements may contribute to creating an immunoregulatory milieu within the gut that suppresses immune responses to food antigens.

To model oral tolerance, the authors employed a delayed-type hypersensitivity (DTH) model, in which wildtype mice are orally exposed to ovalbumin, an antigen arising from egg whites, during a tolerization phase before being immunized then challenged with the ovalbumin at a distal site. To test the contribution of reverse transcriptase (RT) towards this process, mice were administered RT inhibitors during tolerization. In response to ovalbumin challenge, both non-tolerized mice and tolerized mice exposed to RT inhibitors mounted inflammatory responses such as skin swelling and granulocyte accumulation at the injection site. Further, flow cytometry analysis of gut tissues from untreated and treated mice revealed a reduction in overall Tregswithin the small intestine upon RT inhibition. To test whether RT inhibition restricted the induction of antigen-specific Tregs, Rivera et al. adoptively transferred T cells from OT-II mice, which contain ovalbumin-specific T cells, into syngeneic wildtype mice prior to the administration of RT inhibitors. RT inhibition restricted the number of OT-II Tregswithin the mesenteric lymph nodes. To confirm this phenotype was not specific to ovalbumin, the authors treated wildtype mice with a diet containing the wheat antigen gliadin with and without RT inhibitors. The frequency of gliadin-specific Tregs within secondary lymphoid organs was reduced upon the addition of RT inhibitors. Together, this work suggests RT supports the induction of food antigen-specific Tregs and drives oral tolerance. Notably, RT inhibition exerted minimal changes on the species abundance and diversity of the microbiota, and studies in germ-free mice recapitulated earlier studies in specific pathogen free mice, indicating this effect was independent of the microbiota.

The authors next characterized changes within the gut upon RT inhibitor exposure. T cell receptor (TCR) single-cell RNA sequencing (scRNAseq) revealed a reduction in shared TCR clonotypes upon RT inhibitor treatment. The authors next evaluated known mediators of Treginduction within the gut: cDC1s and CD103+ cDC2s. Both cDC subsets exhibited lower TGF-ß and retinoic acid production upon RT inhibition. scRNAseq of cDC subsets revealed a downregulation of antigen processing genes in cDC1s and cytokine and chemokine genes in CD103+ cDC2s upon RT inhibition, cumulatively suggesting that RT supports the induction and clonal expansion of food antigen-specific Tregs by inducing tolerogenic programs in cDC subsets.

Next, the authors returned to their DTH model and exposed mice to inhibitors of either STING or the IFN-I receptor IFNAR. Systemic inhibition of STING or IFN-I receptor IFNAR phenocopied the results obtained in earlier experiments of RT inhibitor-treated mice, implicating RT-STING-IFN-I axis in the establishment of oral tolerance.

Transcriptomic analysis of gut tissues revealed endogenous retroelements were constitutively expressed in the small intestine by epithelial cells and cDC1s. Select retroelement proteins MLV and L1 ORF1p were mostly highly expressed by gut epithelial cells followed by cDC1s from flow cytometry analyses. Thus, the authors next investigated the contribution of STING signalling in gut epithelial cells during the induction of oral tolerance using STING Villin x Cre ER mice, which recapitulated the results obtained upon treatment with RT, STING, or IFNAR inhibitors. Collectively, this preprint describes a role of retroelement-encoded RT in mediating tolerogenic cDC functions that, in turn, promote the induction and clonal expansion of food antigen-specific Tregswithin the small intestine.

Limitations

•  The authors posit retroelement-encoded RT activates STING signalling in gut epithelial cells, yet cDC1s express greater retroelement transcripts than gut epithelial cells in Fig. 4A-B. Performing oral tolerance experiments in mice harbouring a specific depletion of STING in cDC1s (such as using the XCR1CreSTINGfl/fl model) would help clarify the contribution of STING signalling in cDC1s towards this process.

• Most retroelement loci in humans have lost their ability to encode functional RT enzymes. Given this work was performed in mouse models, the use of human tissues for subsequent studies would help expand the clinical relevance of these findings.

• The specific retroelement loci implicated in this process were not identified. Using computational tools to predict which highly expressed retroelements encode functional RT would help further elucidate which retroelement-expressing cell types are implicated in this response.

Significance/Novelty

Once dismissed as ‘junk DNA’, retroelements comprise nearly half the human genome and impose a significant metabolic cost to cells. This preprint by Rivera et al. contributes to the growing body of literature that reverses this paradigm by defining how these elements participate in normal physiology, such as maintaining tolerogenic responses in the gut.

Credit

Reviewed by Laura Rosenberg as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute, the University of Texas MD Anderson, and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.