1000: A Deep-learning Based Approach Uncovers Novel Mediators of Micro-rna Restraint of type-2 Immunity

Background/Purpose: MicroRNAs, such as miR-24 and miR-27, co-expressed within the Mirc11 and Mirc22 clusters, orchestrate a regulatory network critical to Th2 cell differentiation and cytokine production, notably Interleukin-4 (IL-4), with emerging roles in immune dysregulation in rheumatic diseases. To probe this network, we developed DeepRNA-Reg, a deep learning-based algorithm leveraging recurrent neural networks for high-fidelity comparative analysis of high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) datasets.

Methods: The DeepRNA-Reg algorithm utilizes deep learning to predict differentially enriched sites in paired HITS-CLIP data sets, employing a recurrent neural network to infer differential enrichment status across the transcriptome. CD4+ T cells were isolated from spleen and lymph nodes and cultured under Th2 conditions. C57BL/6 male and female age and sex matched mice between 5 to 12 weeks of age were used for all experiments. To generate “miR-23, 24, 27 KO” T cells lacking expression of all Mirc11 cluster miRNAs (miR- 23a, miR-24-2, and miR-27a) and Mirc22 cluster miRNAs (miR-23b, miR-24-1, and miR-27b), we intercrossed previously generated conditionally mutant Mirc22^tm1.1Mtm mice and Mirc11^tm1.1Mtm mice and with Cd4-Cre transgenic mice (Tg(CD4-cre)1Cwi; 4196, Taconic) to induce Cre-mediated deletion in T cells. Ago2 HITS- CLIP was performed using an anti-Ago2 monoclonal antibody. Lastly, differential protein expression was evaluated in CD4+ T Cells harvested from mice bearing selective microRNA cluster knock-out.

Results: This analysis of miR-24, 27 targets uncovered a suite of transcriptional regulators that are known to affect IL-4 expression, including Gata3 and Ikzf1 but also Rora, Tcf7, Runx1, Bcl11b, Foxo1, Nfat5, Tsc22d3 and Klf11. In addition, this study uncovered the cell surface costimulatory receptor CD28 as a novel target of miR-24 and miR-27. CD28 is a key regulator of T cell activation, differentiation and cytokine production. Flow cytometric analysis of CD28 protein expression confirmed that miR-23,24,27 KO Th2 cells express a larger quantity of CD28 on their cell surface, consistent with direct targeting by miR-24 and/or miR- 27.

Conclusion: These findings demonstrate that DeepRNA-Reg analysis of comparative Ago2 HITS-CLIP data can both confirm prior knowledge and uncover novel nodes in post-transcriptional regulatory networks. In tested data sets, DeepRNA-Reg uncovered novel mediators in the mechanism of microRNA-mediated restraint of type-2 immunity in T-Helper 2 cells, a pattern of immune response implicated in the pathophysiology.