Identification of RISC-associated microRNAs and their targets during CD8⁺ T cell activation

MicroRNAs (miRNAs) are short (~22 nucleotide long) single-stranded noncoding RNAs that regulate gene expression post-transcriptionally in the RNA-induced silencing complex (RISC). miRNAs play an important role in immune cell function and affect many aspects of T cell immunity. Activation of naive T cells induces dramatic changes in the expression of miRNAs and RISC-associated proteins. We studied these changes in expression of miRNAs in CD8+ T cells using the OT-I transgenic T-cell receptor (TCR) mouse model, in which all T cells are CD8+ and respond to ovalbumin peptides. Upon in vitro activation, we saw dynamic changes in the expression of individual miRNAs, which were influenced by whether the T cells responded to high or low affinity peptides and whether they were differentiating to effector or memory cells. It was recently shown that in naive T cells, miRNAs are predominantly found in a low molecular weight (LMW) RISC composed of Argonaute (Ago)-proteins and miRNAs. Upon activation of T cells, biologically active miRNAs interacting with their target messenger RNAs (mRNAs) were shown to redistribute to a high molecular weight (HMW) RISC, which additionally contains RNA metabolism factors and Ago-interacting proteins such as GW182. We followed the development of HMW and LMW complexes in activated CD8⁺ T cells in order to determine their role and to identify the miRNAs and their targets present in both. We confirmed that GW182 protein was induced upon CD8⁺ T cell activation and associated with Ago-2, forming HMW complexes. To study the distribution of miRNAs between HMW and LMW RISC, we undertook small RNA sequencing of the associated miRNAs. From these data we identified specific miRNAs that were enriched in HMW RISC in activated CD8⁺ T cells. We also found that miRNA abundance did not always reflect its association with HMW RISC. Lastly, to discover miRNA targets, we used a novel method called cross-linking, ligation and sequencing of hybrids (CLASH), which directly identifies miRNAs and their targets by immunoprecipitation of RISC and RNA sequencing. From these data we found potential novel targets for key miRNAs in CD8⁺ T cells. Expanding our knowledge of the role of miRNAs in T cell activation beyond observations of miRNA expression changes, by focusing on biologically active miRNAs and their targets in HMW RISC will deepen our understanding of the mechanism of action of miRNAs as well as the signalling pathways surrounding T cell activation

miR-7 is recruited to the High Molecular Weight 1 RNA-induced silencing complex in CD8+ T cells upon activation and suppresses IL-2 signaling

Increasing evidence suggests mammalian Argonaute (Ago) proteins partition into distinct complexes within cells, but there is still little biochemical or functional understanding of the miRNAs differentially associated with these complexes. In naïve T cells, Ago2 is found almost exclusively in low molecular weight (LMW) complexes which are associated with miRNAs but not their target mRNAs. Upon T-cell activation, a proportion of these Ago2 complexes move into a newly formed high molecular weight (HMW) RNA-induced silencing complex (RISC), which is characterized by the presence of the GW182 protein that mediates translational repression. Here, we demonstrate distinct partitioning of miRNAs and isomiRs in LMW versus HMW RISCs upon antigen-mediated activation of CD8 + T cells. We identify miR-7 as highly enriched in HMW RISC and demonstrate that miR-7 inhibition leads to increased production of IL-2 and up-regulation of the IL-2 receptor, the transferrin receptor, CD71 and the amino acid transporter, CD98. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signaling and the metabolic processes regulated by IL-2. </p

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites

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HpARI protein secreted by a helminth parasite suppresses interleukin-33

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy. Osbourn et al identified HpARI, a protein secreted by a helminth parasite that is capable of suppressing allergic responses. HpARI binds to IL-33 (a critical inducer of allergy) and nuclear DNA, preventing the release of IL-33 from necrotic epithelial cells