Extracellular RNA was a hot topic of discussion at Immunology 2016, the annual meeting of the American Association of Immunologists (AAI), held at the Washington State Convention Center in Seattle, Washington May 13-17th, 2016. The National Cancer Institute (NCI) sponsored a symposium on “Extracellular RNA in the Immune System”, co-chaired by Dr. Kevin Howcroft (Division of Cancer Biology, Cancer Immunology, Hematology, and Etiology Branch, NCI) and K. Mark Ansel (University of California San Francisco – your faithful blogger). Four invited speakers presented and participated in lively discussion with an audience of gathered experts and curious newcomers to the field of extracellular RNA.
Dr. Gyongyi Szabo (University of Massachusetts) opened the symposium with a presentation of her laboratory’s work on extracellular vesicles and miRNAs in innate immune cell communication in the liver. Alcohol exposure induces liver inflammation, marked by release of pro-inflammatory cytokines and activation of myeloid cells, including Kupffer cells, the resident macrophages of the liver. In a mouse model, alcohol consumption increased expression of miR-155 in both macrophages and hepatocytes via TLR4 and NFκB-driven transcription. Inhibition or genetic deletion of miR-155 in this model blunted macrophage activation and cytokine production. Exosomes loaded with miR-155 mimetics could be delivered to hepatocytes and other liver cells to correct some of the defects observed in miR-155-deficient animals. Remarkably, endogenous miR-155 and miR-122 were elevated in serum collected after controlled “binge-drinking” in human study subjects, and these exosomes also conveyed information to cultured monocytes, altering their production of TNF and IL-1. Together these data suggest that extracellular communication between hepatocytes and innate immune cells via exosomal miRNAs regulates inflammation in response to alcohol consumption.
The theme of regulation of inflammatory responses by miRNA-containing exosomes was extended by Dr. Ryan O’Connell (University of Utah). His pioneering work on miR-155 and miR-146 demonstrated their opposing roles in inflammatory processes mediated by various cell types in several tissues and disease settings. Recent work in his laboratory showed that both of these miRNAs are released by bone-marrow-derived dendritic cells in a fashion dependent on Rab27 and neutral sphingomyelinase (N-SMase) activity, and that these miRNAs could be exchanged between cells separated by a filter that prevents cell-cell contact. Transferred miR-146a reduced recipient cells’ response to bacterial lipopolysaccharide, a classical innate immune stimulant in vitro and in vivo. In addition, transferred miR-155 was found to directly repress the 3’ UTR of target genes in recipient cells, supporting the possibility that functional miRNA transfer via exosomes could be used as a therapeutic modality for regulating inflammation. Getting these miRNAs to the right cell types in vivo remains an important challenge to bringing this technology to the clinic.
In addition to exosomes, high density lipoprotein (HDL) particles carry miRNAs and other extracellular RNAs in blood. Abnormal pro-inflammatory HDL is associated with systemic lupus erythematosus (SLE). Dani Michell (Vanderbilt University), a postdoctoral fellow in Kasey Vickers’ laboratory, discussed her work, conducted in collaboration with Amy Major’s laboratory, on miRNAs in HDL in SLE. HDL from subjects with SLE contained increased levels of miR-22-3p and miR-192-5p compared with HDL from healthy control subjects. Blocking miR-22 with locked nucleic acid inhibitors in vivo reduced spleen size and interferon production, and affected some clinical features in a mouse model of lupus. Experiments aimed at defining source and recipient cells in this system indicated that monocytes are much better than T lymphocytes at taking up HDL-associated miRNAs. It will be interesting to learn how HDL-associated miRNAs regain gene regulatory function in recipient cells.
The final presentation focused on lymphocytes as source cells for naturally occurring exRNAs in body fluids. Immuno-compromised mice with a mutation that specifically blocks lymphocyte development exhibit altered serum extracellular miRNA profiles. In support of the idea that lymphocytes themselves are an important source of ex-miRNAs, the most reduced exRNA species detected was miR-150, a miRNA highly expressed by lymphocytes. Activated T lymphocytes secrete vesicles that are enriched for tRNA fragments and miRNAs including miR-150. Rigorous purification revealed that these vesicles have characteristics of exosomes, including defined density, size, and protein markers including the tetraspanin CD9. Cellular fractionation also revealed tRNA fragment and miRNA enrichment in membrane fractions containing multivesicular bodies. Whether these extracellular lymphocyte-derived RNAs mediate cell-to-cell communication or not, signal-mediated reduction of cellular miRNAs certainly alters gene regulation in activated T lymphocytes. Thus, exRNA secretion may have important roles in regulating inflammatory processes in both source and recipient cells.
These topics will certainly remain on the mind of immunologists that attended the exRNA symposium — at least until Immunology 2017, to be held in Washington DC next May.