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The proinflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). Global knockout (KO) of miR-155 in mice confers resistance to a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. However, cell-intrinsic roles for miR-155 during EAE have not been formally determined. In this study, we use single-cell RNA sequencing and cell-specific conditional miR-155 KOs to determine the importance of miR-155 expression in distinct immune cell populations. Time-course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells (DCs) in global miR-155 KO mice compared with wild-type controls at day 21 after EAE induction. Deletion of miR-155 in T cells, driven by CD4 Cre, significantly reduced disease severity similar to global miR-155 KOs. CD11c Cre-mediated deletion of miR-155 in DCs also resulted in a modest yet significant reduction in the development of EAE, with both T cell– and DC-specific KOs showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in infiltrating macrophages during EAE, deletion of miR-155 using LysM Cre did not impact disease severity. Taken together, these data show that although miR-155 is highly expressed in most infiltrating immune cells, miR-155 has distinct roles and requirements depending on the cell type, and we have demonstrated this using the gold standard conditional KO approach. This provides insights into which functionally relevant cell types should be targeted by the next generation of miRNA therapeutics.

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SUMMARYRegulation of the microbiota is critical to intestinal health yet the mechanisms employed by innate immunity remain unclear. Here we show that mice deficient in the C-Type-lectin receptor, Clec12a developed severe colitis, which was dependent on the microbiota. Fecal-microbiota-transplantation (FMT) studies into germfree mice revealed a colitogenic microbiota formed within Clec12a-/-mice that was marked by expansion of the gram-positive organism,Faecalibaculum rodentium. Treatment withF. rodentiumwas sufficient to worsen colitis in wild-type mice. Macrophages within the gut express the highest levels of Clec12a. Cytokine and sequencing analysis in Clec12a-/-macrophages revealed heighten inflammation but marked reduction in genes associated with phagocytosis. Indeed, Clec12a-/-macrophages are impaired in their ability to uptakeF. rodentium.Purified Clec12a had higher binding to gram-positive organisms such asF. rodentium. Thus, our data identifies Clec12a as an innate immune surveillance mechanism to control expansion of potentially harmful commensals without overt inflammation.

Estrogen receptor α (ER) mutations occur in up to 30% of metastatic ER-positive breast cancers. Recent data has shown that ER mutations impact the expression of thousands of genes not typically regulated by wildtype ER. While the majority of these altered genes can be explained by constant activity of mutant ER or genomic changes such as altered ER binding and chromatin accessibility, as much as 33% remain unexplained, indicating the potential for post-transcriptional effects. Here we explored the role of microRNAs in mutant ER-driven gene regulation and identified several microRNAs that are dysregulated in ER mutant cells. These differentially regulated microRNAs target a significant portion of mutant-specific genes involved in key cellular processes. When the activity of microRNAs is altered using mimics or inhibitors, significant changes are observed in gene expression and cellular proliferation related to mutant ER. An in-depth evaluation of miR-301b led us to discover an important role forPRKD3in the proliferation of ER mutant cells. Our findings show that microRNAs contribute to mutant ER gene regulation and cellular effects in breast cancer cells.

Intercellular communication is critical for maintaining homeostasis in mammalian organ systems, including the gastrointestinal (GI) tract. Exosomes are small nanoscale lipid extracellular vesicles that mediate communication between a wide variety of cell types. Notably, the roles played by immune cell exosomes in regulating homeostasis and inflammation in the GI tract are largely uncharacterized. Here, we describe an unknown mechanism of exosome-mediated immune regulation within the GI tract. By generating novel mouse strains, we demonstrate that disruption of exosome signaling via the deletion of Rab27a in CD11c+ myeloid cells exacerbates colitis, which is reversed through administration of dendritic cell (DC)-derived exosomes. Profiling small noncoding RNAs within exosomes from colonic tissues revealed a distinct subset of microRNAs (miRNAs) carried by both gut- and DC-derived exosomes. Among the DC-enriched exosomal miRNAs, miR-146a was transferred from gut CD11c+ cells to myeloid and T cells through a Rab27-dependent mechanism to decrease macrophage-mediated inflammation during colitis. Clinically, RAB27A is also expressed at lower levels in a cohort of ulcerative colitis patients versus healthy controls. Together, this work reveals an exosome-mediated modulatory mechanism of gut inflammation, paving the way for exosomes containing miRNA cargo as a potential therapeutic for inflammatory bowel disease. Supported by National Institutes of Health Grants T32 AI 138945-1 (K.M.B.) and AI123106-01A1 (R.M.O.)

The pro-inflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). The whole-body deletion of miR-155 in mice confers resistance to a mouse model of MS, EAE, by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. Here we utilize single-cell RNA sequencing and cell-specific conditional miR-155 knockouts to determine the importance of miR-155 expression in distinct cell populations. Time course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells in whole-body miR-155 knockout mice compared with wild-type controls at day 21. Deletion of miR-155 in T cells, driven by CD4 cre, reduced disease severity similar to whole-body miR-155 knockouts. CD11c cre-driven deletion of miR-155 in dendritic cells also significantly reduced EAE disease score, with both T cell and dendritic cell-specific knockouts showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in macrophages during EAE, deletion miR-155 by LysM cre in did not affect disease severity.

Summary Intestinal epithelial cells (IECs) have long been understood to express high levels of major histocompatibility complex class II (MHC class II) molecules but are not considered canonical antigen-presenting cells, and the impact of IEC-MHC class II signaling on gut homeostasis remains enigmatic. As IECs serve as the primary barrier between underlying host immune cells, we reasoned that IEC-intrinsic antigen presentation may play a role in responses toward the microbiota. Mice with an IEC-intrinsic deletion of MHC class II (IECΔMHC class II) are healthy but have fewer microbial-bound IgA, regulatory T cells (Tregs), and immune repertoire selection. This was associated with increased interindividual microbiota variation and altered proportions of two taxa in the ileum where MHC class II on IECs is highest. Intestinal mononuclear phagocytes (MNPs) have similar MHC class II transcription but less surface MHC class II and are capable of acquiring MHC class II from IECs. Thus, epithelial-myeloid interactions mediate development of adaptive responses to microbial antigens within the gastrointestinal tract.