The epithelial C15ORF48/miR-147-NDUFA4 axis is an essential regulator of gut inflammation, energy metabolism, and the microbiome.

Chronic inflammation is epidemiologically linked to the pathogenesis of gastrointestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer (CRC). However, our understanding of the molecular mechanisms controlling gut inflammation remains insufficient, hindering the development of targeted therapies for IBD and CRC. In this study, we uncovered C15ORF48/miR-147 as a negative regulator of gut inflammation, operating through the modulation of epithelial cell metabolism. C15ORF48/miR-147 encodes two molecular products, C15ORF48 protein and miR-147-3p microRNA, which are predominantly expressed in the intestinal epithelium. C15ORF48/miR-147 ablation leads to gut dysbiosis and exacerbates chemically induced colitis in mice. C15ORF48 and miR-147-3p work together to suppress colonocyte metabolism and inflammation by silencing NDUFA4, a subunit of mitochondrial complex IV (CIV). Interestingly, the C15ORF48 protein, a structural paralog of NDUFA4, contains a unique C-terminal α-helical domain crucial for displacing NDUFA4 from CIV and its subsequent degradation. NDUFA4 silencing hinders NF-κB signaling activation and consequently attenuates inflammatory responses. Collectively, our findings have established the C15ORF48/miR-147-NDUFA4 molecular axis as an indispensable regulator of gut homeostasis, bridging mitochondrial metabolism and inflammation.

A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages.

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

The aryl hydrocarbon receptor instructs the immunomodulatory profile of a subset of Clec4a4+ eosinophils unique to the small intestine.

C-type lectin domain family 4, member a4 (Clec4a4) is a C-type lectin inhibitory receptor specific for glycans thought to be exclusively expressed on murine CD8α− conventional dendritic cells. Using newly generated Clec4a4-mCherry knock-in mice, we identify a subset of Clec4a4-expressing eosinophils uniquely localized in the small intestine lamina propria. Clec4a4+ eosinophils evinced an immunomodulatory signature, whereas Clec4a4− eosinophils manifested a proinflammatory profile. Clec4a4+ eosinophils expressed high levels of aryl hydrocarbon receptor (Ahr), which drove the expression of Clec4a4 as well as other immunomodulatory features, such as PD-L1. The abundance of Clec4a4+ eosinophils was dependent on dietary AHR ligands, increased with aging, and declined in inflammatory conditions. Mice lacking AHR in eosinophils expanded innate lymphoid cells of type 2 and cleared Nippostrongylus brasiliensis infection more effectively than did wild-type mice. These results highlight the heterogeneity of eosinophils in response to tissue cues and identify a unique AHR-dependent subset of eosinophils in the small intestine with an immunomodulatory profile.

Whole-genome profiling of DNA methylation and hydroxymethylation identifies distinct regulatory programs among innate lymphocytes.

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized functions. DNA methylation and hydroxymethylation are essential for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine (5hmC) in mouse innate lymphoid cell 3 (ILC3), ILC2 and natural killer (NK) cells. We identified differentially methylated regions (DMRs) and differentially hydroxymethylated regions (DHMRs) between ILC and NK cell subsets and correlated them with transcriptional signatures. We associated lineage-determining transcription factors (LDTFs) with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions (OCRs), histone modifications and TF-binding sites. We further identified an association between hydroxymethylation and NK cell superenhancers (SEs). Using mice lacking the DNA hydroxymethylase TET2, we showed the requirement for TET2 in optimal production of hallmark cytokines by ILC3s and interleukin-17A (IL-17A) by inflammatory ILC2s. These findings provide a powerful resource for studying innate lymphocyte epigenetic regulation and decode the regulatory logic governing their identity.

microRNA-142 guards against autoimmunity by controlling Treg cell homeostasis and function.

Regulatory T (Treg) cells are critical in preventing aberrant immune responses. Posttranscriptional control of gene expression by microRNA (miRNA) has recently emerged as an essential genetic element for Treg cell function. Here, we report that mice with Treg cell-specific ablation of miR-142 (hereafter Foxp3CremiR-142fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T-cell tolerance. Foxp3CremiR-142fl/fl mice displayed a significant decrease in the abundance and suppressive capacity of Treg cells. Expression profiling of miR-142-deficient Treg cells revealed an up-regulation of multiple genes in the interferon gamma (IFNγ) signaling network. We identified several of these IFNγ-associated genes as direct miR-142-3p targets and observed excessive IFNγ production and signaling in miR-142-deficient Treg cells. Ifng ablation rescued the Treg cell homeostatic defect and alleviated development of autoimmunity in Foxp3CremiR-142fl/fl mice. Thus, our findings implicate miR-142 as an indispensable regulator of Treg cell homeostasis that exerts its function by attenuating IFNγ responses.

Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders.

The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS.

MicroRNA-142 Is Critical for the Homeostasis and Function of Type 1 Innate Lymphoid Cells.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-ß (TGF-ß) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

miR-146a-Traf6 regulatory axis controls autoimmunity and myelopoiesis, but is dispensable for hematopoietic stem cell homeostasis and tumor suppression.

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a-mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a-Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a-/- mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a-/- phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a-/- mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.

Altered lymphopoiesis and immunodeficiency in miR-142 null mice.

MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.