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.

RNA m6A reader YTHDF2 facilitates precursor miR-126 maturation to promote acute myeloid leukemia progression.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

Acquired miR-142 deficit in leukemic stem cells suffices to drive chronic myeloid leukemia into blast crisis.

The mechanisms underlying the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC) are not fully elucidated. Here, we show lower levels of miR-142 in CD34+CD38- blasts from BC CML patients than in those from CP CML patients, suggesting that miR-142 deficit is implicated in BC evolution. Thus, we create miR-142 knockout CML (i.e., miR-142-/-BCR-ABL) mice, which develop BC and die sooner than miR-142 wt CML (i.e., miR-142+/+BCR-ABL) mice, which instead remain in CP CML. Leukemic stem cells (LSCs) from miR-142-/-BCR-ABL mice recapitulate the BC phenotype in congenic recipients, supporting LSC transformation by miR-142 deficit. State-transition and mutual information analyses of "bulk" and single cell RNA-seq data, metabolomic profiling and functional metabolic assays identify enhanced fatty acid ß-oxidation, oxidative phosphorylation and mitochondrial fusion in LSCs as key steps in miR-142-driven BC evolution. A synthetic CpG-miR-142 mimic oligodeoxynucleotide rescues the BC phenotype in miR-142-/-BCR-ABL mice and patient-derived xenografts.

Paradoxical attenuation of neuroinflammatory response upon LPS challenge in miR-146b deficient mice.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b (miR-146a/b), both of which are known to suppress immune responses in a variety of conditions. Here, we studied how constitutive deficiency of miR-146b (Mir146b-/-) affects lipopolysaccharide (LPS)-induced neuroinflammation in mice. Our experiments demonstrated that miR-146b deficiency results in the attenuation of LPS-induced neuroinflammation, as it was evidenced by the reduction of sickness behavior, a decrease in the inflammatory status of microglia, and the loss of morphological signs of microglial activation in the hippocampus. Gene expression analysis revealed that LPS-induced upregulation of hippocampal pro-inflammatory cytokines is attenuated in Mir146b-/- mice, compared to wild-type (WT) mice. In addition, reduced expression of the NF-κB nuclear protein p65, reduced miR-146 family target TLR4 expression and relatively stronger upregulation of miR-146a was found in Mir146b-/- mice as compared to WT mice upon LPS challenge. Compensatory upregulation of miR-146a can explain the attenuation of the LPS-induced neuroinflammation. This was supported by experiments conducted with miR-146a/b deficient mice (Mir146a/b-/-), which demonstrated that additional deletion of the miR-146a led to the restoration of LPS-induced sickness behavior and proinflammatory cytokines. Our experiments also showed that the observed upregulation of miR-146a in Mir146b-/- mice is due to the overexpression of a miR-146a transcription inducer, interferon regulatory factor 7 (Irf7). Altogether, our results show the existence of crosstalk between miR-146a and mir-146b in the regulation of LPS-induced neuroinflammation.

Enhanced Cognition and Neurogenesis in miR-146b Deficient Mice.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b, which are both known to suppress a variety of immune responses. Here in this study, we show that miR-146b is abundantly expressed in neuronal cells, while miR-146a is mainly expressed in microglia and astroglia of adult mice. Accordingly, miR-146b deficient (Mir146b-/-) mice exhibited anxiety-like behaviors and enhanced cognition. Characterization of cellular composition of Mir146b-/- mice using flow cytometry revealed an increased number of neurons and a decreased abundancy of astroglia in the hippocampus and frontal cortex, whereas microglia abundancy remained unchanged. Immunohistochemistry showed a higher density of neurons in the frontal cortex of Mir146b-/- mice, enhanced hippocampal neurogenesis as evidenced by an increased proliferation, and survival of newly generated cells with enhanced maturation into neuronal phenotype. No microglial activation or signs of neuroinflammation were observed in Mir146b-/- mice. Further analysis demonstrated that miR-146b deficiency is associated with elevated expression of glial cell line-derived neurotrophic factor (Gdnf) mRNA in the hippocampus, which might be at least in part responsible for the observed neuronal expansion and the behavioral phenotype. This hypothesis is partially supported by the positive correlation between performance of mice in the object recognition test and Gdnf mRNA expression in Mir146b-/- mice. Together, these results show the distinct function of miR-146b in controlling behaviors and provide new insights in understanding cell-specific function of miR-146b in the neuronal and astroglial organization of the mouse brain.

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.

Dual role of the miR-146 family in rhinovirus-induced airway inflammation and allergic asthma exacerbation.

Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA-146a and microRNA-146b (miR-146a/b) are anti-inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF-κB) pathway and inhibit pro-inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR-146a/b could regulate cellular responses to RVs in HBECs and airways during RV-induced asthma exacerbation. We demonstrated that expression of miR-146a/b and pro-inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell-penetrating peptide (CPP)-miR-146a nanocomplexes before infection with RV significantly reduced the expression of the pro-inflammatory chemokines CCL5, IL-8 and CXCL1, increased interferon-λ production, and attenuated infection with the green fluorescent protein (GFP)-expressing RV-A16 in HBECs. Concordantly, compared to wild-type (wt) mice, Mir146a/b-/- mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV-A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)-induced allergic airway inflammation and RV-induced exacerbation models. Interestingly, intranasal administration of CPP-miR-146a nanocomplexes reduced HDM-induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild-type mice. In conclusion, the overexpression of miR-146a has a strong anti-inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR-146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV-induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP-miR-146a nanocomplexes has therapeutic potential for targeting airway inflammation.

microRNA-146a controls age-related bone loss.

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here, we identify microRNA-146a (miR-146a) as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a-deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallels bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a-deficient mice are protected from ovariectomy-induced bone loss. In humans, the levels of miR-146a are increased in patients suffering fragility fractures in comparison with those who do not. These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age-related bone dysfunctions such as the development of bone marrow adiposity and osteoporosis.

Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Myeloid cell-targeted miR-146a mimic inhibits NF-κB-driven inflammation and leukemia progression in vivo.

NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.

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.

Molecular Moirai: Long Noncoding RNA Mediators of HSC Fate.

PURPOSE OF REVIEW: Hematopoiesis is an ordered developmental process that requires dynamic regulation to warrant proper response to physiological challenges and prevent malignancies. Long noncoding RNAs are emerging as key, multi-faceted regulators of gene expression. This review explores the function of lncRNAs in the control of HSC homeostasis and hematopoietic differentiation. RECENT FINDINGS: Multiple lncRNAs have been implicated in maintaining HSC stemness and enabling progenitors to carry out the correct programs of lineage differentiation. Specific lncRNAs have been identified that regulate the differentiation of multipotent progenitors into terminally differentiated blood cells. These lncRNAs predominantly act by assisting master regulators that drive specific differentiation programs, either by enhancing or repressing the transcription of particular genomic loci. SUMMARY: Long noncoding RNAs contribute to the correct differentiation and maturation of various hematopoietic lineages by assisting with the activation of transcriptional programs in a time- and cell-dependent manner.

miR-143/145 differentially regulate hematopoietic stem and progenitor activity through suppression of canonical TGFß signaling.

Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs), but activation of progenitor cells (HPCs). We identify components of the transforming growth factor ß (TGFß) pathway as key targets of miR-143/145. Enforced expression of the TGFß adaptor protein and miR-145 target, Disabled-2 (DAB2), recapitulates the HSC defect seen in miR-143/145-/- mice. Despite reduced HSC activity, older miR-143/145-/- and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy, associated with expansion of HPC. Thus, miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFß/DAB2 activation, and loss of these miRNAs creates a preleukemic state.

MicroRNA-146a controls functional plasticity in γδ T cells by targeting NOD1.

γδ T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-γ (IFN-γ), which segregate with CD27 expression. In the periphery, CD27- γδ (γδ27-) T cells can be induced under inflammatory conditions to coexpress IL-17 and IFN-γ; the molecular basis of this functional plasticity remains to be determined. On the basis of differential microRNA (miRNA) expression analysis and modulation in γδ T cell subsets, we identified miR-146a as a thymically imprinted post-transcriptional brake to limit IFN-γ expression in γδ27- T cells in vitro and in vivo. On the basis of biochemical purification of Argonaute 2-bound miR-146a targets, we identified Nod1 to be a relevant mRNA target that regulates γδ T cell plasticity. In line with this, Nod1-deficient mice lacked multifunctional IL-17+ IFN-γ+ γδ27- cells and were more susceptible to Listeria monocytogenes infection. Our studies establish the miR-146a/NOD1 axis as a key determinant of γδ T cell effector functions and plasticity.

miR-146a modulates autoreactive Th17 cell differentiation and regulates organ-specific autoimmunity.

Autoreactive CD4 T cells that differentiate into pathogenic Th17 cells can trigger autoimmune diseases. Therefore, investigating the regulatory network that modulates Th17 differentiation may yield important therapeutic insights. miR-146a has emerged as a critical modulator of immune reactions, but its role in regulating autoreactive Th17 cells and organ-specific autoimmunity remains largely unknown. Here, we have reported that miR-146a-deficient mice developed more severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS). We bred miR-146a-deficient mice with 2D2 T cell receptor-Tg mice to generate 2D2 CD4 T cells that are deficient in miR-146a and specific for myelin oligodendrocyte glycoprotein (MOG), an autoantigen in the EAE model. miR-146a-deficient 2D2 T cells induced more severe EAE and were more prone to differentiate into Th17 cells. Microarray analysis revealed enhancements in IL-6- and IL-21-induced Th17 differentiation pathways in these T cells. Further study showed that miR-146a inhibited the production of autocrine IL-6 and IL-21 in 2D2 T cells, which in turn reduced their Th17 differentiation. Thus, our study identifies miR-146a as an important molecular brake that blocks the autocrine IL-6- and IL-21-induced Th17 differentiation pathways in autoreactive CD4 T cells, highlighting its potential as a therapeutic target for treating autoimmune diseases.

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.

miR-146b Probably Assists miRNA-146a in the Suppression of Keratinocyte Proliferation and Inflammatory Responses in Psoriasis.

miR-146a inhibits inflammatory responses in human keratinocytes and in different mouse models of skin inflammation. Little is known about the role of miR-146b in the skin. In this study, we confirmed the increased expression of miR-146a and miR-146b (miR-146a/b) in the lesional skin of patients with psoriasis. The expression of miR-146a was approximately twofold higher than that of miR-146b in healthy human skin, and it was more strongly induced by stimulation of proinflammatory cytokines in keratinocytes and fibroblasts. miR-146a/b target genes regulating inflammatory responses or proliferation were altered in the skin of patients with psoriasis, among which FERMT1 was verified as a direct target of miR-146a. In silico analysis of genome-wide data from >4,000 psoriasis cases and >8,000 controls confirmed a moderate association between psoriasis and genetic variants in the miR-146a encoding gene. Transfection of miR-146a/b suppressed and inhibition enhanced keratinocyte proliferation and the expression of psoriasis-related target genes. Enhanced expression of miR-146a/b-influenced genes was detected in cultured keratinocytes from miR-146a-/- and skin fibroblasts from miR-146a-/- and miR-146b-/- mice stimulated with psoriasis-associated cytokines as compared with wild-type mice. Our results indicate that besides miR-146a, miR-146b is expressed and might be capable of modulation of inflammatory responses and keratinocyte proliferation in psoriatic skin.

MicroRNA-146a governs fibroblast activation and joint pathology in arthritis.

Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.

Identification of targets of tumor suppressor microRNA-34a using a reporter library system.

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3' UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks.

Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ+ Th1/Tregs.

RATIONALE: Forkhead box P3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. OBJECTIVE: Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe-/- mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. METHODS AND RESULTS: We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a-/- Tregs, we demonstrate that elevated IFNγ+ Mir146a-/- Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe-/- mice, in comparison to Mir146a+/+ Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. CONCLUSIONS: Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.