miR-146a Deficiency Research Articles

MiR-146a alleviates inflammatory bowel disease in mice through systematic regulation of multiple genetic networks.

Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy. Whether there is a single gene that systematically regulates IBD is not yet known. MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed. RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background. The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates. Bulk RNA-sequencing, RT-qPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon. Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice. We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD. DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice. The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a-/- and WT mice. Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes. Quantitative RT-PCR or immunostaining confirmed that Ccl3, Saa3, Csf3, Lcn2, Serpine1, Serpine2, MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, p-P65, and IRAK1 were regulated by miR-146a in DSS induced IBD. Among them, MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A were involved in the active stage of IBD in humans. Our date demonstrated that miR-146a acts as a top regulator in C57/BL6N mice to systematically repress multiple genetic regulatory networks involved in immune response of intestine to environment factors, and combinatory treatment with miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through down-regulating multiple genetic regulatory networks which were increased in colon tissue from IBD patients. Our findings suggests that miR-146a is a top inhibitor of IBD, and that miR-146a-5p and miR-146a-3p mimics might be potential drug for IBD.

Abstract 3000: Mir-146a Deficiency Differentially Regulates Sexual Dimorphism Of Abdominal Aortic Aneurysms In Mice

Background and Objective: Abdominal aortic aneurysms (AAAs) are permanent aortic dilations with 80% mortality after rupture. AAA exhibits sexual dimorphism, with males at higher risk in both mice and humans. In spite of lower incidence, women acquire aggressive aneurysm growth and rupture at smaller sizes, compared to men. Despite this oddity, mechanisms driving the sexual dimorphism of AAA have not been defined. Previously, we demonstrated that Lysyl Oxidase (LOX) inhibition abolishes sexual dimorphism of AAA in mice. Recent clinical studies showed elevated miR-146a levels, an anti-inflammatory miRNA, in plasma and AAA tissues from patients. In our preliminary studies, we found that aortic miR-146a is significantly lower in females compared to male mice. However, the contribution of miR-146a in the sexual dimorphism of AAAs is unknown. In this study, we examined the role of miR-146a in AAA sexual dimorphism in mice by administering β-aminopropionitrile (BAPN), a LOX inhibitor and Angiotensin II (AngII) concurrently. Methods and Results: 8 -10 week old male and female C57BL/6J mice that were either miR146a wild type (WT) or deficient (KO; n=18-24/per group) were infused with saline or Ang II (1,000 ng/kg/min) via subcutaneous osmotic mini-pump for 28 days. In addition, mice were co-administered with BAPN (1 mg/ml; 0.15g/kg/d in drinking water) from day 0-14. miR-146a deficiency had no effect on body weight and blood pressure in both male and female mice. AngII+BAPN-administration increased AAA in both male (82%;19/23) and female (72%;18/25) mice compared to saline+BAPN controls. Interestingly, miR-146a deficiency profoundly but significantly increased AAA in males (WT=1.5 ± 0.1 versus KO=2.3 ± 0.1 mm, P<0.05), but significantly decreased AAA in females (WT=1.5 ± 0.1 versus KO=1.2 ± 0.1 mm, P<0.05). By RNA sequencing and qPCR, we identified several differentially expressed genes in the absence of miR-146a, with RFLNA upregulated and ELAVL2 downregulated in male KO mice, and SLC38A5, CCND2, DUSP10 genes upregulated in female KO mice. Ongoing studies aim to delineate the role of these genes in miR-146a-mediated sexual dimorphism of AAA. Conclusion: These findings demonstrate that miR-146a plays a critical role in mediating sexual dimorphism of AAA in mice.

MiR-146a Maintains Immune Tolerance of Kupffer Cells and Facilitates Hepatitis B Virus Persistence in Mice.

Kupffer cells (KCs), the largest tissue-resident macrophage population in the body, play a central role in maintaining a delicate balance between immune tolerance and immunity in the liver. However, the underlying molecular mechanism remains elusive. In this study, we show that KCs express high levels of miR-146a, which is under control of the PU.1 transcription factor. miR-146a deficiency promoted KCs differentiation toward a proinflammatory phenotype; conversely, miR-146a overexpression suppressed this phenotypic differentiation. We found that hepatitis B virus (HBV) persistence or HBV surface Ag treatment significantly upregulated miR-146a expression and thereby impaired polarization of KCs toward a proinflammatory phenotype. Furthermore, in an HBV carrier mouse model, KCs depletion by clodronate liposomes dramatically promoted HBV clearance and enhanced an HBV-specific hepatic CD8+ T cell and CD4+ T cell response. Consistent with this finding, miR-146a knockout mice cleared HBV faster and elicited a stronger adaptive antiviral immunity than wild-type mice. In vivo IL-12 blockade promoted HBV persistence and tempered the HBV-specific CTL response in the liver of miR-146a knockout mice. Taken together, our results identified miR-146a as a critical intrinsic regulator of an immunosuppressive phenotype in KCs under inflammatory stimuli, which may be beneficial in maintenance of liver homeostasis under physiological condition. Meanwhile, during HBV infection, miR-146a contributed to viral persistence by inhibiting KCs proinflammatory polarization, highlighting its potential as a therapeutic target in HBV infection.

MiR-146a regulates regulatory T cells to suppress heart transplant rejection in mice

Regulatory T cells (Tregs), which characteristically express forkhead box protein 3 (Foxp3), are essential for the induction of immune tolerance. Here, we investigated microRNA-146a (miR-146a), a miRNA that is widely expressed in Tregs and closely related to their homeostasis and function, with the aim of enhancing the function of Tregs by regulating miR-146a and then suppressing transplant rejection. The effect of the absence of miR-146a on Treg function in the presence or absence of rapamycin was detected in both a mouse heart transplantation model and cell co-cultures in vitro. The absence of miR-146a exerted a mild tissue-protective effect by transiently prolonging allograft survival and reducing the infiltration of CD4+ and CD8+ T cells into the allografts. Meanwhile, the absence of miR-146a increased Treg expansion but impaired the ability of Tregs to restrict T helper cell type 1 (Th1) responses. A miR-146a deficiency combined with interferon (IFN)-γ blockade repaired the impaired Treg function, further prolonged allograft survival, and alleviated rejection. Importantly, miR-146a regulated Tregs mainly through the IFN-γ/signal transducer and activator of transcription (STAT) 1 pathway, which is implicated in Treg function to inhibit Th1 responses. Our data suggest miR-146a controls a specific aspect of Treg function, and modulation of miR-146a may enhance Treg efficacy in alleviating heart transplant rejection in mice.

Downregulation of microRNA-146a in diabetes, obesity and hypertension may contribute to severe COVID-19

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is able to produce an excessive host immune reaction and may leads to severe disease- a life-threatening condition occurring more often in patients suffering from comorbidities such as hypertension, diabetes and obesity. Infection by human corona viruses highly depends on host microRNA (miR) involved in regulation of host innate immune response and inflammation-modulatory miR-146a is among the first miRs induced by immune reaction to a virus. Moreover, recent analysis showed that miR-146 is predicted to target at the SARS-CoV-2 genome. As the dominant regulator of Toll-like receptors (TLRs) downstream signaling, miR-146a may limit excessive inflammatory response to virus. Downregulation of circulating miR-146a was found in diabetes, obesity and hypertension and it is reflected by enhanced inflammation and fibrosis, systemic effects accompanying severe COVID-19. Thus it could be hypothesized that miR-146a deficiency may contribute to severe COVID-19 state observed in diabetes, obesity and hypertension but further investigations are needed.

MiR-146a is a pivotal regulator of neutrophil extracellular trap formation promoting thrombosis.

Neutrophil extracellular traps (NET) induce a procoagulant response linking inflammation and thrombosis. Low levels of miR-146a, a brake of inflammatory response, are involved in higher risk of cardiovascular events, but the mechanisms explaining how miR-146a exerts its function remain largely undefined. The aim of this study was to explore the impact of miR-146a deficiency in NETosis both in sterile and non-sterile models in vivo, and to investigate the underlying mechanism. Two models of inflammation were used: (i) Ldlr-/- mice transplanted with bone marrow from miR-146a-/- or wild-type mice were fed a high-fat diet, generating an atherosclerosis model; and (ii) an acute inflammation model was generated by injecting lipopolysaccharide (1 mg/kg) into miR-146a-/- and wildtype mice. miR-146a deficiency increased NETosis in both models. Accordingly, miR-146a-/- mice showed significantly reduced carotid occlusion time and elevated levels of NET in thrombi following FeCl3-induced thrombosis. Infusion of DNAse I abolished arterial thrombosis in both WT and miR-146a-/- mice. Interestingly, miR-146a-deficient mice have aged, hyperreactive and pro-inflammatory neutrophils in their circulation which are more prone to form NET independently of the stimulus. Furthermore, we demonstrated that patients with community-acquired pneumonia with reduced miR-146a levels associated with the T variant of the functional rs2431697 had an increased risk of cardiovascular events due, in part, to an increased generation of NET.

MiR-146a in Myasthenia Gravis Thymus Bridges Innate Immunity With Autoimmunity and Is Linked to Therapeutic Effects of Corticosteroids.

Toll-like receptor (TLR)-mediated innate immune responses are critically involved in the pathogenesis of myasthenia gravis (MG), an autoimmune disorder affecting neuromuscular junction mainly mediated by antiacetylcholine receptor antibodies. Considerable evidence indicate that uncontrolled TLR activation and chronic inflammation significantly contribute to hyperplastic changes and germinal center (GC) formation in the MG thymus, ultimately leading to autoantibody production and autoimmunity. miR-146a is a key modulator of innate immunity, whose dysregulation has been associated with autoimmune diseases. It acts as inhibitor of TLR pathways, mainly by targeting the nuclear factor kappa B (NF-κB) signaling transducers, interleukin 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6); miR-146a is also able to target c-REL, inducible T-cell costimulator (ICOS), and Fas cell surface death receptor (FAS), known to regulate B-cell function and GC response. Herein, we investigated the miR-146a contribution to the intrathymic MG pathogenesis. By real-time PCR, we found that miR-146a expression was significantly downregulated in hyperplastic MG compared to control thymuses; contrariwise, IRAK1, TRAF6, c-REL, and ICOS messenger RNA (mRNA) levels were upregulated and negatively correlated with miR-146a levels. Microdissection experiments revealed that miR-146a deficiency in hyperplastic MG thymuses was not due to GCs, but restricted to the GC-surrounding medulla, characterized by IRAK1 overexpression. We also showed higher c-REL and ICOS mRNA levels, and lower FAS mRNA levels, in GCs than in the remaining medulla, according to the contribution of these molecules in GC formation. By double immunofluorescence, an increased proportion of IRAK1-expressing dendritic cells and macrophages was found in hyperplastic MG compared to control thymuses, along with GC immunoreactivity for c-REL. Interestingly, in corticosteroid-treated MG patients intrathymic miR-146a and mRNA target levels were comparable to those of controls, suggesting that immunosuppressive therapy may restore the microRNA (miRNA) levels. Indeed, an effect of prednisone on miR-146a expression was demonstrated in vitro on peripheral blood cells. Serum miR-146a levels were lower in MG patients compared to controls, indicating dysregulation of the circulating miRNA. Our overall findings strongly suggest that defective miR-146a expression could contribute to persistent TLR activation, lack of inflammation resolution, and hyperplastic changes in MG thymuses, thus linking TLR-mediated innate immunity to B-cell-mediated autoimmunity. Furthermore, they unraveled a new mechanism of action of corticosteroids in inducing control of autoimmunity in MG via miR-146a.

MiR-146a Deficiency Accelerates Hepatic Inflammation Without Influencing Diet-induced Obesity in Mice

miR-146a, an anti-inflammatory microRNA, is shown to be a negative regulator of adipocyte inflammation. However, the functional contribution of miR-146a in the development of obesity is not defined. In order to determine whether miR-146a influences diet-induced obesity, mice that were either wild type (WT) or miR-146a deficient (KO) were fed with high (60% kcal) fat diet (HFD) for 16 weeks. Deficiency of miR-146a did not influence obesity measured as HFD-induced body weight and fat mass gain, or metabolism of glucose and insulin tolerance. In addition, adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively, were comparable between the two groups of mice. Although, miR-146a deficiency had no influence on HFD-induced hepatic lipid accumulation, interestingly, it significantly increased obesity-induced inflammatory responses in liver tissue. The present study demonstrates that miR-146a deficiency had no influence on the development of HFD-induced obesity and adipose tissue remodeling, whereas it significantly increased hepatic inflammation in obese mice. This result suggests that miR-146a regulates hepatic inflammation during development of obesity.

MiR-146a attenuates apoptosis and modulates autophagy by targeting TAF9b/P53 pathway in doxorubicin-induced cardiotoxicity

Clinical therapy of doxorubicin (DOX) is limited due to its cardiotoxicity. miR-146a was proved as a protective factor in many cardiovascular diseases, but its role in chronic DOX-induced cardiotoxicity is unclear. The objective of this study was to demonstrate the role of miR-146a in low-dose long-term DOX-induced cardiotoxicity. Experiments have shown that DOX intervention caused a dose-dependent and time-dependent cardiotoxicity involving the increased of apoptosis and dysregulation of autophagy. The cardiotoxicity was inhibited by overexpressed miR-146a and was more severe when miR-146a was downgraded. Further research proved that miR-146a targeted TATA-binding protein (TBP) associated factor 9b (TAF9b), a coactivator and stabilizer of P53, indirectly destroyed the stability of P53, thereby inhibiting apoptosis and improving autophagy in cardiomyocytes. Besides, miR-146a knockout mice were used for in vivo validation. In the DOX-induced model, miR-146a deficiency made it worse whether in cardiac function, cardiomyocyte apoptosis or basal level of autophagy, than wild-type. In conclusion, miR-146a partially reversed the DOX-induced cardiotoxicity by targeting TAF9b/P53 pathway to attenuate apoptosis and adjust autophagy levels.

MiR-146a deficiency does not aggravate muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is a genetic disease evoked by a mutation in the dystrophin gene. It is associated with progressive muscle degeneration and increased inflammation. Up to this date, mainly anti-inflammatory treatment is available for patients suffering from DMD. miR-146a is known to diminish inflammation and fibrosis in different tissues by downregulating the expression of proinflammatory cytokines. However, its role in DMD has not been studied so far.In our work, we have generated mice globally lacking both dystrophin and miR-146a (miR-146a−/−mdx) and examined them together with wild-type, single miR-146a knockout and dystrophic (mdx—lacking dystrophin) mice in a variety of aspects associated with DMD pathophysiology (muscle degeneration, inflammatory reaction, muscle satellite cells, muscle regeneration, and fibrosis).We have shown that miR-146a level is increased in dystrophic muscles in comparison to wild-type mice. Its deficiency augments the expression of proinflammatory cytokines (IL-1β, CCL2, TNFα). However, muscle degeneration was not significantly worsened in mdx mice lacking miR-146a up to 24 weeks of age, although some aggravation of muscle damage and inflammation was evident in 12-week-old animals, though no effect of miR-146a deficiency was visible on quantity, proliferation, and in vitro differentiation of muscle satellite cells isolated from miR-146a−/−mdx mice vs. mdx. Similarly, muscle regeneration and collagen deposition were not changed by miR-146a deficiency. Nevertheless, the lack of miR-146a is associated with decreased Vegfa and increased Tgfb1.Overall, the lack of miR-146a did not aggravate significantly the dystrophic conditions in mdx mice, but its effect on DMD in more severe conditions warrants further investigation.

Abstract 138: TNBC induces immune suppressive tumor microenvironments through microRNAs

Abstract Metastatic breast cancer is the second leading cause of cancer-related deaths for US women. The triple negative breast cancer subtype (TNBC) is one of the major contributors to breast cancer mortality, among all types of breast cancer. There are no effective treatments available for metastatic tumors. Therefore, identifying and targeting novel tumorigenesis pathways will facilitate developing effective therapeutic approaches that could ultimately lead to effective treatment for breast cancer, particularly metastatic cancer and TNBC. Our results demonstrate that miR-146a promotes tumorigenesis and metastasis of breast cancer. We found that miR-146a was markedly overexpressed in TNBC cell lines. Notably, miR-146a deficiency in a spontaneous mammary tumor model leads to reduced tumor growth and metastasis. Notably, miR-146a shifts the polarization of macrophages into immunosuppressive phenotypes. Our studies suggest that miR-146a may contribute to tumor development through modulation of tumor microenvironments. Note: This abstract was not presented at the meeting. Citation Format: Beichu Guo, Melvin Kantono, Wei Sun. TNBC induces immune suppressive tumor microenvironments through microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 138.

MiR-146a deficiency in hematopoietic cells is not involved in the development of atherosclerosis.

BackgroundAtherosclerosis involves activation of the IRAK1/TRAF6/NF-κB inflammatory cascade, which is negatively regulated by miR146a. Previous studies showed that the TT genotype of rs2431697, located near the miR-146a gene, drives lower miR-146a transcription and predicts adverse cardiovascular events in anticoagulated atrial fibrillation patients. Moreover, systemic miR-146a administration protects mice from atherosclerosis. Here we evaluated the ability of miR-146a expression in the hematopoietic component to regulate atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr-/-).Methods and resultsLethally-irradiated Ldlr-/- mice transplanted with bone marrow from wild-type or miR-146a-null mice were fed an atherogenic diet for 8 and 20 weeks. Irak1, Traf6 and MIR146A expression were quantified in thoracic aorta by qRT-PCR and Western blot. Aortic plaque size and composition were characterized by Oil-Red staining and immunohistochemistry and leukocyte recruitment by intravital microscopy. Blood cell counts were similar in fat-fed Ldlr-/-mice with or without hematopoietic miR-146a expression. However, plasma cholesterol decreased in fat-fed Ldlr-/-mice transplanted with bone marrow deficient for miR-146a. Finally, aortic atherosclerosis burden and recruitment of leukocytes into the vessel wall were undistinguishable between the two groups, despite higher levels of Irak1 and Traf6 mRNA and protein in the aorta of fat-fed mice lacking hematopoietic miR-146a expression.ConclusionsmiR-146a deficiency exclusively in hematopoietic cells modulates cholesterol levels in plasma and the expression of its targets in the artery wall of fat-fed Ldlr-/- mice, but does not accelerate atherosclerosis. Atheroprotection upon systemic miR-146a administration may therefore be caused by specific effects on vascular cells.

MicroRNA-146a Deficiency Protects against Listeria monocytogenes Infection by Modulating the Gut Microbiota.

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes. High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes, Blautia, Coprococcus_1, and Ruminococcus_1. Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes, indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Mice with miR-146a deficiency develop severe gouty arthritis via dysregulation of TRAF 6, IRAK 1 and NALP3 inflammasome

BackgroundMicroRNAs (miRNAs) serve as important regulators of inflammatory and immune responses and are implicated in several immune disorders including gouty arthritis. The expression of miR-146a is upregulated in the peripheral blood mononuclear cells of patients with inter-critical gout when compared to normouricemic and hyperuricemic controls and those patients with acute gout flares. However, the role of miR-146a in the development of gout remains unknown. Here, we used miR-146a knockout (KO) mice to test miR-146a function in a monosodium urate (MSU)-induced gouty arthritis model.MethodsThe footpad or ankle joint of miR-146a KO and wild-type (WT) mice were injected with an MSU suspension to induce acute gouty arthritis. Bone marrow-derived macrophages (BMDMs) were stimulated with MSU and the gene expression of miR-146a; interleukin 1 beta (IL-1β); tumor necrosis factor-α (TNF-α); and the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome was evaluated. TNF-α and IL-1β protein levels in BMDMs were assessed by fluorescence-activated cell sorting and western blot analyses. Gene and protein levels of TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase (IRAK1), the targets of miR-146a, were also measured.ResultsSignificantly increased paw swelling and index and ankle joint swelling were observed in miR-146a KO mice compared to WT controls after MSU treatment. MiR-146a expression in BMDMs from WT mice was dramatically upregulated at 4 h following MSU stimulation. Additionally, the expression of IL-1β, TNF-α, and NALP3 was higher in BMDMs from miR-146a KO mice after exposure to MSU crystals compared to those from WT mice. Consistent with the observed gene expression, the IL-1β and TNF-α proteins were upregulated in miR-146a KO mice. Additionally quantitative RT-PCR and western blot demonstrated that TRAF6 and IRAK1 were dramatically upregulated in BMDMs from miR-146 KO mice compared to those from WT mice.ConclusionsCollectively, these observations suggest that miR-146a provides negative feedback regulation of gouty arthritis development and lack of miR-146a enhances gouty arthritis via upregulation of TRAK6, IRAK-1, and the NALP3 inflammasome function.

MiR-146a restricts germinal center B cell responses through targeting CD40 signaling pathway

Abstract The germinal center (GC) reaction is the hallmark of B cell-mediated immune responses to T cell-dependent (TD) antigens. Engagement of CD40 by its ligand, CD154, which is expressed on activated CD4+ T cells, follicular helper T (Tfh) cells in particular, is crucial for TD humoral immunity. Previously, it has been demonstrated that miR-146a serves as a molecular break in limiting Tfh cells and the resultant GC B cell responses. Here, by taking the mixed bone marrow chimeras as well as the cell-type-specific miR-146a deletion approaches, we show that like Tfh cells, elevated levels of miR-146a in B cells are equally important in controlling humoral immunity as miR-146a deficiency leads to increased GC B cell differentiation. Moreover, deletion of miR-146a specifically in isotype-switched B cells also resulted in augmented GC responses, suggesting that continued miR-146a expression in GC B cells is required for the maintenance of optimal GC reactions. Mechanistically, we found that miR-146a-ablated B cells exhibit elevated CD40 expression and that enhanced CD40 signaling is accounted for the dysregulated GC responses in mice with B cell-specific miR-146a ablation. Together, our study demonstrates an indispensable role of miR-146a in restricting GC B cell responses and suggests that miR-146a is a critical negative regulator in controlling both T and B cells, two key arms in orchestrating protective humoral immunity.

Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy.

Inflammation has a critical role in the pathogenesis of diabetic complications, including diabetic nephropathy (DN). MicroRNAs have recently emerged as important regulators of DN. However, the role of microRNAs in the regulation of inflammation during DN is poorly understood. Here, we examined the in vivo role of microRNA-146a (miR-146a), a known anti-inflammatory microRNA, in the pathogenesis of DN. In a model of streptozotocin-induced diabetes, miR-146a(-/-) mice showed significantly exacerbated proteinuria, renal macrophage infiltration, glomerular hypertrophy, and fibrosis relative to the respective levels in control wild-type mice. Diabetes-induced upregulation of proinflammatory and profibrotic genes was significantly greater in the kidneys of miR-146a(-/-) than in the kidneys of wild-type mice. Notably, miR-146a expression increased in both peritoneal and intrarenal macrophages in diabetic wild-type mice. Mechanistically, miR-146a deficiency during diabetes led to increased expression of M1 activation markers and suppression of M2 markers in macrophages. Concomitant with increased expression of proinflammatory cytokines, such as IL-1β and IL-18, markers of inflammasome activation also increased in the macrophages of diabetic miR-146a(-/-) mice. These studies suggest that in early DN, miR-146a upregulation exerts a protective effect by downregulating target inflammation-related genes, resulting in suppression of proinflammatory and inflammasome gene activation. Loss of this protective mechanism in miR-146a(-/-) mice leads to accelerated DN. Taken together, these results identify miR-146a as a novel anti-inflammatory noncoding RNA modulator of DN.

Recipient Dendritic Cells Are Regulated By MiR-146a during Acute GvHD

Acute Graft-versus-host disease (GvHD) is a major immunological complication following allogeneic hematopoietic cell transplantation (allo-HCT) occurring in 30-40% of patients. Although immunosuppression with corticosteroids serves as standard first-line therapy in acute GvHD, sustained responses are achieved in less than 50% of patients. Therefore, the identification of risk factors and a better understanding of the molecular mechanisms underlying the disease are indispensable to develop novel therapeutic strategies. MicroRNAs control various key biological processes, including innate and adaptive immune responses. In particular, microRNA-146a (miR-146a) is emerging as a central regulator of innate immune cell signaling.Here we found that a G/C single nucleotide polymorphism (SNP) within the pre-miR-146a gene, which reduces the amount of miR-146a produced from the C allele, was strongly associated with the risk to develop severe acute GvHD. Patients with the rs2910164 CC genotype had a significantly higher risk to develop acute GvHD grade III-IV than all other allo-HCT recipients (P =0.002) as well as an overall increased histopathological GvHD severity. In order to functionally dissect the role of miR-146a in recipient cells during GvHD, we used gene targeted (miR-146a-/-) mice and a major histocompatibility complex (MHC)-mismatched experimental model for acute GvHD. MiR-146a deficiency of the recipient mice resulted in increased GvHD severity, elevated serum levels of pro-inflammatory cytokines and accelerated mortality compared to wild type (WT) recipients after allo-HCT. Chimeric mice with a lack of miR-146a specifically in the hematopoietic system showed an even more distinct disease exacerbation, whereas miR-146a deficiency only in non-hematopoietic recipient cells did not affect GvHD severity. Moreover, co-transplantation of recipient type miR-146a-/-dendritic cells (DCs) elicited a more aggressive course of GvHD, while overexpression of miR-146a in DCs using a specific miR-146a mimic ameliorated disease. Phenotypically, miR-146a-/- DCs displayed a more activated phenotype with increased MHC class II, CD80 and CD86 surface expression levels after endotoxin stimulation when compared to WT DCs. Global gene expression analysis revealed upregulation of the JAK-STAT signaling pathway in miR-146a-/- DCs, which we confirmed at the protein and phosphoprotein level using Western blot and flow cytometry. Importantly, inhibition of JAK-STAT signaling in DCs using the JAK1/2 inhibitor ruxolitinib prevented miR-146a-/- DC-induced GvHD exacerbation.In conclusion, our findings indicate that miR-146a acts as a central regulator of recipient type DCs during GvHD, by dampening cytokine receptor signaling via the JAK-STAT pathway. We detected the SNP rs2910164 as a marker to identify patients at high risk for GvHD before allo-HCT, facilitating the opportunity to apply pre-emptive interventions. DisclosuresNo relevant conflicts of interest to declare.

Identification of Novel Mir-34a Targets in a c-Myc Murine Model

MicroRNAs (miRNAs) serve a fundamental function in the post-transcriptional regulation of gene expression. MiRNA-mediated repression is mediated by the RNA-induced silencing complex (RISC) and is generally thought to be predicted by base-pairing in the seed region of the microRNA. In predicting the effects of miRNAs on gene expression, it is important to consider that the transcriptional context will result in differential targeting of transcripts, and necessitates the study of miRNA function in the cell type of interest. Here, to study tumor suppressive functions of two miRNAs, miR-146a and miR-34a, we utilized the Eμ-Myc transgenic mouse bred to targeted knockouts for these two miRNAs. We found that miR-146a deficiency led to more aggressive B-cell neoplasms that showed a more differentiated B-cell phenotype. Transcriptional changes were fairly limited and were not enriched for miR-146a targets as a class. However, the changes included differences that were consistent with regulation of the transcription factor Egr1, which was demonstrated to be a direct target in human cells. In miR-34a deficient mice with the Eμ-Myc transgene, we did not observe changes in survival or in the phenotype of the neoplasms that developed. However, transcriptional profiling revealed changes in many predicted miR-34a targets, as determined by gene set enrichment analyses (GSEA). Novel predicted targets are being analyzed by luciferase assays. These analyses reveal the importance of assessing cell-type and disease-specific miRNA targeting, which can reveal novel and previously unknown miRNA targets and facilitate our understanding of leukemia and lymphoma pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-146a modulates B-cell oncogenesis by regulating Egr1.

miR-146a is a NF-κB induced microRNA that serves as a feedback regulator of this critical pathway. In mice, deficiency of miR-146a results in hematolymphoid cancer at advanced ages as a consequence of constitutive NF-κB activity. In this study, we queried whether the deficiency of miR-146a contributes to B-cell oncogenesis. Combining miR-146a deficiency with transgenic expression of c-Myc led to the development of highly aggressive B-cell malignancies. Mice transgenic for c-Myc and deficient for miR-146a were characterized by significantly shortened survival, increased lymph node involvement, differential involvement of the spleen and a mature B-cell phenotype. High-throughput sequencing of the tumors revealed significant dysregulation of approximately 250 genes. Amongst these, the transcription factor Egr1 was consistently upregulated in mice deficient for miR-146a. Interestingly, transcriptional targets of Egr1 were enriched in both the high-throughput dataset and in a larger set of miR-146a-deficient tumors. miR-146a overexpression led to downregulation of Egr1 and downstream targets with concomitant decrease in cell growth. Direct targeting of the human EGR1 by miR-146a was seen by luciferase assay. Together our findings illuminate a bona fide role for miR-146a in the modulation of B-cell oncogenesis and reveal the importance of understanding microRNA function in a cell- and disease-specific context.

MicroRNA-146a regulates ICOS-ICOSL signalling to limit accumulation of T follicular helper cells and germinal centres.

Tight control of T follicular helper (Tfh) cells is required for optimal maturation of the germinal centre (GC) response. The molecular mechanisms controlling Tfh-cell differentiation remain incompletely understood. Here we show that microRNA-146a (miR-146a) is highly expressed in Tfh cells and peak miR-146a expression marks the decline of the Tfh response after immunization. Loss of miR-146a causes cell-intrinsic accumulation of Tfh and GC B cells. MiR-146a represses several Tfh-cell-expressed messenger RNAs, and of these, ICOS is the most strongly cell autonomously upregulated target in miR-146a-deficient T cells. In addition, miR-146a deficiency leads to increased ICOSL expression on GC B cells and antigen-presenting cells. Partial blockade of ICOS signalling, either by injections of low dose of ICOSL blocking antibody or by halving the gene dose of Icos in miR-146a-deficient T cells, prevents the Tfh and GC B-cell accumulation. Collectively, miR-146a emerges as a post-transcriptional brake to limit Tfh cells and GC responses.