The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell type-specific target genes.

The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3' RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.

The miR-183/96/182 cluster is a checkpoint for resident immune cells and shapes the cellular landscape of the cornea.

PURPOSE: The conserved miR-183/96/182 cluster (miR-183C) regulates both corneal sensory innervation and corneal resident immune cells (CRICs). This study is to uncover its role in CRICs and in shaping the corneal cellular landscape at a single-cell (sc) level. METHODS: Corneas of naïve, young adult [2 and 6 months old (mo)], female miR-183C knockout (KO) mice and wild-type (WT) littermates were harvested and dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit. CD45+ CRICs were enriched by Magnetic Activated Cell Sorting (MACS). scRNA libraries were constructed and sequenced followed by comprehensive bioinformatic analyses. RESULTS: The composition of major cell types of the cornea stays relatively stable in WT mice from 2 to 6 mo, however the compositions of subtypes of corneal cells shift with age. Inactivation of miR-183C disrupts the stability of the major cell-type composition and age-related transcriptomic shifts of subtypes of corneal cells. The diversity of CRICs is enhanced with age. Naïve mouse cornea contains previously-unrecognized resident fibrocytes and neutrophils. Resident macrophages (ResMφ) adopt cornea-specific function by expressing abundant extracellular matrix (ECM) and ECM organization-related genes. Naïve cornea is endowed with partially-differentiated proliferative ResMφ and contains microglia-like Mφ. Resident lymphocytes, including innate lymphoid cells (ILCs), NKT and γδT cells, are the major source of innate IL-17a. miR-183C limits the diversity and polarity of ResMφ. CONCLUSION: miR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.

Host-microbe interactions in cornea.

Corneal infections result through interaction between microbes and host innate immune receptors. Damage to the cornea occurs as a result of microbial virulence factors and is often exacerbated by lack of a controlled host immune response; the latter contributing to bystander damage to corneal structure. Understanding mechanisms involved in host microbial interactions is critical to development of novel therapeutic targets, ultimate control of microbial pathogenesis, and restoration of tissue homeostasis. Studies on these interactions continue to provide exciting findings directly related to this ultimate goal.

Glycyrrhizin Interacts with TLR4 and TLR9 to Resolve P. aeruginosa Keratitis.

This study tests the mechanism(s) of glycyrrhizin (GLY) protection against P. aeruginosa keratitis. Female C57BL/6 (B6), TLR4 knockout (TLR4KO), myeloid specific TLR4KO (mTLR4KO), their wildtype (WT) littermates, and TLR9 knockout (TLR9KO) mice were infected with P. aeruginosa KEI 1025 and treated with GLY or PBS onto the cornea after infection. Clinical scores, photography with a slit lamp, RT-PCR and ELISA were used. GLY effects on macrophages (Mϕ) and polymorphonuclear neutrophils (PMN) isolated from WT and mTLR4KO and challenged with KEI 1025 were also tested. Comparing B6 and TLR4KO, GLY treatment reduced clinical scores and improved disease outcome after infection and decreased mRNA expression levels in cornea for TLR4, HMGB1, and RAGE in B6 mice. TLR9 mRNA expression was significantly reduced by GLY in both mouse strains after infection. GLY also significantly reduced HMGB1 (B6 only) and TLR9 protein (both B6 and TLR4KO). In TLR9KO mice, GLY did not significantly reduce clinical scores and only slightly improved disease outcome after infection. In these mice, GLY significantly reduced TLR4, but not HMGB1 or RAGE mRNA expression levels after infection. In contrast, in the mTLR4KO and their WT littermates, GLY significantly reduced corneal disease, TLR4, TLR9, HMGB1, and RAGE corneal mRNA expression after infection. GLY also significantly reduced TLR9 and HMGB1 corneal protein levels in both WT and mTLR4KO mice. In vitro, GLY significantly lowered mRNA expression levels for TLR9 in both Mϕ and PMN isolated from mTLR4KO or WT mice after incubation with KEI 1025. In conclusion, we provide evidence to show that GLY mediates its effects by blocking TLR4 and TLR9 signaling pathways and both are required to protect against disease.

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa-Induced Keratitis.

Purpose: Previously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential. Methods: Eight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid-modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-ß-III tubulin antibody. Results: Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1ß at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII+ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression. Conclusions: Prophylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.

The miR-183/96/182 Cluster Regulates the Functions of Corneal Resident Macrophages.

Tissue-resident macrophages (ResMϕ) play important roles in the normal development and physiological functions as well as tissue repair and immune/inflammatory response to both internal and external insults. In cornea, ResMϕ are critical to the homeostasis and maintenance, wound healing, ocular immune privilege, and immune/inflammatory response to injury and microbial infection. However, the roles of microRNAs in corneal ResMϕ are utterly unknown. Previously, we demonstrated that the conserved miR-183/96/182 cluster (miR-183/96/182) plays important roles in sensory neurons and subgroups of both innate and adaptive immune cells and modulates corneal response to bacterial infection. In this study, we provide direct evidence that the mouse corneal ResMϕ constitutively produce both IL-17f and IL-10. This function is regulated by miR-183/96/182 through targeting Runx1 and Maf, key transcriptional regulators for IL-17f and IL-10 expression, respectively. In addition, we show that miR-183/96/182 has a negative feedback regulation on the TLR4 pathway in mouse corneal ResMϕ. Furthermore, miR-183/96/182 regulates the number of corneal ResMϕ. Inactivation of miR-183/96/182 in mouse results in more steady-state corneal resident immune cells, including ResMϕ, and leads to a simultaneous early upregulation of innate IL-17f and IL-10 production in the cornea after Pseudomonas aeruginosa infection. Its multiplex regulations on the simultaneous production of IL-17f and IL-10, TLR4 signaling pathway and the number of corneal ResMϕ place miR-183/96/182 in the center of corneal innate immunity, which is key to the homeostasis of the cornea, ocular immune privilege, and the corneal response to microbial infections.

Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction.

Improved in situ hybridization methods for mRNA detection in tissues have been developed based on the hybridization chain reaction (HCR). We show that in situ HCR methods can be used for the detection of microRNAs in tissue sections from mouse retinas. In situ HCR can be used for the detection of two microRNAs simultaneously or for the combined detection of microRNA and mRNA. In addition, miRNA in situ HCR can be combined with immunodetection of proteins. We use these methods to characterize cells expressing specific microRNAs in the mouse retina. We find that miR-181a is expressed in amacrine cells during development and in adult retinas, and it is present in both GABAergic and glycinergic amacrine cells. The detection of microRNAs with in situ HCR should facilitate studies of microRNA function and gene regulation in the retina and other tissues.

Mutation Screening in the miR-183/96/182 Cluster in Patients With Inherited Retinal Dystrophy.

Inherited retinal dystrophy (IRD) is a heterogenous blinding eye disease and affects more than 200,000 Americans and millions worldwide. By far, 270 protein-coding genes have been identified to cause IRD when defective. However, only one microRNA (miRNA), miR-204, has been reported to be responsible for IRD when a point-mutation occurs in its seed sequence. Previously, we identified that a conserved, polycistronic, paralogous miRNA cluster, the miR-183/96/182 cluster, is highly specifically expressed in all photoreceptors and other sensory organs; inactivation of this cluster in mice resulted in syndromic IRD with multi-sensory defects. We hypothesized that mutations in the miR-183/96/182 cluster in human cause IRD. To test this hypothesis, we perform mutation screening in the pre-miR-183, -96, -182 in >1000 peripheral blood DNA samples of patients with various forms of IRD. We identified six sequence variants, three in pre-miR-182 and three in pre-miR-96. These variants are in the pre-miRNA-182 or -96, but not in the mature miRNAs, and are unlikely to be the cause of the IRD in these patients. In spite of this, the nature and location of these sequence variants in the pre-miRNAs suggest that some may have impact on the biogenesis and maturation of miR-182 or miR-96 and potential roles in the susceptibility to diseases. Although reporting on negative results so far, our study established a system for mutation screening in the miR-183/96/182 cluster in human for a continued effort to unravel and provides deeper insight into the potential roles of miR-183/96/182 cluster in human diseases.

miR-183-96-182 Cluster Is Involved in Invariant NKT Cell Development, Maturation, and Effector Function.

The development, differentiation and function of invariant NKT (iNKT) cells require a well-defined set of transcription factors, but how these factors are integrated to each other and the detailed signaling networks remain poorly understood. Using a Dicer-deletion mouse model, our previous studies have demonstrated the critical involvement of microRNAs (miRNAs) in iNKT cell development and function, but the role played by individual miRNAs in iNKT cell development and function is still not clear. In this study, we show the dynamic changes of miRNA 183 cluster (miR-183C) expression during iNKT cell development. Mice with miR-183C deletion showed a defective iNKT cell development, sublineage differentiation, and cytokine secretion function. miRNA target identification assays indicate the involvement of multiple target molecules. Our study not only confirmed the role of miR-183C in iNKT cell development and function but also demonstrated that miR-183C achieved the regulation of iNKT cells through integrated targeting of multiple signaling molecules and pathways.

MicroRNAs in Ocular Infection.

MicroRNAs (miRNAs) are small, non-coding, regulatory RNA molecules and constitute a newly recognized, important layer of gene-expression regulation at post-transcriptional levels. miRNAs quantitatively fine tune the expression of their downstream genes in a cell type- and developmental stage-specific fashion. miRNAs have been proven to play important roles in the normal development and function as well as in the pathogenesis of diseases in all tissues and organ systems. miRNAs have emerged as new therapeutic targets and biomarkers for treatment and diagnosis of various diseases. Although miRNA research in ocular infection remains in its early stages, a handful of pioneering studies have provided insight into the roles of miRNAs in the pathogenesis of parasitic, fungal, bacterial, and viral ocular infections. Here, we review the current status of research in miRNAs in several major ocular infectious diseases. We predict that the field of miRNAs in ocular infection will greatly expand with the discovery of novel miRNA-involved molecular mechanisms that will inform development of new therapies and identify novel diagnostic biomarkers.

The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses.

Although primary humoral responses are vital to durable immunity, fine-tuning is critical to preventing catastrophes such as autoimmunity, chronic inflammation, and lymphomagenesis. MicroRNA (miRNA)-mediated regulation is particularly well suited for fine-tuning roles in physiology. Expression of clustered paralogous miR-182, miR-96, and miR-183 (collectively, 183c) is robustly induced upon B cell activation, entry into the germinal center, and plasmablast differentiation. 183cGT/GT mice lacking 183c miRNA expression exhibit largely normal primary humoral responses, encompassing class switch recombination, affinity maturation, and germinal center reaction, as well as plasmablast differentiation. Our rigorous analysis included ex vivo class switch recombination and plasmablast differentiation models as well as in vivo immunization with thymus-dependent and thymus-independent Ags. Our work sways the debate concerning the role of miR-182 in plasmablast differentiation, strongly suggesting that 183c miRNAs are dispensable. In the process, we present a valuable framework for systematic evaluation of primary humoral responses. Finally, our work bolsters the notion of robustness in miRNA:target interaction networks and advocates a paradigm shift in miRNA studies.

The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their production of reactive nitrogen (RNS) and oxygen species (ROS) and their inflammatory response to Pseudomonas aeruginosa (PA) infection and/or lipopolysaccharide (LPS) treatment. We show that knockdown of miR-183/96/182 results in decreased production of multiple proinflammatory cytokines in response to PA or LPS treatment in Mϕ-like Raw264.7 cells. Consistently, peritoneal Mϕ from miR-183/96/182-knockout versus wild-type mice are less responsive to PA or LPS, although their basal levels of proinflammatory cytokines are increased. In addition, overexpression of miR-183/96/182 results in decreased production of nitrite and ROS in Raw264.7 cells. We also provide evidence that DAP12 and Nox2 are downstream target genes of miR-183/96/182. These data suggest that miR-183/96/182 imposes global regulation on various aspects of Mϕ function through different downstream target genes.

The microRNA-183/96/182 Cluster is Essential for Stereociliary Bundle Formation and Function of Cochlear Sensory Hair Cells.

The microRNA (miR)-183/96/182 cluster plays important roles in the development and functions of sensory organs, including the inner ear. Point-mutations in the seed sequence of miR-96 result in non-syndromic hearing loss in both mice and humans. However, the lack of a functionally null mutant has hampered the evaluation of the cluster's physiological functions. Here we have characterized a loss-of-function mutant mouse model (miR-183CGT/GT), in which the miR-183/96/182 cluster gene is inactivated by a gene-trap (GT) construct. The homozygous mutant mice show profound congenital hearing loss with severe defects in cochlear hair cell (HC) maturation, alignment, hair bundle formation and the checkboard-like pattern of the cochlear sensory epithelia. The stereociliary bundles retain an immature appearance throughout the cochlea at postnatal day (P) 3 and degenerate soon after. The organ of Corti of mutant newborn mice has no functional mechanoelectrical transduction. Several predicted target genes of the miR-183/96/182 cluster that are known to play important roles in HC development and function, including Clic5, Rdx, Ezr, Rac1, Myo1c, Pvrl3 and Sox2, are upregulated in the cochlea. These results suggest that the miR-183/96/182 cluster is essential for stereociliary bundle formation, morphogenesis and function of the cochlear HCs.

Intraocular Delivery of miR-146 Inhibits Diabetes-Induced Retinal Functional Defects in Diabetic Rat Model.

Purpose: Previously, we showed that microRNA-146 (miR-146) is a pivotal negative feedback regulator of multiple nuclear factor kappa-B (NF-κB) activation pathways in retinal endothelial cells (RECs). We hypothesized that miR-146 plays an important role in diabetic retinopathy (DR) by inhibiting diabetes-induced inflammatory response in the retina. The purpose of the current study is to test this hypothesis in vivo. Methods: Lentiviruses expressing rno-miR-146a, lenti-miR-146a, and negative control oligonucleotide with scrambled sequence, lenti-miR-neg ctl, were produced. Young male Sprague-Dawley rats were injected with a single dose of streptozotocin ([STZ] 65 mg/kg) to induce diabetes. One week after diabetes, animals were injected with lentivirus intravitreally (4 µl, ∼106 CFU/mL). Three months after diabetes, retinal microvascular leakage was tested by Evans blue assay; retinal function by electroretinogram (ERG). Total RNA and protein lysate were isolated from the retina for quantitative (q)RT-PCR and Western blot analyses. Results: Lenti-miR-146a robustly transduced human retinal endothelial cells (HRECs) and increased the expression of miR-146a in vitro. In vivo, intravitreal injection of lenti-miR-146a increased the expression of miR-146a in the retina, while its key downstream target genes, including CARD10, IRAK1, and TRAF6, were downregulated. Intravitreal delivery of miR-146 inhibited diabetes-induced upregulation of NF-κB downstream gene, Intercellular Adhesion Molecule 1 (ICAM1), as well as microvascular leakage and retinal functional defects. Conclusions: Intravitreal delivery of miR-146 inhibited diabetes-induced NF-κB activation and retinal microvascular and neuronal functional defects in a diabetic rat model.

A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper cells.

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4(+) T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and function.

The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression.

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.

Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PURPOSE: The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions. METHODS: Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays. RESULTS: MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease. CONCLUSIONS: MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

At the interface of sensory and motor dysfunctions and Alzheimer's disease.

Recent evidence indicates that sensory and motor changes may precede the cognitive symptoms of Alzheimer's disease (AD) by several years and may signify increased risk of developing AD. Traditionally, sensory and motor dysfunctions in aging and AD have been studied separately. To ascertain the evidence supporting the relationship between age-related changes in sensory and motor systems and the development of AD and to facilitate communication between several disciplines, the National Institute on Aging held an exploratory workshop titled "Sensory and Motor Dysfunctions in Aging and AD." The scientific sessions of the workshop focused on age-related and neuropathologic changes in the olfactory, visual, auditory, and motor systems, followed by extensive discussion and hypothesis generation related to the possible links among sensory, cognitive, and motor domains in aging and AD. Based on the data presented and discussed at this workshop, it is clear that sensory and motor regions of the central nervous system are affected by AD pathology and that interventions targeting amelioration of sensory-motor deficits in AD may enhance patient function as AD progresses.

MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.

PURPOSE: Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions. METHODS: Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. RESULTS: We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. CONCLUSIONS: We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR.