The gut microbiota regulates white adipose tissue inflammation and obesity via a family of microRNAs.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, dysregulation of the gut microbiota-miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota-derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.

In Vivo Imaging of Small Molecular Weight Peptides for Targeted Renal Drug Delivery: A Study in Normal and Polycystic Kidney Diseased Mice.

Autosomal dominant polycystic kidney disease (ADPKD) is a leading monogenetic cause of end-stage renal disease with limited therapeutic repertoire. A targeted drug delivery strategy that directs a small molecule to renal niches around cysts could increase the safety margins of agents that slow the progression of ADPKD but are poorly tolerated due to extrarenal toxicity. Herein, we determined whether previously characterized lysine-based and glutamic acid-based megalin-binding peptides can achieve renal-specific localization in the juvenile cystic kidney (JCK) mouse model of polycystic kidney disease and whether the distribution is altered compared with control mice. We performed in vivo optical and magnetic resonance imaging studies using peptides conjugated to the VivoTag 680 dye and demonstrated that megalin-interacting peptides distributed almost exclusively to the kidney cortex in both normal and JCK mice. Confocal analysis demonstrated that the peptide-dye conjugate distribution overlapped with megalin-positive renal proximal tubules. However, in the JCK mouse, the epithelium of renal cysts did not retain expression of the proximal tubule markers aquaporin 1 and megalin, and therefore these cysts did not retain peptide-dye conjugates. Furthermore, human kidney tumor tissues were evaluated by immunohistochemistry and revealed significant megalin expression in tissues from patients with renal cell carcinoma, raising the possibility that these tumors could be treated using this drug delivery strategy. Taken together, our data suggest that linking a small-molecule drug to these carrier peptides could represent a promising opportunity to develop a new platform for renal enrichment and targeting in the treatment of ADPKD and certain renal carcinomas.

Increased Expression of Resistin in MicroRNA-155-Deficient White Adipose Tissues May Be a Possible Driver of Metabolically Healthy Obesity Transition to Classical Obesity.

We reported that microRNA-155 (miR-155) deficiency in ApoE-/- mice yields a novel metabolically healthy obese (MHO) model, which exhibits improved atherosclerosis but results in obesity, non-alcoholic fatty liver disease (NAFLD) without insulin resistance. Using experimental data mining approaches combined with experiments, we found that, among 109 miRNAs, miR-155, and miR-221 are significantly modulated in all four hyperlipidemia-related diseases (HRDs), namely atherosclerosis, NAFLD, obesity and type II diabetes (T2DM). MiR-155 is significantly upregulated in atherosclerosis and decreased in other HRDs. MiR-221 is increased in three HRDs but reduced in obesity. These findings led to our new classification of types I and II MHOs, which are regulated by miR-221 and miR-155, respectively. Western blots showed that the proinflammatory adipokine, resistin, is significantly increased in white adipose tissues (WAT) of the MHO mice, revealing our newly proposed, miR-155-suppressed "secondary wave inflammatory state (SWIS)," characteristic of MHO transition to classical obesity (CO). Taken together, we are first to show that MHO may have heterogeneity in comorbidities, and is therefore classified into type I, and type II MHOs; and that increased expression of resistin in miR-155-/- white adipose tissues may be a driver for SWIS in MHO transition to CO. Our findings provide novel insights into the pathogenesis of MHO, MHO transition to CO, hyperlipidemic pathways related to cancer, and new therapeutic targets.

Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA.

Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.

MicroRNA-155 Deficiency Leads to Decreased Atherosclerosis, Increased White Adipose Tissue Obesity, and Non-alcoholic Fatty Liver Disease: A NOVEL MOUSE MODEL OF OBESITY PARADOX.

Obesity paradox (OP) describes a widely observed clinical finding of improved cardiovascular fitness and survival in some overweight or obese patients. The molecular mechanisms underlying OP remain enigmatic partly due to a lack of animal models mirroring OP in patients. Using apolipoprotein E knock-out (apoE-/-) mice on a high fat (HF) diet as an atherosclerotic obesity model, we demonstrated 1) microRNA-155 (miRNA-155, miR-155) is significantly up-regulated in the aortas of apoE-/- mice, and miR-155 deficiency in apoE-/- mice inhibits atherosclerosis; 2) apoE-/-/miR-155-/- (double knock-out (DKO)) mice show HF diet-induced obesity, adipocyte hypertrophy, and present with non-alcoholic fatty liver disease; 3) DKO mice demonstrate HF diet-induced elevations of plasma leptin, resistin, fed-state and fasting insulin and increased expression of adipogenic transcription factors but lack glucose intolerance and insulin resistance. Our results are the first to present an OP model using DKO mice with features of decreased atherosclerosis, increased obesity, and non-alcoholic fatty liver disease. Our findings suggest the mechanistic role of reduced miR-155 expression in OP and present a new OP working model based on a single miRNA deficiency in diet-induced obese atherogenic mice. Furthermore, our results serve as a breakthrough in understanding the potential mechanism underlying OP and provide a new biomarker and novel therapeutic target for OP-related metabolic diseases.

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan.

Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway.

Interleukin-17 (IL-17)-secreting T helper 17 cells were recently identified as a CD4(+) T helper subset and implicated in various inflammatory and autoimmune diseases. The issues of whether and by what mechanism hyperlipidemic stress induces IL-17A to activate aortic endothelial cells (ECs) and enhance monocyte adhesion remained largely unknown. Using biochemical, immunological, microarray, experimental data mining analysis, and pathological approaches focused on primary human and mouse aortic ECs (HAECs and MAECs) and our newly generated apolipoprotein E (ApoE)(-/-)/IL-17A(-/-) mice, we report the following new findings. 1) The hyperlipidemia stimulus oxidized low density lipoprotein up-regulated IL-17 receptor(s) in HAECs and MAECs. 2) IL-17A activated HAECs and increased human monocyte adhesion in vitro. 3) A deficiency of IL-17A reduced leukocyte adhesion to endothelium in vivo. 3) IL-17A activated HAECs and MAECs via up-regulation of proinflammatory cytokines IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine CXC motif ligand 1 (CXCL1), and CXCL2. 4) IL-17A activated ECs specifically via the p38 mitogen-activated protein kinases (MAPK) pathway; the inhibition of p38 MAPK in ECs attenuated IL-17A-mediated activation by ameliorating the expression of the aforementioned proinflammatory cytokines, chemokines, and EC adhesion molecules including intercellular adhesion molecule 1. Taken together, our results demonstrate for the first time that IL-17A activates aortic ECs specifically via p38 MAPK pathway.

Early hyperlipidemia promotes endothelial activation via a caspase-1-sirtuin 1 pathway.

OBJECTIVE: The role of receptors for endogenous metabolic danger signals-associated molecular patterns has been characterized recently as bridging innate immune sensory systems for danger signals-associated molecular patterns to initiation of inflammation in bone marrow-derived cells, such as macrophages. However, it remains unknown whether endothelial cells (ECs), the cell type with the largest numbers and the first vessel cell type exposed to circulating danger signals-associated molecular patterns in the blood, can sense hyperlipidemia. This report determined whether caspase-1 plays a role in ECs in sensing hyperlipidemia and promoting EC activation. APPROACH AND RESULTS: Using biochemical, immunologic, pathological, and bone marrow transplantation methods together with the generation of new apoplipoprotein E (ApoE)(-/-)/caspase-1(-/-) double knockout mice, we made the following observations: (1) early hyperlipidemia induced caspase-1 activation in ApoE(-/-) mouse aorta; (2) caspase-1(-/-)/ApoE(-/-) mice attenuated early atherosclerosis; (3) caspase-1(-/-)/ApoE(-/-) mice had decreased aortic expression of proinflammatory cytokines and attenuated aortic monocyte recruitment; and (4) caspase-1(-/-)/ApoE(-/-) mice had decreased EC activation, including reduced adhesion molecule expression and cytokine secretion. Mechanistically, oxidized lipids activated caspase-1 and promoted pyroptosis in ECs by a reactive oxygen species mechanism. Caspase-1 inhibition resulted in accumulation of sirtuin 1 in the ApoE(-/-) aorta, and sirtuin 1 inhibited caspase-1 upregulated genes via activator protein-1 pathway. CONCLUSIONS: Our results demonstrate for the first time that early hyperlipidemia promotes EC activation before monocyte recruitment via a caspase-1-sirtuin 1-activator protein-1 pathway, which provides an important insight into the development of novel therapeutics for blocking caspase-1 activation as early intervention of metabolic cardiovascular diseases and inflammations.

Metabolomic analyses for atherosclerosis, diabetes, and obesity.

Insulin resistance associated with type 2 diabetes mellitus (T2DM), obesity, and atherosclerosis is a global health problem. A portfolio of abnormalities of metabolic and vascular homeostasis accompanies T2DM and obesity, which are believed to conspire to lead to accelerated atherosclerosis and premature death. The complexity of metabolic changes in the diseases presents challenges for a full understanding of the molecular pathways contributing to the development of these diseases. The recent advent of new technologies in this area termed "Metabolomics" may aid in comprehensive metabolic analysis of these diseases. Therefore, metabolomics has been extensively applied to the metabolites of T2DM, obesity, and atherosclerosis not only for the assessment of disease development and prognosis, but also for the biomarker discovery of disease diagnosis. Herein, we summarize the recent applications of metabolomics technology and the generated datasets in the metabolic profiling of these diseases, in particular, the applications of these technologies to these diseases at the cellular, animal models, and human disease levels. In addition, we also extensively discuss the mechanisms linking the metabolic profiling in insulin resistance, T2DM, obesity, and atherosclerosis, with a particular emphasis on potential roles of increased production of reactive oxygen species (ROS) and mitochondria dysfunctions.

An evolving new paradigm: endothelial cells--conditional innate immune cells.

Endothelial cells (ECs) are a heterogeneous population that fulfills many physiological processes. ECs also actively participate in both innate and adaptive immune responses. ECs are one of the first cell types to detect foreign pathogens and endogenous metabolite-related danger signals in the bloodstream, in which ECs function as danger signal sensors. Treatment with lipopolysaccharide activates ECs, causing the production of pro-inflammatory cytokines and chemokines, which amplify the immune response by recruiting immune cells. Thus, ECs function as immune/inflammation effectors and immune cell mobilizers. ECs also induce cytokine production by immune cells, in which ECs function as immune regulators either by activating or suppressing immune cell function. In addition, under certain conditions, ECs can serve as antigen presenting cells (antigen presenters) by expressing both MHC I and II molecules and presenting endothelial antigens to T cells. These facts along with the new concept of endothelial plasticity suggest that ECs are dynamic cells that respond to extracellular environmental changes and play a meaningful role in immune system function. Based on these novel EC functions, we propose a new paradigm that ECs are conditional innate immune cells. This paradigm provides a novel insight into the functions of ECs in inflammatory/immune pathologies.

Identification of novel pretranslational regulatory mechanisms for NF-κB activation.

NF-κB-controlled transcriptional regulation plays a central role in inflammatory and immune responses. Currently, understanding about NF-κB activation mechanism emphasizes IκB-tethered complex inactivation in the cytoplasm. In the case of NF-κB activation, IκB phosphorylation leads to its degradation, followed by NF-κB relocation to the nucleus and trans-activation of NF-κB-targeted genes. Pretranslational mechanism mediated NF-κB activation remains poorly understood. In this study, we investigated NF-κB pretranslational regulation by performing a series of database mining analyses and using six large national experimental databases (National Center of Biotechnology Information UniGene expressed sequence tag profile database, Gene Expression Omnibus database, Transcription Element Search System database, AceView database, and Epigenomics database) and TargetScan software. We reported the following findings: 1) NF-κB-signaling genes are differentially expressed in human and mouse tissues; 2) heart and vessels are the inflammation-privileged tissues and less easy to be inflamed because lacking in key NF-κB-signaling molecular expression; 3) NF-κB-signaling genes are induced by cardiovascular disease risk factors oxidized phospholipids and proinflammatory cytokines in endothelial cells; 4) transcription factors CCAAT/enhancer-binding proteins and NF-κB have higher binding site frequencies in the promoters of proinflammatory cytokine-induced NF-κB genes; 5) most NF-κB-signaling genes have multiple alternative promoters and alternatively spliced isoforms; 6) NF-κB family genes can be regulated by DNA methylation; and 7) 27 of 38 NF-κB-signaling genes can be regulated by microRNAs. Our findings provide important insight into the mechanism of NF-κB activation, which may contribute to cardiovascular disease, inflammatory diseases, and immunological disorders.

MicroRNAs and toll-like receptor/interleukin-1 receptor signaling.

The discovery of miRNAs has revolutionized the way we examine the genome, RNA products, and the regulation of transcription and translation. Their ability to modulate protein expression through mRNA degradation and translation repression resulted in avid scientific interest in miRNAs over the past decade. This research has led to findings that indicate miRNAs can regulate an array of cellular functions such as cellular apoptosis, proliferation, differentiation, and metabolism. Specifically, the capability of miRNAs to finely-tune gene expression naturally lends itself to immune system regulation which requires precise control for proper activity. In fact, abnormal miRNAs expression is often seen with inflammatory disorders like rheumatoid arthritis, systemic lupus erthematosus, experimental autoimmune encephalomyelitis, and inflammatory cancers. As a result, research investigating miRNAs modulation of immune cell proliferation, differentiation, and cellular signaling has yielded fruitful results. Specifically, in this review, we will examine the impact of miRNAs on toll-like receptor (TLRs) and interleukin-1ß (IL-1ß) signaling, which are integral in the proper functioning of the innate immune system. These signaling pathways share several key downstream signaling adaptors and therefore produce similar downstream effects such as the production of pro-inflammatory cytokines, chemokines, and interferons. This review will examine in depth the specific interactions of miRNAs with receptors, adaptor molecules, and regulator molecules within these cellular pathways. In addition, we will discuss the modulation of miRNAs' expression by TLR and IL-1R signaling through positive and negative feedback loops.

Endothelial progenitor cells in atherosclerosis.

Endothelial progenitor cells (EPCs) are involved in the maintenance of endothelial homoeostasis and in the process of new vessel formation. Experimental and clinical studies have shown that atherosclerosis is associated with reduced numbers and dysfunction of EPCs; and that medications alone are able to partially reverse the impairment of EPCs in patients with atherosclerosis. Therefore, novel EPC-based therapies may provide enhancement in restoring EPCs' population and improvement of vascular function. Here, for a better understanding of the molecular mechanisms underlying EPC impairment in atherosclerosis, we provide a comprehensive overview on EPC characteristics, phenotypes, and the signaling pathways underlying EPC impairment in atherosclerosis.

IL-35 is a novel responsive anti-inflammatory cytokine--a new system of categorizing anti-inflammatory cytokines.

It remains unknown whether newly identified anti-inflammatory/immunosuppressive cytokine interleukin-35 (IL-35) is different from other anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-ß in terms of inhibition of inflammation initiation and suppression of full-blown inflammation. Using experimental database mining and statistical analysis methods we developed, we examined the tissue expression profiles and regulatory mechanisms of IL-35 in comparison to other anti-inflammatory cytokines. Our results suggest that in contrast to TGF-ß, IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. We also provide structural evidence that AU-rich element (ARE) binding proteins and microRNAs target IL-35 subunit transcripts, by which IL-35 may achieve non-constitutive expression status. Furthermore, we propose a new system to categorize anti-inflammatory cytokines into two groups: (1) the house-keeping cytokines, such as TGF-ß, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL-35 suppress inflammation in full-blown stage. Our in-depth analyses of molecular events that regulate the production of IL-35 as well as the new categorization system of anti-inflammatory cytokines are important for the design of new strategies of immune therapies.

MicroRNAs and other mechanisms regulate interleukin-17 cytokines and receptors.

Interleukin-17 cytokines are a family of pro-inflammatory cytokines. Our current studies found: i) IL-17 cytokines are not ubiquitously expressed, but several receptors and TRAF3IP2 are ubiquitously expressed in tissues with a few exceptions; ii) heart and vascular tissue are in the second tier of readiness to respond to IL-17 cytokine stimulation; iii) alternative transcription starting sites and alternative spliced isoforms are found in IL-17 cytokine and receptor transcripts; iv) higher hypomethylation status is associated with higher expressions of IL-17 receptors; v) the binding sites of several RNA binding proteins are found in the 3'UTRs of the mRNAs of IL-17 cytokines and receptors; and vi) numerous microRNA binding sites are statistically equivalent to that of experimentally verified microRNAs-mRNA interactions in the 3'UTRs of IL-17 cytokine and receptor mRNAs. These results suggest that mechanisms including alternative promoters, alternative splicing, RNA binding proteins, and microRNAs regulate the structures and expressions of IL-17 cytokines and receptors. These results provide an insight into the roles of IL-17 in mediating inflammation and immunity.

Regulatory T cells and Atherosclerosis.

Atherosclerosis is a chronic autoimmune inflammatory disease. The involvement of both innate and adaptive immune responses in the pathogenesis of the disease has been well recognized. Tregs are an essential part of the immune system and have indispensable functions in maintaining immune system homeostasis, mediating peripheral tolerance, preventing autoimmune diseases, and suppressing inflammatory and proatherogenic immune response. Tregs carry out their immunosuppressive functions via several mechansims. One of the well-documented suppressive mechanisms of Tregs is the secretion of anti-inflammatory cytokines including IL-10, TGF-ß, and IL-35. Studies have found that IL-10 and TGF-ß have atheroprotective properties. In addition, Tregs can suppress the activity of proatherogenic effector T cells, suggesting an atheroprotective role. In fact, fewer Tregs are found in atherogenic ApoE-/- mice comparing to wild-type mice, suggesting an uncontrolled balance between weakened Tregs and effector T cells in atherogenesis. Some clinical studies of autoimmune diseases also suggest that decreased Tregs numbers are associated with increased disease activity. The importance of Tregs in many autoimmune diseases and experimental atherosclerosis has been established in in vivo and in vitro studies. However, the roles of Tregs in atherosclerosis in the clinical setting remains to be further characterized.

Structural evidence of anti-atherogenic microRNAs.

Our research attempted to address two important questions--how microRNAs modulate atherogenic inflammatory genes from a panoramic viewpoint and whether their augmented expression results from reduced microRNAs suppression. To resolve these knowledge gaps, we employed a novel database mining technique in conjunction with statistical analysis criteria established from experimentally verified microRNAs. We found that the expression of 33 inflammatory genes up-regulated in atherosclerotic lesions contain structural features in the 3'UTR of their mRNAs for potential microRNAs regulation. Additionally, the binding features governing the interactions between the microRNAs and the inflammatory gene mRNA were statistically identical to the features of experimentally verified microRNAs. Furthermore, 21 of the 33 inflammatory genes (64%) were targeted by highly expressed microRNAs and 10 of these (48%) were targeted by a single microRNA, suggesting microRNA regulation specificity. Supplementing our findings, 7 out of the 20 unique microRNAs (35%) were previously confirmed to be down-regulated when treated with pro-atherogenic factors. These results indicate a critical role of anti-inflammatory microRNAs in suppressing pro-atherogenic inflammatory gene expression.

The Role of Immunogenicity in Cardiovascular Disease.

Recently, many of the complexities associated with cardiovascular diseases (CVD) have been unlocked. However, despite these breakthroughs, CVD and its related complications are the leading contributors of morbidity and mortality worldwide, which indicates the shortcomings of current treatment regimens and the need for continued research. Published data within the field clearly indicates that CVD are built on inflammation and autoimmune platforms, though a strong, fundamental understanding of the mechanisms remains elusive. Areas such as the mechanisms underlying increased immunogenicity of self-proteins in the cardiovascular system, the roles of immunogenic auto-antigens in eliciting inflammatory autoimmune responses, and the immunosuppressive mechanisms involved in controlling inflammatory and autoimmune cardiovascular diseases remain to be well-understood. We will delve into these topics and the advancements made within the field in this review. Specifically, we will concentrate on the innate and adaptive immune responses mediating immunogenicity; the mechanisms of inflammation and autoimmunity in atherogenesis; the mechanisms of inflammation and autoimmunity in diabetic atherosclerosis; immunogenicity and stem cell therapy; as well as immunogenicity and immunosuppression. In depth examination and comprehension of these topics will provide insight into the recent progress of the field and bring to the forefront potentially novel therapeutic avenues.

An interactive framework for developing simulation models of hospital accident and emergency services.

Discrete-event simulation can be a valuable tool in modelling health care systems. This paper describes an interactive framework to model and simulate a hospital accident and emergency department. An interactive spreadsheet (Excel) facilitated the user-friendly input of data such as patient pathways, arrival times, service times and resources into the discrete event simulation package (SIMUL8). The framework was enhanced further by configuring SIMUL8 to visually show patient flow and activity on a schematic plan of an A&E. The patient flow and activity information included patient icons flowing along A&E corridors and pathways, processes undertaken in A&E work areas and queue activity. One major benefit of visually showing patient flow and activity was that modellers and decision makers could visually gain a dynamic insight into the performance of the overall system and visually see changes over the model run cycle. Another key benefit of the interactive framework was the ability to quickly and easily change model parameters to trial, test and compare different scenarios.