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Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Leveraging spatial molecular information to construct a phylogeographic map of tumor evolution can reveal individualized growth trajectories with diagnostic and therapeutic potential. Integrative analysis of spatial multi-omic data from 31 colorectal specimens revealed simultaneous microenvironmental and clonal alterations as a function of progression. Copy number variation served to re-stratify microsatellite stable and unstable tumors into chromosomally unstable (CIN+) and hypermutated (HM) classes. Phylogeographical maps classified tumors by their evolutionary dynamics, and clonal regions were placed along a global pseudotemporal progression trajectory. Cell-state discovery from a single-cell cohort revealed recurring epithelial gene signatures and infiltrating immune states in spatial transcriptomics. Charting these states along progression pseudotime, we observed a transition to immune exclusion in CIN+ tumors as characterized by a novel gene expression signature comprised ofDDR1, TGFBI, PAK4,andDPEP1. We demonstrated how these genes and their protein products are key regulators of extracellular matrix components, are associated with lower cytotoxic immune infiltration, and show prognostic value in external cohorts. Through high-dimensional data integration, this atlas provides insights into co-evolution of tumors and their microenvironments, serving as a resource for stratification and targeted treatment of CRC.

Background Little is known about how benign prostatic hyperplasia (BPH) develops and why patients respond differently to medical therapy designed to reduce lower urinary tract symptoms (LUTS). The Medical Therapy of Prostatic Symptoms (MTOPS) trial randomized men with symptoms of BPH and followed response to medical therapy for up to 6 years. Treatment with a 5α-reductase inhibitor (5ARI) or an alpha-adrenergic receptor antagonist (α-blocker) reduced the risk of clinical progression, while men treated with combination therapy showed a 66% decrease in risk of progressive disease. However, medical therapies for BPH/LUTS are not effective in many patients. The reasons for nonresponse or loss of therapeutic response in the remaining patients over time are unknown. A better understanding of why patients fail to respond to medical therapy may have a major impact on developing new approaches for the medical treatment of BPH/LUTS. Prostaglandins (PG) act on G-protein-coupled receptors (GPCRs), where PGE2 and PGF2 elicit smooth muscle contraction. Therefore, we measured PG levels in the prostate tissue of BPH/LUTS patients to assess the possibility that this signaling pathway might explain the failure of medical therapy in BPH/LUTS patients. Method Surgical BPH (S-BPH) was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy and underwent surgical intervention to relieve LUTS. Control tissue was termed Incidental BPH (I-BPH). I-BPH was TZ obtained from men undergoing radical prostatectomy for low-volume, low-grade prostatic adenocarcinoma (PCa, Gleason score ≤ 7) confined to the peripheral zone. All TZ tissue was confirmed to be cancer-free. S-BPH patients divided into four subgroups: patients on α-blockers alone, 5ARI alone, combination therapy (α-blockers plus 5ARI), or no medical therapy (none) before surgical resection. I-BPH tissue was subgrouped by prior therapy (either on α-blockers or without prior medical therapy before prostatectomy). We measured prostatic tissue levels of prostaglandins (PGF2α , PGI2 , PGE2 , PGD2 , and TxA2 ), quantitative polymerase chain reaction levels of mRNAs encoding enzymes within the PG synthesis pathway, cellular distribution of COX1 (PTGS1) and COX2 (PTGS2), and tested the ability of PGs to contract bladder smooth muscle in an in vitro assay. Results All PGs were significantly elevated in TZ tissues from S-BPH patients (n = 36) compared to I-BPH patients (n = 15), regardless of the treatment subgroups. In S-BPH versus I-BPH, mRNA for PG synthetic enzymes COX1 and COX2 were significantly elevated. In addition, mRNA for enzymes that convert the precursor PGH2 to metabolite PGs were variable: PTGIS (which generates PGI2 ) and PTGDS (PGD2 ) were significantly elevated; nonsignificant increases were observed for PTGES (PGE2 ), AKR1C3 (PGF2α ), and TBxAS1 (TxA2 ). Within the I-BPH group, men responding to α-blockers for symptoms of BPH but requiring prostatectomy for PCa did not show elevated levels of COX1, COX2, or PGs. By immunohistochemistry, COX1 was predominantly observed in the prostatic stroma while COX2 was present in scattered luminal cells of isolated prostatic glands in S-BPH. PGE2 and PGF2α induced contraction of bladder smooth muscle in an in vitro assay. Furthermore, using the smooth muscle assay, we demonstrated that α-blockers that inhibit alpha-adrenergic receptors do not appear to inhibit PG stimulation of GPCRs in bladder muscle. Only patients who required surgery to relieve BPH/LUTS symptoms showed significantly increased tissue levels of PGs and the PG synthetic enzymes. Conclusions Treatment of BPH/LUTS by inhibition of alpha-adrenergic receptors with pharmaceutical α-blockers or inhibiting androgenesis with 5ARI may fail because of elevated paracrine signaling by prostatic PGs that can cause smooth muscle contraction. In contrast to patients who fail medical therapy for BPH/LUTS, control I-BPH patients do not show the same evidence of elevated PG pathway signaling. Elevation of the PG pathway may explain, in part, why the risk of clinical progression in the MTOPS study was only reduced by 34% with α-blocker treatment.

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single cell-based RNA-seq of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) has not been previously compared to that of T1DN. We analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26fsTRAP; eNOS-/-; db/db mice and compared it to streptozotocin-induced T1DN in podocin-Cre; Rosa26fsTRAP; eNOS-/- mice utilizing Translating Ribosome Affinity Purification (TRAP) and RNA-seq. Over 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN compared to T1DN. TGF-β signaling pathway genes were upregulated while MAPK pathway genes were downregulated only in T2DN while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number diabetes-induced genes not previously been linked to podocyte injury were confirmed in both mouse and human DN. Differences and similarities in the podocyte translatome in T2DN and T1DN can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury.

Summary Single-cell RNA sequencing data require several processing procedures to arrive at interpretable results. While commercial platforms can serve as “one-stop shops” for data analysis, they relinquish the flexibility required for customized analyses and are often inflexible between experimental systems. For instance, there is no universal solution for the discrimination of informative or uninformative encapsulated cellular material; thus, pipeline flexibility takes priority. Here, we demonstrate a full data analysis pipeline, constructed modularly from open-source software, including tools that we have contributed. For complete details on the use and execution of this protocol, please refer to Petukhov et al. (2018), Heiser et al. (2020), and Heiser and Lau (2020)., Graphical Abstract, Highlights • An open-source, containerized pipeline for processing scRNA-seq data • The provided Singularity and Conda files permit rapid software installation • Multiple methods detailed for matrix quality control are provided for modular usage • Data entry points are highlighted for flexibility with other pipelines, Single-cell RNA sequencing data require several processing procedures to arrive at interpretable results. While commercial platforms can serve as “one-stop shops” for data analysis, they relinquish the flexibility required for customized analyses and are often inflexible between experimental systems. For instance, there is no universal solution for the discrimination of informative or uninformative encapsulated cellular material; thus, pipeline flexibility takes priority. Here, we demonstrate a full data analysis pipeline, constructed modularly from open-source software, including tools that we have contributed.

Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SSLs). The origins and molecular landscapes of these histologically distinct pre-cancerous polyps remain incompletely understood. Here, we present an atlas at single-cell resolution of sporadic conventional tubular/tubulovillous ADs, SSLs, hyperplastic polyps (HPs), microsatellite stable (MSS) and unstable (MSI-H) CRC, and normal colonic mucosa. Using single-cell transcriptomics and multiplex imaging, we studied 69 datasets from 33 participants. We also examined separate sets of 66 and 274 polyps for RNA and targeted gene sequencing, respectively. We performed multiplex imaging on a tissue microarray of 14 ADs and 15 CRCs, and we integrated pre-cancer polyp data with published single-cell and The Cancer Genome Atlas (TCGA) bulk CRC data to establish potential polyp-cancer relationships. Striking differences were observed between ADs and SSLs that extended to MSS and MSI-H CRCs, respectively, reflecting their distinct origins and trajectories. ADs arose from WNT pathway dysregulation in stem cells, which aberrantly expanded and expressed a Hippo and ASCL2 regenerative program. In marked contrast, SSLs were depleted of stem cell-like populations and instead exhibited a program of gastric metaplasia in the setting of elevated cytotoxic inflammation. Using subtype-specific gene regulatory networks and shared genetic variant analysis, we implicated serrated polyps, including some HPs conventionally considered benign, as arising from a metaplastic program in committed absorptive cells. ADs and SSLs displayed distinct patterns of immune cell infiltration that may influence their natural history. Our multi-omic atlas provides novel insights into the malignant potential of colorectal polyps and serves as a framework for precision surveillance and prevention of sporadic CRC.

ObjectivesTo determine if plasma microbial small RNAs (sRNAs) are altered in patients with rheumatoid arthritis (RA) compared with control subjects, associated with RA disease-related features, and altered by disease-modifying antirheumatic drugs (DMARDs).MethodssRNA sequencing was performed on plasma from 165 patients with RA and 90 matched controls and a separate cohort of 70 patients with RA before and after starting a DMARD. Genome alignments for RA-associated bacteria, representative bacterial and fungal human microbiome genomes and environmental bacteria were performed. Microbial genome counts and individual sRNAs were compared across groups and correlated with disease features. False discovery rate was set at 0.05.ResultsGenome counts of Lactobacillus salivarius, Anaerobaculum hydrogeniformans, Staphylococcus epidermidis, Staphylococcus aureus, Paenisporosarcina spp, Facklamia hominis, Sphingobacterium spiritivorum, Lentibacillus amyloliquefaciens, Geobacillus spp, and Pseudomonas fluorescens were significantly decreased in the plasma of RA compared with control subjects. Three microbial transfer RNA-derived sRNAs were increased in RA versus controls and inversely associated with disease activity. Higher total microbial sRNA reads were associated with lower disease activity in RA. Baseline total microbial sRNAs were threefold higher among patients who improved with DMARD versus those who did not but did not change significantly after 6 months of treatment.ConclusionPlasma microbial sRNA composition is altered in RA versus control subjects and associated with some measures of RA disease activity. DMARD treatment does not alter microbial sRNA abundance or composition, but increased abundance of microbial sRNAs at baseline was associated with disease activity improvement at 6 months.

Background The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Results Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. Conclusions Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Background and aims Cardiovascular disease (CVD) is the leading cause of death in chronic kidney disease (CKD) patients, however, the underlying mechanisms that link CKD and CVD are not fully understood and limited treatment options exist in this high-risk population. microRNAs (miRNA) are critical regulators of gene expression for many biological processes in atherosclerosis, including endothelial dysfunction and inflammation. We hypothesized that renal injury-induced endothelial miRNAs promote atherosclerosis. Here, we demonstrate that dual inhibition of endothelial miRNAs inhibits atherosclerosis in the setting of renal injury. Methods Aortic endothelial miRNAs were analyzed in apolipoprotein E-deficient (Apoe−/-) mice with renal damage (5/6 nephrectomy, 5/6Nx) by real-time PCR. Endothelial miR-92a-3p and miR-489-3p were inhibited by locked-nucleic acid (LNA) miRNA inhibitors complexed to HDL. Results Renal injury significantly increased endothelial miR-92a-3p levels in Apoe−/-;5/6Nx mice. Dual inhibition of miR-92a-3p and miR-489-3p in Apoe−/−;5/6Nx with a single injection of HDL + LNA inhibitors significantly reduced atherosclerotic lesion area by 28.6% compared to HDL + LNA scramble (LNA-Scr) controls. To examine the impact of dual LNA treatment on aortic endothelial gene expression, total RNA sequencing was completed, and multiple putative target genes and pathways were identified to be significantly altered, including the STAT3 immune response pathway. Among the differentially expressed genes, Tgfb2 and Fam220a were identified as putative targets of miR-489-3p and miR-92a-3p, respectively. Both Tgfb2 and Fam220a were significantly increased in aortic endothelium after miRNA inhibition in vivo compared to HDL + LNA-Scr controls. Furthermore, Tgfb2 and Fam220a were validated with gene reporter assays as direct targets of miR-489-3p and miR-92a-3p, respectively. In human coronary artery endothelial cells, over-expression and inhibition of miR-92a-3p decreased and increased FAM220A expression, respectively. Moreover, miR-92a-3p overexpression increased STAT3 phosphorylation, likely through direct regulation of FAM220A, a negative regulator of STAT3 phosphorylation. Conclusions These results support endothelial miRNAs as therapeutic targets and dual miRNA inhibition as viable strategy to reduce CKD-associated atherosclerosis.

Background & Aims Countries endemic for parasitic infestations have a lower incidence of Crohn’s disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. Methods We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn’s-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. Results Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. Conclusions We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

INTRODUCTION AND OBJECTIVE:Approximately 30% of men with lower urinary tract symptoms (LUTS) are resistant to medical therapies and progress to surgical intervention. The pathological changes produ...

Colesevelam is a bile acid sequestrant approved to treat both hyperlipidemia and type 2 diabetes, but the mechanism for its glucose-lowering effects is not fully understood. The aim of this study was to investigate the role of hepatic microRNAs (miRNAs) as regulators of metabolic disease and to investigate the link between the cholesterol and glucose-lowering effects of colesevelam. To quantify the impact of colesevelam treatment in rodent models of diabetes, metabolic studies were performed in Zucker diabetic fatty (ZDF) rats and db/db mice. Colesevelam treatments significantly decreased plasma glucose levels and increased glycolysis in the absence of changes to insulin levels in ZDF rats and db/db mice. High-throughput sequencing and real-time PCR were used to quantify hepatic miRNA and mRNA changes, and the cholesterol-sensitive miR-96/182/183 cluster was found to be significantly increased in livers from ZDF rats treated with colesevelam compared with vehicle controls. Inhibition of miR-182 in vivo attenuated colesevelam-mediated improvements to glycemic control in db/db mice. Hepatic expression of mediator complex subunit 1 (MED1), a nuclear receptor coactivator, was significantly decreased with colesevelam treatments in db/db mice, and MED1 was experimentally validated to be a direct target of miR-96/182/183 in humans and mice. In summary, these results support that colesevelam likely improves glycemic control through hepatic miR-182–5p, a mechanism that directly links cholesterol and glucose metabolism. NEW & NOTEWORTHY Colesevelam lowers systemic glucose levels in Zucker diabetic fatty rats and db/db mice and increases hepatic levels of the sterol response element binding protein 2-responsive microRNA cluster miR-96/182/183. Inhibition of miR-182 in vivo reverses the glucose-lowering effects of colesevelam in db/db mice. Mediator complex subunit 1 (MED1) is a novel, direct target of the miR-96/182/183 cluster in mice and humans.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

MicroRNAs (miRNA) are shown to be involved in the progression of several types of kidney diseases. Podocytes maintain the integrity of the glomerular basement membrane. Extracellular vesicles (EV) are important in cell-to-cell communication as they can transfer cellular content between cells, including miRNA. However, little is known about how extracellular signals from the glomerular microenvironment regulate podocyte activity. Using a non-contact transwell system, communication between glomerular endothelial cells (GEnC) and podocytes was characterised in-vitro. Identification of transferred EV-miRNAs from GEnC to podocytes was performed using fluorescence cell tracking and miRNA mimetics. To represent kidney disease, podocyte molecular profiling and functions were analysed after EV treatments derived from steady state or activated GEnC. Our data shows activation of GEnC alters EV-miRNA loading, but activation was not found to alter EV secretion. EV delivery of miRNA to recipient podocytes altered cellular miRNA abundance and effector functions in podocytes, including decreased secretion of VEGF and increased mitochondrial stress which lead to altered cellular metabolism and cytoskeletal rearrangement. Finally, results support our hypothesis that miRNA-200c-3p is transfered by EVs from GEnC to podocytes in response to activation, ultimately leading to podocyte dysfunction.

BackgroundMale lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often use LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a pro-inflammatory and pro-fibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to pro-inflammatory stimuli and identified downstream targets of OPN in prostate stromal cells.MethodsImmunohistochemistry was performed on prostate sections obtained from the transition zone (TZ) of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS i.e. surgical BPH (S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging system and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by ELISA. qPCR was used to measure gene expression changes in these cells in response to OPN.ResultsOPN immunostaining (p=0.0107) and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression ofCXCL1,CXCL2,CXCL8,PTGS2andIL6in BHPrS-1, but not in epithelial cell lines.ConclusionsOPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by pro-inflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of pro-inflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Here, we engineered a new dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. We overcame the index-hopping issue, demonstrated significant improvements in base-calling accuracy, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior library structures. Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Extracellular microRNAs (miRNAs) are a new class of biomarkers for cellular phenotypes and disease, and are bioactive signals within intercellular communication networks. Previously, we reported that miRNAs are secreted from macrophage to high-density lipoproteins (HDL) and delivered to recipient cells to regulate gene expression. Despite the potential importance of HDL-miRNAs, regulation of HDL-miRNA export from cells has not been fully studied. Here, we report that pancreatic islets and beta cells abundantly export miR-375-3p to HDL and this process is inhibited by cellular mechanisms that promote insulin secretion. Small RNA sequencing and PCR approaches were used to quantify beta cell miRNA export to HDL. Strikingly, high glucose conditions were found to inhibit HDL-miR-375-3p export, which was dependent on extracellular calcium. Likewise, stimulation of cAMP was found to repress HDL-miR-375-3p export. Furthermore, we found that beta cell ATP-sensitive potassium channel (KATP) channels are required for HDL-miRNA export as chemical inhibition (tolbutamide) and global genetic knockout (Abcc8−/−) approaches inhibited HDL-miR-375-3p export. This process is not likely associated with cholesterol flux, as gain-of-function and loss-of-function studies for cholesterol transporters failed to alter HDL-miR-375-3p export. In conclusion, results support that pancreatic beta cells export miR-375-3p to HDL and this process is inversely regulated to insulin secretion.

Cellular microRNAs (miRNA) have proven to be critical regulators of inflammatory gene expression across many pathways within autoimmunity. Circulating miRNAs serve as a new class of disease biomarkers. Nevertheless, the functional roles of miRNAs, particularly extracellular miRNAs, in systemic lupus erythematosus (SLE) remain poorly understood. Therefore, we aimed to link changes in extracellular miRNAs to lymphocyte gene regulation and the pathophysiology of SLE. Here, we demonstrate that circulating miR-22-3p levels are associated with SLE, and miR-22-3p regulates T and B cell function and SLE-associated kidney disease. Based on high-throughput small RNA sequencing and real-time PCR, extracellular miR-22-3p levels were found to be significantly increased in whole plasma in human SLE subjects. To determine the functional impact of miR-22-3p in SLE, miR-22-3p loss-of-function studies were performed in a mouse model of SLE (B6.SLE1.2.3). We found that in vivo administration of locked-nucleic acid inhibitors of miR-22-3p (LNA-22) reduced lymphocyte accumulation in both the spleen and lymph nodes compared to LNA scramble (LNA-Scr) control-treated mice. Strikingly, LNA-22-3p treatments reduced kidney disease pathology and glomerular IgG deposition compared to LNA-Scr treatments in SLE mice. Moreover, miR-22-3p inhibition reduced the proportion of T effector memory IFN-γ producing CD4+ T cells, suggesting that miR-22-3p regulates Th1 T cell differentiation. We also found that miR-22 inhibition in mice reduced STAT1 phosphorylation in the kidney which was correlated with loss of IFN-γ production by splenic CD4+ T cells. In conclusion, our findings suggest that miR-22-3p is a critical regulator of SLE-associated CD4+ T cell immunity and kidney disease. These results provide therapeutic potential for limiting splenic Th1 signaling and preventing the progression of lupus nephritis.Key FindingsExtracellular miR-22-3p levels are significantly increased in plasma from human SLE subjects.Inhibition of miR-22-3p in vivo significantly reduced lymphocyte accumulation in both the spleen and lymph nodes in a mouse model of SLE, thus reducing splenomegaly and lymphadenopathy.miR-22-3p inhibition significantly reduced IFN-γ expression and secretion from splenic T cell subsets.Inhibition of miR-22-3p in vivo resulted in decreased IgG deposition in the kidney, decreased STAT1 phosphorylation, and decreased kidney disease in a mouse model of SLE.

Atherosclerosis is a chronic inflammatory disease, and despite resounding success in lipid management to reduce cardiovascular events, most heart attacks in the US occur in patients with clinically normal LDL-C levels. The next generation of therapies for cardiovascular disease (CVD) will include inflammation-based targets; however, systemic immune suppression has limitations. Thus, the critical barrier for successful targeting of inflammation in CVD is the identification of vascular ligands that drive immune cell activation. Previously, we demonstrated that low-density lipoproteins (LDL) transport microRNAs. Here, we report that LDL traffic a wide-diversity of small non-coding RNAs (sRNA) in circulation, many of which are derived from exogenous species, e.g. bacteria and fungi. Moreover, using high-throughput sRNA sequencing, we found that LDL-sRNA signatures are influenced primarily by environmental microbiota, as opposed to commensal bacteria in the gut microbiome. Using a mouse model of compromised mucosal immunity, we identified a process by which LDL accumulates bacterial sRNAs. Next, we demonstrated that native LDL potently stimulated cytokine release from macrophages through activation of single-stranded RNA (ssRNA)-sensing toll-like receptor (TLR) signaling, which was blunted by partial silencing of ssRNA-sensing TLRs in macrophages. Most importantly, preliminary results in mice suggest that targeting ssRNA-sensing TLRs is a potential strategy to suppress atherosclerosis. Taken together, we put forth a novel paradigm in which, LDL scavenge microbial sRNAs to promote clearance during health, yet serve as TLR-ligands that propagate macrophage inflammation in hypercholesterolemia, which likely contributes to the underlying inflammation in the pathogenesis of CVD.

High-throughput small RNA sequencing (sRNA-seq) has facilitated the discovery of many classes of small RNAs (sRNA) and helped establish the field of extracellular RNA (exRNA). Although several tools are available for sRNA-seq analysis, exRNAs present unique analytical challenges that are not met by current software. Therefore, we developed a novel data analysis pipeline specifically for exRNAs entitled, Tools for Integrative Genome analysis of Extracellular sRNAs (TIGER). To demonstrate the power of this tool, sRNA-seq was performed on high-density lipoproteins (HDL), apolipoprotein B-containing particles (APOB), bile, urine, and liver samples collected from wild-type (WT) and scavenger receptor BI knockout (SR-BI KO) mice. TIGER was able to account for approximately 60% of reads on lipoproteins and >85% of reads in liver, bile, and urine, a significant advance compared to existing software, largely due to the identification of non-host sRNAs in these datasets. A key advance for the TIGER pipeline is the ability to analyze host and non-host sRNAs across many classes at the genome, parent RNA, and individual fragment levels. Moreover, disparate sample types were compared at each level using hierarchical clustering, correlations, betadispersions, principal coordinate analysis, and permutational multivariate analysis of variance. TIGER analysis was also used to quantify distinct features of exRNAs, including 5-prime microRNA (miRNA) variants, 3-prime miRNA non-templated additions, parent RNA positional coverage, and length distributions by RNA class. Results suggest that the majority of sRNAs on lipoproteins are non-host sRNAs derived from bacterial sources in the microbiome and environment, specifically rRNA-derived sRNAs from proteobacteria. Here, we report novel discoveries of lipoprotein sRNAs that were facilitated by the new sRNA-seq analysis pipeline, TIGER, which has tremendous applicability for the field of exRNA.

Cardiovascular disease (CVD) is a leading cause of mortality in developed countries and is a frequent comorbidity of numerous metabolic and inflammatory diseases that demands more effective therapies. Dyslipidemias are a classical risk factor for CVD, but emerging alternative functions of lipoproteins have implicated them in novel narratives for the pathophysiology of many diseases that warrant further study. Our lab has identified functional, intercellular gene regulatory networks mediated by extracellular transport of microRNAs (miRNA) by lipoproteins. Here, we quantified the landscape of small RNAs (sRNA) on human and animal lipoproteins and discovered that most lipoprotein-sRNAs are derived from microorganisms of multiple kingdoms, primarily bacteria. Based on these observations, our over-arching hypothesis is that lipoprotein-sRNA signatures are shaped by the interface of host tissues with resident, environmental and dietary microbiota, and likely participate in unique gene regulation networks that contribute to complex (patho)physiological traits. To investigate this hypothesis, we developed a sRNA-sequencing analysis pipeline that identifies and quantifies both host and non-host sRNAs. Using this bioinformatic tool, we identified and validated a number of lipoprotein-sRNAs derived from bacteria that are similar in size to miRNAs with identical seed regions, termed Doppelganger (Dopl)-miRNAs. We hypothesized that mucosal immunity contributes to non-host sRNAs on lipoproteins, as mucosal linings are a primary interface between host tissues and microorganisms. To this end, we investigated the lipoprotein-sRNAs of mice lacking the polymeric immunoglobulin receptor (pIgR -/- ), which is devoid of polymeric IgA and IgM at mucosal linings and models human respiratory and intestinal diseases. We report a global increase in lipoprotein-miRNAs juxtaposed with a profound decrease in bacterial sRNA and Dopl-miRNAs from specific taxa. This work unfurls novel links between microbiota, mucosal immunity, and lipoprotein-sRNA gene networks and emphasizes the potential for novel nucleic-acid based therapeutics.