A high fiber diet or supplementation with Lactococcus lactis subspecies cremoris to pregnant mice confers protection against intestinal injury in adult offspring.

The diet during pregnancy, or antenatal diet, influences the offspring's intestinal health. We previously showed that antenatal butyrate supplementation reduces injury in adult murine offspring with dextran sulfate sodium (DSS)-induced colitis. Potential modulators of butyrate levels in the intestine include a high fiber diet or dietary supplementation with probiotics. To test this, we supplemented the diet of pregnant mice with high fiber, or with the probiotic bacteria Lactococcus lactis subspecies cremoris or Lactobacillus rhamnosus GG. We then induced chronic colitis with DSS in their adult offspring. We demonstrate that a high fiber antenatal diet, or supplementation with Lactococcus lactis subspecies cremoris during pregnancy diminished the injury from DSS-induced colitis in offspring. These data are evidence that antenatal dietary interventions impact offspring gut health and define the antenatal diet as a therapeutic modality to enhance offspring intestinal health.

Accelerated atherosclerosis in beta-thalassemia.

Children with beta-thalassemia (BT) present with an increase in carotid intima-medial thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. To evaluate this, wild-type (WT, littermates) and BT (Hbbth3/+) mice, both male and female, were placed on a 3-mo high-fat diet with low-density lipoprotein receptor suppression via overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation (D377Y). Mechanistically, we hypothesize that heme-mediated oxidative stress creates a proatherogenic environment in BT because BT is a hemolytic anemia that has increased free heme and exhausted hemopexin, heme's endogenous scavenger, in the vasculature. We evaluated the effect of hemopexin (HPX) therapy, mediated via an adeno-associated virus, to the progression of atherosclerosis in BT and a phenylhydrazine-induced model of intravascular hemolysis. In addition, we evaluated the effect of deferiprone (DFP)-mediated iron chelation in the progression of atherosclerosis in BT mice. Aortic en face and aortic root lesion area analysis revealed elevated plaque accumulation in both male and female BT mice compared with WT mice. Hemopexin therapy was able to decrease plaque accumulation in both BT mice and mice on our phenylhydrazine (PHZ)-induced model of hemolysis. DFP decreased atherosclerosis in BT mice but did not provide an additive benefit to HPX therapy. Our data demonstrate for the first time that the underlying pathophysiology of BT leads to accelerated atherosclerosis and shows that heme contributes to atherosclerotic plaque development in BT.NEW & NOTEWORTHY This work definitively shows for the first time that beta-thalassemia leads to accelerated atherosclerosis. We demonstrated that intravascular hemolysis is a prominent feature in beta-thalassemia and the resulting increases in free heme are mechanistically relevant. Adeno-associated virus (AAV)-hemopexin therapy led to decreased free heme and atherosclerotic plaque area in both beta-thalassemia and phenylhydrazine-treated mice. Deferiprone-mediated iron chelation led to deceased plaque accumulation in beta-thalassemia mice but provided no additive benefit to hemopexin therapy.

Microbial Changes occurring during oronasal fistula wound healing.

The oral microbiome is a complex community that matures with dental development while oral health is also a recognized risk factor for systemic disease. Despite the oral cavity having a substantial microbial burden, healing of superficial oral wounds occurs quickly and with little scarring. By contrast, creation of an oro-nasal fistula (ONF), often occurring after surgery to correct a cleft palate, is a significant wound healing challenge that is further complicated by a connection of the oral and nasal microbiome. In this study, we characterized the changes in the oral microbiome of mice following a freshly inflicted wound in the oral palate that results in an open and unhealed ONF. Creation of an ONF in mice significantly lowered oral microbiome alpha diversity, with concurrent blooms of Enterococcus faecalis, Staphylococcus lentus, and Staphylococcus xylosus in the oral cavity. Treatment of mice with oral antibiotics one week prior to ONF infliction resulted in a reduction in the alpha diversity, prevented E. faecalis and S. lentus, and S. xylosus blooms, but did not impact ONF healing. Strikingly, delivery of the beneficial microbe Lactococcus lactis subsp. cremoris (LLC) to the wound bed of the freshly inflicted ONF via a PEG-MAL hydrogel vehicle resulted in rapid healing of the ONF. Healing of the ONF was associated with the maintenance of relatively high microbiome alpha diversity, and limited the abundance of E. faecalis and S. lentus, and S. xylosus in the oral cavity. These data demonstrate that a freshly inflicted ONF in the murine palate is associated with a dysbiotic oral microbiome state that may prevent ONF healing, and a bloom of opportunistic pathogens. The data also demonstrate that delivery of a specific beneficial microbe, LLC, to the ONF can boost wound healing, can restore and/or preserve oral microbiome diversity, and inhibit blooms of opportunistic pathogens.

Intestinal barrier dysfunction in murine sickle cell disease is associated with small intestine neutrophilic inflammation, oxidative stress, and dysbiosis.

The intestinal microbiome has emerged as a potential contributor to the severity of sickle cell disease (SCD). We sought to determine whether SCD mice exhibit intestinal barrier dysfunction, inflammation, and dysbiosis. Using the Townes humanized sickle cell mouse model, we found a 3-fold increase in intestinal permeability as assessed via FITC-dextran (4 kDa) assay in SS (SCD) mice compared to AA (wild type) mice (n = 4, p < 0.05). This was associated with 25 to 50% decreases in claudin-1, 3, and 15 and zonula occludens-1 gene expression (n = 8-10, p < 0.05) in the small intestine. Increased Ly6G staining demonstrated more neutrophils in the SS small intestine (3-fold, n = 5, p < 0.05) associated with increased expression of TNFα, IL-17A, CXCL1, and CD68 (2.5 to 5-fold, n = 7-10, p < 0.05). In addition, we observed 30 to 55% decreases in superoxide dismutase-1, glutathione peroxidase-1, and catalase antioxidant enzyme expression (n = 7-8, p < 0.05) concomitant to an increase in superoxide (2-fold, n = 4, p < 0.05). Importantly, all significant observations of a leaky gut phenotype and inflammation were limited to the small intestine and not observed in the colon. Finally, characterization of the composition of the microbiome within the small intestine revealed dysbiosis in SS mice compared to their AA littermates with 47 phyla to species-level significant alterations in amplicon sequence variants. We conclude that the intestinal barrier is compromised in SCD, associated with decreased gene expression of tight junction proteins, enhanced inflammation, oxidative stress, and gut microbiome dysbiosis, all specific to the small intestine.

Butyrate supplementation to pregnant mice elicits cytoprotection against colonic injury in the offspring.

BACKGROUND: Maternal diet during pregnancy can impact progeny health and disease by influencing the offspring's gut microbiome and immune development. Gut microbial metabolism generates butyrate, a short-chain fatty acid that benefits intestinal health. Here we assess the effects of antenatal butyrate on the offspring's gastrointestinal health. We hypothesized that antenatal butyrate supplementation will induce protection against colitis in the offspring. METHODS: C57BL/6 mice received butyrate during pregnancy and a series of experiments were performed on their offspring. RNA sequencing was performed on colonic tissue of 3-week-old offspring. Six-8-week-old offspring were subjected to dextran sulfate sodium-induced colitis. Fecal microbiome analysis was performed on the 6-8-week-old offspring. RESULTS: Antenatal butyrate supplementation dampened transcript enrichment of inflammation-associated colonic genes and prevented colonic injury in the offspring. Antenatal butyrate increased the offspring's stool microbiome diversity and expanded the prevalence of specific gut microbes. CONCLUSIONS: Antenatal butyrate supplementation resulted in downregulation of genes in the offspring's colon that function in inflammatory signaling. In addition, antenatal butyrate supplementation was associated with protection against colitis and an expanded fecal microbiome taxonomic diversity in the offspring. IMPACT: Dietary butyrate supplementation to pregnant mice led to downregulation of colonic genes involved in inflammatory signaling and cholesterol synthesis, changes in the fecal microbiome composition of the offspring, and protection against experimentally induced colitis in the offspring. These data support the mounting evidence that the maternal diet during pregnancy has enduring effects on the offspring's long-term health and disease risk. Although further investigations are needed to identify the mechanism of butyrate's effects on fetal gut development, the current study substantiates the approach of dietary intervention during pregnancy to optimize the long-term gastrointestinal health of the offspring.

Type 2 and type 17 effector cells are increased in the duodenal mucosa but not peripheral blood of patients with functional dyspepsia.

Background: Functional dyspepsia is characterised by chronic symptoms of post-prandial distress or epigastric pain not associated with defined structural pathology. Increased peripheral gut-homing T cells have been previously identified in patients. To date, it is unknown if these T cells were antigen-experienced, or if a specific phenotype was associated with FD. Objective: This study aimed to characterise T cell populations in the blood and duodenal mucosa of FD patients that may be implicated in disease pathophysiology. Methods: We identified duodenal T cell populations from 23 controls and 49 Rome III FD patients by flow cytometry using a surface marker antibody panel. We also analysed T cell populations in peripheral blood from 37 controls and 61 patients. Where available, we examined the number of duodenal eosinophils in patients and controls. Results: There was a shift in the duodenal T helper cell balance in FD patients compared to controls. For example, patients had increased duodenal mucosal Th2 populations in the effector (13.03 ± 16.11, 19.84 ± 15.51, p=0.038), central memory (23.75 ± 18.97, 37.52 ± 17.51, p=0.007) and effector memory (9.80±10.50 vs 20.53±14.15, p=0.001) populations. Th17 populations were also increased in the effector (31.74±24.73 vs 45.57±23.75, p=0.03) and effector memory (11.95±8.42 vs 18.44±15.63, p=0.027) subsets. Peripheral T cell populations were unchanged between FD and control. Conclusion: Our findings identify an association between lymphocyte populations and FD, specifically a Th2 and Th17 signature in the duodenal mucosa. The presence of effector and memory cells suggest that the microinflammation in FD is antigen driven.

Microbial metabolite delta-valerobetaine is a diet-dependent obesogen.

Obesity and obesity-related metabolic disorders are linked to the intestinal microbiome. However, the causality of changes in the microbiome-host interaction affecting energy metabolism remains controversial. Here, we show the microbiome-derived metabolite δ-valerobetaine (VB) is a diet-dependent obesogen that is increased with phenotypic obesity and is correlated with visceral adipose tissue mass in humans. VB is absent in germ-free mice and their mitochondria but present in ex-germ-free conventionalized mice and their mitochondria. Mechanistic studies in vivo and in vitro show VB is produced by diverse bacterial species and inhibits mitochondrial fatty acid oxidation through decreasing cellular carnitine and mitochondrial long-chain acyl-coenzyme As. VB administration to germ-free and conventional mice increases visceral fat mass and exacerbates hepatic steatosis with a western diet but not control diet. Thus, VB provides a molecular target to understand and potentially manage microbiome-host symbiosis or dysbiosis in diet-dependent obesity.

Lactobacillus rhamnosus GG Orchestrates an Antitumor Immune Response.

BACKGROUND & AIMS: In colorectal cancer, approximately 95% of patients are refractory to immunotherapy because of low antitumor immune responses. Therefore, there is an exigent need to develop treatments that increase antitumor immune responses and decrease tumor burden to enhance immunotherapy. METHODS: The gut microbiome has been described as a master modulator of immune responses. We administered the human commensal, Lactobacillus rhamnosus GG (LGG), to mice and characterized the changes in the gut immune landscape. Because the presence of lactobacilli in the gut microbiome has been linked with decreased tumor burden and antitumor immune responses, we also supplemented a genetic and a chemical model of murine intestinal cancer with LGG. For clinical relevance, we therapeutically administered LGG after tumors had formed. We also tested for the requirement of CD8 T cells in LGG-mediated modulation of gut tumor burden. RESULTS: We detected increased colonic CD8 T-cell responses specifically in LGG-supplemented mice. The CD8 T-cell induction was dependent on dendritic cell activation mediated via Toll-like receptor-2, thereby describing a novel mechanism in which a member of the human microbiome induces an intestinal CD8 T-cell response. We also show that LGG decreased tumor burden in the murine gut cancer models by a CD8 T-cell-dependent manner. CONCLUSIONS: These data support the potential use of LGG to augment antitumor immune responses in colorectal cancer patients and ultimately for increasing the breadth and efficacy of immunotherapy.

At the heart of microbial conversations: endocannabinoids and the microbiome in cardiometabolic risk.

Cardiometabolic syndrome encompasses intertwined risk factors such as hypertension, dyslipidemia, elevated triglycerides, abdominal obesity, and other maladaptive metabolic and inflammatory aberrations. As the molecular mechanisms linking cardiovascular disease and metabolic disorders are investigated, endocannabinoids have emerged as molecules of interest. The endocannabinoid system (ECS) of biologically active lipids has been implicated in several conditions, including chronic liver disease, osteoporosis, and more recently in cardiovascular diseases. The gut microbiome is a major regulator of inflammatory and metabolic signaling in the host, and if disrupted, has the potential to drive metabolic and cardiovascular diseases. Extensive studies have unraveled the impact of the gut microbiome on host physiology, with recent reports showing that gut microbes exquisitely control the ECS, with significant influences on host metabolic and cardiac health. In this review, we outline how modulation of the gut microbiome affects host metabolism and cardiovascular health via the ECS, and how these findings could be exploited as novel therapeutic targets for various metabolic and cardiac diseases.

Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis.

BACKGROUND & AIMS: The intestinal epithelium must be resilient to physiochemical stress to uphold the physiological barrier separating the systemic compartment from the microbial and antigenic components of the gut lumen. Identifying proteins that mediate protection and enhancing their expression is therefore a clear approach to promote intestinal health. We previously reported that oral ingestion of the probiotic Lactobacillus rhamnosus GG not only induced the expression of several recognized cytoprotective factors in the murine colon, but also many genes with no previously described function, including the gene encoding proline-rich acidic protein 1 (PRAP1). PRAP1 is a highly expressed protein in the epithelium of the gastrointestinal tract and we sought to define its function in this tissue. METHODS: Purified preparations of recombinant PRAP1 were analyzed biochemically and PRAP1 antisera were used to visualize localization in tissues. Prap1-/- mice were characterized at baseline and challenged with total body irradiation, then enteroids were generated to recapitulate the irradiation challenge ex vivo. RESULTS: PRAP1 is a 17-kilodalton intrinsically disordered protein with no recognizable sequence homology. PRAP1 expression levels were high in the epithelia of the small intestine. Although Prap1-/- mice presented only mild phenotypes at baseline, they were highly susceptible to intestinal injury upon challenge. After irradiation, the Prap1-/- mice showed accelerated death with a significant increase in apoptosis and p21 expression in the small intestinal epithelium. CONCLUSIONS: PRAP1 is an intrinsically disordered protein highly expressed by the gastrointestinal epithelium and functions at exposed surfaces to protect the barrier from oxidative insult.

Galectin-9 Is a Novel Regulator of Epithelial Restitution.

Increasingly, the ß-galactoside binding lectins, termed galectins, are being recognized as critical regulators of cell function and organismal homeostasis. Within the context of the mucosal surface, galectins are established regulators of innate and adaptive immune responses, microbial populations, and several critical epithelial functions, including cell migration, proliferation, and response to injury. However, given their complex tissue distribution and expression patterns, their role within specific processes remains poorly understood. We took a genetic approach to understand the role of endogenous galectin-9 (Gal-9), a mucosal galectin that has been linked to inflammatory bowel disease, within the context of the murine intestine. Gal-9-deficient (Gal9-/-, also known as Lgals9-/-) animals show increased sensitivity to chemically induced colitis and impaired proliferation in the setting of acute injury. Moreover, Gal9-/--derived enteroids showed impaired growth ex vivo. Consistent with a model in which endogenous Gal-9 controls epithelial growth and repair, Gal9-/- animals showed increased sensitivity to intestinal challenge in multiple models of epithelial injury, including acute irradiation injury and ectopic wound biopsies. Finally, regenerating crypts from patient biopsies showed increased expression of Gal-9, indicating these processes may be conserved in humans. Taken together, these studies implicate Gal-9 in the regulation of cellular proliferation and epithelial restitution after intestinal epithelial injury.

Tetraspanin CD9 is Regulated by miR-518f-5p and Functions in Breast Cell Migration and In Vivo Tumor Growth.

Breast cancer is the most commonly diagnosed and the second leading cause of cancer-related mortality among women worldwide. miR-518f-5p has been shown to modulate the expression of the metastasis suppressor CD9 in prostate cancer. However, the role of miR-518f-5p and CD9 in breast cancer is unknown. Therefore, this study aimed to elucidate the role of miR-518f-5p and the mechanisms responsible for decreased CD9 expression in breast cancer, as well as the role of CD9 in de novo tumor formation and metastasis. miR-518f-5p function was assessed using migration, adhesion, and proliferation assays. miR-518f-5p was overexpressed in breast cancer cell lines that displayed significantly lower CD9 expression as well as less endogenous CD9 3'UTR activity, as assessed using qPCR and dual luciferase assays. Transfection of miR-518f-5p significantly decreased CD9 protein expression and increased breast cell migration in vitro. Cd9 deletion in the MMTV/PyMT mouse model impaired tumor growth, but had no effect on tumor initiation or metastasis. Therefore, inhibition of miR-518f-5p may restore CD9 expression and aid in the treatment of breast cancer metastasis.

Lactococcus lactis Subspecies cremoris Elicits Protection Against Metabolic Changes Induced by a Western-Style Diet.

BACKGROUND & AIMS: A Western-style diet, which is high in fat and sugar, can cause significant dyslipidemia and nonalcoholic fatty liver disease; the diet has an especially strong effect in women, regardless of total calorie intake. Dietary supplementation with beneficial microbes might reduce the detrimental effects of a Western-style diet. We assessed the effects of Lactococcus lactis subspecies (subsp) cremoris on weight gain, liver fat, serum cholesterol, and insulin resistance in female mice on a high-fat, high-carbohydrate diet. METHODS: Female C57BL/6 mice were fed either a high-fat, high-carbohydrate (Western-style) diet that contained 40% fat (mostly milk fat) and 43% carbohydrate (mostly sucrose) or a calorie-matched-per-gram control diet. The diets of mice were supplemented with 1 × 109 colony-forming units of L lactis subsp cremoris ATCC 19257 or Lactobacillus rhamnosus GG ATCC 53103 (control bacteria) 3 times per week for 16 weeks. Body weights were measured, and fecal, blood, and liver tissues were collected and analyzed. Livers were analyzed for fat accumulation and inflammation, and blood samples were analyzed for cholesterol and glucose levels. Mice were housed within Comprehensive Lab Animal Monitoring System cages, and respiratory exchange ratio and activity were measured. Hepatic lipid profiles of L lactis subsp cremoris-supplemented mice were characterized by lipidomics mass spectrometry analysis. RESULTS: Mice fed L lactis subsp cremoris while on the Western-style diet gained less weight, developed less hepatic steatosis and inflammation, and had a lower mean serum level of cholesterol and body mass index than mice fed the control bacteria. Mice fed the L lactis subsp cremoris had increased glucose tolerance while on the Western-style diet compared to mice fed control bacteria and had alterations in hepatic lipids, including oxylipins. CONCLUSIONS: Dietary supplementation with L lactis subsp cremoris in female mice on a high-fat, high-carbohydrate (Western-style) diet caused them to gain less weight, develop less liver fat and inflammation, reduce serum cholesterol levels, and increase glucose tolerance compared with mice on the same diet fed control bacteria. L lactis subsp cremoris is safe for oral ingestion and might be developed for persons with metabolic and liver disorders caused by a Western-style diet.

Lactobacillus rhamnosus GG-induced Expression of Leptin in the Intestine Orchestrates Epithelial Cell Proliferation.

BACKGROUND & AIMS: Identifying the functional elements that mediate efficient gut epithelial growth and homeostasis is essential for understanding intestinal health and disease. Many of these processes involve the Lactobacillus-induced generation of reactive oxygen species by NADPH oxidase (Nox1). However, the downstream signaling pathways that respond to Nox1-generated reactive oxygen species and mediate these events have not been described. METHODS: Wild-type and knockout mice were fed Lactobacillus rhamnosus GG and the transcriptional and cell signaling pathway responses in the colon measured. Corroboration of data generated in mice was done using in organoid tissue culture and in vivo gut injury models. RESULTS: Ingestion of L rhamnosus GG induces elevated levels of leptin in the gut epithelia, which as well as functioning in the context of metabolism, has pleiotropic activity as a chemokine that triggers cell proliferation. Consistently, using gut epithelial-specific knockout mice, we show that L rhamnosus GG-induced elevated levels of leptin is dependent on a functional Nox1 protein in the colonic epithelium, and that L rhamnosus GG-induced cell proliferation is dependent on Nox1, leptin, and leptin receptor. We also show that L rhamnosus GG induces the JAK-STAT signaling pathway in the gut in a Nox1, leptin, and leptin receptor-dependent manner. CONCLUSIONS: These results demonstrate a novel role for leptin in the response to colonization by lactobacilli, where leptin functions in the transduction of signals from symbiotic bacteria to subepithelial compartments, where it modulates intestinal growth and homeostasis.

Neutrophil-Derived Reactive Oxygen Orchestrates Epithelial Cell Signaling Events during Intestinal Repair.

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP-binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell-signaling events functioning in intestinal repair.

Lactococcus Lactis Subsp. cremoris Is an Efficacious Beneficial Bacterium that Limits Tissue Injury in the Intestine.

The use of beneficial bacteria to promote health is widely practiced. However, experimental evidence corroborating the efficacy of bacteria promoted with such claims remains limited. We address this gap by identifying a beneficial bacterium that protects against tissue damage and injury-induced inflammation in the gut. We first employed the Drosophila animal model to screen for the capacity of candidate beneficial bacteria to protect the fly gut against injury. From this screen, we identified Lactococcus lactis subsp. cremoris as a bacterium that elicited potent cytoprotective activity. Then, in a murine model, we demonstrated that the same strain confers powerful cytoprotective influences against radiological damage, as well as anti-inflammatory activity in a gut colitis model. In summary, we demonstrate the positive salutary effects of a beneficial bacterium, namely, L. lactis subsp. cremoris on intestinal tissue and propose the use of this strain as a therapeutic to promote intestinal health.

Trends in the Incidence of First Acute Myocardial Infarction in Metropolitan and Regional Areas of the Hunter Region.

INTRODUCTION: There is conflicting information regarding the contemporary incidence of first acute myocardial infarction (AMI) in Australia. We sought to document the regional variations in first AMI incidence in a large health district. METHODS: We identified all patients presenting with first AMI in the Hunter region of New South Wales from 2004 to 2013. We calculated age and gender adjusted incidence of AMI and evaluated differences between patients from regional and metropolitan areas. We assessed 30-day and 12-month outcomes, including mortality, through linkage with the NSW Registry of Births Deaths and Marriages. RESULTS: The incidence of first AMI in regional areas was persistently higher throughout the study compared to metropolitan areas (IRR 1.244; 95% CI 1.14-1.35; p≤0.001). There were no significant differences between regional and metropolitan areas in 30-day and 12-month outcomes following presentation with first AMI. CONCLUSIONS: The study demonstrates persistently higher rates in regional compared to metropolitan areas, supporting the need for implementation of targeted intervention and prevention strategies.

Extracellular vesicles with altered tetraspanin CD9 and CD151 levels confer increased prostate cell motility and invasion.

To facilitate intercellular communication, cells release nano-sized, extracellular vesicles (EVs) to transfer biological cargo to both local and distant sites. EVs are enriched in tetraspanins, two of which (CD9 and CD151) have altered expression patterns in many solid tumours, including prostate cancer, as they advance toward metastasis. We aimed to determine whether EVs from prostate cells with altered CD9 and CD151 expression could influence cellular behaviour and increase the metastatic capabilities of non-tumourigenic prostate cells. EVs were isolated by ultrafiltration and characterised for their tetraspanin expression and size distribution. iTRAQ was used to identify differences between RWPE1 and tetraspanin-modified RWPE1 EV proteomes, showing an enrichment in protein degradation pathways. Addition of EVs from RWPE1 cells with reduced CD9 or increased CD151 abundance resulted in increased invasion of RWPE1 cells, and increased migration in the case of high CD151 abundance. We have been able to show that alteration of CD9 and CD151 on prostate cells alters the proteome of their resultant EVs, and that these EVs can enhance the migratory and invasive capabilities of a non-tumourigenic prostate cellular population. This work suggests that cellular tetraspanin levels can alter EVs, potentially acting as a driver of metastasis in prostate cancer.

miR-518f-5p decreases tetraspanin CD9 protein levels and differentially affects non-tumourigenic prostate and prostate cancer cell migration and adhesion.

Tetraspanin CD9 is generally considered to be a metastasis suppressor, with decreased levels associated with progression and metastasis in many advanced stage cancers. Little is known about the cause of CD9 dysregulation in prostate cancer, however there are several miRNA-binding sites in the 3´UTR of the transcript suggesting it could be post-transcriptionally regulated. Using microarrays and luciferase assays in tumourigenic and non-tumourigenic prostate cell lines we identified miR-518f-5p as a regulator of the CD9 3'UTR gene expression, and decreased expression of endogenous CD9 in non-tumorigenic prostate RWPE1 and prostate cancer DU145 cells. This resulted in differential functional effects, in which RWPE1 cells showed increased migration and decreased adhesion to extracellular matrix substrates, whereas DU145 cells showed decreased migration and increased adhesion. Moreover, overexpression of miR-518f-5p significantly increased proliferation between 48h and 72h in normal RWPE1 cells, with no effect on tumourigenic DU145 cell proliferation. These results show that tetraspanin CD9 is regulated by miRNAs in prostate cell lines and that due to differential functional effects in non-tumourigenic versus tumourigenic prostate cells, miR-518f-5p may be an effective biomarker and/or therapeutic target for prostate cancer progression.

Characterization of the early molecular changes in the glomeruli of Cd151 -/- mice highlights induction of mindin and MMP-10.

In humans and FVB/N mice, loss of functional tetraspanin CD151 is associated with glomerular disease characterised by early onset proteinuria and ultrastructural thickening and splitting of the glomerular basement membrane (GBM). To gain insight into the molecular mechanisms associated with disease development, we characterised the glomerular gene expression profile at an early stage of disease progression in FVB/N Cd151 -/- mice compared to Cd151 +/+ controls. This study identified 72 up-regulated and 183 down-regulated genes in FVB/N Cd151 -/- compared to Cd151 +/+ glomeruli (p < 0.05). Further analysis highlighted induction of the matrix metalloprotease MMP-10 and the extracellular matrix protein mindin (encoded by Spon2) in the diseased FVB/N Cd151 -/- GBM that did not occur in the C57BL/6 diseased-resistant strain. Interestingly, mindin was also detected in urinary samples of FVB/N Cd151 -/- mice, underlining its potential value as a biomarker for glomerular diseases associated with GBM alterations. Gene set enrichment and pathway analysis of the microarray dataset showed enrichment in axon guidance and actin cytoskeleton signalling pathways as well as activation of inflammatory pathways. Given the known function of mindin, its early expression in the diseased GBM could represent a trigger of both further podocyte cytoskeletal changes and inflammation, thereby playing a key role in the mechanisms of disease progression.