Hepatitis C Virus Alters Macrophage Cholesterol Metabolism Through Interaction with Scavenger Receptors.

Lipid accumulation and inflammation act together to induce, sustain, and further development of chronic liver disease. Hepatitis C virus (HCV) infection induces metabolic and immune changes in liver macrophages, promoting lipid accumulation and inflammation that synergize and culminate in the development of steatohepatitis and fibrogenesis. Chronic HCV patients have increased liver macrophages with disruptions in cholesterol metabolism and alterations in inflammatory mediators. While HCV-induced changes in inflammatory mediators are well documented, how HCV triggers metabolic change in macrophages is unknown. In this report, we examined the mechanism of macrophage sensing of HCV to cause metabolic impairment and subsequent immune dysfunction. We demonstrate that HCV protein and RNA kinetics in macrophages are distinct from hepatocytes. In macrophages, HCV RNAs and protein accumulate rapidly after exposure but internalized RNAs quickly decline to a low-level set point. Notably, exposure of macrophages to HCV resulted in increased lipids and cholesterol and activation of cholesterol-sensing, immunomodulatory liver X receptors (LXRs). Furthermore, we provide evidence that HCV RNA accumulation in macrophages occurs through scavenging receptors. These results suggest that HCV released from infected hepatocytes stimulates accumulation of lipids and activation of LXR in macrophages contributing to metabolic changes involved in HCV-induced chronic liver disease. Our results provide novel insight into mechanisms through which impaired lipid metabolism in macrophages associated with HCV infection promotes development of liver steatohepatitis and fibrosis.

Novel insights into the molecular mechanisms underlying risk of colorectal cancer from smoking and red/processed meat carcinogens by modeling exposure in normal colon organoids.

Tobacco smoke and red/processed meats are well-known risk factors for colorectal cancer (CRC). Most research has focused on studies of normal colon biopsies in epidemiologic studies or treatment of CRC cell lines in vitro. These studies are often constrained by challenges with accuracy of self-report data or, in the case of CRC cell lines, small sample sizes and lack of relationship to normal tissue at risk. In an attempt to address some of these limitations, we performed a 24-hour treatment of a representative carcinogens cocktail in 37 independent organoid lines derived from normal colon biopsies. Machine learning algorithms were applied to bulk RNA-sequencing and revealed cellular composition changes in colon organoids. We identified 738 differentially expressed genes in response to carcinogens exposure. Network analysis identified significantly different modules of co-expression, that included genes related to MSI-H tumor biology, and genes previously implicated in CRC through genome-wide association studies. Our study helps to better define the molecular effects of representative carcinogens from smoking and red/processed meat in normal colon epithelial cells and in the etiology of the MSI-H subtype of CRC, and suggests an overlap between molecular mechanisms involved in inherited and environmental CRC risk.

Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 µmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed (P < 2.2E-16). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment (P = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as TRABD2A (q = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for EGFR and PTGES2, the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. PREVENTION RELEVANCE: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.

Colon Crypts of Subjects With Familial Adenomatous Polyposis Show an Increased Number of LGR5+ Ectopic Stem Cells.

INTRODUCTION: Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer (CRC) syndrome characterized by accelerated adenoma development due to inherited (or de novo) mutations in the APC regulator of WNT signaling pathway (APC) gene. The mechanism underlying this accelerated polyp development in subjects with FAP has not been defined. Given that LGR5+ stem cells drive crypt cell proliferation, we hypothesized that FAP crypts would demonstrate aberrant leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) staining patterns. METHODS: Biopsies were taken from 11 healthy subjects, 7 subjects with Lynch syndrome, 4 subjects with FAP, and 1 subject with MUTYH-associated polyposis syndrome during routine screening or surveillance colonoscopy. Crypt staining was evaluated by immunohistochemistry of paraffin-embedded tissue sections. Stem cell numbers were estimated by immunofluorescence staining of isolated crypts using antibodies against LGR5 and other proteins. RESULTS: Subjects with FAP exhibited a greater number of LGR5+ stem cells in their crypts than healthy subjects and subjects with Lynch syndrome and MUTYH-associated polyposis syndrome. Most crypts of subjects with FAP harbored LGR5+ cells located above the lower third of the crypts. DISCUSSION: These findings support a model in which inactivation of one copy of APC leads to increased numbers of LGR5+ stem cells, many of which are ectopic, in colon crypts of subjects with FAP. Overabundant and ectopic LGR5+ stem cells could lead to an expanded proliferative zone of dividing cells more likely to develop mutations that would contribute to the accelerated adenoma development observed in FAP.

Ethanol exposure drives colon location specific cell composition changes in a normal colon crypt 3D organoid model.

Alcohol is a consistently identified risk factor for colon cancer. However, the molecular mechanism underlying its effect on normal colon crypt cells remains poorly understood. We employed RNA-sequencing to asses transcriptomic response to ethanol exposure (0.2% vol:vol) in 3D organoid lines derived from healthy colon (n = 34). Paired regression analysis identified 2,162 differentially expressed genes in response to ethanol. When stratified by colon location, a far greater number of differentially expressed genes were identified in organoids derived from the left versus right colon, many of which corresponded to cell-type specific markers. To test the hypothesis that the effects of ethanol treatment on colon organoid populations were in part due to differential cell composition, we incorporated external single cell RNA-sequencing data from normal colon biopsies to estimate cellular proportions following single cell deconvolution. We inferred cell-type-specific changes, and observed an increase in transit amplifying cells following ethanol exposure that was greater in organoids from the left than right colon, with a concomitant decrease in more differentiated cells. If this occurs in the colon following alcohol consumption, this would lead to an increased zone of cells in the lower crypt where conditions are optimal for cell division and the potential to develop mutations.

Oncogenic Features in Histologically Normal Mucosa: Novel Insights Into Field Effect From a Mega-Analysis of Colorectal Transcriptomes.

INTRODUCTION: Colorectal cancer is a common malignancy that can be cured when detected early, but recurrence among survivors is a persistent risk. A field effect of cancer in the colon has been reported and could have implications for surveillance, but studies to date have been limited. A joint analysis of pooled transcriptomic data from all available bulk RNA-sequencing data sets of healthy, histologically normal tumor-adjacent, and tumor tissues was performed to provide an unbiased assessment of field effect. METHODS: A novel bulk RNA-sequencing data set from biopsies of nondiseased colon from screening colonoscopy along with published data sets from the Genomic Data Commons and Sequence Read Archive were considered for inclusion. Analyses were limited to samples with a quantified read depth of at least 10 million reads. Transcript abundance was estimated with Salmon, and downstream analysis was performed in R. RESULTS: A total of 1,139 samples were analyzed in 3 cohorts. The primary cohort consisted of 834 independent samples from 8 independent data sets, including 462 healthy, 61 tumor-adjacent, and 311 tumor samples. Tumor-adjacent gene expression was found to represent an intermediate state between healthy and tumor expression. Among differentially expressed genes in tumor-adjacent samples, 1,143 were expressed in patterns similar to tumor samples, and these genes were enriched for cancer-associated pathways. DISCUSSION: Novel insights into the field effect in colorectal cancer were generated in this mega-analysis of the colorectal transcriptome. Oncogenic features that might help explain metachronous lesions in cancer survivors and could be used for surveillance and risk stratification were identified.

Modeling the effect of prolonged ethanol exposure on global gene expression and chromatin accessibility in normal 3D colon organoids.

In this study we aimed to explore the potential biological effect of ethanol exposure on healthy colon epithelial cells using normal human colon 3D organoid "mini-gut" cultures. In numerous published studies ethanol use has been shown to be an environmental risk factor for colorectal cancer (CRC) development; however, the influence of ethanol exposure on normal colon epithelial cell biology remains poorly understood. We investigated the potential molecular effects of ethanol exposure in normal colon 3D organoids in a small pilot study (n = 3) using RNA-seq and ATAC-seq. We identify 1965 differentially expressed genes and 2217 differentially accessible regions of chromatin in response to ethanol treatment. Further, by cross-referencing our results with previously published analysis in colorectal cancer cell lines, we have not only validated a number of reported differentially expressed genes, but also identified several novel candidates for future investigation. In summary, our data highlights the potential importance for the use of normal colon 3D organoid models as a novel tool for the investigation of the relationship between the effects of environmental risk factors associated with colorectal cancer and the molecular mechanisms through which they confer this risk.

IL-1ß/TNF-α/IL-6 inflammatory cytokines promote STAT1-dependent induction of CH25H in Zika virus-infected human macrophages.

Zika virus (ZIKV)3 is an enveloped, single-stranded, positive-sense RNA virus of the Flaviviridae family that has emerged as a public health threat because of its global transmission and link to microcephaly. Currently there is no vaccine for this virus. Conversion of cholesterol to 25-hydroxycholesterol by cholesterol 25-hydroxylase (CH25H) has been shown to have broad antiviral properties. However, the molecular basis of induction of CH25H in humans is not known. Elucidation of signaling and transcriptional events for induction of CH25H expression is critical for designing therapeutic antiviral agents. In this study, we show that CH25H is induced by ZIKV infection or Toll-like receptor stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines, including IL-1ß, tumor necrosis factor α, and IL-6, and this induction depends on the STAT1 transcription factor. Additionally, we observed that cAMP-dependent transcription factor (ATF3) weakly binds to the CH25H promoter, suggesting cooperation with STAT1. However, ZIKV-induced CH25H was independent of type I interferon. These findings provide important information for understanding how the Zika virus induces innate inflammatory responses and promotes the expression of anti-viral CH25H protein.

Short Communication: Accumulation of Neutral Lipids in Liver and Aorta of Nef-Transgenic Mice.

HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.

Expression of scavenger receptor-AI promotes alternative activation of murine macrophages to limit hepatic inflammation and fibrosis.

The liver maintains an immunologically tolerant environment as a result of continuous exposure to food and bacterial constituents from the digestive tract. Hepatotropic pathogens can take advantage of this niche and establish lifelong chronic infections causing hepatic fibrosis and hepatocellular carcinoma. Macrophages (Mϕ) play a critical role in regulation of immune responses to hepatic infection and regeneration of tissue. However, the factors crucial for Mϕ in limiting hepatic inflammation or resolving liver damage have not been fully understood. In this report, we demonstrate that expression of C-type lectin receptor scavenger receptor-AI (SR-AI) is crucial for promoting M2-like Mϕ activation and polarization during hepatic inflammation. Liver Mϕ uniquely up-regulated SR-AI during hepatotropic viral infection and displayed increased expression of alternative Mϕ activation markers, such as YM-1, arginase-1, and interleukin-10 by activation of mer receptor tyrosine kinase associated with inhibition of mammalian target of rapamycin. Expression of these molecules was reduced on Mϕ obtained from livers of infected mice deficient for the gene encoding SR-AI (msr1). Furthermore, in vitro studies using an SR-AI-deficient Mϕ cell line revealed impeded M2 polarization and decreased phagocytic capacity. Direct stimulation with virus was sufficient to activate M2 gene expression in the wild-type (WT) cell line, but not in the knockdown cell line. Importantly, tissue damage and fibrosis were exacerbated in SR-AI-/- mice following hepatic infection and adoptive transfer of WT bone-marrow-derived Mϕ conferred protection against fibrosis in these mice. CONCLUSION: SR-AI expression on liver Mϕ promotes recovery from infection-induced tissue damage by mediating a switch to a proresolving Mϕ polarization state. (Hepatology 2017;65:32-43).

Immunometabolic Signaling Pathways Contribute to Macrophage and Dendritic Cell Function.

Understanding of antigen-presenting cell (APC) participation in tissue inflammation and metabolism has advanced through numerous studies using systems biology approaches. Previously unrecognized connections between these research areas have been elucidated in the context of inflammatory disease involving innate and adaptive immune responses. A new conceptual framework bridges APC biology, metabolism, and cytokines in the generation of effective T-cell responses. Exploring these connections is paramount to addressing the rising tide of multi-organ system diseases, particularly chronic diseases associated with metabolic syndrome, infection, and cancer. Focused research in these areas will aid the development of strategies to harness and manipulate innate immunology to improve vaccine development, anti-viral, anti-inflammatory, and anti-tumor therapies. This review highlights recent advances in APC "immunometabolism" specifically related to chronic viral and metabolic disease in humans. The goal of this review is to develop an abridged and consolidated outlook on recent thematic updates to APC immunometabolism in the areas of regulation and crosstalk between metabolic and inflammatory signaling and the integrated stress response and how these signals dictate APC function in providing T-cell activation Signal 3.

Expression profile of heat shock response factors during hookworm larval activation and parasitic development.

When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress.

HIV-1 protein Nef inhibits activity of ATP-binding cassette transporter A1 by targeting endoplasmic reticulum chaperone calnexin.

HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux.

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.

Stimulation of the liver X receptor pathway inhibits HIV-1 replication via induction of ATP-binding cassette transporter A1.

Cholesterol plays an important role in the HIV life cycle, and infectivity of cholesterol-depleted HIV virions is significantly impaired. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits the activity of the major cellular cholesterol transporter ATP binding cassette transporter A1 (ABCA1), suggesting that the virus may use this mechanism to get access to cellular cholesterol. In this study, we investigated the effect on HIV infection of a synthetic liver X receptor (LXR) ligand, N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (TO-901317), which is a potent stimulator of ABCA1 expression. We demonstrate that TO-901317 restores cholesterol efflux from HIV-infected T lymphocytes and macrophages. TO-901317 potently suppressed HIV-1 replication in both cell types and inhibited HIV-1 replication in ex vivo cultured lymphoid tissue and in RAG-hu mice infected in vivo. This anti-HIV activity was dependent on ABCA1, because the effect of the drug was significantly reduced in ABCA1-defective T cells from a patient with Tangier disease, and RNA interference-mediated inhibition of ABCA1 expression eliminated the effect of TO-901317 on HIV-1 replication. TO-901317-mediated inhibition of HIV replication was due to reduced virus production and reduced infectivity of produced virions. The infectivity defect was in part due to reduced fusion activity of the virions, which was directly linked to reduced viral cholesterol. These results describe a novel approach to inhibiting HIV infection by stimulating ABCA1 expression.