Multi-omic network analysis identified betacellulin as a novel target of omega-3 fatty acid attenuation of western diet-induced nonalcoholic steatohepatitis.

Clinical and preclinical studies established that supplementing diets with ω3 polyunsaturated fatty acids (PUFA) can reduce hepatic dysfunction in nonalcoholic steatohepatitis (NASH) but molecular underpinnings of this action were elusive. Herein, we used multi-omic network analysis that unveiled critical molecular pathways involved in ω3 PUFA effects in a preclinical mouse model of western diet induced NASH. Since NASH is a precursor of liver cancer, we also performed meta-analysis of human liver cancer transcriptomes that uncovered betacellulin as a key EGFR-binding protein upregulated in liver cancer and downregulated by ω3 PUFAs in animals and humans with NASH. We then confirmed that betacellulin acts by promoting proliferation of quiescent hepatic stellate cells, inducing transforming growth factor-ß2 and increasing collagen production. When used in combination with TLR2/4 agonists, betacellulin upregulated integrins in macrophages thereby potentiating inflammation and fibrosis. Taken together, our results suggest that suppression of betacellulin is one of the key mechanisms associated with anti-inflammatory and anti-fibrotic effects of ω3 PUFA on NASH.

Enriched dietary saturated fatty acids induce trained immunity via ceramide production that enhances severity of endotoxemia and clearance of infection.

Trained immunity is an innate immune memory response that is induced by a primary inflammatory stimulus that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Dietary fatty acids can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli to induce trained immunity. Here we find mice fed a diet enriched exclusively in saturated fatty acids (ketogenic diet; KD) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and hyperglycemia. We find KD alters the composition of the hematopoietic stem cell compartment and enhances the response of bone marrow macrophages, monocytes, and splenocytes to secondary LPS challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in KD-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA induces a hyper-inflammatory response to LPS in macrophages, and this was dependent on the synthesis of ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality in response to systemic LPS, and this phenotype was not reversible for up to 7 days post-PA-exposure. Conversely, we find PA exposure enhanced clearance of Candida albicans in Rag1-/- mice. Lastly, we show that oleic acid, which depletes intracellular ceramide, reverses PA-induced hyper-inflammation in macrophages and enhanced mortality in response to LPS. These implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory and highlight the plasticity of this innate immune reprogramming by dietary constituents.

Microbiota and adipocyte mitochondrial damage in type 2 diabetes are linked by Mmp12+ macrophages.

Microbiota contribute to the induction of type 2 diabetes by high-fat/high-sugar (HFHS) diet, but which organs/pathways are impacted by microbiota remain unknown. Using multiorgan network and transkingdom analyses, we found that microbiota-dependent impairment of OXPHOS/mitochondria in white adipose tissue (WAT) plays a primary role in regulating systemic glucose metabolism. The follow-up analysis established that Mmp12+ macrophages link microbiota-dependent inflammation and OXPHOS damage in WAT. Moreover, the molecular signature of Mmp12+ macrophages in WAT was associated with insulin resistance in obese patients. Next, we tested the functional effects of MMP12 and found that Mmp12 genetic deficiency or MMP12 inhibition improved glucose metabolism in conventional, but not in germ-free mice. MMP12 treatment induced insulin resistance in adipocytes. TLR2-ligands present in Oscillibacter valericigenes bacteria, which are expanded by HFHS, induce Mmp12 in WAT macrophages in a MYD88-ATF3-dependent manner. Thus, HFHS induces Mmp12+ macrophages and MMP12, representing a microbiota-dependent bridge between inflammation and mitochondrial damage in WAT and causing insulin resistance.

Xanthohumol Requires the Intestinal Microbiota to Improve Glucose Metabolism in Diet-Induced Obese Mice.

SCOPE: The polyphenol xanthohumol (XN) improves dysfunctional glucose and lipid metabolism in diet-induced obesity animal models. Because XN changes intestinal microbiota composition, the study hypothesizes that XN requires the microbiota to mediate its benefits. METHODS AND RESULTS: To test the hypothesis, the study feeds conventional and germ-free male Swiss Webster mice either a low-fat diet (LFD, 10% fat derived calories), a high-fat diet (HFD, 60% fat derived calories), or a high-fat diet supplemented with XN at 60 mg kg-1 body weight per day (HXN) for 10 weeks, and measure parameters of glucose and lipid metabolism. In conventional mice, the study discovers XN supplementation decreases plasma insulin concentrations and improves Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). In germ-free mice, XN supplementation fails to improve these outcomes. Fecal sample 16S rRNA gene sequencing analysis suggests XN supplementation changes microbial composition and dramatically alters the predicted functional capacity of the intestinal microbiota. Furthermore, the intestinal microbiota metabolizes XN into bioactive compounds, including dihydroxanthohumol (DXN), an anti-obesogenic compound with improved bioavailability. CONCLUSION: XN requires the intestinal microbiota to mediate its benefits, which involves complex diet-host-microbiota interactions with changes in both microbial composition and functional capacity. The study results warrant future metagenomic studies which will provide insight into complex microbe-microbe interactions and diet-host-microbiota interactions.

Transkingdom interactions between Lactobacilli and hepatic mitochondria attenuate western diet-induced diabetes.

Western diet (WD) is one of the major culprits of metabolic disease including type 2 diabetes (T2D) with gut microbiota playing an important role in modulating effects of the diet. Herein, we use a data-driven approach (Transkingdom Network analysis) to model host-microbiome interactions under WD to infer which members of microbiota contribute to the altered host metabolism. Interrogation of this network pointed to taxa with potential beneficial or harmful effects on host's metabolism. We then validate the functional role of the predicted bacteria in regulating metabolism and show that they act via different host pathways. Our gene expression and electron microscopy studies show that two species from Lactobacillus genus act upon mitochondria in the liver leading to the improvement of lipid metabolism. Metabolomics analyses revealed that reduced glutathione may mediate these effects. Our study identifies potential probiotic strains for T2D and provides important insights into mechanisms of their action.

Gut-resident CX3CR1hi macrophages induce tertiary lymphoid structures and IgA response in situ.

Intestinal mononuclear phagocytes (MPs) are composed of heterogeneous dendritic cell (DC) and macrophage subsets necessary for the initiation of immune response and control of inflammation. Although MPs in the normal intestine have been extensively studied, the heterogeneity and function of inflammatory MPs remain poorly defined. We performed phenotypical, transcriptional, and functional analyses of inflammatory MPs in infectious Salmonella colitis and identified CX3CR1+ MPs as the most prevalent inflammatory cell type. CX3CR1+ MPs were further divided into three distinct populations, namely, Nos2 +CX3CR1lo, Ccr7 +CX3CR1int (lymph migratory), and Cxcl13 +CX3CR1hi (mucosa resident), all of which were transcriptionally aligned with macrophages and derived from monocytes. In follow-up experiments in vivo, intestinal CX3CR1+ macrophages were superior to conventional DC1 (cDC1) and cDC2 in inducing Salmonella-specific mucosal IgA. We next examined spatial organization of the immune response induced by CX3CR1+ macrophage subsets and identified mucosa-resident Cxcl13 +CX3CR1hi macrophages as the antigen-presenting cells responsible for recruitment and activation of CD4+ T and B cells to the sites of Salmonella invasion, followed by tertiary lymphoid structure formation and the local pathogen-specific IgA response. Using mice we developed with a floxed Ccr7 allele, we showed that this local IgA response developed independently of migration of the Ccr7 +CX3CR1int population to the mesenteric lymph nodes and contributed to the total mucosal IgA response to infection. The differential activity of intestinal macrophage subsets in promoting mucosal IgA responses should be considered in the development of vaccines to prevent Salmonella infection and in the design of anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease.

Transkingdom network reveals bacterial players associated with cervical cancer gene expression program.

Cervical cancer is the fourth most common cancer in women worldwide with human papillomavirus (HPV) being the main cause the disease. Chromosomal amplifications have been identified as a source of upregulation for cervical cancer driver genes but cannot fully explain increased expression of immune genes in invasive carcinoma. Insight into additional factors that may tip the balance from immune tolerance of HPV to the elimination of the virus may lead to better diagnosis markers. We investigated whether microbiota affect molecular pathways in cervical carcinogenesis by performing microbiome analysis via sequencing 16S rRNA in tumor biopsies from 121 patients. While we detected a large number of intra-tumor taxa (289 operational taxonomic units (OTUs)), we focused on the 38 most abundantly represented microbes. To search for microbes and host genes potentially involved in the interaction, we reconstructed a transkingdom network by integrating a previously discovered cervical cancer gene expression network with our bacterial co-abundance network and employed bipartite betweenness centrality. The top ranked microbes were represented by the families Bacillaceae, Halobacteriaceae, and Prevotellaceae. While we could not define the first two families to the species level, Prevotellaceae was assigned to Prevotella bivia. By co-culturing a cervical cancer cell line with P. bivia, we confirmed that three out of the ten top predicted genes in the transkingdom network (lysosomal associated membrane protein 3 (LAMP3), STAT1, TAP1), all regulators of immunological pathways, were upregulated by this microorganism. Therefore, we propose that intra-tumor microbiota may contribute to cervical carcinogenesis through the induction of immune response drivers, including the well-known cancer gene LAMP3.

Interplay between viruses and bacterial microbiota in cancer development.

During the last few decades we have become accustomed to the idea that viruses can cause tumors. It is much less considered and discussed, however, that most people infected with oncoviruses will never develop cancer. Therefore, the genetic and environmental factors that tip the scales from clearance of viral infection to development of cancer are currently an area of active investigation. Microbiota has recently emerged as a potentially critical factor that would affect this balance by increasing or decreasing the ability of viral infection to promote carcinogenesis. In this review, we provide a model of microbiome contribution to the development of oncogenic viral infections and viral associated cancers, give examples of this process in human tumors, and describe the challenges that prevent progress in the field as well as their potential solutions.

Differentially correlated genes in co-expression networks control phenotype transitions.

BACKGROUND: Co-expression networks are a tool widely used for analysis of "Big Data" in biology that can range from transcriptomes to proteomes, metabolomes and more recently even microbiomes. Several methods were proposed to answer biological questions interrogating these networks. Differential co-expression analysis is a recent approach that measures how gene interactions change when a biological system transitions from one state to another. Although the importance of differentially co-expressed genes to identify dysregulated pathways has been noted, their role in gene regulation is not well studied. Herein we investigated differentially co-expressed genes in a relatively simple mono-causal process (B lymphocyte deficiency) and in a complex multi-causal system (cervical cancer). METHODS: Co-expression networks of B cell deficiency (Control and BcKO) were reconstructed using Pearson correlation coefficient for two mus musculus datasets: B10.A strain (12 normal, 12 BcKO) and BALB/c strain (10 normal, 10 BcKO). Co-expression networks of cervical cancer (normal and cancer) were reconstructed using local partial correlation method for five datasets (total of 64 normal, 148 cancer). Differentially correlated pairs were identified along with the location of their genes in BcKO and in cancer networks. Minimum Shortest Path and Bi-partite Betweenness Centrality where statistically evaluated for differentially co-expressed genes in corresponding networks.    Results: We show that in B cell deficiency the differentially co-expressed genes are highly enriched with immunoglobulin genes (causal genes). In cancer we found that differentially co-expressed genes act as "bottlenecks" rather than causal drivers with most flows that come from the key driver genes to the peripheral genes passing through differentially co-expressed genes. Using in vitro knockdown experiments for two out of 14 differentially co-expressed genes found in cervical cancer (FGFR2 and CACYBP), we showed that they play regulatory roles in cancer cell growth. CONCLUSION: Identifying differentially co-expressed genes in co-expression networks is an important tool in detecting regulatory genes involved in alterations of phenotype.

Reverse enGENEering of Regulatory Networks from Big Data: A Roadmap for Biologists.

Omics technologies enable unbiased investigation of biological systems through massively parallel sequence acquisition or molecular measurements, bringing the life sciences into the era of Big Data. A central challenge posed by such omics datasets is how to transform these data into biological knowledge, for example, how to use these data to answer questions such as: Which functional pathways are involved in cell differentiation? Which genes should we target to stop cancer? Network analysis is a powerful and general approach to solve this problem consisting of two fundamental stages, network reconstruction, and network interrogation. Here we provide an overview of network analysis including a step-by-step guide on how to perform and use this approach to investigate a biological question. In this guide, we also include the software packages that we and others employ for each of the steps of a network analysis workflow.

Ménage à trois: an evolutionary interplay between human papillomavirus, a tumor, and a woman.

Cervical cancer is the third most common cancer in women with human papillomavirus (HPV) being a key etiologic factor of this devastating disease. In this article, we describe modern advances in the genomics and transcriptomics of cervical cancer that led to uncovering the key gene drivers. We also introduce, herein, a model of cervical carcinogenesis that explains how the interplay between virus, tumor, and woman results in the selection of clones that simultaneously harbor genomic amplifications for genes that drive cell cycle, antiviral response, and inhibit cell differentiation. The new model may help researchers understand the controversies in antiviral therapy and immunogenetics of this cancer and may provide a basis for future research directions in early diagnostics and personalization of therapy.

Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer.

Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.

Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut.

Using a systems biology approach, we discovered and dissected a three-way interaction between the immune system, the intestinal epithelium and the microbiota. We found that, in the absence of B cells, or of IgA, and in the presence of the microbiota, the intestinal epithelium launches its own protective mechanisms, upregulating interferon-inducible immune response pathways and simultaneously repressing Gata4-related metabolic functions. This shift in intestinal function leads to lipid malabsorption and decreased deposition of body fat. Network analysis revealed the presence of two interconnected epithelial-cell gene networks, one governing lipid metabolism and another regulating immunity, that were inversely expressed. Gene expression patterns in gut biopsies from individuals with common variable immunodeficiency or with HIV infection and intestinal malabsorption were very similar to those of the B cell-deficient mice, providing a possible explanation for a longstanding enigmatic association between immunodeficiency and defective lipid absorption in humans.

Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection.

T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.

New approach reveals CD28 and IFNG gene interaction in the susceptibility to cervical cancer.

Cervical cancer is a complex disease with multiple environmental and genetic determinants. In this study, we sought an association between polymorphisms in immune response genes and cervical cancer using both single-locus and multi-locus analysis approaches. A total of 14 single nucleotide polymorphisms (SNPs) distributed in CD28, CTLA4, ICOS, PDCD1, FAS, TNFA, IL6, IFNG, TGFB1 and IL10 genes were determined in patients and healthy individuals from three independent case/control sets. The first two sets comprised White individuals (one group with 82 cases and 85 controls, the other with 83 cases and 85 controls) and the third was constituted by non-white individuals (64 cases and 75 controls). The multi-locus analysis revealed higher frequencies in cancer patients of three three-genotype combinations [CD28+17(TT)/IFNG+874(AA)/TNFA-308(GG), CD28+17(TT)/IFN+847(AA)/PDCD1+7785(CT), and CD28 +17(TT)/IFNG+874(AA)/ICOS+1564(TT)] (P < 0.01, Monte Carlo simulation). We hypothesized that this two-genotype [CD28(TT) and IFNG(AA)] combination could have a major contribution to the observed association. To address this question, we analyzed the frequency of the CD28(TT), IFNG(AA) genotype combination in the three groups combined, and observed its increase in patients (P = 0.0011 by Fisher's exact test). The contribution of a third polymorphism did not reach statistical significance (P = 0.1). Further analysis suggested that gene-gene interaction between CD28 and IFNG might contribute to susceptibility to cervical cancer. Our results showed an epistatic effect between CD28 and IFNG genes in susceptibility to cervical cancer, a finding that might be relevant for a better understanding of the disease pathogenesis. In addition, the novel analytical approach herein proposed might be useful for increasing the statistical power of future genome-wide multi-locus studies.

Differential expression of new LTA splice variants upon lymphocyte activation.

Lymphotoxin alpha (LTA) is a member of the TNF cytokine superfamily, produced principally by lymphocytes. It plays an important role in immune and inflammatory responses. Many TNF superfamily members have functionally important isoforms generated by alternative splicing but alternative splicing of LTA has never been studied. The known LTA protein is encoded by a transcript containing four exons. Here we report seven new LTA splice variants, three of them evolutionary conserved. We demonstrate their presence in cytoplasmic RNA suggesting that they could be translated into new LTA isoforms. We observed that their expression is differentially regulated upon activation of peripheral blood mononuclear cells and lymphocyte subpopulations (CD4+, CD8+, and CD19+). Our data suggest that the new LTA splice variants might play a role in the regulation of the immune response.

Microarrays for cancer diagnosis and classification.

Microarray analysis has yet to be widely accepted for diagnosis and classification of human cancers, despite the exponential increase in microarray studies reported in the literature. Among several methods available, a few refined approaches have evolved for the analysis of microarray data for cancer diagnosis. These include class comparison, class prediction and class discovery. Using as examples some of the major experimental contributions recently provided in the field of both hematological and solid tumors, we discuss the steps required to utilize microarray data to obtain general and reliable gene profiles that could be universally used in clinical laboratories. As we show, microarray technology is not only a new tool for the clinical lab but it can also improve the accuracy of the classical diagnostic techniques by suggesting novel tumor-specific markers. We then highlight the importance of publicly available microarray data and the development of their integrated analysis that may fulfill the promise that this new technology holds for cancer diagnosis and classification.

A novel strategy for defining haplotypes by selective depletion using restriction enzymes.

Various single nucleotide polymorphisms (SNPs) have been investigated regarding association with gene expression levels or human diseases. Although different SNPs within one gene are frequently analyzed individually, it is highly probable that in the majority of the cases, a precise combination of SNP alleles, i.e., haplotype, determines a functional trait. Methods commonly used for haplotype determination, involving studies in families, cloning, or somatic cell hybrids, are expensive and time-consuming. We herein suggest a novel and simple strategy for haplotype determination, involving selective haplotype depletion with a restriction enzyme, followed by sequencing. We studied 11 LTA gene polymorphisms in 102 Brazilian individuals, and we applied this novel methodology for haplotyping 67 out of 70 LTA heterozygous individuals. We concluded that the method is rapid and efficient, and, as it includes only simple and widespread-used techniques, it could be used in most of the laboratories without further investment in equipments. The wider usage of haplotyping could be important to clarify contradictory results frequently observed among studies that focus on a single SNP.

Identification of new alternative splice events in the TCIRG1 gene in different human tissues.

Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.

Polimorfismo do sistema HLA em uma amostra de mestiços da população de Teresina, Piauí / HLA polymorphism in a racially admixed sample of the population of Teresina, Piauí

OBJETIVO: Estabelecer as freqüências das especificidades HLA-A, B, DRB1 e DQB1 numa amostra de mestiços da cidade de Teresina, Piauí, para caracterizar a sua composição genética. MÉTODOS: A reação em cadeia da polimerase de seqüência de "primers" específicos (PCR-SSP) foi utilizada para determinar as especificidades HLA-A, B, DRB1 e DQB1 de 97 indivíduos mestiços, saudáveis e não relacionados da cidade de Teresina. A freqüência genotípica foi estimada e comparada com aquelas descritas em amostras de brasileiros caucasianos, portugueses, negros e índios utilizando a Análise de Componente Principal (PCA) e a Análise de Agrupamento Hierárquico (HCA). RESULTADOS: A freqüência das especificidades HLA-A, B, DRB1 e DQB1 observada na amostra de Teresina foi intermediária entre os caucasianos e negros e não foi observada freqüência elevada das especificidades típicas de populações ameríndias. A PCA e HCA demonstraram que os mestiços de Teresina estão muito próximos aos caucasianos e negros e não apresentam similaridades com os índios. CONCLUSAO: A composição genética do mestiço de Teresina é predominantemente bi-híbrida de genes de origem caucasiana e negra com pouca participação de genes indígenos.