Therapeutic Metabolic Reprograming Using microRNAs: From Cancer to HIV Infection.

MicroRNAs (miRNAs) are crucial regulators of cellular processes, including metabolism. Attempts to use miRNAs as therapeutic agents are being explored in several areas, including the control of cancer progression. Recent evidence suggests fine tuning miRNA activity to reprogram tumor cell metabolism has enormous potential as an alternative treatment option. Indeed, cancer growth is known to be linked to profound metabolic changes. Likewise, the emerging field of immunometabolism is leading to a refined understanding of how immune cell proliferation and function is governed by glucose homeostasis. Different immune cell types are now known to have unique metabolic signatures that switch in response to a changing environment. T-cell subsets exhibit distinct metabolic profiles which underlie their alternative differentiation and phenotypic functions. Recent evidence shows that the susceptibility of CD4+ T-cells to HIV infection is intimately linked to their metabolic activity, with many of the metabolic features of HIV-1-infected cells resembling those found in tumor cells. In this review, we discuss the use of miRNA modulation to achieve metabolic reprogramming for cancer therapy and explore the idea that the same approach may serve as an effective mechanism to restrict HIV replication and eliminate infected cells.

Molecular cloning and characterisation of chicken IL-18 binding protein.

The human IL-1 receptor family is comprised of 11 membrane bound or soluble receptors and the IL-18 binding protein (IL-18BP). These receptors are dispersed across seven genomic loci, with the majority at a single locus. Direct orthologues were identified in the chicken at conserved genomic loci; however, the IL-18BP remained absent from the first four builds of the chicken genome sequence. Subsequent assemblies identified the gene at a locus syntenic with mammals; however, these predicted sequences differed between genome builds and contained multiple errors. A partial IL-18BP-like sequence in the NCBI EST database was used to clone the full-length cDNA. A splice variant, which lacks the exon that encodes part of the signal peptide, was also cloned. Human IL-18BP is differentially spliced to produce a number of variants, which are all secreted. By contrast, the spliced chicken isoform was predicted to be intracellular, and we identified similar variants with the same exon missing in a limited number of divergent vertebrate species. Mammalian and viral IL-18BPs inhibit IL-18 activity by directly binding to this cytokine. Full-length and intracellular chicken IL-18BPs were equally effective at inhibiting IL-18-mediated IFN-γ release from an avian B-cell line. Analysis of the predicted chIL-18BP protein sequence revealed two crucial residues, which account for 50% of the binding affinity between human IL-18 and IL-18BP, are conserved in the chicken and a fowlpox-encoded homologue, fpv214. This suggests specific fowlpox viruses used in humans as a vaccine vector have the potential to dampen anti-viral host immune responses.

Two Lineages of KLRA with Contrasting Transcription Patterns Have Been Conserved at a Single Locus during Ruminant Speciation.

Cattle possess the most diverse repertoire of NK cell receptor genes among all mammals studied to date. Killer cell receptor genes encoded within the NK complex and killer cell Ig-like receptor genes encoded within the leukocyte receptor complex have both been expanded and diversified. Our previous studies identified two divergent and polymorphic KLRA alleles within the NK complex in the Holstein-Friesian breed of dairy cattle. By examining a much larger cohort and other ruminant species, we demonstrate the emergence and fixation of two KLRA allele lineages (KLRA*01 and -*02) at a single locus during ruminant speciation. Subsequent recombination events between these allele lineages have increased the frequency of KLRA*02 extracellular domains. KLRA*01 and KLRA*02 transcription levels contrasted in response to cytokine stimulation, whereas homozygous animals consistently transcribed higher levels of KLRA, regardless of the allele lineage. KLRA*02 mRNA levels were also generally higher than KLRA*01 Collectively, these data point toward alternative functional roles governed by KLRA genotype and allele lineage. On a background of high genetic diversity of NK cell receptor genes, this KLRA allele fixation points to fundamental and potentially differential function roles.

Lipid and Non-lipid Factors Affecting Macrophage Dysfunction and Inflammation in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease and a leading cause of human mortality. The lesional microenvironment contains a complex accumulation of variably oxidized lipids and cytokines. Infiltrating monocytes become polarized in response to these stimuli, resulting in a broad spectrum of macrophage phenotypes. The extent of lipid loading in macrophages influences their phenotype and consequently their inflammatory status. In response to excess atherogenic ligands, many normal cell processes become aberrant following a loss of homeostasis. This can have a direct impact upon the inflammatory response, and conversely inflammation can lead to cell dysfunction. Clear evidence for this exists in the lysosomes, endoplasmic reticulum and mitochondria of atherosclerotic macrophages, the principal lesional cell type. Furthermore, several intrinsic cell processes become dysregulated under lipidotic conditions. Therapeutic strategies aimed at restoring cell function under disease conditions are an ongoing coveted aim. Macrophages play a central role in promoting lesional inflammation, with plaque progression and stability being directly proportional to macrophage abundance. Understanding how mixtures or individual lipid species regulate macrophage biology is therefore a major area of atherosclerosis research. In this review, we will discuss how the myriad of lipid and lipoprotein classes and products used to model atherogenic, proinflammatory immune responses has facilitated a greater understanding of some of the intricacies of chronic inflammation and cell function. Despite this, lipid oxidation produces a complex mixture of products and with no single or standard method of derivatization, there exists some variation in the reported effects of certain oxidized lipids. Likewise, differences in the methods used to generate macrophages in vitro may also lead to variable responses when apparently identical lipid ligands are used. Consequently, the complexity of reported macrophage phenotypes has implications for our understanding of the metabolic pathways, processes and shifts underpinning their activation and inflammatory status. Using oxidized low density lipoproteins and its oxidized cholesteryl esters and phospholipid constituents to stimulate macrophage has been hugely valuable, however there is now an argument that only working with low complexity lipid species can deliver the most useful information to guide therapies aimed at controlling atherosclerosis and cardiovascular complications.

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.

The chicken IL-1 family: evolution in the context of the studied vertebrate lineage.

The interleukin-1 gene family encodes a group of related proteins that exhibit a remarkable pleiotropy in the context of health and disease. The set of indispensable functions they control suggests that these genes should be found in all eukaryotic species. The ligands and receptors of this family have been primarily characterised in man and mouse. The genomes of most non-mammalian animal species sequenced so far possess all of the IL-1 receptor genes found in mammals. Yet, strikingly, very few of the ligands are identifiable in non-mammalian genomes. Our recent identification of two further IL-1 ligands in the chicken warranted a critical reappraisal of the evolution of this vitally important cytokine family. This review presents substantial data gathered across multiple, divergent metazoan genomes to unambiguously trace the origin of these genes. With the hypothesis that all of these genes, both ligands and receptors, were formed in a single ancient ancestor, extensive database mining revealed sufficient evidence to confirm this. It therefore suggests that the emergence of mammals is unrelated to the expansion of the IL-1 family. A thorough review of this cytokine family in the chicken, the most extensively studied amongst non-mammalian species, is also presented.

Identification, cloning, and functional characterization of the IL-1 receptor antagonist in the chicken reveal important differences between the chicken and mammals.

The human IL-1 family contains 11 genes encoded at three separate loci. Nine, including IL-1R antagonist (IL-1RN), are present at a single locus on chromosome 2, whereas IL-18 and IL-33 lie on chromosomes 11 and 9, respectively. There are currently only two known orthologs in the chicken, IL-1ß and IL-18, which are encoded on chromosomes 22 and 24, respectively. Two novel chicken IL-1 family sequences were identified from expressed sequence tag libraries, representing secretory and intracellular (icIL-1RN) structural variants of the IL-1RN gene, as seen in mammals. Two further putative splice variants (SVs) of both chicken IL-1RN (chIL-1RN) structural variants were also isolated. Alternative splicing of human icIL-1RN gives three different transcripts; there are no known SVs for human secretory IL-1RN. The chicken icIL-1RN SVs differ from those found in human icIL-1RN in terms of the rearrangements involved. In mammals, IL-1RN inhibits IL-1 activity by physically occupying the IL-1 type I receptor. Both full-length structural variants of chIL-1RN exhibited biological activity similar to their mammalian orthologs in a macrophage cell line bioassay. The four SVs, however, were not biologically active. The chicken IL-1 family is more fragmented in the genome than those of mammals, particularly in that the large multigene locus seen in mammals is absent. This suggests differential evolution of the family since the divergence of birds and mammals from a common ancestor, and makes determination of the full repertoire of chicken IL-1 family members more challenging.

Identification of chicken granulocyte colony-stimulating factor (G-CSF/CSF3): the previously described myelomonocytic growth factor is actually CSF3.

The current chicken genome build contains only a single colony-stimulating factor (CSF) gene, granulocyte/macrophage (GM)-CSF (CSF2). However, genes encoding receptors for two other CSFs, G-CSF (CSF3) and M-CSF (CSF1), are present in the genome. Another apparently chicken-specific CSF, myelomonocytic growth factor (MGF), shares substantial sequence homology with mammalian CSF3 but is absent from the genome. The putative region of the chicken genome that should contain the CSF3 ortholog, while not currently mapped to a specific chromosome, exhibits considerable conserved synteny with loci containing this gene in several other species. In silico analysis of the predicted CSF3 location revealed a large region homologous with the MGF promoter, upstream of a large sequence gap. In view of the many structural and functional features common to both MGF and huCSF3, we predicted that MGF is in fact CSF3 and its gene would be located within the sequence gap. To validate this hypothesis, a primer walking strategy was used to bridge the genomic sequence gap. Full-length sequencing of the entire region and thorough, detailed analysis of the coding region confirmed that the MGF gene lay within this sequence gap, and therefore that it should be renamed CSF3.