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Although genetic and epidemiological evidence indicates vitamin D insufficiency contributes to multiple sclerosis (MS), and serum levels of vitamin D increase on treatment with cholecalciferol, recent metanalyses indicate that this vitamin D form does not ameliorate disease. Genetic variation in genes regulating vitamin D, and regulated by vitamin D, affect MS risk. We evaluated if the expression of vitamin D responsive MS risk genes could be used to assess vitamin D response in immune cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls and people with MS treated with dimethyl fumarate. We assayed changes in expression of vitamin D responsive MS risk (VDRMS) genes in response to treatment with 25 hydroxy vitamin D in the presence or absence of inflammatory stimuli. Expression of CYP24A1 and other VDRMS genes was significantly altered in PBMCs treated with vitamin D in the homeostatic and inflammatory models. Gene expression in MS samples had similar responses to controls, but lower initial expression of the risk genes. Vitamin D treatment abrogated these differences. Expression of CYP24A1 and other MS risk genes in blood immune cells indicate vitamin D response and could enable assessment of immunological response to vitamin D in clinical trials and on therapy.

Direct acting antiviral therapies rapidly clear chronic hepatitis C virus (HCV) infection and restore natural killer (NK) cell function. We investigated NK-cell memory formation following HCV clearance by examining NK-cell phenotype and responses from control and chronic HCV patients before and after therapy following sustained virologic response at 12 weeks post therapy (SVR12). NK-cell phenotype at SVR12 differed significantly from paired pretreatment samples, with an increase in maturation markers CD16, CD57, and KLRG1. HCV patients possessed stronger cytotoxic responses against HCV-infected cells as compared to healthy controls; a response that further increased following SVR12. The antigen-specific response was mediated by KLRG1+ NK cells, as demonstrated by increased degranulation and proliferation in response to HCV antigen only. Our data suggest that KLRG1+ HCV-specific memory NK cells develop following viral infection, providing insight into their role in HCV clearance and relevance with regard to vaccine design.

Background Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. Methods We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. Results These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p

Multiple Sclerosis (MS) is a neurological condition driven in part by immune cells from the peripheral circulation, the targets for current successful therapies. The autoimmune and MS risk gene ZMIZ1 is underexpressed in blood in people with MS. We show that, from three independent sets of transcriptomic data, expression of ZMIZ1 is tightly correlated with that of hundreds of other genes. Further we show expression is partially heritable (heritability 0.26), relatively stable over time, predominantly in plasmacytoid dendritic cells and non-classical monocytes, and that levels of ZMIZ1 protein expression are reduced in MS. ZMIZ1 gene expression is increased in response to calcipotriol (1,25 Vitamin D3) (p < 0.0003) and associated with Epstein Barr Virus (EBV) EBNA-1 antibody titre (p < 0.004). MS therapies fingolimod and dimethyl fumarate altered blood ZMIZ1 gene expression compared to untreated MS. The phenotype indicates susceptibility to MS, and may correspond with clinical response and represent a novel clinical target.

The data presented in this article are related to the research article entitled “The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis” Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), PE(-100) for all. Of the nearly 11 000 VDR-binding peaks identified across the genome in DC1s, 1317 were shared with DC2s (91% of DC2 sites) and 1579 with monocytes (83% of monocyte sites). Latitude-dependent autoimmune disease risk polymorphisms were highly overrepresented within 5 kb of the peaks. Several transcription factor recognition motifs were highly overrepresented in the peaks, including those for the autoimmune risk gene, BATF. This evidence indicates that VDR regulates hundreds of myeloid cell genes and that the molecular pathways controlled by VDR in these cells are important in maintaining tolerance.

ObjectiveVaccination against hepatitis B virus (HBV) confers protection from subsequent infection through immunological memory that is traditionally considered the domain of the adaptive immune system. This view has been challenged following the identification of antigen-specific memory natural killer cells (mNKs) in mice and non-human primates. While the presence of mNKs has been suggested in humans based on the expansion of NK cells following pathogen exposure, evidence regarding antigen-specificity is lacking. Here, we demonstrate the existence of HBV-specific mNKs in humans after vaccination and in chronic HBV infection.DesignNK cell responses were evaluated by flow cytometry and ELISA following challenge with HBV antigens in HBV vaccinated, non-vaccinated and chronic HBV-infected individuals.ResultsNK cells from vaccinated subjects demonstrated higher cytotoxic and proliferative responses against autologous hepatitis B surface antigen (HBsAg)-pulsed monocyte-derived dendritic cells (moDCs) compared with unvaccinated subjects. Moreover, NK cell lysis of HBsAg-pulsed moDCs was significantly higher than that of hepatitis B core antigen (HBcAg)-pulsed moDCs (non-vaccine antigen) or tumour necrosis factor α-activated moDCs in a NKG2D-dependent manner. The mNKs response was mediated by CD56dim NK cells coexpressing CD57, CD69 and KLRG1. Further, mNKs from chronic hepatitis B patients exhibited greater degranulation against HBcAg-pulsed moDCs compared with unvaccinated or vaccinated patients. Notably, mNK activity was negatively correlated with HBV DNA levels.ConclusionsOur data support the presence of a mature mNKs following HBV antigen exposure either through vaccination or infection. Harnessing these antigen specific, functionally active mNKs provides an opportunity to develop novel treatments targeting HBV in chronic infection.

Natural killer (NK) cells are known to exert strong antiviral activity. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is expressed by terminally differentiated NK cells and KLRG1-expressing lymphocytes are known to expand following chronic viral infections. We aimed to elucidate the previously unknown role of KLRG1 in the pathogenesis of chronic hepatitis B (CHB).KLRG1+ NK cells were taken from the blood and liver of healthy individuals and patients with CHB. The phenotype and function of these cells was assessed using flow cytometry and in vitro stimulation.Patients with CHB had a higher frequency of KLRG1+ NK cells compared to healthy controls (blood 13.4 vs. 2.3%, p 0.0001 and liver 23.4 vs. 2.6%, p 0.01). KLRG1+ NK cells were less responsive to K562 and cytokine stimulation, but demonstrated enhanced cytotoxicity (9.0 vs. 4.8%, p 0.05) and IFN-γ release (8.0 vs. 1.5%, p 0.05) via antibody dependent cellular cytotoxicity compared to their KLRG1- counterparts. KLRG1+ NK cells possessed a mature phenotype, demonstrating stronger cytolytic activity and IFN-γ secretion against hepatic stellate cells (HSCs) than KLRG1- NK cells. Moreover, KLRG1+ NK cells more effectively induced primary HSC apoptosis in a TRAIL-dependent manner. Increased KLRG1+ NK cell frequency in the liver and blood was associated with lower fibrosis stage (F0/F1) in patients with CHB. Finally, the expression of CD44, degranulation and IFN-γ production were all increased in KLRG1+ NK cells following stimulation with osteopontin, the CD44 ligand, suggesting that HSC-derived osteopontin may cause KLRG1+ NK cell activation.KLRG1+ NK cells likely play an antifibrotic role during the natural course of CHB infection. Harnessing this antifibrotic function may provide a novel therapeutic approach to treat liver fibrosis in patients with CHB.Individuals that are chronically infected with hepatitis B virus (HBV) possess an increased number of immune cells, called natural killer (NK) cells expressing the surface marker KLRG1 in the blood and liver. Here, we demonstrate that these specific NK cells are able to kill activated stellate cells in the liver. Because activated stellate cells contribute to liver scarring, i.e. fibrosis, and subsequent liver dysfunction in individuals with chronic HBV infection, KLRG1+ NK cells are a novel immune cell type that can limit liver scarring.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

Importance Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo. Objective To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease. Design, Setting, and Participants This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December 31, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of interleukin 2/interleukin 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration. Main Outcomes and Measures In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93Amice, as well as the association of Treg amplification with disease onset and survival time in mice. Results The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+subset) and rate of disease progression (R = −0.40,P = .002). Expansion of the effector Treg population in the SOD1G93Amice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c–treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days;P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93Amice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves. Conclusions and Relevance These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.

Background Research into gene expression enables scientists to decipher the complex regulatory networks that control fundamental biological processes. Quantitative real-time PCR (qPCR) is a powerful and ubiquitous method for interrogation of gene expression. Accurate quantification is essential for correct interpretation of qPCR data. However, conventional relative and absolute quantification methodologies often give erroneous results or are laborious to perform. To overcome these failings, we developed an accurate, simple to use, universal calibrator, AccuCal. Results Herein, we show that AccuCal quantification can be used with either dye- or probe-based detection methods and is accurate over a dynamic range of ≥105 copies, for amplicons up to 500 base pairs (bp). By providing absolute quantification of all genes of interest, AccuCal exposes, and circumvents, the well-known biases of qPCR, thus allowing objective experimental conclusions to be drawn. Conclusion We propose that AccuCal supersedes the traditional quantification methods of PCR. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0256-y) contains supplementary material, which is available to authorized users.

Single-nucleotide polymorphisms near the interferon lambda 3 (IFNL3) gene predict outcomes to infection and anti-viral treatment in hepatitis C virus (HCV) infection. To identify IFNL3 genotype effects on peripheral blood, we collected phenotype data on 400 patients with genotype 1 chronic hepatitis C (CHC). The IFNL3 responder genotype predicted significantly lower white blood cells (WBCs), as well as lower absolute numbers of monocytes, neutrophils and lymphocytes for both rs8099917 and rs12979860. We sought to define the WBC subsets driving this association using flow cytometry of 67 untreated CHC individuals. Genotype-associated differences were seen in the ratio of CD4CD45RO+ to CD4CD45RO-; CD8CD45RO+ to CD8CD45RO-, NK CD56 dim to bright and monocyte numbers and percentages. Whole blood expression levels of IFNL3, IFNLR1 (interferon lambda receptor 1), IFNLR1-mem (a membrane-associated receptor), IFNLR1-sol (a truncated soluble receptor), MxA and T- and NK (natural killer) cell transcription factors TBX21, GATA3, RORC, FOXP3 and EOMES in two subjects were also determined. CHC patients demonstrated endogenous IFN activation with higher levels of MxA, IFNLR1, IFNLR1-mem and IFNLR1-sol, and IFNL3 genotype-associated differences in transcription factors. Taken together, these data provide evidence of an IFNL3 genotype association with differences in monocyte, T- and NK cell levels in the peripheral blood of patients with CHC. This could underpin genotype associations with spontaneous and treatment-induced HCV clearance and hepatic necroinflammation.

Multiple Sclerosis (MS) is an autoimmune disease treated by therapies targeting peripheral blood cells. We previously identified that expression of two MS-risk genes, the transcription factors EOMES and TBX21 (ET), was low in blood from MS and stable over time. Here we replicated the low ET expression in a new MS cohort (p < 0.0007 for EOMES, p < 0.028 for TBX21) and demonstrate longitudinal stability (p < 10− 4) and high heritability (h2 = 0.48 for EOMES) for this molecular phenotype. Genes whose expression correlated with ET, especially those controlling cell migration, further defined the phenotype. CD56 + cells and other subsets expressed lower levels of Eomes or T-bet protein and/or were under-represented in MS. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with ET expression, but HLA-DRB1*1501 genotype was. ET expression was normalised to healthy control levels with natalizumab, and was highly variable for glatiramer acetate, fingolimod, interferon-beta, dimethyl fumarate.

Host response biomarkers can accurately distinguish between influenza and bacterial infection. However, published biomarkers require the measurement of many genes, thereby making it difficult to implement them in clinical practice. This study aims to identify a single-gene biomarker with a high diagnostic accuracy equivalent to multi-gene biomarkers.In this study, we combined an integrated genomic analysis of 1071 individuals with in vitro experiments using well-established infection models.We identified a single-gene biomarker, IFI27, which had a high prediction accuracy (91%) equivalent to that obtained by multi-gene biomarkers. In vitro studies showed that IFI27 was upregulated by TLR7 in plasmacytoid dendritic cells, antigen-presenting cells that responded to influenza virus rather than bacteria. In vivo studies confirmed that IFI27 was expressed in influenza patients but not in bacterial infection, as demonstrated in multiple patient cohorts (n=521). In a large prospective study (n=439) of patients presented with undifferentiated respiratory illness (aetiologies included viral, bacterial and non-infectious conditions), IFI27 displayed 88% diagnostic accuracy (AUC) and 90% specificity in discriminating between influenza and bacterial infections.IFI27 represents a significant step forward in overcoming a translational barrier in applying genomic assay in clinical setting; its implementation may improve the diagnosis and management of respiratory infection.