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Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell-dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA. [ABSTRACT FROM AUTHOR]

The West Nile virus (WNV) is an emerging infection of biodefense concern and there are no available treatments or vaccines. Here we used a high-throughput method based on a novel gene expression analysis, RNA-Seq, to give a global picture of differential gene expression by primary human macrophages of 10 healthy donors infected in vitro with WNV. From a total of 28 million reads per sample, we identified 1,514 transcripts that were differentially expressed after infection. Both predicted and novel gene changes were detected, as were gene isoforms, and while many of the genes were expressed by all donors, some were unique. Knock-down of genes not previously known to be associated with WNV resistance identified their critical role in control of viral infection. Our study distinguishes both common gene pathways as well as novel cellular responses. Such analyses will be valuable for translational studies of susceptible and resistant individuals—and for targeting therapeutics—in multiple biological settings. [ABSTRACT FROM AUTHOR]

Background: RNA-Seq technology measures the transcript abundance by generating sequence reads and counting their frequencies across different biological conditions. To identify differentially expressed genes between two conditions, it is important to consider the experimental design as well as the distributional property of the data. In many RNA-Seq studies, the expression data are obtained as multiple pairs, e.g., pre- vs. post-treatment samples from the same individual. We seek to incorporate paired structure into analysis. Results: We present a Bayesian hierarchical mixture model for RNA-Seq data to separately account for the variability within and between individuals from a paired data structure. The method assumes a Poisson distribution for the data mixed with a gamma distribution to account variability between pairs. The effect of differential expression is modeled by two-component mixture model. The performance of this approach is examined by simulated and real data. Conclusions: In this setting, our proposed model provides higher sensitivity than existing methods to detect differential expression. Application to real RNA-Seq data demonstrates the usefulness of this method for detecting expression alteration for genes with low average expression levels or shorter transcript length. [ABSTRACT FROM AUTHOR]

The Th17 cytokine, IL-22, regulates host immune responses to extracellular pathogens. Whether IL-22 plays a role in viral infection, however, is poorly understood. We report here that Il22-/- mice were more resistant to lethal West Nile virus (WNV) encephalitis, but had similar viral loads in the periphery compared to wild type (WT) mice. Viral loads, leukocyte infiltrates, proinflammatory cytokines and apoptotic cells in the central nervous system (CNS) of Il22-/- mice were also strikingly reduced. Further examination showed that Cxcr2, a chemokine receptor that plays a non-redundant role in mediating neutrophil migration, was significantly reduced in Il22- /- compared to WT leukocytes. Expression of Cxcr2 ligands, cxcl1 and cxcl5, was lower in Il22-/- brains than wild type mice. Correspondingly, neutrophil migration from the blood into the brain was attenuated following lethal WNV infection of Il22-/- mice. Our results suggest that IL-22 signaling exacerbates lethal WNV encephalitis likely by promoting WNV neuroinvasion. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV), a mosquito-borne, single-stranded RNA flavivirus, causes significant human morbidity and mortality in the older population; thus, we investigated the effects of aging on infection with WNV in dendritic cells (DCs). We infected DCs with WNV in vitro and quantified cytokines and chemokines (type I IFN and CXCL10), pathogen recognition receptors RIG-I, and Toll-like receptors 3 and 7. The production of type I IFN was significantly lower in DCs from older donors, compared with younger donors. Although we observed no significant age-related difference in expression or nuclear translocation of signaling molecules in initial antiviral responses, DCs from older donors have diminished induction of late-phase responses (eg, STAT1, IRF7, and IRF1), suggesting defective regulation of type I IFN. Our results identify deficits in critical regulatory pathways in the antiviral response that may contribute to the enhanced susceptibility to viral infections observed in aging. [ABSTRACT FROM PUBLISHER]

Polymorphonuclear leukocytes (PMNs) are key in innate immunity, but their role in viral pathogenesis is incompletely understood. In infection due to West Nile virus (WNV), we found that expression of 2 PMNattracting chemokines, Cxcl1 and Cxcl2, was rapidly and dramatically elevated in macrophages. PMNs are rapidly recruited to the site of WNV infection in mice and support efficient replication of WNV. Mice depleted of PMNs after WNV inoculation developed higher viremia and experienced earlier death, compared with the control group, which suggest a protective role for PMNs. In contrast, when PMNs were depleted prior to infection with WNV, and in mice deficient in Cxcr2 (a chemokine receptor gene), viremia was reduced and survival was enhanced. Collectively, these data suggest that PMNs have a biphasic response to WNV infection, serving as a reservoir for replication and dissemination in early infection and later contributing to viral clearance. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Our data show that interleukin-10 (IL-10) is dramatically elevated both in vitro and in vivo following WNV infection. Consistent with an etiologic role of IL-10 in WNV pathogenesis, we find that WNV infection is markedly diminished in IL-10 deficient (IL-10-/-) mice, and pharmacologic blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice. Increased production of antiviral cytokines in IL-10-/- mice is associated with more efficient control of WNV infection. Moreover, CD4+ T cells produce copious amounts of IL-10, andmay be an important cellular source of IL-10 during WNV infection in vivo. In conclusion, IL-10 signaling plays a negative role in immunity against WNV infection, and blockade of IL-10 signaling by genetic or pharmacologic means helps to control viral infection, suggesting a novel anti-WNV therapeutic strategy. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus–host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV–human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets. [ABSTRACT FROM AUTHOR]

Ixodes scapularis ticks transmit many pathogens, including Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Vaccines directed against arthropod proteins injected into the host during tick engorgement could prevent numerous infectious diseases. Salp14, a salivary anticoagulant poses a key target for such intervention. Salp14 is the prototypic member of a family of potential l. scapularis anticoagulants, expressed and secreted in tick saliva during tick feeding. RNA interference was used to assess the role of Salp14 in tick feeding. Salp14 and its paralogs were silenced, as demonstrated by the reduction of mRNA and protein specific for these antigens. Tick salivary glands lacking Salp14 had reduced anticoagulant activity, as revealed by a 60-80% reduction of anti-factor Xa activity. Silencing the expression of salp14 and its paralogs also reduced the ability of l. scapularis to feed, as demonstrated by a 50-70% decline in the engorgement weights. Because ticks have several anticoagulants, it is likely that the expression of multiple anticoagulants in I. scapularis saliva would have to be ablated simultaneously to abolish tick feeding. These studies demonstrate that RNA interference can silence I. scapularis genes and disrupt their physiologic function in vivo, and they identify vaccine candidates that can alter vector engorgement. [ABSTRACT FROM AUTHOR]

The killing of Borrelia burgdorferi by intact human polymorphonuclear leukocytes (PMNL) and by individual PMNL components was compared. Intact PMNL killed B. burgdorferi 6.5-fold more efficiently and 5-fold more completely when spirochetes were opsonized with specific antibodies. U-cytoplasts, which have activatable oxidase, killed opsonized B. burgdorferi with an efficiency similar to that of intact PMNL in killing unopsonized B. burgdorferi. Although B. burgdorferi were susceptible to H(2)O(2) and nitric oxide, PMNL lysates killed B. burgdorferi nearly as well as intact PMNL killed opsonized B. burgdorferi, suggesting a critical role for granule contents. B. burgdorferi were killed by the PMNL antimicrobial components elastase, LL-37, bactericidal/permeability-increasing protein, and human neutrophil peptide-1. B. burgdorferi had limited susceptibility to killing by lysozyme and were not killed by azurocidin, proteinase 3, or lactoferrin. The efficient killing of B. burgdorferi by a variety of PMNL mechanisms highlights the paradoxical persistence of spirochetes in vivo. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the activation of macrophages during Lyme disease infections. Details on Lyme disease; Methodology; Results and discussion.

West Nile virus (WNV) is a mosquito-borne virus which causes symptomatic disease in a minority of infected humans. To identify novel genetic variants associated with severe disease, we utilized data from an existing case-control study of WNV and included population controls for an expanded analysis. We conducted imputation and gene-gene interaction analysis in the largest and most comprehensive genetic study conducted to date for West Nile neuroinvasive disease (WNND). Within the imputed West Nile virus dataset (severe cases n = 381 and asymptomatic/mild controls = 441), we found novel loci within the MCF.2 Cell Line Derived Transforming Sequence Like (MCF2L) gene (rs9549655 and rs2297192) through the individual loci analyses, although none reached statistical significance. Incorporating population controls from the Wisconsin Longitudinal Study on Aging (n = 9012) did not identify additional novel variants, a possible reflection of the cohort's inclusion of individuals who could develop mild or severe WNV disease upon infection. Many of the top gene-gene interaction results were intergenic, with currently undefined biological roles, highlighting the need for further investigation into these regions and other identified gene targets in severe WNND. Further studies including larger sample sizes and more diverse populations reflective of those at risk are needed to fully understand the genetic architecture of severe WNDD and provide guidance on viable targets for therapeutic and vaccine development. [ABSTRACT FROM AUTHOR]