Cellular dissection of psoriasis for transcriptome analyses and the post-GWAS era

Abstract Background Genome-scale studies of psoriasis have been used to identify genes of potential relevance to disease mechanisms. For many identified genes, however, the cell type mediating disease activity is uncertain, which has limited our ability to design gene functional studies based on genomic findings. Methods We identified differentially expressed genes (DEGs) with altered expression in psoriasis lesions (n = 216 patients), as well as candidate genes near susceptibility loci from psoriasis GWAS studies. These gene sets were characterized based upon their expression across 10 cell types present in psoriasis lesions. Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding. Results Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%). In contrast, psoriasis GWAS candidates tended to have highest expression in immune cells (71%), with a significant fraction showing maximal expression in neutrophils (24%, P < 0.001). By identifying candidate cell types for genes near susceptibility loci, we could identify and prioritize SNPs at which susceptibility variants are predicted to influence transcription factor binding. This led to the identification of potentially causal (non-coding) SNPs for which susceptibility variants influence binding of AP-1, NF-κB, IRF1, STAT3 and STAT4. Conclusions These findings underscore the role of innate immunity in psoriasis and highlight neutrophils as a cell type linked with pathogenetic mechanisms. Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.http://deepblue.lib.umich.edu/bitstream/2027.42/109537/1/12920_2013_Article_485.pd

Evaluation of TruCount Absolute-Count Tubes for Determining CD4 and CD8 Cell Numbers in Human Immunodeficiency Virus-Positive Adults

A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was −8% and −3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of −1% for both CD4 and CD8 with 6-h samples and −2% and −3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter- and intralaboratory variability in determining absolute CD4 and CD8 counts

Donor dependent, interferon-γ induced HLA-DR expression on human neutrophils in vivo

Neutrophils are effector cells of innate immune responses. Stimulated by interferon-γ (IFN-γ) to express HLA-DR, neutrophils acquire accessory cell functions for superantigen-mediated T cell activation. In vitro HLA-DR induction on neutrophils varies in a functionally relevant way as levels of MHC class II expression and magnitude of neutrophil induced T cell responses are correlated functions. The aim of this study was to assess whether IFN-γ induces HLA-DR on human neutrophils in a donor dependent fashion in vivo and to define regulatory events operative in MHC class II expression of neutrophils. In vivo administration of rhIFN-γ in 55 patients with renal cell carcinoma resulted in a varying increase of HLA-DR on neutrophils. By setting a cut-off for response at>10% HLA-DR positive neutrophils, HLA-DR responders (51%) were as frequent as nonresponders (49%). In vivo kinetic studies revealed a peak expression of HLA-DR on neutrophils 48 h after rhIFN-γ application, while nonresponders remained HLA-DR negative over a 72-h period. In vitro IFN-γ stimulated neutrophils recapitulated the response profiles observed in vivo. No differences in IFN-γ dependent CD64 and invariant chain expression, and IFN-γ serum levels were observed among the response subgroups. HLA-DR mRNA was detected in neutrophils from rhIFN-γ treated responders and nonresponders, HLA-DR protein solely in lysates of responder neutrophils. IFN-γ stimulated HLA-DR expression on neutrophils is subject to donor dependent variations in vivo, which result from rather post-transcriptional than transcriptional regulation. Due to their abundance in inflammatory reactions heterogeneous HLA-DR expression by neutrophils could determine the outcome of superantigen-driven diseases