Analysis of Innate Immune Signaling Networks

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We established the PSIIM Molecular and Cell Biology group in August 2008, and lab personnel have been hired for this project only recently, however progress has been made in evaluating RAW and THP1 cell responses to a variety of TLR ligands. Using RAW and THP1 reporter cell lines developed for screening applications, we have determined kinetic time courses and dose response data for the activation of NFkB and TNF alpha transcription for eight TLR ligands (LPS, Pam2CSK4, Pam3CSK4, FSL-1, Peptidoglycan, Resiquimod 848, CpG DNA, and poly I:C). We find the THP1 cell line is minimally responsive to LPS in its undifferentiated monocyte state, but it becomes highly responsive to LPS at 1ng/ml 72 hr after treatment with 5ng/ml phorbol ester (PMA). This suggests that PMA differentiation is inducing a more macrophage-like state in this cell line. We are currently attempting to correlate the kinetics of NFkB activation by each ligand with the subsequent induction of TNF alpha promoter activity. Previous reports have described models for oscillation of NFkB activity based on phasic expression and degradation of negative feedback regulators. We have observed oscillatory kinetics for both p65/RelA and TNF alpha promoter activation in our cell lines, and we are currently assessing whether these data might provide an initial framework for identifying similarities and differences between varied stimuli. We have also begun our efforts to collect data on the phosphoprotein profile in TLR-activated macrophages. We will compare data from quantitative western blotting with the xMAP (multi-analyte profiling) technology developed by Luminex. A key feature of the xMAP approach is the ability to assess numerous targets (up to 100) in a single sample, which reduces the noise in the data introduced by sample-to-sample variability. We will also evaluate this technology for the measurement of specific transcripts and secreted cytokines in an effort to generate a broad dataset that more comprehensively describes the cellular state induced by TLR activation.