Project description:Background: Moderate hypothermia (32oC for 12 â 72 hours) has therapeutic applications, but the mechanisms by which it affects cellular function are unclear. We tested the hypothesis that moderate hypothermia produces broad changes in gene expression by human cells at the level of mRNA. Methods: Acute monocytic leukemia THP-1 cells were incubated under control conditions (37oC) or moderate hypothermia (32oC) for 24 hours. Cellular mRNA was extracted and a cold stress response was confirmed by examining the expression of the cold-inducible gene CIRBP by reverse transcription PCR (RT-PCR). Gene expression analysis was performed on seven sets of samples with Affymetrix U133A chips, using established statistical methods. Sequences were considered to be affected by cold if they showed statistically significant changes in expression and also met published post-hoc filter criteria (changes in geometric mean expression of 2-fold or greater and detection calls of âpresentâ or âmarginalâ in at least half of the experiments). The changes in expression of a select number of these sequences were further confirmed by PCR. Results: 167 sequences met our prospectively defined criteria for a change in expression; 67 showed increases in expression and 100 showed decreases in expression. Consistent with prior literature, the cold-inducible genes CIRBP and RBM3 showed increases in expression. Functional categories affected by cold stress included genes involved in immune responses, cell growth, proliferation, and differentiation, and metabolism and biosynthesis. Several heat shock proteins (HSPs) showed decreases in expression. Conclusions: Moderate hypothermia produces substantial changes in cellular gene expression, in categories potentially of importance to systemic function. Interestingly, cold exposure without re-warming decreased the expression of several HSPs. Our in vitro findings suggest that moderate hypothermia in vivo might produce physiologically important changes in gene expression by circulating leukocytes. Experiment Overall Design: Seven sets of paired THP-1 samples, grown under either control (37oC) or hypothermic (32oC) conditions for 24 hours

Project description:MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related-1 molecule (MR1). Activated MAIT cells can exert regulatory, pro-inflammatory, or cytotoxic responses that enable the direct killing of infected cells. While tremendous progress regarding the multifaceted roles of MAIT cells has been made in the last decade, our understanding of the MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR-1 ligand stimulations. Translation of both cell types was more efficiently induced by 5-OP-RU in comparison to Ac-6-FP, which correlated with higher conjugation frequencies and CD3 polarization at immunological synapses. In addition to known effector responses, protein translations uncovered type I and type II Interferon-driven protein expression profiles in both 5-OP-RU-stimulated MAIT and THP-1 cells. Interestingly, the translatome of THP-1 macrophages suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

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Evaluation of proteomics strategies for low cell numbers: SP3- and C18-based protocols combined with five cell lysis methods

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