ABSTRACT: A time-course (T0, 15, 30 min, 1, 3, 6 or 16 hours) of mRNA abundance changes was studied in the human Hep3B hepatoma cell line challenged with a pro-inflammatory cytokine-enriched medium (CM,conditionned medium) vs control medium (NCM,non conditionned medium). Paired cultures prepared from a same batch of trypsinized cells were challenged with CM vs NCM for a given length of time and this was repeated in three independent experiments (S1,S2,S3).
Project description:A time-course (T0, 15, 30 min, 1, 3, 6 or 16 hours) of mRNA abundance changes was studied in the human Hep3B hepatoma cell line challenged with a pro-inflammatory cytokine-enriched medium (CM,conditionned medium) vs control medium (NCM,non conditionned medium). Paired cultures prepared from a same batch of trypsinized cells were challenged with CM vs NCM for a given length of time and this was repeated in three independent experiments (S1,S2,S3). Keywords: time-course
Project description:Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. We examined global gene expression patterns by microarray during fatal murine CM (FMCM) and non-cerebral malaria (NCM). There was differential expression of a number of genes, including some not yet characterized in the pathogenesis of FMCM. Some gene induction was observed during Plasmodium infection regardless of the development of CM and there was a predominance of genes linked to IFN responses, even in NCM. However, upon real-time PCR validation and quantitation, these genes were much more highly expressed in FMCM than in NCM. The observed changes included genes belonging to pathways such as interferon (IFN) signaling, MHC processing and presentation, apoptosis, immunomodulatory and anti-microbial processes. We further characterized differentially expressed genes by examining the cellular source of their expression as well as their temporal expression patterns during the course of malaria infection. These data identify a number of novel genes that represent interesting candidates for further investigation in FMCM. Keywords: disease state analysis 5 individual mouse brains were collected for each group (Uninfected control, PbA(6), PbK(6), PbK(14). RNA was extracted from these mice and then pooled to create a single sample for hybridisation. Comparisons were made between the experimental groups (PbA(6), PbK(6), PbK(14)) and the reference group (Uninfected control).
Project description:Primary Graft Dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular non-classical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) are mobilized from the spleen and, upon entry into the allograft, permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that pharmacological or genetic depletion of donor NCM abrogated CM recruitment, single-cell RNAseq identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of human and murine tissues combined with murine knockouts and chimeras indicated that while IL1β was secreted by multiple cell lineages, donor NCM were responsible for the early activation of AM and CCL2 release. IL1β production by NCM was NLRP3 inflammasome-dependent and inhibited by donor treatment using a clinically approved sulphonylurea, Glyburide. Production of CCL2 in the donor AM occurred through IL1R-dependent activation of PKC and NFκB-pathway. Accordingly, we show that IL1β-dependent paracrine interaction between donor NCM and AM leads to recruitment of host AM necessary for PGD. Since depletion of donor NCM, IL1β or IL1R antagonism, and inflammasome inhibition, abrogated recruitment of CM as well as PGD and are feasible using FDA-approved compounds, our findings have potential for immediate clinical translation.
Project description:Classical monocytes (CMs) can be converted into conventional dominant Nr4a1-dependent nonclassical monocytes (N-NCMs). Upon activation of Nod2 signaling, CM also can be converted into noncanonical Nod2-dependent inducible NCMs (I-NCMs). The transcriptional profiles and typical markers specific for those two NCM subsets yet to be determined. To transcriptionally characterize N-NCM and I-NCM, we performed transcriptional profiling analysis using RNAseq data obtained from the flow-sorted NCM subsets. Through the comprehensive RNAseq analysis, we identified typical transcriptional markers specific to N-NCM and I-NCM. By virtue of those specific markers, we identified the potential I-NCM populations in multiple cellular settings.
Project description:Monocytes are ephemeral myeloid immune cells that arise from adult hematopoiesis and circulate in the blood. They comprise two main subsets, in mice defined as classical and non-classical monocytes (CM, NCM). Recent fate mapping and transcriptomic analyses revealed that CM themselves are heterogeneous. Here, we report surface markers that allow segregation of murine GMP- and MDP-derived CM, as well as their functional characterization, including fate definition following adoptive cell transfer. GMP-Mo and MDP- Mo gave equal rise to homeostatic CM progeny, such as blood NCM and gut macrophages; the cells however differentially seeded selected other tissues, including the dura mater and lung. Specifically, GMP-Mo and MDP-Mo gave rise to distinct interstitial lung macrophages, linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide evidence for the existence of two functionally distinct CM subsets in the mouse, which differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge.
Project description:Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. We examined global gene expression patterns by microarray during fatal murine CM (FMCM) and non-cerebral malaria (NCM). There was differential expression of a number of genes, including some not yet characterized in the pathogenesis of FMCM. Some gene induction was observed during Plasmodium infection regardless of the development of CM and there was a predominance of genes linked to IFN responses, even in NCM. However, upon real-time PCR validation and quantitation, these genes were much more highly expressed in FMCM than in NCM. The observed changes included genes belonging to pathways such as interferon (IFN) signaling, MHC processing and presentation, apoptosis, immunomodulatory and anti-microbial processes. We further characterized differentially expressed genes by examining the cellular source of their expression as well as their temporal expression patterns during the course of malaria infection. These data identify a number of novel genes that represent interesting candidates for further investigation in FMCM. Keywords: disease state analysis
Project description:Monocytes are short-lived myeloid immune cells that arise from adult hematopoiesis and circulate for a short time in the blood. They comprise two main subsets, in mice defined as classical Ly6Chigh and non-classical Ly6Clow monocytes (CM, NCM). Recent fate mapping and transcriptomic analyses revealed that CM themselves are heterogeneous. Here, we report surface markers that allow segregation of murine GMP- and MDP-derived CM in the BM and blood. Functional characterization, including fate definition following adoptive cell transfer, established that GMP-Mo and MDP-Mo could equal rise to homeostatic CM progeny, such as NCM in blood and gut macrophages, but differentially seeded selected other tissues. Specifically, GMP-Mo and MDP-Mo gave rise to distinct interstitial lung macrophages, thus linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide comprehensive evidence for the existence of two functionally distinct CM subsets in the mouse, which differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge. Our findings are indicative of impact of monocyte ontogeny on in situ differentiation.
Project description:In this analysis, sorted classical (CM), intermediate (IM) and non-classical (NCM) monocyte subsets from children under steady state (healthy, H) and dengue febrile illness (Dengue, D) were analyzed for their transcriptional profiles using RNA seq. The monocyte subsets were sorted from peripheral blood cells after excluding CD3, CD19, CD20, CD56, CD66b and NKp30 positive cells and then gating on HLADR positive population to identify CM, IM and NCM subsets based on surface expression of CD16 and CD14. The transcriptional profile of the three monocyte subsets was separately compared in healthy children, in dengue febrile children and in dengue versus healthy states. This study highlights hierarchy of gene expression in classical, intermediate and non-classical monocytes in healthy and dengue febrile conditions.
Project description:Uveal melanoma (UM) is the most common intraocular tumor in adults, and its pathogenesis is only partly understood. UM can develop de novo from normal choroidal melanocytes (NCMs) or from pre-existing nevi that stem from NCMs and are thought to harbour UM-initiating mutations, most commonly in GNAQ or GNA11. However, there is a complete lack of commercially available NCM cell lines and no detailed protocol for developing oncogene-mutated CM line (MutCM) to study UM development. The purpose of this study was to establish and characterize premalignant CM models from human donor eyes to recapitulate the cell populations at the origin of UM.
Project description:Emerging evidence suggests that the nutrient availability in the tumour cell microenvironment and the dimensionality of in vitro tumour cells impact cell proteome and drug responsiveness. An improved physiological medium, called Melbourne Medium (MM), has been developed most recently by our research group. MM was designed to emulate low molecular weight components of human plasma, so it is a more physiologically relevant medium compared to conventional medium such as DMEM (CM). Global proteomics experiments were conducted to compare the protein profiles of MCF-7 and MDA-MB-231 breast cancer cells cultured in MM versus CM and in different experimental conditions (3D versus 2D).