ABSTRACT: Hypoxia promotes macrophage polarization triggered by IL-6.In an attempt to gain speciﬁc insight into the mechanism of macrophage polarization during hypoxia. We used microarrays to detail the global programme of gene expression and identified distinct classes of changed genes during this process. Four groups of RAW264.7 cells were harvested for RNA extraction and hybridization on Affymetrix microarrays (normoxia control, normoxia+IL-6,hypoxia control,hypoxia+IL-6).
Project description:Hypoxia promotes macrophage polarization triggered by IL-6.In an attempt to gain speciﬁc insight into the mechanism of macrophage polarization during hypoxia. We used microarrays to detail the global programme of gene expression and identified distinct classes of changed genes during this process. Overall design: Four groups of RAW264.7 cells were harvested for RNA extraction and hybridization on Affymetrix microarrays (normoxia control, normoxia+IL-6,hypoxia control,hypoxia+IL-6).
Project description:Purification of mouse mRNAs encoding signal transducer and activator of transcription 3 (Stat3) identified many miRNAs using multiplex miRNA array in RAW264.7 cells. To investigate the miRNAs targeting to mouse Stat3 gene in macrophage cells, we performed a protocol miRIP to affinity purify the miRNAs from an endogenous segment of Stat3 mRNA using nucleic acid hybridization and use multiplex miRNA array to identify the associated miRNAs.
Project description:Toxoplasma strains are known to inhibit the expression of several interferon-gamma induced genes, and a type II strain was shown to dysregulate genome-wide responses to interferon-gamma in human fibroblasts (Kim et al., 2007, J Immunol.). In this study we aimed to determine the effect of infection with three clonal lineages of Toxoplasma, type I, II, and III strains on genome-wide interferon-gamma induced transcription in murine macrophages. We also assessed the effect of the two main Toxoplasma modulators of mouse macrophage transcription, ROP16 and GRA15 (Jensen et al., 2011, Cell Host Microbe). We used Affymetrix microarrays to analyze host cell transcription after Toxoplasma infection and interferon-gamma stimulation. RAW264.7 murine macrophages were left uninfected or infected with type I (RH), type I ∆rop16 (RH ∆rop16), type II (Pru), type II ∆gra15 (Pru ∆gra15), or type II (CEP) parasites at an MOI ~5 for 18 hours and subsequently stimulated with murine IFN-γ for six hours. Plaque assays were done to assess parasite viability. Total RNA was isolated and hybridized to Affymetrix Mouse 430A 2.0 gene chips.
Project description:The polarization of macrophages into an anti-inflammatory or regulatory phenotype plays an important role in resolving inflammation. PGE2 regulates macrophage polarization via a PKA dependent pathway. PKA phosphorylates SIKs, inhibiting their ability to phosphorylate CRTC3 in cells. This in turn allows CRTC3 to translocate to the nucleus where it acts as a co-activator with the transcription factor CREB to induce IL-10 transcription. In line with this we find that either genetic or pharmacological inhibition of SIKs mimics the effect of PGE2 on IL-10 production. We show here that PGE2, in combination with LPS, is able to promote a regulatory like phenotype in macrophages characterized by high expression of IL-10 as well as the regulatory markers SPHK1 and LIGHT. Gene expression profiles of primary bone marrow derived mouse macrophages treated with LPS or LPS+PGE and untreated controls were generated using Affymetrix mouse gene 1.1 ST arrays. In total, 12 arrays were generated from the three groups (LPS, LPS+PGE and control) with 4-replicates in each group. One sample from LPS group was removed due to QC issues and the remaining 11 that were used in our analyses are submitted here.
Project description:Botulinum neurotoxin type A (BoNT/A) is one of the most potent protein toxins, which makes it a possible biological weapon and therapeutic. Using microarray analysis we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. Time dependent expression profiles after treatment of 1nM or 5nM Botulinum neurotoxin A
Project description:Macrophages acquire distinct phenotypes during tissue stress and inflammatory responses. Classically, macrophages are categorized into two different subsets named inflammatory M1 and anti-inflammatory M2 macrophages. We herein identified a unique pathogenic macrophage subpopulation, named M17, driven by IL-23 with a distinct gene expression profile including cytokines and membrane molecules. In contrast to M1 and M2-polarized macrophages, M17 macrophages express high levels of CXCR5 on the cell surface and produce large amounts of IL-17A, IL-22 and IFN-γ. IL-23 induces IL-17A expression in macrophages through the signal transducer and activator of transcription 3 (STAT3)-retinoid related orphan receptor-γ T (RORγT) pathway, while induces IFN-γ through T-bet. Importantly, IL-23-induced M17 macrophage polarization promotes the pathogenesis of psoriasis in vivo. The characteristics and significances of M17 macrophage subpopulation in the physiological status and pathogenesis caused by infections, tumors and graft rejection needs to be explored. Overall design: To further demonstrate whether M17 macrophage polarization represents a distinct polarization of macrophages, we detected gene expression profiles of M1, M2 and M17 polarized macrophages by microarray methods.
Project description:In response to microenvironmental signals macrophages undergo different activation, indicated as classic/M1 and alternative/M2 polarization. C-Myc transcription factor could be an essential player in M2 polarization. Functional relevance of c-Myc in M2 macrophage biology is investigated by evaluating the effect of 100-58F4, on the transcriptional profile induced on human macrophages by IL-4. Human monocytes were obtained from normal donor buffy coats by two-step gradient centrifugation using Ficoll (Biochrom) and Percoll (Amersham). Non-adherent cells were discarded, and the purified monocytes were incubated for 7 days in RPMI 1640 (Biochom) supplemented with 10% FCS (HyClone) and 100 ng/mL M-CSF to obtain resting macrophages. Macrophage polarization was obtained by removing the culture medium and culturing cells in RPMI 1640 supplemented with 10% FCS and 100 ng/mL LPS plus 20 ng/mL IFN-gamma (M1 polarization) or 20 ng/mL IL-4 (M2 polarization) for 24 h. When needed, chemical inhibitors were added with IL-4.
Project description:Bone marrow derived macrophages from C57BL/6 mice were stimulated into M1 and M2 polarization state. Analysis of BMDMs from LysMcre;FoxO1Fl/FL mice and control littermates. Results provide insight into the regulatory role of FoxO1 during macrophage polarization. BMDMs were stimulated with 100ng/ml LPS plus 20ng/ml IFN-γ into M1 polarization, and stimulated with 10ng/ml IL-4 plus 10ng/ml IL-13 into M2 polarization. Both for 24 hours. Unstimulated cells as M0 state. Overall design: Six samples, including 3 polarization state from FoxO1 deficient BMDMs and WT BMDMs, separately.
Project description:Macrophages are innate immune cells characterized by their plasticity and their ability to react to various environmental stimuli. These cells are involved in a multiple number of tissular functions in homeostasis and pathological contexts. According to their environment these cells could be polarized toward different states of activation which determine their functional orientation. A large part of the macrophage biology field is devoted to better define what polarizations are, from a molecular point of view. It is now accepted that a multidimensional model of polarization is needed to grasp the broad phenotype repertoire depending on various environmental signals. Oxygen tension is one of these tissular environmental parameters. We designed this study to obtain a proteomic signature of various polarizations in human monocytes derived macrophages. We also seek to explore how environmental oxygen tension varying from an atmospheric composition (18.6% O2) to a “tissular normoxia” (3% O2) could modify our classification of macrophages’ polarization. We have obtained various polarization specific proteins and oxygen sensors for human macrophages. One example is arachidonate 15-lipoxygenase (ALOX15) which is a IL4/IL13 polarization specific proteins up regulated under low oxygen exposure associated to an increase of the phagocytosis rate of apoptotic cells. These results illustrate the necessity to take into account physicochemical parameters like oxygen when macrophage polarization is studied to correctly assess their functions in tissues.
Project description:In this study we compared the response of human monocyte-derived macrophages differentiated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) to the most common activation stimuli: LPS plus interferon-γ to induce macrophage polarization towards the M1 type or IL-4 to induce macrophage polarization towards the M2a type. Additionally, IL-10 was used to drive M-CSF-primed macrophages into the M2c state. We used the the whole-human genome microarray to determine genes that were up- or downregulated by the activation stimuli in both macrophage lineages, with focus on genes implicated in immune response. Overall design: We generated 7 different macrophage subtypes in three biological replicates: Isolated monocytes of three healthy donors (biological replicates) were differentiated with either 25ng/ml GM-CSF or 50ng/ml M-CSF for 7d. GM-CSF-differentiated macrophages were then either mock-activated with culture medium only (control; condition 1) or activated with 100 ng/ml LPS + 25ng/ml IFNγ (condition 2), or 20 ng/ml IL-4 (condition 3) for 2d (48 h). This activation regime was also used to activate M-CSF-differentiated macrophages, which were in additionally activated with 20 ng/ml IL-10 (condition 4) for 2d (48 h).