Next Generation Sequencing and Analysis of the Transcriptomes of Wild-type and Alkbh5-/- Neutrophils
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ABSTRACT: By performing transcriptome-wide RNA sequencing (RNA-seq) analysis on the peritoneal neutrophils from Alkbh5-deficient mice (Alkbh5-/-) and Wild-type littermates (Alkbh5+/+) at 12h or 36h after mild cecal ligation and puncture (CLP), respectively, we want to identify potential targets of ALKBH5 and characterize the transcriptional landscape in neutrophils during antibacterial innate defense. Gene Ontology biological processes enrichment analysis of the significantly differentially expressed genes (DEGs) showed that neutrophil migration made up the most significantly enriched biological processes with annotations of neutrophil association upon loss of ALKBH5 in neutrophils, at both 12h and 36h after CLP. Many significantly DEGs also encompassed transcriptional signatures related to neutrophils, specifically to neutrophil influx into the infection site, including chemotaxis, response to chemokine, extravasation, ERK1 and ERK2 cascade, homeostasis of neutrophils. ALKBH5 deletion led to significantly decreased transcript expression of neutrophil migration-promoting Cxcr2 and Nlrp12; while increased transcript expression of neutrophil migration-suppressive Ptger4, Tnc, and Wnk1. These results demonstrated that ALKBH5 imprints migration-promoting transcriptional landscape in neutrophils to enable their migration into the site of infection for antibacterial innate defense.
Project description:By performing RNA-seq analysis on bone marrow neutrophils from the Alkbh5-deficient mice and Wild-type littermates undergoing CLP-induced sepsis, we want to investigate the effect of ALKBH5 on transcriptional landscape of mouse bone marrow neutrophils during bacterial infection. Then, we performed gene expression profiling and Gene Ontology enrichment analysis of the significantly differentially expressed genes using data obtained from RNA-seq.
Project description:Neutrophil accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. Based on recent evidence that mucosal metabolic changes influence disease outcomes, we hypothesized that transmigrating neutrophils influence the transcriptional profile of intestinal epithelia. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by neutrophil-epithelial crosstalk. Real-time O2 sensing indicated that transmigrating neutrophils rapidly deplete microenvironmental O2 sufficient enough to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). Utilizing HIF reporter mice in a TNBS colitis model, we investigated the relative contribution of neutrophils and the respiratory burst to M-bM-^@M-^\inflammatory hypoxiaM-bM-^@M-^] in vivo. Gp91phox-null mice, which mirror human chronic granulomatous disease, developed accentuated colitis compared to control with exaggerated neutrophil infiltration and diminished inflammatory hypoxia. In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils modulates the host response to inflammation. Likewise, the respiratory burst contributes fundamentally to localized O2 depletion, resultant microenvironmental hypoxia and effective inflammatory resolution. Two models were employed, direct and indirect. The M-bM-^@M-^\DirectM-bM-^@M-^] migration model entailed establishing a chemotactic gradient (using fMLP) across monolayers of T84 intestinal epithelial cells grown on the underside of permeable supports (3um pore). Neutrophils (PMN) were induced to migrate in the physiologically relevant the physiologically relevant basolateral-to-apical direction. Following migration, T84s were rested in complete media and 2hrs later harvested for RNA isolation. In the Indirect model, PMN were applied to T84s as with the direct model. After migration, conditioned supernatants were collected, cells pelleted and supernatants filtered through 0.2um pore. Conditioned supernatants were transferred to naive T84 monolayers for 2hrs, followed by RNA harvest. Each model was exposed to neutrophils (PMN) or not. All monolayers contained chemotactic peptide fMLP on the apical side. Total of 12 samples, 4 conditions in triplicate: Direct migration without neutrophils (T84 +fMLP -PMN), Direct migration with neutrophils (T84 +fMLP +PMN), Indirect migration without neutrophils (T84 +fMLP -PMN), Indirect migration with neutrophils (T84 +fMLP +PMN)
Project description:By performing RNA-seq analysis on ALKBH5-deficient (ALKBH5-/-, KO) and Wild-type (WT) dHL-60 human neutrophils infected with Escherichia coli, we want to investigate the effect of ALKBH5 on transcriptional landscape of human neutrophils during bacterial infection. Then, we performed gene expression profiling and Gene Ontology enrichment analysis of the significantly differentially expressed genes using data obtained from RNA-seq.
Project description:Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. Several studies have shown that the presence of TGF-β in the tumor microenvironment contributes to the skewing of neutrophils to have a more pro-tumor phenotype. However, the direct effects of TGF-β on neutrophil signaling and migration are unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether TGF-β directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil migration in either a transwell or an underagarose migration assay. However, TGF-β1 does activate signals canonically through SMAD3 and noncanonically through ERK1/2 in neutrophils in a time and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) is responsible for SMAD3 activation. Moreover, we discovered that TCM from aggressive breast cancer cells induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying neutrophil recruitment. However, we found that TGF-β1 alone does not induce secretion of LTB4. We next performed RNA-sequencing to evaluate the effects of TGF-β1 and TCM on the neutrophil transcriptome. We found that TGF-β1 and TCM result in changes in gene transcription in HL-60 cells, specifically of two pro-tumor genes OSM and VEGFA. Together, our findings characterize the effects of TGF-β1 on neutrophil signaling, migration, and gene expression that can be applied to understanding the changes in neutrophils that occur in the tumor microenvironment.
Project description:[Background] TNFa-induced adipose-related protein (TIARP) is a six-transmembrane protein that is expressed on macrophages, neutrophils and synoviocytes. We have recently reported that TIARP deficient mice (TIARP-/-) spontaneously developed arthritis, and had the high susceptibility to collagen-induced arthritis (CIA) with enhanced interleukin (IL)-6 production. However, the effect of TIARP to neutrophils and fibroblast-like syonoviocytes (FLS) has not been clearly elucidated. [Methods] We analyzed the roles of TIARP in K/BxN serum transfer model using TIARP-/- mice. We characterized the differences of neutrophils between WT and TIARP-/- mice by DNA microarray. Transmigration assays of TIARP-/- neutrophils were performed in vitro and in vivo. FLS were cultured with TNF? and the production of CXCL2 (a specific ligand of CXCR1 and CXCR2) and IL-6 were measured by ELISA. Moreover, TIARP-/- mice transferred with K/BxN serum were treated with anti-IL-6R antibodies. [Results] Arthritis in TIARP-/- mice transferred with K/BxN serum was significantly exacerbated. We identified overexpression of CXCR1 and CXCR2 in TIARP-/- neutrophils by DNA microarray. Neutrophils from TIARP-/- mice showed strong migration activity. The enhancement of chomotactic activity of TIARP-/- neutrophil was greatly facilitated by CXCL2 in vitro and in vivo. In addition, TIARP-/-FLS has enhanced the production of CXCL2 and IL-6 and the cell proliferation in the presence of TNFa, and the blockade of IL-6R significantly attenuated arthritis in vivo. [Conclusion] Our findings indicate that TIARP might down-regulate the production of CXCL2 and IL-6 in FLS, and the expression of chemokine receptors (CXCR1 and CXCR2) in neutrophils, resulting in the protective ability of neutrophils migration into arthritic joints. Mice were treated with thiogycollate medium intraperitoneally. After 3 days, peritoneal macrophages were isolated from three WT or TIARP-deficient mice, and these cells were stimulated by TNF? for 24 hours. Ly6G+ Neutrophils were isolated from splenocytes by MACS.
Project description:Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow can be shaped within the circulation remains vague. Phenotypic changes of circulating neutrophils caused by systemic inflammation are thought to result from several factors, including a “left shift” of the neutrophil compartment towards younger bone marrow-derived subsets. However, experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile the human and murine neutrophil transcriptome and proteome during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to plastic shifts dominated by conserved upregulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB-signaling dependent upregulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Importantly, blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by upregulation of distinct effector molecules upon infection. In summary, these data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation. This adds to the understanding of competing modes of adaptation to inflammatory challenges by the neutrophil compartment.
Project description:Analysis of neutrophil proteomic alterations induced by migration towards inflamed joints in juvenile idiopathic arthritis (JIA). In this experiment neutrophil proteomes were investigated after migration towards JIA synovial fluid in an in vitro model of a synovial membrane, compared to neutrophils incubated in synovial fluid without migration. The migration model consisted of transwell inserts with human knee synoviocytes on the undersides and HMEC endothelial cells on the insides, placed in wells containing medium with 10 % JIA synovial fluid.
Project description:Neutrophils are essential innate immune cells with unusual anti-microbial properties while dysfunctions of neutrophils lead to severe health problems such as lethal infections. Generation of neutrophils from human induced pluripotent stem cells (hiPSCs) is highly promising to produce off-the-shelf neutrophils for transfusion therapies. However, the anti-microbial potencies of hiPSCs derived neutrophils (iNEUs) remain less documented. Here, we develop a scalable approach to generate iNEUs in a chemical defined condition. iNEUs display typical neutrophil characters in terms of phagocytosis, migration, formation of neutrophil extracellular traps (NETs), etc. Importantly, iNEUs display a strong killing potency against various bacteria such as K.pneumoniae, P.aeruginosa, E.coli and S.aureus. Moreover, transfusions of iNEUs in mice with neutrophil dysfunction largely enhance their survival in lethal infection of different bacteria. Together, our data show that hiPSCs derived neutrophils hold strong anti-microbial potencies to protect severe infections under neutrophil dysfunction conditions.
Project description:Sepsis is a highly heterogeneous syndrome that impacts immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct immune cell functional phenotypes. Employing organ-on-chip for neutrophil functional analysis, we identified three distinct sepsis neutrophil phenotypes based on adhesion and migration patterns across human lung endothelial cells in response to cytokine activation (cytomix, TNF/IL-1β/IFNγ). The phenotypes were categorized as: a Hyperimmune phenotype characterized by enhanced cytomix-induced neutrophil adherence and migration, a Hypoimmune phenotype that was unresponsive to cytomix treatment, and a Hybrid phenotype with increased adherence but blunted migration in response to stimulation. Proteomic analysis identified both unique and shared proteins between the three phenotypes as compared to healthy controls. Proteins associated with neutrophil adherence were significantly upregulated in the Hyperimmune and Hybrid neutrophils, while the Hypoimmune group showed significant downregulation of these proteins. Clinically, the Hypoimmune group had significantly fewer patients requiring mechanical ventilation (29%) compared to the Hyperimmune group (70%). The Hypoimmune group and Hybrid group had significantly shorter ICU length of stay (LOS) than the Hyperimmune group. The Hypoimmune group also showed a trend for a lower mortality rate (35.7%) compared to the Hyperimmune group (50%). Thus, we identified associations between neutrophil phenotypes and important clinical outcomes, such as mechanical ventilation requirements, ICU LOS, and possibly mortality. Classification of sepsis patient phenotypes with diverse functional neutrophil responses and proteomic signatures can help distinguish patients who would benefit from specific treatments, such as immunosuppressive therapies and those who may be negatively impacted.
Project description:Adult-onset Still’s disease (AOSD) is a rare autoinflammatory disease, characterized by fever, rash, arthritis and other systemic inflammatory manifestations like hepatosplenomegaly and serositis. Neutrophil activation is the hallmark of AOSD. Here we aimed to identify candidate biomarker and to validate its association with clinical manifestations in AOSD. Transcriptome analysis of neutrophils from treatment-naïve active AOSD patients and healthy donors was performed. Using cut-offs of >2.0 fold change with p<0.05, we identified 864 DEGs in neutrophils from patients with active AOSD compared to healthy donors, including 442 and 422 upregulated and downregulated genes in active AOSD, respectively. By gene ontology (GO) analysis, we identified several strongly enriched terms of biological process. The most significantly indicated GO term was neutrophil degranulation.