Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of mouse bone-marrow–derived macrophages and osteoclast precursors (BMM, RANKL-16h, RANKL-36h) from wild-type and Senp6 conditional knockout mice

ABSTRACT: To examine the impaired osteoclastogenic program in Senp6^LysM BMMs, we performed RNA-seq on WT and Senp6^LysM BMMs stimulated with RANKL for 0 h (R0), 16 h (R16), and 36 h (R36), corresponding to sequential stages of early osteoclastogenesis.

INSTRUMENT(S): NA, Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Ye Liu

PROVIDER: E-MTAB-16140 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Invasive candidiasis is a serious healthcare problem with high mortality rates, particularly in immunocompromised patients. Understanding how the immune response towards Candida albicans is mounted and controlled is fundamental to developing new therapeutic strategies. Protein tyrosine phosphatase 1B (PTP1B) is a key phosphatase regulating various immunoreceptor signalling cascades including inflammatory and metabolic responses and a pharmaceutical target. This study uncovers the importance of PTP1B in driving protective antifungal immune cell responses post-infection. Following systemic C. albicans infection, mice lacking PTP1B in myeloid cells (LysM PTP1B-/-) were significantly more susceptible to infection, with lower survival rates, greater body weight loss and higher fungal burdens in brain, kidney, and liver. LysM PTP1B-/- infected mice exhibited increased expression of organ inflammatory cytokines TNF, IL-10, IL-6 and IL-1β and increased kidney tubular inflammation, 1- and 3-days post-infection. Kidneys from infected LysM PTP1B-/- mice also demonstrated significantly increased chemokine expression and leukocyte infiltration, collectively promoting immunopathology. PTP1B-/- neutrophils infiltrating into kidneys were, however, less mature and in vitro produced less ROS. Bone marrow-derived macrophages from LysM PTP1B-/- mice, through proteomic analysis, show enhancement of immune response and type I interferon signalling pathways, and they were less able to phagocytose and kill C. albicans relating to changes in their metabolism and viability when fighting infection. The decrease in phagocytosis and viability in PTP1B-/- macrophages following infection translated to human macrophages treated with a pharmacological PTP1B inhibitor. Our data provides new insights connecting the critical role of myeloid cell PTP1B in regulating immune defence against C. albicans through modulating levels of inflammatory cytokines, neutrophil clearance mechanisms and macrophage functional and metabolic responses, implying that boosting specific PTP1B-dependent signalling pathways could have therapeutic implications for combatting systemic fungal infection.

Project description:Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts, and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule is the enigmatic coupling factor. We have identified Cthrc1, a protein secreted by mature bone-resorbing osteoclasts, that targets stromal cells so as to stimulate osteogenesis. The expression of Cthrc1 is robustly induced when mature osteoclasts are placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increases in a high turnover state, such as that which is induced by RANKL injections in vivo, whereas it decreases with aging or following alendronate treatment, conditions associated with suppressed bone turnover. The targeted deletion of the Cthrc1 gene eliminates Cthrc1 expression in bone, whereas its deficiency in osteoblasts does not exert any significant effect. Osteoclast-specific deletion of the Cthrc1 gene results in osteopenia due to reduced bone formation: it also impairs the coupling process following resorption induced by RANKL injections, with a resultant impairment of bone mass recovery. Thus, Cthrc1 is an osteoclast-secreted “coupling factor” that regulates bone remodeling and hence, skeletal integrity. Total bone marrow cells were prepared from the femurs and tibias of 8-10-week-old C57BL/6 mice and cultured in the presence of M-CSF (100ng/ml) for 3 days as described previously (Takeshita et al., 2000 JBMR 15:1477-1488). Cells were harvested with 0.02% EDTA/PBS and used as bone marrow macrophages (BMMs). These BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (100ng/ml) for 2 days. TRAP positive mononuclear cells were harvested and used as pre-osteoclasts (pOC). These pOC cells were further cultured in the presence of M-CSF and RANKL for 2 days in normal plastic plate or on dentin slices. After 2 days, multinucleated TRAP positive mature osteoclasts were generated as mature osteoclasts on plate (mOCp) and mature resorbing osteoclasts on dentin (mOCd), respectively. RNAs were extracted from four different stages of osteoclast lineage cells; BMMs, pOC, mOCp and mOCd, and used for microarray analysis.

Dataset Information

RNA-seq of mouse bone-marrow–derived macrophages and osteoclast precursors (BMM, RANKL-16h, RANKL-36h) from wild-type and Senp6 conditional knockout mice

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