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Effect of recombinant human Annexin A5 on gene expression of bone marrow-derived macrophages differentiating into osteoclasts

ABSTRACT: Primary BMMs were obtained from male C57BL/6 miceaged 4-6 weeks. The mice were euthanized, and femurs and tibias were removed. BMMs were flushed out of the bones using a cold culture medium, filtered through a 70-um cell strainer , and centrifuged at 1500 rpm for 5 min. The cells were suspended in cold culture medium supplemented with 20 ng/ml M-CSF and cultured for five days, with half of the medium being replaced on day three and full replacement on day 5 before being used for subsequent experiments.The culture medium was alpha-MEM supplemented with 10% FBS and 1% penicillin-streptomycin-gentamicin solution. Mature BMDMs were double-stained using flow cytometry antibodies CD16/32 and F4/80 for identification. The recombinant protein AnxA5 used in this study was produced by SEME Cell Technology Co., Ltd, China. To investigate the role of AnxA5 in osteoclast differeation, 100000 BMDMs were seeded in 12 well plates. After 24 hours of cell seeding, the regular medium was replaced with culture medium (30ng/ml m-csf, control group, A-1, A-2,A-3 and A-4), culture medium containing 50 ng/mL RankL (model group, B-1, B-2, B-3 amd B-4), culture medium containing 50 ng/mL RankL and 2 ug/mL of Annexin A5 (Treat group, C-1, C-2,C-3 and C-4) for 5 days.

ORGANISM(S): Mus musculus

PROVIDER: GSE254430 | GEO | 2025/12/01

REPOSITORIES: GEO

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Project description:Purpose: To characterize the processes defining the end dendritic cell (DC) phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from human donors using Ficoll-Paque gradient centrifugation. CD14+ monocytes were isolated from PBMCs by magnetic-activated cell sorting (MACS) and cultured in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 µM 2-Mercaptoethanol, 1% Penicillin-Streptomycin at 37°C and 5% CO2. During the differentiation stage, complete medium was added 150 U/mL recombinant human IL-4 and 160 U/mL recombinant human GM-CSF to obtain monocyte-derived dendritic cells (moDCs). Medium was replaced after 3 days of culturing, and moDCs were used 6 days after the start of culture. The cells were on average >70% CD1a-positive, and with <3% CD14+CD16+ cells, based on flow cytometry, and responded with high levels of IL-12p70 production upon stimulation with LPS and IFN-y (mean IL-12p70 production of 4641.8 pg/mL; SD = 688.1 pg/mL). Cells were harvested and rested for 1 hour at 37°C and 5% CO2 before stimulation. At this point, moDCs were supplemented with complete medium containing TSLP (to final concentration of 25 ng/mL), IL-25 (25 ng/mL), TSLP+IL-25 (both 25 ng/mL), helminth PCF (100 μg dry matter/mL, tested in different doses in pre-study experiments), butyrate (2 mM), or succinate (0.5 mM and 2 mM, di-sodium succinate hexahydrate). Unstimulated cells were supplemented with complete medium alone. Immediately afterwards, medium containing Escherichia coli O26:B6 LPS (final concentration of 100 ng/mL) and IFN-γ (10 ng/mL) was added. Cells were stimulated for 4 or 20 h at 37°C, 5% CO2, and 95% humidity. Results: Our data show that helminth PCF and butyrate treatment suppress the Th1-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS+IFN-γ-matured DCs by downregulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in TLR4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, while transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were upregulated. Conclusion: Collectively, our results further our understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

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Effect of recombinant human Annexin A5 on gene expression of bone marrow-derived macrophages differentiating into osteoclasts

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