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<p> <?tk 2?>THP-1 cell lines are routinely utilized as an in vitro model to investigate monocyte and macrophage functions. In this study, the effectiveness of producing pro-inflammatory cytokines; Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor alpha (TNF-α) through the differentiation of THP-1 cells with phorbol 12-myristate 13-acetate (PMA) followed by treatment with lipopolysaccharides (LPS) were evaluated in vitro. A minimum amount of 10&#xa0;ng/ml of PMA for THP-1 cell differentiation was sufficient to induce adherence, and this was determined through morphological assessment as well as evaluation of the percentage of cell attachment. Real-time Polymerase Chain Reaction (RT-qPCR) analysis was carried out to substantiate the macrophage-like characteristic acquisition of THP-1 cells post-PMA treatment through measurement of the macrophage surface-specific antigen <i>CD14</i> gene. Results showed a distinct upregulation of gene expression after 48&#xa0;h of PMA treatment compared to untreated. Secondly, <i>NFKB-1</i> gene expression began to increase six hours post-LPS treatment with a prior rest phase of 24&#xa0;h both in serum-containing and serum-free conditions after PMA treatment. Nevertheless, serum-free conditions showed a markedly higher expression as compared to serum-containing conditions. Additionally, RT-qPCR results showed a consistently marked upregulation of pro-inflammatory cytokines 24&#xa0;h after LPS treatment. Furthermore, western blot analysis ascertained the upregulation of the pro-inflammatory cytokines’ levels 24&#xa0;h post-LPS treatment. Collectively, results showed that pro-inflammatory cytokines can be effectively produced from THP-1 cells in vitro via diligent monitoring of PMA and LPS concentrations as well as manipulating cell growth media conditions in addition to measurement of <i>CD14</i> and <i>NFKB-1</i> gene levels. In summary, <i>CD14</i> and <i>NFKB-1</i> genes act as important check points for production of THP-1 cells derived pro-inflammatory cytokines.</p>

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CD14 and NFKB-1 Serve as Essential Gatekeepers for Monitoring In Vitro Production of Pro-inflammatory Cytokines in THP-1 Monocytic Cells

  • 期刊论文

  • 发表日期：

    2025年4月16日

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  • Aaron Kok Zhi Yang,
  • Narazah Mohd Yusoff,
  • Emmanuel Jairaj Moses

摘要

THP-1 cell lines are routinely utilized as an in vitro model to investigate monocyte and macrophage functions. In this study, the effectiveness of producing pro-inflammatory cytokines; Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor alpha (TNF-α) through the differentiation of THP-1 cells with phorbol 12-myristate 13-acetate (PMA) followed by treatment with lipopolysaccharides (LPS) were evaluated in vitro. A minimum amount of 10 ng/ml of PMA for THP-1 cell differentiation was sufficient to induce adherence, and this was determined through morphological assessment as well as evaluation of the percentage of cell attachment. Real-time Polymerase Chain Reaction (RT-qPCR) analysis was carried out to substantiate the macrophage-like characteristic acquisition of THP-1 cells post-PMA treatment through measurement of the macrophage surface-specific antigen CD14 gene. Results showed a distinct upregulation of gene expression after 48 h of PMA treatment compared to untreated. Secondly, NFKB-1 gene expression began to increase six hours post-LPS treatment with a prior rest phase of 24 h both in serum-containing and serum-free conditions after PMA treatment. Nevertheless, serum-free conditions showed a markedly higher expression as compared to serum-containing conditions. Additionally, RT-qPCR results showed a consistently marked upregulation of pro-inflammatory cytokines 24 h after LPS treatment. Furthermore, western blot analysis ascertained the upregulation of the pro-inflammatory cytokines’ levels 24 h post-LPS treatment. Collectively, results showed that pro-inflammatory cytokines can be effectively produced from THP-1 cells in vitro via diligent monitoring of PMA and LPS concentrations as well as manipulating cell growth media conditions in addition to measurement of CD14 and NFKB-1 gene levels. In summary, CD14 and NFKB-1 genes act as important check points for production of THP-1 cells derived pro-inflammatory cytokines.