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Background <p>Cellular immunity studies in Syrian hamsters have been hampered by the lack of species-specific immunological reagents, particularly cytokines. To overcome this limitation, we characterized the biological activity of recombinant hamster cytokines IFN-γ, TGF-β, IL-6, IL-10 and TNF-α previously produced in <i>Escherichia coli</i> in a mammalian cell model (CHO-K1 cells).</p> Methods <p>Recombinant proteins were purified, detoxified and their activity assessed by metabolic activity (MTT) and gene expression (RT-qPCR) assays in CHO-K1 cells. The suitability of this cell model for this purpose was also determined through RT-qPCR, validating the expression of cytokine receptors and associated signaling pathway markers.</p> Results <p>All cytokines except rIFN-γ increased metabolic activity at 24&#xa0;h, whereas rIFN-γ induced a dose-dependent reduction without evidence of cytotoxicity, suggesting a potential cell cycle arrest rather than cell death. At 48&#xa0;h, all cytokines induced a decrease in metabolic activity. CHO-K1 cells expressed all protein receptor genes evaluated, supporting their relevance as a model to determine recombinant cytokine activity. Transcriptional analysis revealed pathway specific effects: rIFN-γ markedly induced <i>STAT1</i>; rTGF-β increased <i>SMAD2/3</i> expression, and rTNF-α upregulated <i>NFκB</i>. Gene expression analysis also revealed that rIL-6 downregulated <i>tnfα</i>, while rIL-10 and rTGF-β reduced <i>tnfα</i> and <i>ifnγ</i> expression. rIFN-γ also suppressed <i>tnfα</i>, with limited effects on other genes. These responses were consistent with the expected signaling dynamics of each cytokine.</p> Conclusions <p>Recombinant hamster cytokines exhibit functional activity in CHO-K1 cells, as shown by metabolic responses, modulation of immune-related genes, and activation of expected signaling mediators. Together with the detection of cytokine receptor transcripts, these results support the use of CHO-K1 cells for evaluating recombinant cytokine bioactivity and broaden the range of validated immunological tools available for Syrian hamster research.</p>

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Production and validation of recombinant hamster cytokines as tools for immunological studies

  • 期刊论文

  • 发表日期：

    2026年1月9日

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  • Laura de V. Maiocchi,
  • Natasha R. de Oliveira,
  • Mara A. C. Maia,
  • Ana C. K. Pedra,
  • Tiffany T. Bunde,
  • Beatriz C. M. Santos,
  • Izani Bonel Acosta,
  • Antonio Sergio Varela Junior,
  • Odir A. Dellagostin,
  • Thais L. O. Bohn

摘要

Background

Cellular immunity studies in Syrian hamsters have been hampered by the lack of species-specific immunological reagents, particularly cytokines. To overcome this limitation, we characterized the biological activity of recombinant hamster cytokines IFN-γ, TGF-β, IL-6, IL-10 and TNF-α previously produced in Escherichia coli in a mammalian cell model (CHO-K1 cells).

Methods

Recombinant proteins were purified, detoxified and their activity assessed by metabolic activity (MTT) and gene expression (RT-qPCR) assays in CHO-K1 cells. The suitability of this cell model for this purpose was also determined through RT-qPCR, validating the expression of cytokine receptors and associated signaling pathway markers.

Results

All cytokines except rIFN-γ increased metabolic activity at 24 h, whereas rIFN-γ induced a dose-dependent reduction without evidence of cytotoxicity, suggesting a potential cell cycle arrest rather than cell death. At 48 h, all cytokines induced a decrease in metabolic activity. CHO-K1 cells expressed all protein receptor genes evaluated, supporting their relevance as a model to determine recombinant cytokine activity. Transcriptional analysis revealed pathway specific effects: rIFN-γ markedly induced STAT1; rTGF-β increased SMAD2/3 expression, and rTNF-α upregulated NFκB. Gene expression analysis also revealed that rIL-6 downregulated tnfα, while rIL-10 and rTGF-β reduced tnfα and ifnγ expression. rIFN-γ also suppressed tnfα, with limited effects on other genes. These responses were consistent with the expected signaling dynamics of each cytokine.

Conclusions

Recombinant hamster cytokines exhibit functional activity in CHO-K1 cells, as shown by metabolic responses, modulation of immune-related genes, and activation of expected signaling mediators. Together with the detection of cytokine receptor transcripts, these results support the use of CHO-K1 cells for evaluating recombinant cytokine bioactivity and broaden the range of validated immunological tools available for Syrian hamster research.