Mycoplasma suppression of THP-1 Cell TLR responses is corrected with antibiotics - PubMed (original) (raw)

Mycoplasma suppression of THP-1 Cell TLR responses is corrected with antibiotics

Ekaterina Zakharova et al. PLoS One. 2010.

Abstract

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 beta was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1beta and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Plasmocin-dependent removal of mycoplasmal contamination of THP-1 cells.

THP-1 cells, chronically infected with mycoplasma, were treated with Plasmocin (25 µg/ml every 3 days) and aliquots were taken every three to four days for detection of mycoplasma by PCR. DNA from mycoplasma negative (−) and positive (+) cells was served as control (Ctr).

Figure 2

Figure 2. Chronic mycoplasma infection inhibits THP-1 cells response to PAMPs.

THP-1 cells, mycoplasma negative (−), positive (+) or treated with Plasmocin and confirmed to be a mycoplasma negative (P) were stimulated either with E. coli LPS or F. novicida (F.n.) for 15 hours in antibiotic-free media. Gene expression was measured in relative copy numbers (RCN) and cytokine concentration in ng/ml. Mycoplasma positive cells show decrease in expression of IL-1β message (A) and mature cytokine release (B), as well as IL-8 mRNA expression (C) and protein release (D). Mycoplasma removal restores cellular function to these PAMPs (P). Data are expressed as mean ± SEM. n = 4 for qPCR of THP-1 treated with LPS and n = 8 for all other samples.

Figure 3

Figure 3. Gene expression in mycoplasma negative and positive THP-1 cells stimulated with LPS or F. novicida.

Mycoplasma negative (Myco −) and positive (Myco +) THP-1 cells were stimulated either with E. coli LPS or F. novicida (F.n.) for 15 hours in antibiotic-free media. Gene expression was measured in relative copy numbers (RCN). Data are expressed as mean ± SEM. n = 6. Asterisk (*) represents a p value<0.05 in comparison between two groups, to show significance of mycoplasma contamination.

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