Mycoplasma Contamination Alters the Expression Profile of Pro-Inflammatory Cytokines by Human Innate Immune Cells

The impact of Mycoplasma contaminations on in vitro test systems has been extensively discussed and in vitro studies have demonstrated various effects on the physiology of the contaminated cell line. Thus far, little is known about the effect of biologically active substances, such as viruses produced in contaminated cell lines on primary immune cells. Therefore, the aim of the present study was to pinpoint the impact of Mycoplasma contamination on human innate immune cells. To address this, myeloid dendritic cells (mDC) and two types of macrophages (type M1 and M2) were in vitro differentiated from healthy donors and treated with different volumes of cell-culture supernatant from Mycoplasma-contaminated VeroE6 cells (SN(M)). Depending on the cell type, Mycoplasma contamination mediated a decrease in viability and the pre-activation of the immune cells. Surprisingly, small amounts of SN(M) were sufficient to induce the secretion of massive amounts of pro-inflammatory interleukin (IL)-6 and, to a lesser extent, tumor necrosis factor (TNF)-α and interleukin (IL)-10. Moreover, SN(M) co-stimulation of cells treated with a variety of toll-like receptor (TLR) ligands and different RNA-encoded viruses differentially altered their activation status, depending on the analyzed markers, cell types, and stimuli. Furthermore, SN(M) co-treatment strongly influenced the induced cytokine profile of mDC as well as M1 and M2 macrophages upon treatment with various stimuli, especially upon viral infection. In line with this, a batch of SARS-CoV-2 produced in Mycoplasma-contaminated VeroE6 cells induced high levels of IL-6 and TNF-α while treatment with the non-contaminated virus stock did not result in expression of cytokines. Our data indicate that Mycoplasma contamination has a major impact on the experimental outcome of infection experiments using human immune cells and therefore can result in misinterpretation of experimental data.