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Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

Heidegger S, Jarosch A, Schmickl M, Endres S, Bourquin C, Hotz C - PLoS ONE (2015)

Bottom Line: Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2.Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment.We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science Munich (CIPSM), Division of Clinical Pharmacology, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, 80336 Munich, Germany.

ABSTRACT
Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

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Innate immune activation by Mycoplasma hyorhinis-infected B16 cells is mediated by a protein factor.Bone marrow cells were cultured in the presence of cell lysates of Mycoplasma hyorhinis-infected B16 cells. Some cell lysates were pre-treated either with DNase, RNase or proteinase K. 18 h later, IL-6 levels in the bone marrow cell culture supernatant were analyzed by ELISA. Protein digestion was performed at 55°C. In one condition, B16 cell lysates were heated to 55°C in the absence of proteinase K. Data give the mean values of triplicate samples + S.E.M. and are representative of two independent experiments. Asterisks indicate statistically significant difference to the appropriate non-inhibitor-treated conditions. ND, not determined.
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pone.0142523.g003: Innate immune activation by Mycoplasma hyorhinis-infected B16 cells is mediated by a protein factor.Bone marrow cells were cultured in the presence of cell lysates of Mycoplasma hyorhinis-infected B16 cells. Some cell lysates were pre-treated either with DNase, RNase or proteinase K. 18 h later, IL-6 levels in the bone marrow cell culture supernatant were analyzed by ELISA. Protein digestion was performed at 55°C. In one condition, B16 cell lysates were heated to 55°C in the absence of proteinase K. Data give the mean values of triplicate samples + S.E.M. and are representative of two independent experiments. Asterisks indicate statistically significant difference to the appropriate non-inhibitor-treated conditions. ND, not determined.

Mentions: Endosomal TLRs are considered to detect mainly bacterial and viral nucleic acids. The ligation of Mycoplasma-derived lipoproteins to TLR2 and its heterodimers with either TLR1 or TLR6 has been shown to be critically dependent on the ligand’s lipid moiety since digestion with lipases but not proteases impaired the lipoproteins’ immunostimulatory capacity in human monocytic cells [23]. To determine the nature of the component responsible for immune cell activation by Mycoplasma hyorhinis-infected cells, B16 cell lysates were treated either with DNase I, RNase A or proteinase K before they were added to a bone marrow cell culture. Digestion of nucleic acids within the B16 lysates did not decrease the capacity of infected cells to induce IL-6 release from immune cells (Fig 3). In contrast, protein digestion with proteinase K at 55°C resulted in strong inhibition of immunostimulation by Mycoplasma-contaminated cell lysates. Proteinase-treated B16 lysates failed to induce IL-6 production in bone marrow cells as determined by ELISA from the culture supernatant (Fig 3). To rule out that a spillover of enzyme to the bone marrow cell culture was responsible for the decrease in IL-6 protein, LPS-stimulated bone marrow cells were additionally supplemented with proteinase K-treated mock supernatants, which did not result in a decrease of IL-6 secretion. To further substantiate that the ligand itself is degraded by protease treatment, IL-6 mRNA expression was determined by quantitative real-time PCR. We found that proteinase K treatment of contaminated B16 cell lysates resulted in strongly impaired upregulation of IL-6 mRNA levels in bone marrow cells (S4 Fig) in contrast to LPS (not shown). In the absence of proteinase K, heating to 55°C resulted in less pronounced reduction in immunostimulatory capacity of Mycoplasma-infected cells. In conclusion, these data demonstrate that Mycoplasma hyorhinis-infected cell lines harbor a factor for which the pro-inflammatory activity is dependent on a protein component.


Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

Heidegger S, Jarosch A, Schmickl M, Endres S, Bourquin C, Hotz C - PLoS ONE (2015)

Innate immune activation by Mycoplasma hyorhinis-infected B16 cells is mediated by a protein factor.Bone marrow cells were cultured in the presence of cell lysates of Mycoplasma hyorhinis-infected B16 cells. Some cell lysates were pre-treated either with DNase, RNase or proteinase K. 18 h later, IL-6 levels in the bone marrow cell culture supernatant were analyzed by ELISA. Protein digestion was performed at 55°C. In one condition, B16 cell lysates were heated to 55°C in the absence of proteinase K. Data give the mean values of triplicate samples + S.E.M. and are representative of two independent experiments. Asterisks indicate statistically significant difference to the appropriate non-inhibitor-treated conditions. ND, not determined.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643973&req=5

pone.0142523.g003: Innate immune activation by Mycoplasma hyorhinis-infected B16 cells is mediated by a protein factor.Bone marrow cells were cultured in the presence of cell lysates of Mycoplasma hyorhinis-infected B16 cells. Some cell lysates were pre-treated either with DNase, RNase or proteinase K. 18 h later, IL-6 levels in the bone marrow cell culture supernatant were analyzed by ELISA. Protein digestion was performed at 55°C. In one condition, B16 cell lysates were heated to 55°C in the absence of proteinase K. Data give the mean values of triplicate samples + S.E.M. and are representative of two independent experiments. Asterisks indicate statistically significant difference to the appropriate non-inhibitor-treated conditions. ND, not determined.
Mentions: Endosomal TLRs are considered to detect mainly bacterial and viral nucleic acids. The ligation of Mycoplasma-derived lipoproteins to TLR2 and its heterodimers with either TLR1 or TLR6 has been shown to be critically dependent on the ligand’s lipid moiety since digestion with lipases but not proteases impaired the lipoproteins’ immunostimulatory capacity in human monocytic cells [23]. To determine the nature of the component responsible for immune cell activation by Mycoplasma hyorhinis-infected cells, B16 cell lysates were treated either with DNase I, RNase A or proteinase K before they were added to a bone marrow cell culture. Digestion of nucleic acids within the B16 lysates did not decrease the capacity of infected cells to induce IL-6 release from immune cells (Fig 3). In contrast, protein digestion with proteinase K at 55°C resulted in strong inhibition of immunostimulation by Mycoplasma-contaminated cell lysates. Proteinase-treated B16 lysates failed to induce IL-6 production in bone marrow cells as determined by ELISA from the culture supernatant (Fig 3). To rule out that a spillover of enzyme to the bone marrow cell culture was responsible for the decrease in IL-6 protein, LPS-stimulated bone marrow cells were additionally supplemented with proteinase K-treated mock supernatants, which did not result in a decrease of IL-6 secretion. To further substantiate that the ligand itself is degraded by protease treatment, IL-6 mRNA expression was determined by quantitative real-time PCR. We found that proteinase K treatment of contaminated B16 cell lysates resulted in strongly impaired upregulation of IL-6 mRNA levels in bone marrow cells (S4 Fig) in contrast to LPS (not shown). In the absence of proteinase K, heating to 55°C resulted in less pronounced reduction in immunostimulatory capacity of Mycoplasma-infected cells. In conclusion, these data demonstrate that Mycoplasma hyorhinis-infected cell lines harbor a factor for which the pro-inflammatory activity is dependent on a protein component.

Bottom Line: Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2.Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment.We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science Munich (CIPSM), Division of Clinical Pharmacology, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, 80336 Munich, Germany.

ABSTRACT
Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

Show MeSH
Related in: MedlinePlus