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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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Plasmacytoid dendritic cells produce IFN-α and IL-6 in response to Mycoplasma hyorhinis-infected B16 cells.Bone marrow-derived plasmacytoid DCs were cultured in the presence of decreasing concentrations of lysates derived from Mycoplasma hyorhinis-infected B16 cells (A, C) or different TLR stimuli (B, D). In some conditions, chloroquine was added to the pDC cultures. After 18 h, IL-6 and IFN-α levels in the pDC culture supernatant were analyzed by ELISA. Data give the mean + S.E.M. of triplicate samples and are representative of at least two independent experiments. Asterisks indicate statistically significant differences to the appropriate wild-type or untreated conditions.
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pone.0142523.g004: Plasmacytoid dendritic cells produce IFN-α and IL-6 in response to Mycoplasma hyorhinis-infected B16 cells.Bone marrow-derived plasmacytoid DCs were cultured in the presence of decreasing concentrations of lysates derived from Mycoplasma hyorhinis-infected B16 cells (A, C) or different TLR stimuli (B, D). In some conditions, chloroquine was added to the pDC cultures. After 18 h, IL-6 and IFN-α levels in the pDC culture supernatant were analyzed by ELISA. Data give the mean + S.E.M. of triplicate samples and are representative of at least two independent experiments. Asterisks indicate statistically significant differences to the appropriate wild-type or untreated conditions.

Mentions: Dendritic cells can be divided into several subpopulations. In contrast to conventional DCs (cDCs), that represent the main population in GM-CSF DC cultures, plasmacytoid DCs (pDC) morphologically resemble plasma cells and their function strongly depends on endosomal pathogen recognition via TLR7 and TLR9 [24]. To elucidate the role of pDCs in innate immune activation by components of Mycoplasma-infected cell lines, we analyzed the triggered cytokine release from bone marrow-derived pDCs. Following incubation with cell lysates from Mycoplasma hyorhinis-infected B16 cells, pDCs showed production of IL-6 that was entirely blocked by chloroquine treatment (Fig 4A). In contrast, pDCs did not respond to stimulation with the synthetic lipoprotein TLR2 ligand FSL-1 (Fig 4B). These data suggest that the immunostimulatory component from Mycoplasma hyorhinis-infected cells is recognized by pDCs within their endosomal compartment and is, as with cDC, distinct from canonical TLR2-dependent Mycoplasma-associated pathogenic patterns.


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)

Plasmacytoid dendritic cells produce IFN-α and IL-6 in response to Mycoplasma hyorhinis-infected B16 cells.Bone marrow-derived plasmacytoid DCs were cultured in the presence of decreasing concentrations of lysates derived from Mycoplasma hyorhinis-infected B16 cells (A, C) or different TLR stimuli (B, D). In some conditions, chloroquine was added to the pDC cultures. After 18 h, IL-6 and IFN-α levels in the pDC culture supernatant were analyzed by ELISA. Data give the mean + S.E.M. of triplicate samples and are representative of at least two independent experiments. Asterisks indicate statistically significant differences to the appropriate wild-type or untreated conditions.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142523.g004: Plasmacytoid dendritic cells produce IFN-α and IL-6 in response to Mycoplasma hyorhinis-infected B16 cells.Bone marrow-derived plasmacytoid DCs were cultured in the presence of decreasing concentrations of lysates derived from Mycoplasma hyorhinis-infected B16 cells (A, C) or different TLR stimuli (B, D). In some conditions, chloroquine was added to the pDC cultures. After 18 h, IL-6 and IFN-α levels in the pDC culture supernatant were analyzed by ELISA. Data give the mean + S.E.M. of triplicate samples and are representative of at least two independent experiments. Asterisks indicate statistically significant differences to the appropriate wild-type or untreated conditions.
Mentions: Dendritic cells can be divided into several subpopulations. In contrast to conventional DCs (cDCs), that represent the main population in GM-CSF DC cultures, plasmacytoid DCs (pDC) morphologically resemble plasma cells and their function strongly depends on endosomal pathogen recognition via TLR7 and TLR9 [24]. To elucidate the role of pDCs in innate immune activation by components of Mycoplasma-infected cell lines, we analyzed the triggered cytokine release from bone marrow-derived pDCs. Following incubation with cell lysates from Mycoplasma hyorhinis-infected B16 cells, pDCs showed production of IL-6 that was entirely blocked by chloroquine treatment (Fig 4A). In contrast, pDCs did not respond to stimulation with the synthetic lipoprotein TLR2 ligand FSL-1 (Fig 4B). These data suggest that the immunostimulatory component from Mycoplasma hyorhinis-infected cells is recognized by pDCs within their endosomal compartment and is, as with cDC, distinct from canonical TLR2-dependent Mycoplasma-associated pathogenic patterns.

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