THP-1 NF-κB-eGFP reporter cells are highly sensitive towards mycoplasma lipoproteins through engagement of TLR2/6.

<p>(A) THP-1 reporter cells pre-treated with a blocking TLR6 antibody or isotype control (30 min; both used at 5 μg/mL) were incubated with the indicated concentrations of the TLR2/6 ligands FSL-1 and MALP-2 or supernatants derived from mycoplasma infected cell cultures for 24 h. Standard LPS served as a negative control. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from triplicates of three independently performed experiments (n = 3). (B) Fluorescent microscopy images of THP-1 reporter cells stimulated for 24 h with different dilutions of supernatants derived from mycoplasma infected cell cultures. Unstimulated cells served as negative control (left panel). Bright field images are shown for comparison (top row). Scale bar: 10 μm. (C) THP-1 NF-κB-eGFP reporter cells and a commercially available mycoplasma detection kit (MycoAlert) were probed with tissue culture supernatants from different cell sources and species: (1) mouse tail cells, (2) human mesotheliom, (3) human melanoma brain metastasis-derived cell line YDFR, (4) human LN229 glioblastoma, (5) human ovarian cancer cells, (6) COS-7 cell line and (7) human skin fibroblasts. Samples above red line are scored as positive. For the Mycoalert detection system the B/A ratio represents the ratio of the luminescence signals measured at two different time points (reading A and B). For THP-1 reporter assay mean and SE were calculated from duplicates. (D) K562 cells were infected with mycoplasma and then subjected to treatment regimens using commercially available mycoplasma removal agents (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178220#sec002" target="_blank">Material and Methods</a>). Supernatants were collected at various time points throughout the treatment course (day 2, 4, 7, 10, 15, 17, 21, 23, 25 and 29) and tested with the THP-1 NF-κB-eGFP reporter cells (upper panel). Indicated samples were also tested using the MycoAlert kit (lower panel).</p

A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

<div><p>Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an <i>E</i>. <i>coli</i> strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.</p></div

THP-1 NF-κB-eGFP reporter cells show a selective sensitivity towards TLR ligands.

<p>(A) THP-1 NF-κB-eGFP cells were incubated with Pam3CSK4 (TLR1/2; 100 nM), FSL-1 (TLR2/6; 100 nM), MALP-2 (TLR2/6; 46,8 nM), Poly I:C (TLR3; 10 μg/ml), standard LPS (TLR2/4; 300 ng/ml), LPS ultrapure (UP) (TLR4; 300ng/ml), flagellin (TLR5; 100 ng/ml), imidazoquinoline (TLR7/8; 10 μg/ml) and CpG ODN 2006 (TLR9; 50 μg/ml). After 24 h, eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from triplicates of five independently performed experiments (n = 5). (B) Representative flow cytometry histograms of reporter gene expression in THP-1 NF-κB-eGFP cells as described in A. Open histograms: TLR-activated reporter cells; filled histograms: unstimulated reporter cells. Numbers show gMFI. (C) Fluorescent microscopy images of reporter cells activated with standard LPS (3 μg/ml) for 24 h (right panel). Unstimulated cells served as negative control (left panel). Bright field images are shown for comparison (top row). Scale bar: 10 μm. (D) Immature human monocyte-derived DCs were incubated with various TLR ligands (used at the same concentrations as in A) for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. Bar graphs show total percentage of gMFI normalized to standard LPS.</p

THP-1 NF-κB-eGFP reporters can detect the presence of mycoplasma in heat-denatured and cryo-preserved samples.

<p>THP-1 reporter cells were incubated with fresh (left panel), frozen (middle panel) or 95°C heat-inactivated (right panel) mycoplasma-containing tissue culture supernatants used at the indicated dilutions. NF-κB-driven eGFP expression was measured at day 1, 2, 3, 4 and 6 after the onset of the assay by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from duplicates.</p

Dose-dependent response of THP-1 NF-κB-eGFP reporter cells towards specific TLR ligands.

<p>(A-E) THP-1 NF-κB-eGFP cells were incubated with increasing concentrations of Pam3CSK4, FSL-1, Flagellin, standard LPS and MALP-2 as indicated. Untreated cells served as control. After 24 h, induction of NF-κB-driven eGFP was measured by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI, top panels). Mean and SE were calculated from triplicates of three independently performed experiments (n = 3). Flow cytometry histograms of a representative experiment are shown for comparison (bottom panels). Open histograms: control cells; filled histograms: TLR-activated reporter cells.</p

T cell responses to bGST and Bet v 1 in BALB/c mice.

<p>(A) Proliferative and cytokine responses of splenocytes from mice immunized with bGST (n = 5) to titrated amounts of bGST, BPE (25 µg/mL) and Bet v 1 (5 µg/mL). Cytokines were induced with 1.25 µg/mL of bGST. (B) Proliferative and cytokine responses of splenocytes from mice immunized with Bet v 1 (n = 5) to Bet v 1, BPE (25 µg/mL) and bGST (1.25 µg/mL). Cytokines were induced with 5 µg/mL of Bet v 1. Δcpm, cpm of unstimulated cultures were subtracted from stimulated cultures (mean background cpm = 4724±742); Δpg/mL, levels in unstimulated cultures were subtracted from stimulated cultures (mean pg/mL of IFN-γ were <135 pg/ml and of remaining cytokines <20 pg/ml).</p

Purity and identity of bGST.

<p>(A) Coomassie-stained SDS-PAGE showing bGST before (lanes 1 and 2) and after Ni²⁺ affinity-chromatography (lane 3). (B) Circular dichroism spectroscopy of bGST. Far UV-measurements of bGST were recorded at 20°C (solid line), 95°C (dashed line) and 20°C after renaturation (dotted line). The mean residue ellipticities (⊖) are shown at given wavelengths. (C) Natural bGST was excised from a Coomassie-stained SDS-PAGE of BPE and analysed by mass spectrometry. Bold characters show aa residues of natural bGST in the sequence of recombinant bGST.</p

No cross-reactivity between bGST and HDM-GST.

<p>(A) IgG1-reactivity of sera (n = 3) collected from bGST-immunized mice at day 0 and day 59 to BPE and HDME. (B) IgE-reactivity of seven Der p 8-sensitized HDM-allergic patients to bGST and HDME. NHS, non-allergic control sera; O.D. optical density; dotted and dashed lines indicate the cut-off for positive IgE-reactivity.</p

IgE-responses of BP-allergic individuals.

<p>(A) IgE-reactivity of 217 BP-allergic patients to BP allergens and bGST. (B) IgE-reactivity to bGST is inhibited by pre-incubation of sera with BPE (gray line) and bGST (black line). One representative example of four is shown. (C) RBL assay with sera from three patients sensitized to bGST and Bet v 1. Degranulation was induced with titrated amounts of bGST (filled circles) and Bet v 1 (open circles). The release of β-hexosaminidase is expressed as percentage of the release from anti-IgE-treated cells.</p

Ab responses to bGST and Bet v 1 in BALB/c mice.

<p>(A) bGST-specific Ig of mice immunized with bGST (n = 5). (B) Bet v 1-specific Ig of mice immunized with Bet v 1 (n = 5). (C) IgG1 responses to BPE from mice immunized with either bGST or Bet v 1. O.D.; optical density, d, day; mean values+standard deviation are shown.</p