The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation.

The clear and unequivocal identification of immune effector functions is essential to understand immune responses. The cytokine IL-10 is a critical immune regulator and was shown, for example, to limit pathology during various lung diseases. However, the clear identification of IL-10-producing cells is challenging and, therefore, reporter mouse lines were developed to facilitate their detection. Several such reporter lines utilize GFP, including the IL-10GFP (VeRT-X) reporter strain studied here. In line with previous reports, we found that this IL-10GFP line faithfully reports on the IL-10 production of lymphoid cells. However, we show that the IL-10GFP reporter is not suitable to analyse IL-10 production of myeloid cells during inflammation. During inflammation, the autofluorescence of myeloid cells increased to an extent that entirely masked the IL-10-specific GFP-signal. Our data illustrate a general and important technical caveat using GFP-reporter lines for the analysis of myeloid cells and suggest that previous reports on effector functions of myeloid cells using such GFP-based reporters might require re-evaluation.

Introduction

Regulatory cytokines are important players during pulmonary inflammation to limit immunopathology without hampering effective pathogen clearance [1]. The regulatory cytokine IL-10 is involved in various lung diseases, like asthma, allergic airway disease (AAD), chronic obstructive pulmonary disease (COPD), and pulmonary infections [1]. IL-10 producing cells in the lung; FoxP3+ and FoxP3- regulatory T cells [1], CD8+ T cells [2], B cells, alveolar macrophages (AM) [3], interstitial macrophages (IM) [4], airway and alveolar epithelial cells [1], and airway-associated dendritic cells (DCs) [1] were shown to limit disease pathology. However, due to the low percentage of IL-10+ cells within most cell populations and due to the low intensity of the flow cytometric staining of intracellular IL-10 the clear identification of IL-10 producing cells is challenging [5]. Therefore, various reporter mouse lines were developed to avoid the need for intracellular cytokine staining to identify IL-10 producing cells [6]. The IL-10GFP (VeRT-X) strain studied here, expresses an (IRES)-enhanced green fluorescent protein (eGFP) fusion protein downstream of the exon 5 of the il10 gene [7], and was reported to enable the identification of IL-10+ lymphoid but also myeloid cells [7]. Several myeloid cells possess an autofluorescence around 525 nm [8], which coincides with the emission maxima of GFP around 530 nm [9]. However, the impact of this myeloid autofluorescence on the detection of GFP-reporter signals has not been fully clarified. Here, using an LPS-induced lung inflammation model, we demonstrate that the autofluorescence of myeloid cells conceals the IL10GFP-specific signal. This was mainly due to a large increase in the myeloid autofluorescence during the inflammation. These data demonstrate that not all GFP-reporter mouse strains are suitable to analyse effector functions of myeloid cells during inflammation.

Results and discussion

Neutrophils were suggested to be the primary source of IL-10 in the lung following Klebsiella pneumoniae ST258 infection [10]. Furthermore, long-term exposure to house-dust mite (HDM) extracts suppressed allergic airway diseases via the IL-10 production by presumably FoxP3+ T cells and alveolar macrophages [3]. In both studies, the ‘shift’ in the GFP-signal in the experimental IL-10GFP group was compared to either the uninfected IL-10GFP group or the naïve C57BL/6 group as control. When we analysed the IL-10 production during lung inflammation, we noticed that the background signal in the GFP-channel (505 nm—550 nm) of leukocytes from the lung, spleen, or mediastinal lymph node (mLN) was comparable in non-inflamed controls of either C57BL/6 (WT) or IL-10GFP mice (). Upon LPS-induced lung inflammation, the GFP-signal clearly increased, in particular in the lungs (). Nevertheless, the GFP-signal from WT and IL-10GFP mouse-derived cells remained comparable for the percentage () or the mean fluorescence intensity (MFI, ), irrespective of the organ analysed and the inflammatory condition. These data suggest that the GFP-signal detected in the IL-10GFP—derived cells was actually a background signal.

The myeloid-derived GFP signal is comparable between IL10GFP and C57BL/6 mice and increases during lung inflammation.

C57BL/6 and IL-10GFP mice were challenged three times (d0, d1, d2) with either PBS or 10 μg LPS per mouse via aspiration. 16–18 hours after the last administration, the single cell suspension from lungs, spleens, and mLNs were stained for CD4+ T cells (CD45+ CD45R/B220- CD3ε+ CD4+ CD8α-), CD8+ T cells (CD45+ CD45R/B220- CD3ε+ CD4- CD8α+), macrophages (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80+), Siglec-F+ cells (eosinophils, alveolar macrophages; CD45+ CD45R/B220- CD3ε- Siglec-F+ F4/80-/+), neutrophils (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G+ CD11b+), monocytes (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- Ly6C+ CD11b+), and ILCs (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- CD90.2+ CD127lo/-). The expression of IL-10 was measured either by GFP+ or by intracellular IL-10 as indicated. (A, B) The frequency of GFP+ cells (live CD45+) in the lungs of (A) PBS or (B) LPS challenged C57BL/6 (red) and IL-10GFP (green) mice are shown. The graph shows combined data from three independent experiments (PBS: n = 9 mice/group, LPS: n = 13–15 mice/group in total). (C) Representative histogram showing the shift in the GFP-signal (505 nm—550 nm) of live CD45+ lung cells upon LPS administration in C57BL/6 and IL-10GFP mice strains.

To clarify which cell types could produce IL-10, we directly compared the IL-10 signal derived from the GFP-signal or from intracellular cytokine staining (ICCS) for CD4+ and CD8+ T cells, neutrophils, macrophages, Siglec-F+ cells (eosinophils, alveolar macrophages), monocytes, and ILCs. Cells from the lungs, spleens, and mLN of PBS (control) and LPS-challenged C57BL/6 and IL-10GFP mice were analysed (). Although intracellular IL-10 staining is challenging, we obtained a clear IL-10 signal with the commercial antibody (). With this side-by-side comparison, we found that the IL-10-signal derived from GFP or ICCS correlated well for CD4+ () and CD8+ T cells (). These data indicate that the IL-10GFP—signal faithfully reports on the IL-10 production of lymphoid cells. However, the GFP-signal from neutrophils (), macrophages (), and Siglec-F+ () cells was significantly higher than the ICCS-derived signal. Representative dot-plots for all cell types and organs are provided in . Furthermore, the additional staining with a secondary αGFP-AF488-antibody was not able to improve the specificity of the IL-10-signal (). Interestingly, the GFP-signal of monocytes () and ILCs () was substantially higher only in the lung tissues, indicating that the increase in the GFP-channel autofluorescence is organ-specific for some cell types.

The myeloid-derived IL-10GFP signal is masked by autofluorescence in granulocytes.

C57BL/6 and IL-10GFP mice were challenged three times (d0, d1, d2) with either PBS or 10 μg LPS per mouse via aspiration. 16–18 hours after the last administration, the single cell suspension from lungs, spleens, and mLNs were stained for CD4+ T cells (CD45+ CD45R/B220- CD3ε+ CD4+ CD8α-), CD8+ T cells (CD45+ CD45R/B220- CD3ε+ CD4- CD8α+), macrophages (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80+), Siglec-F+ cells (eosinophils, alveolar macrophages; CD45+ CD45R/B220- CD3ε- Siglec-F+ F4/80-/+), neutrophils (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G+ CD11b+), monocytes (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- Ly6C+ CD11b+), and ILCs (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- CD90.2+ CD127lo/-). The expression of IL-10 was measured either by GFP+ or by intracellular IL-10 as indicated. (A-G) Comparison of the IL-10 signal derived from intracellular cytokine staining (ICCS) or from the GFP-signal (IL10GFP) of IL10GFP mice in (D) CD4+ T cells, (E) CD8+ T cells, (F) neutrophils, (G) macrophages, (H) Siglec-F+ cells (eosinophils, alveolar macrophages), (I) monocytes, and (J) ILCs from indicated organs. The graph shows combined data from two independent experiments (PBS: n = 6 mice/group, LPS: n = 7–9 mice/group in total).

An overlay of the GFP-signals of CD4+ T cells, CD8+ T cells, neutrophils, macrophages, Siglec-F+ cells, monocytes, and ILCs from control C57BL/6 lung () and spleen () indicated that the background GFP-signal was mainly derived from granulocytes. According to the background GFP-signal, cell types ranked as Siglec-F > macrophage > neutrophil > monocyte > ILC. A similar increase in the GFP-channel autofluorescence upon inflammation was noted when alveolar macrophages were analysed ().

The cell-type-specific autofluorescence in C57BL/6 mice in control conditions.

Representative histograms from the (A) lung and the (B) spleen of PBS challenged C57BL/6 mice, demonstrating the increase in autofluorescence in the GFP-channel for myeloid cells. The graphs show representative data from three independent experiments (PBS: n = 9 mice/group, LPS: n = 13–15 mice/group in total).

The autofluorescence of alveolar macrophages increases during lung inflammation.

C57BL/6 and IL-10GFP mice were subjected to three daily administrations (d0, d1, d2) of 10 μg LPS per mouse via aspiration. 16–18 hours after the last administration, the single-cell suspensions from the lungs were analysed. Representative histogram showing the GFP-signal from alveolar macrophages (CD45+ CD45R/B220- CD3ε- Siglec-F+ F4/80+ CD11b-) from C57BL/6 and IL10GFP mice as indicated. The data shown are representative of three independent experiments (PBS: n = 9 mice/group, LPS: n = 13–15 mice/group in total).

In conclusion, our data indicate that the IL-10GFP (VeRT-X) reporter strain is not suitable to analyse IL-10 production of myeloid cells during inflammation, due to the strong increase in the autofluorescence, which masks the IL-10-specific GFP-signal. Although we only analysed lung, spleen, and mLN of the VeRT-X IL-10GFP reporter strain, it appears likely that this problem will also be relevant to other organs. Importantly, the strong increase of the granulocytic autofluorescence in the GFP-channels was independent of the strain analysed. Therefore, it is expected that other fluorescent reporter lines that utilize the GFP-channel would face similar problems at distinguishing the reporter-specific signal from the autofluorescence signal when analysing granulocytes. Our data indicate that granulocytes are the source of the background GFP-signal, in line with previous publications reporting high levels of autofluorescence [11], although other sources, like collagen deposition [12,13], cannot be excluded. This large increase in false-positive GFP-signals for myeloid cells during inflammation could have been missed previously due to a focus on lymphoid cells or due to a lack of the WT-treated controls. Our data illustrate a general and important technical caveat using GFP-reporter lines for the analysis of myeloid cells and suggest that previous reports on effector functions of myeloid cells using such GFP-based reporters might require re-evaluation.

Material and methods

Mice

C57BL/6J mice and IL-10GFP (also known as Vert-X) B6(Cg)-Il10tm1.1Karp/J mice were originally obtained from Jackson Laboratory (Bar Harbor, USA). All mice were housed in the vivarium of the Izmir Biomedicine and Genome Center (IBG, Izmir, Turkey) in accordance with the respective institutional animal care committee guidelines. All mouse experiments were performed with prior approval by the institutional ethic committee (‘Izmir Biomedicine and Genome Center’s Ethical Committee on Animal Experimentation’), in accordance with national laws and policies. All the methods were carried out in accordance with the approved guidelines and regulations.

Reagents and monoclonal antibodies

Monoclonal antibodies against the following mouse antigens were used in this study: CD3ε (145.2C11), CD4 (RM4-5), CD8α (53–6.7), CD11b (M1/70), CD11c (N418), CD45 (30-F11), CD45R/B220 (RA3-6B2), CD90.2 (30-H12), CD127 (A7R34), CD170/Siglec-F (E50-2440), F4/80 (BM8), IL-10 (JESS-16E3), Ly6C (HK1.4), Ly6G (1A8). Antibodies were purchased from BD Biosciences (San Diego, USA), BioLegend (San Diego, USA), or ThermoFisher Scientific (Carlsbad, USA). Antibodies were conjugated to Pacific Blue, Brilliant Violet 421, V500, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 785, Brilliant Violet 786, FITC, Alexa Fluor 488, PerCP-Cy5.5, PerCP-eFluor 710, PE, PE-CF594, PE-Cy7, APC, Alexa Fluor 647, Alexa Fluor 700, APC-Cy7, or APC-Fire750. Details on the antibody used in this study are given in the . Anti-mouse CD16/32 antibody (2.4G2) and Zombie UV Dead Cell Staining Kit were obtained from Tonbo Biosciences (San Diego, USA) and from BioLegend, respectively. Unconjugated mouse and rat IgG antibodies were purchased from Jackson ImmunoResearch (West Grove, USA).

LPS-induced lung inflammation

Lung inflammation was induced by three daily administrations (d0, d1, d2) with 10 μg LPS (#L6386, Sigma-Aldrich, St. Louis, USA) per mouse via pharyngeal/laryngeal aspiration. 16–18 hours after the last challenge, the mice were sacrificed, and lungs, spleens, and mediastinal lymph nodes were collected.

Cell preparation

Lungs were removed and minced into smaller pieces in a 6-well plate (Greiner, Germany). The digestion mixture, composed of 1 mg/mL collagenase D and 0.1 mg/mL DNase I (both from Roche, Switzerland) in complete RPMI medium (Gibco, USA), was added to the samples and incubated for 45 min at 37°C on a lateral shaker. The lung samples were filtered through 100 μm mesh with PBS, washed twice, and the red blood cells were eliminated by ACK lysis buffer (Lonza, USA). Spleens and mediastinal lymph nodes were homogenized by filtering through a 76 μm mesh with ice-cold PBS (Lonza), washed twice, and red blood cells were eliminated by ACK lysis buffer (Lonza).

In vitro stimulation

Lymphocytes were stimulated in vitro with PMA (50 ng/mL) and ionomycin (1 μg/mL) (both Sigma-Aldrich, St. Louis, MO) for four hours at 37°C in the presence of both Brefeldin A (GolgiPlug) and Monensin (GolgiStop) in complete RPMI medium (RPMI 1640 medium (Life Technologies); supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) Pen-Strep-Glutamine (10.000 U/ml penicillin, 10.000 μg/ml streptomycin, 29.2 mg/ml L-glutamine (Life Technologies)) and 50 μM β-mercaptoethanol (Sigma)). As GolgiPlug and GolgiStop (both BD Biosciences) were used together, half the amount recommended by the manufacturer were used.

Flow cytometry

Flow cytometry was performed as described [14]. In brief, for staining of cell surface molecules, cells were suspended in staining buffer (PBS, 1% BSA, 0.01% NaN3) and stained with fluorochrome-conjugated antibodies (0.1–1 μg/106 cells, or according to the manufacturer’s recommendations) for 15 min in a total volume of 50 μl at 4°C for 30 min. FcεR-blocking antibody αCD16/32 (2.4G2) and unconjugated rat and mouse IgG (Jackson ImmunoResearch) were added to prevent non-specific binding. If biotin-conjugated antibodies were used, cell-bound antibodies were detected with streptavidin conjugates (1:200, or according to the manufacturer’s recommendations) in a second incubation step. Dead cells were labelled with a commercially available Zombie UV Dead Cell Staining Kit (BioLegend). For the analysis of intracellular cytokines, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 10 min at 37°C. Cells were washed twice and incubated overnight at 4°C with the fluorochrome-conjugated antibodies and unconjugated rat and mouse IgG in Perm/Wash solution (BD Biosciences), which was followed by additional 5 min incubation in Perm/Wash solution without antibodies. For the analysis of the GFP signal, the cells were fixed with freshly prepared 2% formaldehyde solution for 40 minutes on ice as described [15]. This fixation allows the detection of the GFP-signal together with other intracellular proteins [15]. Then, the cells were permeabilized using the BD Perm/Wash solution. Cells were washed twice and incubated overnight at 4°C with the fluorochrome-conjugated antibodies and unconjugated rat and mouse IgG in Perm/Wash solution, which was followed by additional 5 min incubation in Perm/Wash solution without antibodies. Cells were analysed with FACSCanto or LSR-Fortessa (BD Biosciences), and data were processed with CellQuest Pro (BD Biosciences) or Flow Jo (Tree Star) software.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). The statistical analysis was performed with GraphPad Prism 7.0 software (GraphPad Software, San Diego, USA). One-way ANOVA followed by Holm-Sidak posthoc test are used to compare p-values regarded as *p≤0.05, **p≤0.01, and ***p≤0.001.

Gating strategy to identify leukocytes from the lung, spleen, and mLN.

A graphic outline (A) and exemplary graphs (B) are given to illustrate the gating strategy employed to identify CD4+ T cells (CD45+ CD45R/B220- CD3ε+ CD4+ CD8α- CD127lo), CD8+ T cells (CD45+ CD45R/B220- CD3ε+ CD4- CD8α+), macrophages (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80+), Siglec-F+ cells (eosinophils, alveolar macrophages; CD45+ CD45R/B220- CD3ε- Siglec-F+ F4/80-/+), neutrophils (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G+ CD11b+), monocytes (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- Ly6C+ CD11b+), and ILCs in the inflamed lung.

(TIF)

Click here for additional data file.

Efficiency of intracellular IL-10 cytokine staining.

Representative flow cytometric dot plots showing the IL-10 staining (ICCS) in splenic CD4+ T cells.

(TIF)

Click here for additional data file.

Representative flow cytometry plots for the data presented in Fig 2.

C57BL/6 and IL-10GFP mice were challenged three times (d0, d1, d2) with either PBS or 10 μg LPS per mouse via aspiration. 16–18 hours after the last administration, the single cell suspension from (A) lungs, (B) spleens, and (C) mLNs were stained for CD4+ T cells (CD45+ CD45R/B220- CD3ε+ CD4+ CD8α-), CD8+ T cells (CD45+ CD45R/B220- CD3ε+ CD4- CD8α+), macrophages (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80+), Siglec-F+ cells (eosinophils, alveolar macrophages; CD45+ CD45R/B220- CD3ε- Siglec-F+ F4/80-/+), neutrophils (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G+ CD11b+), monocytes (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- Ly6C+ CD11b+), and ILCs (CD45+ CD45R/B220- CD3ε- Siglec-F- F4/80- Ly6G- CD90.2+ CD127lo/-). The expression of IL-10 was measured either by intracellular IL-10 staining (left panel) or GFP—expression (right panel).

(TIF)

Click here for additional data file.

The staining with a secondary αGFP-AF488 antibody does not improve the resolution of the myeloid IL10GFP signal.

Representative histograms from the spleen of PBS (left) or LPS (right) challenged C57BL/6 and IL-10GFP mouse, demonstrating the overall GFP-signal detected with or without labelling with secondary αGFP-AF488.

(TIF)

Click here for additional data file.

(TIF)

Click here for additional data file.

26 Mar 2021

PONE-D-21-05124

The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation

PLOS ONE

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Reviewer #1: Ozkan et al describe a potential caveat of utilising IL-10 reporter mice (VeRT-X) to identify myeloid-derived IL-10 production during an experimental model of LPS-induced lung inflammation.

The manuscript is well written, and the data is presented in a concise manner. The following comments are suggestions which I believe may add clarity to the overall message of the manuscript.

Major comments:

1. Can the authors comment on how the VeRT-X IL-10 reporter line and their results compare to any of the other eight IL-10 reporter lines, described by Bouabe 2012 (https://doi.org/10.1111/j.1365-3083.2012.02695.x)?

2. The original paper describing the VeRT-X mice (Madan et al., 2009) shows IL-10 GFP expression by B cells. Have the authors measured B cell derived IL-10 GFP levels in their model of LPS-induced lung inflammation?

3. It would be helpful to provide representative FACS plots as part of Supplementary Figure 1, to complement the gating strategy.

4. Figure 2 nicely illustrates IL-10 detection by ICCS vs GFP. It would be helpful to show representative FACS plots as part of this figure or as a Supplementary figure.

5. The figure legend for Figure 2 states: “The graph shows combined data from two independent experiments (PBS: n = 6 mice/group. LPS: n=7-9 mice/group)”. Can the authors confirm whether 6-9 mice/group were used in each individual experiment? Or whether the 6-9mice/group refers to the pooled data shown in Figure 2?

Minor comments:

Line 31: please remove ‘s’ from inflammations

Line 49: please change where to were

Line 54: please insert reference 7 after myeloid cells.

Line 54: please check reference 8, since the autofluorescence of myeloid cells was not mentioned in this reference.

Line 90-91: please clarify why data is not shown from WT controls. It might be informative to show this data as a supplementary figure.

Line 103: please remove ‘s’ from inflammations

Line 108: please remove ‘also’

Line 110: please change ‘analysis’ to analysing

Line 114: please remove ‘s’ from inflammations

Line 145: please add ‘d’ to purchase

Line 318: please superscript IL10GFP

Line 321 – 322: please superscript CD45+ CD45R/B220- CD3- Siglec-F+ F4/80+ CD11b- and IL10GFP

Reviewer #2: In this paper, the authors have proposed that IL-10 GFP reporter strain is not an appropriate model to detect IL-10 production by granulocytes. Using C57BL/6 and IL-10 GFP mice and by employing flowcytometry, the authors show that GFP signal seen in granulocytes post LPS challenge is not the actual GFP signal due to IL-10 production. Rather, it is attributable to autofluorescence. I think the authors have used proper controls in the study and results from the study illustrates important technical caveat in using GFP reporter mice for analysing myeloid cells. Overall, the study is good. However, I have few minor suggestions:

1. Line 31: Please change " during inflammations" to during inflammation. Please make this change elsewhere as well.

2. Line 39-40: Please rephrase this sentence as the meaning is not clear.

3. Line 49: Please change "lines where" to lines were.

4. Line 54: Please give reference when you discuss that IL-10 GFP strain was reported to enable identification of IL-10+

myeloid cells.

5. Line 110: Please change"analysis" to analysing.

6. Line 170: Change "where" to were.

7. Line 277-279 and 295-296: Please rephrase these two sentences as they seem incomplete.

8. Line 337-339: I think this sentence " C) Representative histograms from the spleen of PBS-------" is not required as it is a repetition of Supplementary Figure 3 legend.

9. Please check the overall grammar and sentence structure throughout the text.

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Reviewer #1: No

Reviewer #2: No

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1 Apr 2021

Please see the point-to-point reply for details.

Submitted filename: Point-to-point response_Ozkan et al.pdf

Click here for additional data file.

27 Apr 2021

The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation

PONE-D-21-05124R1

Dear Dr. Wingender,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

3 May 2021

PONE-D-21-05124R1

The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation

Dear Dr. Wingender:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Academic Editor

PLOS ONE