Tracking of Inhaled Near-Infrared Fluorescent Nanoparticles in Lungs of SKH‑1 Mice with Allergic Airway Inflammation

Molecular imaging of inflammatory lung diseases, such as asthma, has been limited to date. The recruitment of innate immune cells to the airways is central to the inflammation process. This study exploits these cells for imaging purposes within the lung, using inhaled polystyrene nanoparticles loaded with the near-infrared fluorescence dye Itrybe (Itrybe-NPs). By means of <i>in vivo</i> and <i>ex vivo</i> fluorescence reflectance imaging of an ovalbumin-based allergic airway inflammation (AAI) model in hairless SKH-1 mice, we show that subsequent to intranasal application of Itrybe-NPs, AAI lungs display fluorescence intensities significantly higher than those in lungs of control mice for at least 24 h. <i>Ex vivo</i> immunofluorescence analysis of lung tissue demonstrates the uptake of Itrybe-NPs predominantly by CD68⁺CD11c⁺ECF-L⁺MHCII^low cells, identifying them as alveolar M2 macrophages in the peribronchial and alveolar areas. The <i>in vivo</i> results were validated by confocal microscopy, overlapping tile analysis, and flow cytometry, showing an amount of Itrybe-NP-containing macrophages in lungs of AAI mice significantly larger than that in controls. A small percentage of NP-containing cells were identified as dendritic cells. Flow cytometry of tracheobronchial lymph nodes showed that Itrybe-NPs were negligible in lung draining lymph nodes 24 h after inhalation. This imaging approach may advance preclinical monitoring of AAI <i>in vivo</i> over time and aid the investigation of the role that macrophages play during lung inflammation. Furthermore, it allows for tracking of inhaled nanoparticles and can hence be utilized for studies of the fate of potential new nanotherapeutics

The hiPSC lines showed pluripotent characteristics.

<p>(A) The three hiPSC lines with typical morphology for human pluripotent stem cells expressed alkaline phosphatase. Scale bars: 100 μm. (B) The three hiPSC lines expressed endogenous pluripotency markers <i>OCT4</i>, <i>NANOG</i>, <i>LIN28</i>, and <i>SOX2</i> at the mRNA level as shown by RT-PCR. <i>GAPDH</i> is included as loading control. HES3 cells and MEFs were included as positive and negative control, respectively. (C) Immunofluorescence staining of the hiPSC lines with Abs against pluripotency markers NANOG, OCT4, SSEA4, and TRA1-60. The cells were counterstained with DAPI (blue). Scale bar: 100 μm. (D) Germ layer-specific genes <i>ALB</i>, <i>α-MHC</i>, and <i>TH</i> were expressed in a developmentally controlled manner during EB differentiation. <i>GAPDH</i> is included as loading control. Analyses were performed at different stages (days 5, 15, or 25) during differentiation of EBs after plating at day 8 (d8). Marker, 100 bp DNA marker.</p

Individual hiPSCs varied in their susceptibility to allogeneic and autologous NK cells and NK cells of individual donors varied in their activity against allogeneic and autologous hiPSCs.

<p>(A) A summary of means of specific lysis and the SEM of D1-iPSC4 cells by allogeneic (allo) and autologous (auto) NK cells is shown. The numbers of individual experiments (n) are indicated in the figure. (B) A summary of means of specific lysis and the SEM of the autologous hiPSC line D1-iPSC4 and the two allogeneic hiPSC lines (D2-iPSC1, D3-iPSC3) by NK cells of donor 1 is shown. (C) A summary of means of specific lysis and the SEM of D2-iPSC1 cells by allogeneic (allo) and autologous (auto) NK cells is shown. (D) A summary of means of specific lysis and the SEM of the autologous hiPSC line D2-iPSC1 and the two allogeneic hiPSC lines (D1-iPSC4, D3-iPSC3) by NK cells of donor 2 is shown. (E) A summary of means of specific lysis and the SEM of D3-iPSC3 by allogeneic (allo) and autologous (auto) NK cells is shown. (F) A summary of means of specific lysis and the SEM of the autologous hiPSC line D3-iPSC3 and the two allogeneic hiPSC lines (D1-iPSC4, D2-iPSC1) by NK cells of donor 3 is shown.</p

Flow cytometric analysis of ligands for activating and inhibitory NK receptors on D1-iPSC4 cells.

<p>Representative histograms of propidium iodide negative D1-iPSC4 cells are shown after staining with mAbs for the indicated molecules or with recombinant receptor molecules (NKG2D, NKp30, NKp44, and NKp46) for the respective ligands. Staining with the respective primary reagent is shown in red and with the secondary Ab only in black. The percentages of specifically stained cells (using the marker shown) and the specific MFI were calculated and are given in the figure.</p

Killing of hiPSC lines is in general dependent on DNAM-1 and for D6-iPSC2 cells also on HLA class I molecules.

<p>(A) A summary of means of specific lysis and the SEM of three hiPSC lines (D1-iPSC4, D2-iPSC1, D6-iPSC2) by IL-2-activated NK cells from the donors 4, 5, and 7 in ⁵¹Cr-release assays is shown after incubation with an isotype control (IgG₁) or blocking mAbs against NKG2D (aNKG2D), DNAM-1 (aDNAM-1), or ICAM-1 (aICAM-1) at a concentration of 10 μg/ml. The significance of differences was calculated by ANOVA and is indicated in the figure. The numbers of individual experiments (n) are indicated in the figure. (B) A summary of means of specific lysis and the SEM of three hiPSC lines (D1-iPSC4, D2-iPSC1, D6-iPSC2) by IL-2-activated NK cells from the donors 4, 5 and 7 is shown after incubation with the W6/32 HL mAb that binds to HLA class I molecules or as control the non-binding variant W6/32 HK at a concentration of 10 μg/ml. (C, D) The data for the three hiPSC lines are shown grouped according to the three NK cell donors. (E, F) The data are shown grouped according to the three hiPSC lines and K562 cells. Significant differences were calculated by 2-way-ANOVA adjusted either for the NK cell donors or the hiPSC lines and are indicated in the figure (** P<0.01, *P<0.05).</p

Gene expression analysis of hiPSC lines indicated mostly low mRNA expression of activating and inhibitory NK receptor ligands.

<p>(A) The expression of the indicated genes was tested by qPCR in PBMCs, K562 cells and three hiPSC lines (D1-iPSC4, D2-iPSC1, and D3-iPSC3). For the hiPSC lines and K562 cells means ± SEM of Δct values (ct target gene [tg] minus ct housekeeping gene [hkg]) of three biological replicates are shown. Negative values indicated a higher expression of the target gene than the housekeeping gene. Therefore, an inverted scale is shown. At the left side, genes encoding for ligands of activating NK receptors and <i>ICAM1</i> are shown. In the middle part classical and non-classical HLA class I genes, <i>B2M</i>, and the HLA class II gene <i>DRA</i> are shown. In the right part, genes involved in antigen processing in the HLA class I pathway are grouped. Genes not expressed in the iPSC lines (ct > 30) are marked by Ø. (B) The gene expression in the iPSC lines is shown as relative expression compared to PBMCs. (C) The gene expression in the hiPSC lines is shown as relative expression compared to K562 cells.</p

Human iPSC lines were killed by purified and IL-2-activated NK cells of various donors but allogeneic effector cells were more efficient than autologous NK cells.

<p>(A) NK cells were stimulated for four days with IL-2 (200 U/ml) and used as effector cells against the reference target cell line K562 in ⁵¹Cr-release assays. Each individual test was done in triplicates. The means of specific lysis and the standard error of the mean (SEM) at different effector:target (E:T) ratios (16:1 to 0.25:1) are shown to summarize these experiments. The numbers of individual experiments (n) are indicated in the figure. (B) A summary of means of specific lysis and the SEM of K562 and three hiPSC lines by IL-2-activated NK cells from five donors (1 to 5) is shown. (C) A summary of means of specific lysis and the SEM of the three hiPSC lines (D1-iPSC4, D2-iPSC1, D3-iPSC3) by NK cells of five different donors is shown. (D) A summary of means of specific lysis and the SEM of the three hiPSC lines (D1-iPSC4, D2-iPSC1, D3-iPSC3) by allogeneic (allo) and autologous (auto) NK cells is shown.</p

NK cells degranulating in response to hiPSC lines are enriched for several NK cell receptors.

<p>(A) The degranulation of IL-2-activated NK cells of donor 7 in response to D6-iPSC2 cells is shown. Without contact to hiPSCs only 2.9% of the NK cells expressed the degranulation marker CD107a. After co-culture with target cells for 2 h 25.2% of the CD56⁺ NK cells expressed CD107a at the plasma membrane. 46.7% of the CD107a^- NK cells were KIR positive. Among the CD107a⁺ NK cells 53.0% were KIR positive. The KIR staining was performed with a mixture of all anti-KIR mAbs indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125544#pone.0125544.t001" target="_blank">Table 1</a> to cover all KIR molecules. (B) A summary of means and the SEM of CD107a⁺ NK cells of donors 4, 5, and 7 after exposure to three hiPSC lines (D1-iPSC4, D2-iPSC1, D6-iPSC2) and K562 cells is shown (n = 3). (C) In the left panel a summary of means and the SEM of NKG2D⁺, NKG2A⁺, DNAM-1⁺, and KIR⁺ cells among all NK cells exposed to the hiPSCs as well as CD107a^- and CD107a⁺ NK cells is shown. In the right panel a summary of means and the SEM of the MFI of NKG2D, NKG2A, DNAM-1, and KIR on all NK cells exposed to the hiPSCs as well as CD107a^- and CD107a⁺ NK cells is shown. Significant differences between CD107a^- and CD107a⁺ NK cells are indicated (n = 26, *** P<0.001, ** P<0.01, <i>t</i>-test after Bonferroni-Holm correction).</p