Functional invariant NKT cells in pig lungs regulate the airway hyperreactivity: a potential animal model

Abstract

Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4(+) cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.

Fig. 1

Cell surface CD1d expression on pig cells. a Flow cytometric analysis of pig BAL cells, lung cells, splenocytes, and PBMCs was performed using a pig CD1d cross-reactive mouse anti-CD1d mAb (filled histogram) and an isotype control mAb (open histogram). b Two-color flow cytometric analysis of lung cells for the cell surface CD1d and cytokeratin expression. Numbers in the quadrant indicate percentage of cells, and the data shown are representative of four to ten pigs from two to three independent experiments

Fig. 2

iNKT cells are present in pigs. a Two-color flow cytometric analysis of fresh pig PBMCs, splenocytes, and lung MNCs using pig anti-CD3ε mAb conjugated with R-PE and APC-conjugated mouse empty or PBS-57-loaded CD1d tetramers. b Pig PBMCs was cultured in vitro in the presence of various concentrations of α-GalCer for 4 days, and iNKT cells were analyzed as described above. Numbers in the quadrant indicate percentage of cells and the data shown are the representative of five to ten pigs from five independent experiments

Fig. 3

Functional iNKT cells are present in pig lungs. Pig lung MNCs and PBMCs (control) were cultured in the presence of α-GalCer (1,000 ng/ml) or vehicle for 12 days. a Immunostained using fluorochrome conjugated anti-pig CD3ε and mouse empty or PBS-57 loaded CD1d tetramers and analyzed by flow cytometry. b Culture supernatants harvested on post-6 and 12 day cultures were analyzed for IFNγ and IL-4 using pig cytokine-specific ELISA. Each bar represents the average cytokines OD from three pigs±SEM and * denote statistically significant difference (P<0.05) in the amount of cytokines secreted by cells cultured in the presence of vehicle vs α-GalCer. The data shown are representative of six pigs in three independent experiments

Fig. 4

Acute AHR-induced pig lungs had petechial hemorrhages with excessive CD4⁺ and myeloid cells infiltration. a A representative pig lung macroscopic picture of vehicle or α-GalCer-inoculated pig (n=3 per group) with petechial hemorrhages on both the dorsal and ventral surfaces (arrows indicate areas of hemorrhages) is shown. b A representative pig lung section was subjected to immunohistochemistry, showing more of infiltrated CD4⁺ cells in α-GalCer received pig lungs compared to vehicle controls. Bar, 10 µm. c A representative pig lung section was subjected to H&E staining, showing massive infiltration of inflammatory leukocytes in α-GalCer received pig lungs. Bar, 5 µm. Frozen sections of vehicle or α-GalCer-inoculated pigs (n=3 per group) were immunostained for pig-specific CD4⁺ cells (arrowheads) and then counterstained with hematoxylin. Similar results were obtained in another independent experiment

Fig. 5

Elevated body temperature and increased pro-inflammatory and Th2-cytokine responses were mediated by lung iNKT cells in the acute AHR-induced pigs. Pigs were intratracheally inoculated with α-GalCer or vehicle. a Body temperature was recorded at 9 and 24 h post-inoculation. Mean temperature from three pigs in each treatment group±SEM is shown, and P value was calculated by nonparametric Kruskal–Wallis test. b BAL cells harvested from pigs were immunostained to analyze CD1d on myeloid cells (CD172⁺) by flow cytometry. The data shown were from a representative pig from vehicle or α-GalCer received pigs (n=3 pigs/group). Numbers in the quadrant indicate percentage of cells. c Lung homogenates prepared from pig lungs 24 h post-inoculation were analyzed for indicated cytokines by ELISA. Each bar represents the average cytokine concentration from three pigs±SEM and * denote statistically significant difference (P<0.05) between vehicle vs α-GalCer-inoculated pigs

Fig. 6

Acute AHR-induced pig iNKT cells present in the PBMCs and lung MNCs were preferentially proliferated into CD4⁺ phenotype and secreted more of IL-4 than IFNγ ex vivo. Pigs were intratracheally inoculated with α-GalCer or vehicle and euthanized at post-24 h of inoculation. a PBMCs and b lung MNCs isolated from pigs were cultured for 8 days in the presence of vehicle, α-GalCer, GCK127, or GCK152 at indicated concentrations. Cells were immunostained using fluorochrome conjugated anti-pig CD3ε, CD4α, and mouse empty or PBS-57-loaded CD1d tetramers and subjected to flow cytometry. CD3⁺ gated cells were analyzed for mouse CD1d tetramer⁺ and CD4α^± iNKT cells. The supernatants harvested from cultures were analyzed for c IL-4 and d IFNγ by ELISA. Each bar represents the ratio (vehicle/α-GalCer received pigs) of average percentage of iNKT cells or amounts of cytokine from three pigs in one independent experiment. A trend line was drawn on the y-axis at “1”. Ratio of < or > one means that iNKT cells frequency or secreted cytokines were that many fold < or > in AHR-induced pigs compared to respective mock control, respectively. Similar results were observed in another independent experiment