Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions

Abstract

Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniaein vitro Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.

Keywords: Mycoplasma hyopneumoniae; air-liquid interface; barrier function; migration; pig bronchial epithelial cells.

FIG 1

Immunofluorescence microscopy and TEM analysis of primary PBECs cultured under ALI conditions for different times. (A) Primary PBECs were cultured under ALI conditions for different times (D5, D10, and D15). At the indicated times, an anti-β-tubulin antibody was used to identify cilia on the luminal surface of ALI-PBECs. The IOD values of β-tubulin staining were analyzed by ImageJ software. Data are presented as the ratios of the IOD values in D10 and D15 cells relative to those in D5 cells. The thickness of the vertical structure of the ALI-PBECs was analyzed by x-y-z axis scanning with confocal microscopy. (B) Primary PBECs were cultured under ALI conditions for 15 days. The cells were then subjected to transmission electron microscopy (TEM) analysis. TJs are indicated by a white arrow in the enlarged local view.

FIG 2

Histological examination and SEM analysis of well-differentiated porcine bronchial epithelial cells and native bronchus. (A) Well-differentiated ALI-PBECs and native bronchus were fixed with 4% polyoxymethylene, followed by histological sectioning and H&E staining for morphological comparison. (B) Well-differentiated ALI-PBECs and native bronchus were fixed with 2.5% glutaraldehyde followed by SEM analysis for the ciliary comparison.

FIG 3

Adherence and invasion of M. hyopneumoniae in well-differentiated porcine bronchial epithelial cells. Well-differentiated ALI-PBECs were infected with the M. hyopneumoniae NJ strain for different times (16, 28, and 40 hpi). At the indicated times, the infected PBECs were immunostained for M. hyopneumoniae both before permeabilization to visualize adherent bacteria (green) and after permeabilization to visualize invasive bacteria (red). In addition, ImageJ software was used to analyze the IOD values of M. hyopneumoniae staining. Data are presented as the ratios of the IOD values in infection cells relative to those in control cells.

FIG 4

Quantitative PCR detection of M. hyopneumoniae that translocated to the basolateral chamber. Well-differentiated ALI-PBECs were infected with the M. hyopneumoniae NJ strain, and the medium in the basolateral chamber was collected at different times (16, 28, and 40 hpi). The extracted DNA was quantified by quantitative real-time PCR using the TaqMan system.

FIG 5

Damage caused by M. hyopneumoniae infection to mucociliary function. Well-differentiated ALI-PBECs were infected with the M. hyopneumoniae NJ strain for different times (16, 28 and 40 hpi). (A and B) At the indicated times, cilia and MUC5B on the luminal surface of ALI-PBECs were identified by anti-β-tubulin and MUC5B antibodies. ImageJ software was used to analyze the IOD values according to β-tubulin or MUC5B staining. Data are presented as the ratios of the IOD values in infection cells relative to those in control cells. (C) At the indicated times, SEM analysis was used to evaluate cilia differentiation. Protrusions formed on the cell surface were indicated by a white arrow.

FIG 6

The damage caused by M. hyopneumoniae infection to tight junction function. Well-differentiated ALI-PBECs were infected with the M. hyopneumoniae NJ strain for different times (16, 28, and 40 hpi). (A) At the indicated times, an anti-ZO-1 antibody was also used to detect the tight junctions of cells subjected to different treatments. The IOD values of ZO-1 staining were analyzed by ImageJ software. Data are presented as the ratios of the IOD values in infection cells relative to those in control cells. (B) Vertical structures of the ALI-PBECs subjected to different treatments were analyzed by x-y-z axis scanning with confocal microscopy. (C) TEER values in infection and control cells were measured at the indicated times (16, 28, and 40 hpi).

FIG 7

Damage caused by M. hyopneumoniae infection to the integrity of the epithelial structure. Well-differentiated ALI-PBECs were infected with the M. hyopneumoniae NJ strain for different times (16, 28 and 40 hpi). At the indicated times, anti-β-tubulin and anti-M. hyopneumoniae P46 antibodies were used to detect the cilia and M. hyopneumoniae of cells subjected to different treatments. (A) The thickness of the vertical structure of the ALI-PBECs was analyzed by x-y-z axis scanning with confocal microscopy. Data are presented as the ratios of the thickness in treated cells relative to those in control cells. (B) Three-dimensional (3D) diagrams of M. hyopneumoniae-infected and control cells.

FIG 8

Assessment of epithelial desquamation. Well-differentiated ALI-PBECs were infected with the M. hyopneumoniae NJ strain for 16 hpi. At the indicated time, HBSS in the apical side of the filter with all treatments were collected for cytospins. Cells collected by cytospin were prepared for IFAs. An anti-M. hyopneumoniae P46 antibody was used to detect the mycoplasmas on the desquamated cells.

FIG 9

Pathway for M. hyopneumoniae invasion across the porcine airway epithelium. M. hyopneumoniae infection could disrupt mucociliary function and tight junctions, increase the permeability, and damage the integrity of the epithelial barrier. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber.