Rhinovirus disrupts the barrier function of polarized airway epithelial cells

Abstract

Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease.

Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection.

Methods: Primary human airway epithelial cells grown at air-liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (R(T)) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting.

Measurements and main results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in R(T) without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease R(T), suggesting a requirement for viral replication. Reduced R(T) was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, IFN-gamma and IL-1beta reversed corresponding cytokine-induced reductions in R(T) but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo.

Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function.

Figure 1.

Morphologic features of primary airway epithelial cells differentiated into mucociliary phenotype. (A) Primary airway epithelial cells were grown in transwells at an air/liquid interface. Cells were fixed in buffered formalin, embedded in agarose-paraffin, and sections were stained with hematoxilin and eosin. (B) Primary airway epithelial cells grown at air/liquid interface were fixed in methanol and stained with zona occludins ZO-1 (green) and β-tubulin (red).

Figure 2.

Rhinovirus (RV) decreases the transepithelial resistance (R_T) of well-differentiated primary airway epithelial cells. Primary cultures grown at air/liquid interface were infected apically with (A) RV or (C) ultraviolet-treated RV at one multiplicity of infection and incubated for 5 hours. Infection medium was then removed, and the cells further incubated for 24 hours. Lactate dehydrogenase activity in the basolateral medium (A) and the cell culture R_T (B and C) were measured. Cell lysate used in (A) was generated from representative media-treated cells. Results represent the mean of three independent experiments performed in triplicate; bars represent SEM. Statistical differences between experimental groups were determined by one-way analysis of variance (*P < 0.05).

Figure 3.

RV decreases the R_T of polarized 16HBE14o- cells in a dose- and time-dependent manner. Cells grown in transwells were infected apically with (A) RV39 or (B) RV1B at doses ranging from 0.25 to 5 MOI and incubated for 1 hour. Infection medium was removed, incubation continued for another 23 hours and R_T was measured. Cytotoxic effect was determined by measuring LDH levels in basolateral media (C). The time course of RV-induced reductions in RT was determined by infecting polarized cell monolayers with RV39 at MOI of 1 for 1 hour, replacing the infection media with fresh cell culture media, and measuring R_T measured at the indicated times (D). Data represent the mean and standard error of four independent experiments carried out in triplicate (P < 0.05, analysis of variance).

Figure 4.

RV-induced reductions in R_T are not mediated by proinflammatory cytokines. Polarized 16HBE14o- human bronchial epithelial cells were either treated with proinflammatory cytokines (A) or infected with RV for 24 hours (B) in the presence or absence of neutralizing antibodies to IFN-γ and TNF-α, neutralizing antibody to IL-1β, or normal IgG. R_T was measured as described above. Results represent the mean of three independent experiments carried out in triplicate; bars represent SEM (P < 0.05, analysis of variance).

Figure 5.

Apoptosis does not contribute to rhinovirus (RV)-induced reductions in transepithelial resistance (R_T). (A) Primary diffentiated airway epithelial cells or (B) polarized 16HBE14o- human bronchial epithelial cells were infected with RV39 or sham or treated with 5 μM thepsigargin for 1 hour. Media-treated cells were used as negative controls. Cells were stained with mixture of FITC-annexin V and propidium iodide and analyzed by flow cytometry. Results represent the mean of three independent experiments performed in triplicate; bars represent mean ± SEM (P < 0.05, analysis of variance).

Figure 6.

Effect of RV on the paracellular permeability of polarized epithelial cells to inulin. Polarized 16HBE14o- cells were infected with RV39 or RV1B at MOI of 1 for 24 hours. FITC labeled-inulin was added to the apical chamber, the basolateral chamber was sampled at different time intervals for fluorescence, and P_app was calculated. Results represent the mean of three independent experiments carried out in quadruplicate; bars represent SEM (P < 0.05, analysis of variance).

Figure 7.

Rhinovirus (RV) infection reduces transepithelial resistance by dissociating zona occludins (ZO)-1 from tight junction complex. Well-differentiated (A and B) primary or (C and D) polarized 16HBE14o- human bronchial epithelial cells were infected with either (A and C) sham or (B and D) RV39 and incubated for 24 hours as described above. Cells were fixed in cold methanol and immunostained with antibody to ZO-1 (green). Nuclei were stained with DAPI (blue) (Sigma-Aldrich, St. Louis, MO). Arrows represent dissociation of ZO-1 from the tight junction complex. NP40 soluble and insoluble fractions from medium-treated, sham, RV39, or RV1B-infected polarized 16HBE14o- cells were subjected to Western blot analysis with (E) antibody to ZO-1. (F) A representative blot from three independent experiments is presented. Group mean data from three independent experiments (bars represent mean ± SEM; P < 0.05, analysis of variance).

Figure 8.

Rhinovirus (RV) promotes transmigration of bacteria by the paracellular route. 16HBE14o- human bronchial epithelial cells grown as polarized monolayers were infected apically with (A) sham or (B) RV39 for 24 hours. NTHi (0.1 ml of 1 × 10⁹ cfu/ml) was added to the apical surface and incubated for 3 hours. Cells were fixed and processed for transmission electron microscopy. Arrows point to bacteria. Cultures were immunostained with antibodies to zona occludins (ZO)1 (green) and nontypeable Haemophilus influenzae (NTHi) (red) and analyzed by confocal microscopy, taking sections every 0.5 μm. (C and D) Apical view of sham- and RV- infected cells, respectively. (E and F) Z-sections of sham- and RV- infected cells. Arrows in D indicate areas devoid of ZO-1. Arrows in F indicate bacteria at the basolateral surface.

Figure 9.

Rhinovirus (RV) disrupts the barrier function of Calu-3 cells. (A) Polarized monolayers of Calu-3 cells were infected with RV39, ultraviolet-irradiated RV39, or sham for 24 hours and transepithelial resistance (R_T) was measured. (B) The time course of reductions in R_T in response to RV infection. (C) In some experiments, monolayers were either treated with proinflammatory cytokines or infected with RV for 24 hours in the presence or absence of neutralizing antibodies to IFN-γ and tumor necrosis factor-α, neutralizing antibody to IL-1β or normal IgG. Calu-3 monolayers infected with either sham or RV39 for 24 hours were incubated apically with nontypeable Haemophilus influenzae isolate 6P5H for 6 hours. (D) Medium from the basolateral chamber was sampled for bacteria at 1, 3, or 6 hours. Monolayers infected with (E) sham and (F) RV39 for 24 hours were immunostained with antibody to zona occludins (ZO)-1 (green) and counterstained with DAPI (blue). Results represent the mean of three independent experiments performed in triplicate; bars represent mean ± SEM (P < 0.05, analysis of variance). Arrows in E and F point to the dissociation of ZO-1 from the periphery of cells.

Figure 10.

Influenza A, but not RSV, causes a cytopathic effect in polarized 16HBE14o- human bronchial epithelial cells. Cells grown in Transwells were infected apically with RSV or influenza A virus and incubated for 1 hour Infection medium was removed, cells were further incubated for 23 hours and R_T of cell cultures (A) and LDH levels in basolateral medium (B) were measured. In some experiments, cells were infected with RSV (C) or influenza A virus (D) as described above, fixed in methanol, and immunostained with antibody to ZO-1. Results represent the mean of three independent experiments performed in triplicate; bars represent mean ± SEM (P < 0.05, analysis of variance). Arrows in panel D show loss of both ZO-1 and nuclei.

Figure 11.

Rhinovirus (RV)1B disrupts airway epithelial tight junctions in vivo. C57BL/6 mice were infected with RV1B (5 × 10⁷ TCID₅₀) or an equal volume of sham. Mice were killed at 24 hours post-infection, and paraffin sections prepared from the lungs were deparaffinized, rehydrated, and immunostained with antibody to ZO-1. The bound antibody was detected by anti-rabbit IgG conjugated with AlexaFluor 488. (A and C) Large and small airways, respectively, from sham-infected mice. (B and D) Large and small airways, respectively, from RV1B-infected mice. Arrows represent absence of dissociation of zona occludins-1 from the tight junction complex.