Effects of Vitamin D on Airway Epithelial Cell Morphology and Rhinovirus Replication

Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)2D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0–69.0] vs. 30 µm [24.5–34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1β) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells

Rhinovirus increases Moraxella catarrhalis adhesion to the respiratory epithelium

Rhinovirus causes many types of respiratory illnesses, ranging from minor colds to exacerbations of asthma. Moraxella catarrhalis is an opportunistic pathogen that is increased in abundance during rhinovirus illnesses and asthma exacerbations and is associated with increased severity of illness through mechanisms that are ill-defined. We used a co-infection model of human airway epithelium differentiated at the air-liquid interface to test the hypothesis that rhinovirus infection promotes M. catarrhalis adhesion and survival on the respiratory epithelium. Initial experiments showed that infection with M. catarrhalis alone did not damage the epithelium or induce cytokine production, but increased trans-epithelial electrical resistance, indicative of increased barrier function. In a co-infection model, infection with the more virulent rhinovirus-A and rhinovirus-C, but not the less virulent rhinovirus-B types, increased cell-associated M. catarrhalis. Immunofluorescent staining demonstrated that M. catarrhalis adhered to rhinovirus-infected ciliated epithelial cells and infected cells being extruded from the epithelium. Rhinovirus induced pronounced changes in gene expression and secretion of inflammatory cytokines. In contrast, M. catarrhalis caused minimal effects and did not enhance RV-induced responses. Our results indicate that rhinovirus-A or C infection increases M. catarrhalis survival and cell association while M. catarrhalis infection alone does not cause cytopathology or epithelial inflammation. Our findings suggest that rhinovirus and M. catarrhalis co-infection could promote epithelial damage and more severe illness by amplifying leukocyte inflammatory responses at the epithelial surface

Effects of vitamin D on airway epithelial cell morphology and rhinovirus replication.

Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)2D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0-69.0] vs. 30 µm [24.5-34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1β) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells

Basal Cells of Differentiated Bronchial Epithelium Are More Susceptible to Rhinovirus Infection

We used an in vitro model of differentiated tracheobronchial epithelium to analyze the susceptibility of different cell types to infection with rhinoviruses (RVs). Primary cells from control subjects were cultured in an air–liquid interface to form differentiated epithelia. Suprabasal and basal fractions were separated after trypsin digestion, and cell suspensions were infected with serotypes RV16 and RV1A. These cell fractions were analyzed for expression of viral capsid protein VP2 (flow cytometry), viral replication (real-time PCR), cytokeratin-14, and intercellular adhesion molecule–1 (ICAM-1). Compared with suprabasal fraction, basal cells had increased percentages of cells staining positive for VP2 (RV1A: 37.8% versus 9.1%, P < 0.01; RV16: 12.0 versus 3.0%, P < 0.05). The average number of viral RNA copies per cell was also higher in basal cells (2.2- and 2.4-fold increase in RV1A- and RV16-infected cells, respectively) compared with suprabasal cells. Furthermore, ICAM-1 was expressed by 33.3% of basal cells, compared with 8.1% of suprabasal cells (P < 0.05). Finally, in culture models of epithelial injury (detached suprabasal cells or scratched surface), there was significantly greater replication of RV1A compared with intact cell layer. These findings demonstrate that basal cells are more susceptible to RV infection than suprabasal cells. For major group RV, this may be in part due to increased expression of ICAM-1; however, minor group RV also replicated more effectively in basal cells. These results suggest the possibility that epithelial cell differentiation is associated with the maturation of antiviral defense mechanisms

Effects of 10(fold upregulation).

*<p>p<0.05.</p>**<p>p≤0.01.</p

Effects of 1,25(OH)₂D on cell morphology.

<p>(A) Hematoxylin and eosin stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 24 days in the presence of 10 nM 1,25(OH)₂D. (B) Staining of paraffin cross-sections from 1,25(OH)₂D-treated primary hBEC cell layers with antibody against cytokeratin 5/6 (CK5/6). (C) Alcian blue stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 24 days in the presence of 10 nM 1,25(OH)₂D. (D) Average thicknesses of primary hBEC layers differentiated in the presence of 10 nM 1,25(OH)₂D for 4 to 6 weeks (n = 8). (E) Average thicknesses of fully differentiated primary hBEC layers after treatment with 10 nM 1,25(OH)₂D for 6 weeks (n = 5).</p

Similar effects of supplemental vitamin D vs. retinoic acid deficiency on cell morphology.

<p>Hematoxylin and eosin-stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 28 days in the presence of 10 nM (deficient) or 50 nM (normal) all-<i>trans</i> retinoic acid, with 0 nM or 10 nM 1,25(OH)₂D.</p

1,25(OH)₂D and 25(OH)D have similar effects on cell growth and differentiation.

<p>Hematoxylin and eosin stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 28 days in the presence of various concentrations of 1,25(OH)₂D or 25(OH)D.</p

Effects of 1,25(OH)₂D on RV replication.

<p>(A) Replication of RV16 at 24 hours in differentiated hBEC cultured at air-liquid interface for 24 days in the presence of various concentrations of 1,25(OH)₂D (n = 7). (B) Replication of RV16 (MOI = 0.1, 1, or 10) at 24 hours in differentiated hBEC cultured at air-liquid interface for 27 days in the presence of 10 nM of 1,25(OH)₂D (n = 3).</p