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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5806fjev1 fj.06-5806fjev2 20/12/2121    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sajjan, U. S. Articles by Hershenson, M. B. Search for Related Content PubMed PubMed Citation Articles by Sajjan, U. S. Articles by Hershenson, M. B.

H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM-1 and TLR3 expression

Umadevi S. Sajjan^*, Yue Jia^*, Dawn C. Newcomb, J. Kelley Bentley^*, Nicholas W. Lukacs, John J. LiPuma^* and Marc B. Hershenson^*,,1

^* Department of Pediatrics and Communicable Diseases,

Department of Molecular and Integrative Physiology, and

Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA

¹Correspondence: University of Michigan, 1150 W. Medical Center Dr., Rm. 3570, MSRBII, Box 0688, Ann Arbor, MI 48109-0688, USA. E-mail: mhershen{at}umich.edu

SPECIFIC AIM

Chronic obstructive pulmonary disease (COPD) is a chronic, progressive disease with intermittent acute exacerbations associated with viral and/or bacterial infections. Nontypeable H. influenzae has been isolated from airways of clinically stable COPD patients, suggesting that it can cause chronic infection. Rhinovirus (RV), a single-stranded RNA virus that is responsible for most of the common colds, is an important trigger of acute exacerbations in COPD patients. Respiratory viruses are more likely to be isolated in patients with a history of frequent COPD exacerbations, suggesting that patients with frequent exacerbations are more susceptible to viral infection. Nevertheless, little information exists regarding potential interactions between viruses and bacteria in the pathogenesis of COPD exacerbations. In the present study, we investigated the cooperative effects of H. influenzae and RV on ELR(+) C-X-C chemokine production from airway epithelial cells. We hypothesized that H. influenzae infection increases expression of airway epithelial cell intercellular adhesion molecule (ICAM)-1 and Toll-like receptor (TLR)-3, leading to increased RV binding and exaggerated RV-induced chemokine responses.

PRINCIPAL FINDINGS

1. H. influenzae increases RV39-stimulated C-X-C chemokine production from airway epithelial cells
Well-differentiated human primary airway epithelial cells grown at air-liquid interface were infected apically with RV39 at an multiplicity of infection (MOI) of 1 and incubated for 24 h. Medium from the basolateral chamber was collected for quantification of interleukin (IL)-8, epithelial-derived neutrophil attractant (ENA)-78, and growth-related oncogene (GRO)- proteins by ELISA. Cultures infected with HeLa cell extract (sham) served as control. RV39 significantly increased the basolateral secretion of all three chemokines compared with controls (Fig. 1 ).

View larger version (13K): [in this window] [in a new window]   Figure 1. H. influenzae potentiates RV-39 induced IL-8 (A), ENA-78 (B), and GRO- (C) expression in primary mucociliary-differentiated human tracheal epithelial cells. Primary human airway epithelial cells differentiated into mucociliary phenotype were infected with H. influenzae at MOI of 1 and incubated for 8 h. Unbound bacteria were removed, and cells were infected with RV39 (MOI of 1 for 1 h) apically. Cultures incubated with equal volumes of sham-infected HeLa cell extracts served as controls. Chemokine release in basolateral chamber was measured 24 h later by ELISA. Bars are mean ± SE of 3 experiments; *different from sham infected, P < 0.05, ANOVA.

To examine the effect of bacterial infection on RV39-stimulated chemokines, well-differentiated airway epithelial cell cultures were infected with H. influenzae at MOI of 1 and incubated for 8 h. Unbound bacteria were removed by washing of the apical surface, and cells were incubated with RV39 as before. Cell cultures infected with both bacteria and RV39 secreted significantly more IL-8, ENA-78, and GRO- than cells infected with RV39 or H. influenzae alone (Fig. 1) .

We found similar cooperative effects of H. influenzae and RV on transcription from the IL-8 promoter in 16HBE14o- human bronchial epithelial cells, suggesting that IL-8 expression in response to virus and bacteria is regulated at the transcriptional level.

2. H. influenzae increases RV39 binding to airway epithelial cells and expression of viral receptors ICAM-1 and TLR3
We hypothesized that the observed increase in RV39-induced IL-8 expression in H. influenzae-infected cells is due to increased binding of RV39. To test this, ³⁵S-labeled RV39 was incubated at 4°C for 1 h with uninfected 16HBE14o- cells or cells infected with H. influenzae at MOI of 1 for 8 h, and cell-associated radioactivity was determined. Maximal RV39 binding was increased in cells infected with H. influenzae compared with uninfected cells. These data are consistent with the notion that infection with H. influenzae increases RV39-induced IL-8 expression by augmenting binding of RV to its epithelial cell receptor.

Next, we examined whether the increased binding of RV39 to H. influenzae-infected cells and the resultant increased production of C-X-C chemokines are associated with altered expression of ICAM-1 and TLR3. Binding of major subgroup RV to ICAM-1 is a prerequisite for airway epithelial cell IL-8 expression. TLR3 mediates immune responses to double-stranded (ds)-RNA. Although RV is a single-stranded RNA virus, during replication it makes dsRNA intermediates. 16 HBE14o- cells were infected with H. influenzae as described above, and the total and cell surface expression of ICAM-1 and TLR3 was determined by flow cytometry. Cells infected with H. influenzae showed increased expression of both ICAM-1 and TLR3 (Fig. 2 A and B). Whereas expression of ICAM-1 increased mainly on the cell surface, TLR3 expression increased mainly in the intracellular compartment (Figs. 2C and D) . Similar to 16HBE14o- cells, H. influenzae infection increased expression of both ICAM-1 and TLR3 in differentiated human primary airway epithelial cell cultures. Confocal micrographs and Z-sections demonstrate that ICAM-1 expression is primarily confined to the apical surface, whereas TLR3 is localized primarily to the cell cytoplasm (Figs. 2E-H ). These results suggest that H. influenzae increases RV39 binding to epithelial cells and potentiates RV39-stimulated chemokine expression by increasing expression of the viral receptors ICAM-1 and TLR3.

View larger version (28K): [in this window] [in a new window]   Figure 2. Increased expression ICAM-1 and TLR3 in H. influenzae-infected cells. 16HBE14o- cells were infected with H. influenzae as described, immunostained with antibodies to ICAM-1 or TLR3, and analyzed by flow cytometry. A and B) Representative histograms of fluorescence intensity of permeabilized 16HBE14o- cells immunostained with ICAM-1 and TLR3 Ab, respectively. Similar results were obtained in 2 additional experiments. Intracellular expression of ICAM-1 (C) and TLR3 (D) was calculated by subtracting the mean fluorescence intensity of nonpermeabilized cells from permeabilized cells. Expression of both ICAM-1 (E) and TLR3 (F) is increased in well-differentiated primary airway epithelial cells infected with H. influenzae compared with uninfected controls, as determined by immunofluorescent confocal microscopy. Z sections show cellular localization of ICAM-1 (G) and TLR3 (H). ICAM-1 is localized primarily to the cell surface, whereas TLR3 is primarily found in the cell cytoplasm.

3. RV39-stimulated IL-8 expression is partially dependent on TLR3; H. influenzae-induced TLR3 expression is mediated by TNF-
Next, we examined to what extent TLR3 contributes to RV39-stimulated IL-8 expression in airway epithelial cells. In the first approach, TLR3 expression in 16HBE14o- cells was partially blocked using specific siRNA, and the effects on H. influenzae- and RV39-induced IL-8 promoter activity were determined. Cells transfected with two different preparations of TLR3 siRNA, but not nontargeting siRNA, showed significantly decreased IL-8 promoter activity when stimulated by either RV39 alone or H. influenzae and RV39. siRNA against TLR3 had no effect on H. influenzae-induced IL-8 expression. These results indicate that TLR3 is required for maximal RV39-induced IL-8 production. In a second approach, human embryonic kidney (HEK)-293 cells stably transfected with TLR3 or empty vector were infected with RV39. These cells do not express TLR3 constitutively. On average, cells expressing TLR3 produced 13-fold higher IL-8 levels on RV infection than untransfected HEK293 cells. Together with the previous data, these data suggest that prior infection with H. influenzae increase RV39-induced IL-8 expression in part by increasing expression of TLR3.

We hypothesized that TNF-, produced by epithelial cells in response to H. influenzae infection, is at least partially responsible for the observed increases in TLR3 expression. 16HBE14o- cells secrete TNF- within 5–15 min of infection with H. influenzae. TNF- also increased the expression of TLR3 approximately fivefold. Incubation with TNF- neutralizing antibody, but not normal IgG, caused dose-dependent inhibition of IL-8 expression induced by the combination of H. influenzae and RV. In contrast, previous work from our laboratory has shown that RV39-induced IL-8 responses do not require airway epithelial cell TNF- production. Together, these data suggest that H. influenzae increases expression of the TLR3 viral receptor via TNF-.

CONCLUSIONS AND SIGNIFICANCE

Little information exists regarding potential interactions between viruses and bacteria in the pathogenesis of COPD exacerbations. We hypothesized that H. influenzae infection increases expression of airway epithelial cell ICAM-1 and TLR3, leading to increased RV binding and exaggerated RV-induced chemokine responses. The requirement of TLR3, an evolutionarily conserved pathogen recognition receptor that recognizes and binds dsRNA, for RV-induced chemokine expression has not yet been tested.

We found that infection with nontypeable H. influenzae increases the expression of ICAM-1 and TLR3 in human airway epithelial cells differentiated into mucociliary phenotype, as well as 16HBE14o- cells growing as monolayers. We further showed that H. influenzae increases binding of RV39 to airway epithelial cells, leading to cooperative increases the RV39-stimulated C-X-C chemokine expression. Together, these data provide a mechanism by which prior infection with nontypeable H. influenzae, as occurs in patients with COPD, may increase susceptibility to RV infection (Fig. 3 ). Although the induction of TLR3 expression by RNA viruses or dsRNA may not be surprising, positive regulation of TLR3 due to bacterial infection of the respiratory epithelium has not been demonstrated previously.

View larger version (37K): [in this window] [in a new window]   Figure 3. Summary schematic depicting a cellular mechanism by which prior infection with H. influenzae increases susceptibility of airway epithelial cells to RV infection, leading to increased chemokine expression and COPD exacerbation.

In addition, we found that TLR3-specific siRNA, which blocked both constitutive and H. influenzae-stimulated TLR3 expression in airway epithelial cells, also inhibited the RV-stimulated IL-8 production. Furthermore, HEK293 cells stably transfected with TLR3 produced high amounts of IL-8 in response to RV infection. Together, these findings show for the first time that TLR3 is required for maximal IL-8 expression and sufficient, at least to some degree, for IL-8 expression in response to RV infection.

Preliminary studies in our laboratory indicate that TLR3 expression is increased in the small airways of COPD patients (U. Sajjan, D. Arenberg, and M. Hershenson, unpublished data). Further investigation addressing potential interactions between viruses and bacteria in the pathogenesis of COPD exacerbations is therefore warranted.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5806fje

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