Controls for lung dendritic cell maturation and migration during respiratory viral infection

Dendritic cells are ideally suited to orchestrate the innate and adaptive immune responses to infection, but we know little about how these cells respond to infection with common respiratory viruses. Paramyxoviral infections are the most frequent cause of serious respiratory illness in childhood and are associated with an increased risk of asthma. We therefore used a high-fidelity mouse model of paramyxoviral respiratory infection triggered by Sendai virus to examine the response of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) in the lung. We found that pDCs are scarce at baseline but become the predominant population of lung dendritic cells during infection. This recruitment allows for a source of IFN-alpha locally at the site of infection. In contrast, cDCs rapidly differentiate into myeloid cDCs and begin to migrate from the lung to draining lymph nodes within 2 h after viral inoculation. These events cause the number of lung cDCs to decrease rapidly and remain decreased at the site of viral infection. Maturation and migration of lung cDCs depends on Ccl5 and Ccr5 signals because these events are significantly impaired in Ccl5(-/-) and Ccr5(-/-) mice. cDCs failure to migrate to draining lymph nodes in Ccl5(-/-) or Ccr5(-/-) mice is associated with impaired up-regulation of CCR7 that would normally direct this process. Our results indicate that pDCs and cDCs respond distinctly to respiratory paramyxoviral infection with patterns of movement that should serve to coordinate the innate and adaptive immune responses, respectively.     

Fibroblast growth factor-9 expression in airway epithelial cells amplifies the type I interferon response and alters influenza A virus pathogenesis

Influenza A virus (IAV) preferentially infects conducting airway and alveolar epithelial cells in the lung. The outcome of these infections is impacted by the host response, including the production of various cytokines, chemokines, and growth factors. Fibroblast growth factor-9 (FGF9) is required for lung development, can display antiviral activity in vitro, and is upregulated in asymptomatic patients during early IAV infection. We therefore hypothesized that FGF9 would protect the lungs from respiratory virus infection and evaluated IAV pathogenesis in mice that overexpress FGF9 in club cells in the conducting airway epithelium (FGF9-OE mice). However, we found that FGF9-OE mice were highly susceptible to IAV and Sendai virus infection compared to control mice. FGF9-OE mice displayed elevated and persistent viral loads, increased expression of cytokines and chemokines, and increased numbers of infiltrating immune cells as early as 1 day post-infection (dpi). Gene expression analysis showed an elevated type I interferon (IFN) signature in the conducting airway epithelium and analysis of IAV tropism uncovered a dramatic shift in infection from the conducting airway epithelium to the alveolar epithelium in FGF9-OE lungs. These results demonstrate that FGF9 signaling primes the conducting airway epithelium to rapidly induce a localized IFN and proinflammatory cytokine response during viral infection. Although this response protects the airway epithelial cells from IAV infection, it allows for early and enhanced infection of the alveolar epithelium, ultimately leading to increased morbidity and mortality. Our study illuminates a novel role for FGF9 in regulating respiratory virus infection and pathogenesis.     

Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

Background

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN) are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-γ-dependent antiviral mechanisms in epithelial cells in the airway.

Methods

Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE) followed by exposure to both CSE and IFN-γ. Epithelial cell cytotoxicity and IFN-γ-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV) were tested without and with CSE exposure.

Results

CSE inhibited IFN-γ-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-γ-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-γ was required to inhibit IFN-γ-induced cell signaling. CSE also decreased the inhibitory effect of IFN-γ on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-γ-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects.

Conclusions

The results indicate that CSE inhibits the antiviral effects of IFN-γ, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.     

Dengue virus induces expression of CXC chemokine ligand 10/IFN-gamma-inducible protein 10, which competitively inhibits viral binding to cell surface heparan sulfate

Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-gamma-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.     

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.     

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.     

Protein kinase R regulates double-stranded RNA induction of TNF-alpha but not IL-1 beta mRNA in human epithelial cells

Epithelial cells represent the initial site of respiratory viral entry and the first line of defense against such infections. This early antiviral response is characterized by an increase in the production of proinflammatory cytokines such as TNF-alpha and IL-1 beta. dsRNA, which is a common factor present during the life cycle of both DNA and RNA viruses, is known to induce TNF-alpha and IL-1 beta in a variety of cells. In this work we provide data showing that dsRNA treatment induces TNF-alpha and IL-1 beta in human lung epithelial cells via two different mechanisms. Our data show that dsRNA activation of dsRNA-activated protein kinase (PKR) is associated with induction of TNF-alpha but not IL-1 beta expression. An inhibitor of PKR activation blocked the dsRNA-induced elevations in TNF-alpha but not IL-1 beta mRNA in epithelial cells. Data obtained from infection of epithelial cells with a vaccinia virus lacking the PKR inhibitory polypeptide, E3L, revealed that PKR activation was essential for TNF-alpha but not for IL-1 beta expression. In this report, we provide experimental support for the differential regulation of proinflammatory cytokine expression by dsRNA and viral infections in human airway epithelial cells.     

Transforming growth factor beta enhances respiratory syncytial virus replication and tumor necrosis factor alpha induction in human epithelial cells

Asthma is characterized as a chronic inflammatory disease associated with significant tissue remodeling. Patients with asthma are more susceptible to virus-induced exacerbation, which subsequently can lead to increased rates of hospitalization and mortality. While the most common cause of asthma-related deaths is respiratory viral infections, the underlying factors in the lung environment which render asthmatic subjects more susceptible to viral exacerbation are not yet identified. Since transforming growth factor beta (TGF-beta) is a critical cytokine for lung tissue remodeling and asthma phenotype, we have focused on the effects of TGF-beta on viral replication and virus-induced inflammation. Treatment of human epithelial cells with TGF-beta increased respiratory syncytial virus (RSV) replication by approximately fourfold. Tumor necrosis factor alpha (TNF-alpha) mRNA and protein expression were also significantly increased above levels with RSV infection alone. The increase in RSV replication and TNF-alpha expression after TGF-beta treatment was concomitant with an increase in virus-induced p38 mitogen-activated protein kinase activation. Our data reveal a novel effect for TGF-beta on RSV replication and provide a potential mechanism for the exaggerated inflammatory response observed in asthmatic subjects during respiratory viral infections.     

Expression of CXC chemokine ligand 10 from the mouse hepatitis virus genome results in protection from viral-induced neurological and liver disease

Using the recombinant murine coronavirus mouse hepatitis virus (MHV) expressing the T cell-chemoattractant CXCL10 (MHV-CXCL10), we demonstrate a potent antiviral role for CXCL10 in host defense. Instillation of MHV-CXCL10 into the CNS of CXCL10-deficient (CXCL10(-/-)) mice resulted in viral infection and replication in both brain and liver. Expression of virally encoded CXCL10 within the brain protected mice from death and correlated with increased infiltration of T lymphocytes, enhanced IFN-gamma secretion, and accelerated viral clearance when compared with mice infected with an isogenic control virus, MHV. Similarly, viral clearance from the livers of MHV-CXCL10-infected mice was accelerated in comparison to MHV-infected mice, yet was independent of enhanced infiltration of T lymphocytes and NK cells. Moreover, CXCL10(-/-) mice infected with MHV-CXCL10 were protected from severe hepatitis as evidenced by reduced pathology and serum alanine aminotransferase levels compared with MHV-infected mice. CXCL10-mediated protection within the liver was not dependent on CXC-chemokine receptor 2 (CXCR2) signaling as anti-CXCR2 treatment of MHV-CXCL10-infected mice did not modulate viral clearance or liver pathology. In contrast, treatment of MHV-CXCL10-infected CXCL10(-/-) mice with anti-CXCL10 Ab resulted in increased clinical disease correlating with enhanced viral recovery from the brain and liver as well as increased serum alanine aminotransferase levels. These studies highlight that CXCL10 expression promotes protection from coronavirus-induced neurological and liver disease.     

Gamma interferon controls mouse polyomavirus infection in vivo

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.     

Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E(2) (PGE(2)) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF-/- donors (GM-CSF-/- BMT) with untransplanted mice. GM-CSF-/- BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF-/- BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF-/- BMT mice overproduced PGE(2), but expression of the inhibitory EP(2) receptor was diminished. As a consequence of decreased EP(2) receptor expression, we found diminished accumulation of cAMP in response to PGE(2) stimulation in GM-CSF-/- BMT AMs compared with control BMT AMs. In addition, GM-CSF-/- BMT AMs retained cysteinyl leukotriene production and normal TNF-alpha response compared with AMs from control BMT mice. GM-CSF-/- BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF-/- recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.     

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.     

Lower respiratory tract infection induced by a genetically modified picornavirus in its natural murine host

Infections with the picornavirus, human rhinovirus (HRV), are a major cause of wheezing illnesses and asthma exacerbations. In developing a murine model of picornaviral airway infection, we noted the absence of murine rhinoviruses and that mice are not natural hosts for HRV. The picornavirus, mengovirus, induces lethal systemic infections in its natural murine hosts, but small genetic differences can profoundly affect picornaviral tropism and virulence. We demonstrate that inhalation of a genetically attenuated mengovirus, vMC(0), induces lower respiratory tract infections in mice. After intranasal vMC(0) inoculation, lung viral titers increased, peaking at 24 h postinoculation with viral shedding persisting for 5 days, whereas HRV-A01a lung viral titers decreased and were undetectable 24 h after intranasal inoculation. Inhalation of vMC(0), but not vehicle or UV-inactivated vMC(0), induced an acute respiratory illness, with body weight loss and lower airway inflammation, characterized by increased numbers of airway neutrophils and lymphocytes and elevated pulmonary expression of neutrophil chemoattractant CXCR2 ligands (CXCL1, CXCL2, CXCL5) and interleukin-17A. Mice inoculated with vMC(0), compared with those inoculated with vehicle or UV-inactivated vMC(0), exhibited increased pulmonary expression of interferon (IFN-α, IFN-β, IFN-λ), viral RNA sensors [toll-like receptor (TLR)3, TLR7, nucleotide-binding oligomerization domain containing 2 (NOD2)], and chemokines associated with HRV infection in humans (CXCL10, CCL2). Inhalation of vMC(0), but not vehicle or UV-inactivated vMC(0), was accompanied by increased airway fluid myeloperoxidase levels, an indicator of neutrophil activation, increased MUC5B gene expression, and lung edema, a sign of infection-related lung injury. Consistent with experimental HRV inoculations of nonallergic, nonasthmatic human subjects, there were no effects on airway hyperresponsiveness after inhalation of vMC(0) by healthy mice. This novel murine model of picornaviral airway infection and inflammation should be useful for defining mechanisms of HRV pathogenesis in humans.     

Nerve growth factor modulates human rhinovirus infection in airway epithelial cells by controlling ICAM-1 expression

Human rhinoviruses (HRV) are the most common agent of upper respiratory infections and an important cause of lower respiratory tract symptoms. Our previous research with other viral pathogens has shown that virus-induced airway inflammation and hyperreactivity involve neurotrophic pathways that also affect tropism and severity of the infection. The goals of this study were to analyze systematically the expression of key neurotrophic factors and receptors during HRV-16 infection of human airway epithelial cells and to test the hypothesis that neurotrophins modulate HRV infection by controlling the expression of a major cellular receptor for this virus, the intercellular adhesion molecule 1 (ICAM-1). Neurotrophins and ICAM-1 expression were analyzed at the mRNA level by real-time PCR and at the protein level by flow cytometry and immunocytochemistry. A small inhibitory RNA (siRNA) or a specific blocking antibody was utilized to suppress nerve growth factor (NGF) expression and measure its effects on viral replication and virus-induced cell death. Nasal and bronchial epithelial cells were most susceptible to HRV-16 infection at 33°C and 37°C, respectively, and a significant positive relationship was noted between expression of NGF and tropomyosin-related kinase A (TrkA) and virus copy number. ICAM-1 expression was dose dependently upregulated by exogenous NGF and significantly downregulated by NGF inhibition with corresponding decrease in HRV-16 replication. NGF inhibition also increased apoptotic death of infected cells. Our results suggest that HRV upregulates the NGF-TrkA pathway in airway epithelial cells, which in turn amplifies viral replication by increasing HRV entry via ICAM-1 receptors and by limiting apoptosis.     

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses

Background

Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.

Methodology/Principal Findings

To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.

Conclusions/Significance

Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.     

Distinctive and critical roles for cellular immunity and immune-inflammatory response in the immunopathology of Sendai virus infection in mice

Respiratory viral infections result in severe pulmonary injury, to which host immune response may be a significant contributor. At present, it is not entirely clear the extent to which lung injury is a necessary consequence of host defense. In this report, we use functional genomics approach to characterize the key roles of cellular immunity and immune-inflammatory response in the immunopathology of Sendai virus infection in resistant C57BL/6J and susceptible DBA/2J mice. Infected mice manifested an immune-inflammatory response characterized by the pulmonary influx of neutrophils and mononuclear cells. DBA/2J mice mounted a vigorous immune response, with significant up-regulation of cytokine/chemokine genes in two successive waves through the course of infection. Whereas, C57BL/6J mice displayed an efficient immune response with less severe pathology and clusters of immune-inflammatory responsive genes were exclusively up-regulated on day 4 in this strain. Overall, DBA/2J mice exhibited a dysregulated hyper-inflammatory cytokine/chemokine cascades that does not limit viral spread resulting in a predisposition to severe lung pathology. This response is similar to severe human respiratory paramyxovirus infections, which will serve as a model for the elucidation of hyper-immune inflammatory response that result to severe immunopathology in respiratory viral infections.     

Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells

Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.     

Deletion of CCR1 attenuates pathophysiologic responses during respiratory syncytial virus infection

The role of chemokines in chronic inflammatory responses are central to the recruitment of particular subsets of leukocytes. In the present studies, we have examined the role of CCR1 in the developing pathogenesis of respiratory syncytial virus (RSV) in the lungs of infected BALB/c mice. Although we did not observe significant differences in clearance of RSV, we were able to identify decreased pathophysiologic responses in CCR1(-/-) mice. CCR1(-/-) mice displayed a significant reduction in both airway hyperresponsiveness and mucus production that corresponded to significant increases in IFN-gamma and CXCL10. The goblet cell hyper/metaplasia and the expression of mucus-associated gene, gob5, were correspondingly reduced in the CCR1(-/-) mice. In addition, the Western blot analysis of gob5 protein indicated that CCR1(-/-) mice have virtually no up-regulation of the protein at day 6 of infection compared with wild-type-infected mice. Results from bone marrow chimeric mice indicated that partial reconstitution of the response could be achieved in the CCR1(-/-) mice with wild-type bone marrow cells, suggesting that these cells have a role in the response. However, transplanting of CCR1(-/-) bone marrow into wild-type mice did demonstrate an incomplete deficit in RSV-induced responses, indicating that CCR1(+) parenchymal cells may also play a significant role in the process. Thus, the presence of CCR1 appears to have a significant role in the development of detrimental airway physiologic responses during RSV infection. These data suggest that CCR1 may be a potential target during detrimental pulmonary responses during infection.