Resveratrol-mediated gamma interferon reduction prevents airway inflammation and airway hyperresponsiveness in respiratory syncytial virus-infected immunocompromised mice

Respiratory syncytial virus (RSV) is the most important cause of severe, lower respiratory tract infections in infants, and RSV infections have been associated with chronic wheezing and asthma during childhood. However, the mechanism of RSV-induced airway inflammation and airway hyperresponsiveness (AHR) is poorly understood. Furthermore, there are presently neither effective vaccines nor drugs available for the prevention or treatment of RSV infections. In this study, we investigated the effect of the plant extract resveratrol as a means of preventing airway inflammation and attenuating RSV-induced AHR. Our data showed that resveratrol reduced RSV lung titers and the number of infiltrating lymphocytes present in bronchoalveolar lavage fluid (BALF) and reduced inflammation. Furthermore, resveratrol attenuated airway responses to methacholine following RSV infection and significantly decreased gamma interferon (IFN-γ) levels in BALF of RSV-infected mice. Data presented in this report demonstrated that resveratrol controlled Toll-like receptor 3 (TLR3) expression, inhibited the TRIF signaling pathway, and induced M2 receptor expression following RSV infection. These data support a role for the use of resveratrol as a means of reducing IFN-γ levels associated with RSV-mediated airway inflammation and AHR, which may be mediated via TLR3 signaling.     

Plasmacytoid dendritic cells limit viral replication, pulmonary inflammation, and airway hyperresponsiveness in respiratory syncytial virus infection

Plasmacytoid dendritic cells (pDC), as major producers of IFN-alpha, are thought not only to be pivotal in antiviral immunity, but also to limit allergic inflammation. In this study, we delineate the role of pDC in a mouse model of respiratory syncytial virus (RSV)-induced airway inflammation. Bone marrow-derived pDC generated high levels of IFN-alpha upon RSV infection, and the percentage of pDC expressing MHC class II and maturation-associated costimulatory molecules was increased. However, their weak Ag-presenting capacity was not enhanced. Furthermore, pDC induced marked levels of IL-10 in T cell cultures irrespective of infection. In vivo, numbers of pDC in the lung increased early after RSV infection and remained elevated throughout the inflammatory phase and the resolution phase of infection. Depletion of pDC resulted in increases in peak RSV titers, pulmonary inflammation, and airway hyperresponsiveness. In contrast, adoptive transfer of activated pDC to the airways reduced RSV copy numbers. In conclusion, RSV infection induces activation of murine pDC with robust IFN-alpha production, limiting replication and accelerating elimination of RSV. In addition to this innate response, pDC also may play an immune regulatory role in reducing pulmonary inflammation and inhibiting the development of airway hyperresponsiveness.     

Alteration of airway neuropeptide expression and development of airway hyperresponsiveness following respiratory syncytial virus infection

The mechanisms by which respiratory syncytial virus (RSV) infection causes airway hyperresponsiveness (AHR) are not fully established. We hypothesized that RSV infection may alter the expression of airway sensory neuropeptides, thereby contributing to the development of altered airway function. BALB/c mice were infected with RSV followed by assessment of airway function, inflammation, and sensory neuropeptide expression. After RSV infection, mice developed significant airway inflammation associated with increased airway resistance to inhaled methacholine and increased tracheal smooth muscle responsiveness to electrical field stimulation. In these animals, substance P expression was markedly increased, whereas calcitonin gene-related peptide (CGRP) expression was decreased in airway tissue. Prophylactic treatment with Sendide, a highly selective antagonist of the neurokinin-1 receptor, or CGRP, but not the CGRP antagonist CGRP(8-37), inhibited the development of airway inflammation and AHR in RSV-infected animals. Therapeutic treatment with CGRP, but not CGRP(8-37) or Sendide, abolished AHR in RSV-infected animals despite increased substance P levels and previously established airway inflammation. These data suggest that RSV-induced airway dysfunction is, at least in part, due to an imbalance in sensory neuropeptide expression in the airways. Restoration of this balance may be beneficial for the treatment of RSV-mediated airway dysfunction.     

Differential cytokine mRNA expression in Dermatophagoides farinae allergen-sensitized and respiratory syncytial virus-infected mice

The interaction between mite allergen sensitization and respiratory syncytial virus (RSV) infection at the level of cytokine mRNA expression was examined in a murine model in the present study. Primary RSV infection enhances expression of inflammatory cytokines such as IL-6, IFN-gamma, and eotaxin in the lung and upregulates the expression of Th2-like cytokines IL-10 and IL-13 in the spleen in BALB/c mice. Mite antigen-sensitized and RSV-infected (ASRSV) mice show enhanced (P < 0.05) total serum IgE compared to antigen-sensitized mice. However, the levels of viral mRNA in the lung tissues are comparable between RSV-infected and ASRSV mice. It is concluded that compartmentalization of cytokine expression following RSV infection plays a role in the augmentation of Th2-like and IgE antibody response to RSV.     

Pharyngeal microflora disruption by antibiotics promotes airway hyperresponsiveness after respiratory syncytial virus infection

Background

Regulatory T cells (Treg cells), which are essential for regulation of immune response to respiratory syncytial virus (RSV) infection, are promoted by pharyngeal commensal pneumococcus. The effects of pharyngeal microflora disruption by antibiotics on airway responsiveness and relative immune responses after RSV infection have not been clarified.

Methods

Female BALB/c mice (aged 3 weeks) were infected with RSV and then treated with either oral antibiotics or oral double distilled water (ddH₂O) from 1 d post infection (pi). Changes in pharyngeal microflora were analyzed after antibiotic treatment for 7 d and 14 d. At 8 d pi and 15 d pi, the inflammatory cells in bronchoalveolar lavage fluid (BALF) were investigated in combination with tests of pulmonary histopathology, airway hyperresponsiveness (AHR), pulmonary and splenic Treg cells responses. Pulmonary Foxp3 mRNA expression, IL-10 and TGF-β1 in BALF and lung homogenate were investigated at 15 d pi. Ovalbumin (OVA) challenge was used to induce AHR after RSV infection.

Results

The predominant pharyngeal commensal, Streptococcus, was cleared by antibiotic treatment for 7 d. Same change also existed after antibiotic treatment for 14 d. After RSV infection, AHR was promoted by antibiotic treatment at 15 d pi. Synchronous decreases of pulmonary Treg cells, Foxp3 mRNA and TGF-β1 were detected. Similar results were observed under OVA challenge.

Conclusions

After RSV infection, antibiotic treatment cleared pharyngeal commensal bacteria such as Streptococcus, which consequently, might induce AHR and decrease pulmonary Treg cells.     

TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice

Steroid-resistant asthma comprises an important source of morbidity in patient populations. T(H)17 cells represent a distinct population of CD4(+) Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of T(H)17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4(+) T cells from DO11.10 OVA-specific TCR-transgenic mice to a T(H)2 or T(H)17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of T(H)2 and T(H)17 cells. In vitro, T(H)17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of T(H)2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas T(H)17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of T(H)17 or T(H)2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the T(H)17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both T(H)2 and T(H)17 cells are able to induce AHR, whereas T(H)17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for T(H)17 cells in steroid-resistant asthma.     

Alcohol reduces airway hyperresponsiveness (AHR) and allergic airway inflammation in mice

There is very limited knowledge about the effects of alcohol on airway hyperresponsiveness and inflammation in asthma. Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that alcohol exposure will alter airway hyperresponsiveness (AHR) and pulmonary inflammation in a mouse model of allergic asthma. To test this hypothesis, BALB/c mice were fed either 18% alcohol or water and then sensitized and challenged with ovalbumin (OVA). AHR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause, respectively. Airway inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluid (BALF), cytokine levels in BALF, lung histology, and serum immunoglobulin E (IgE) levels. Alcohol feeding significantly blocked methacholine-induced increases in AHR compared with water-fed controls. Alcohol feeding significantly reduced total cell numbers (64%) as well as the number of eosinophils (84%) recruited to the lungs of these mice. Modest changes in lung pathology were also observed. Alcohol exposure led to a reduction of IgE in the serum of the EtOH OVA mice. These data demonstrate that alcohol exposure blunts AHR and dampens allergic airway inflammation indices in allergic mice and suggest that there may be an important role for alcohol in the modulation of asthma. These data provide an in vivo basis for previous clinical observations in humans substantiating the bronchodilator properties of alcohol and for the first time demonstrates an alcohol-induced reduction of allergic inflammatory cells in a mouse model of allergic asthma.     

Alternative mechanisms of respiratory syncytial virus clearance in perforin knockout mice lead to enhanced disease

Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzyme-mediated lysis of infected cells is thought to be the major molecular mechanism used by CD8(+) cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.     

Chlamydophila pneumoniae induces a sustained airway hyperresponsiveness and inflammation in mice

Background

It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated.

Methods

In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21.

Results

We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness.

Conclusion

Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.     

Pediatrics: ribavirin and respiratory syncytial virus infections

The Scientific Board of the California Medical Association presents the following inventory of items of progress in pediatrics. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, research workers, or scholars to stay abreast of these items of progress in pediatrics that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.The items of progress listed below were selected by the Advisory Panel to the Section on Pediatrics of the California Medical Association and the summaries were prepared under its direction.     

IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection, which induces an immune response dominated by IFN-gamma, results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways, both of which are prevented by pretreatment with anti-IL-5 Ab. To delineate the role of IL-5, IL-4, and IFN-gamma in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice deficient in each of these cytokines to develop these symptoms of RSV infection. Mice deficient in either IL-5, IL-4, or IFN-gamma were administered infectious RSV intranasally, and 6 days later, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and production of IFN-gamma, IL-4, and IL-5 by mononuclear cells from peribronchial lymph nodes were monitored. RSV infection resulted in airway eosinophilia and AHR in both IL-4- and IFN-gamma-deficient mice, but not in IL-5-deficient mice. Reconstitution of IL-5-deficient mice with IL-5 restored these responses and enhanced the responses in IL-4-deficient mice. Anti-VLA-4 (very late Ag-4) treatment prevented lung eosinophilia and AHR following RSV infection and IL-5 reconstitution. We conclude that in response to RSV, IL-5 is essential for the influx of eosinophils into the lung and that eosinophils in turn are critical for the development of AHR. IFN-gamma and IL-4 are not essential for these responses to RSV infection.     

Cytotoxic T-cell response to respiratory syncytial virus in mice.

The role of the humoral and cellular arms of the immune response in protection against respiratory syncytial virus (RSV) infection and in the pathogenesis of the severe forms of this disease is poorly understood. The recent demonstration that some inbred mouse strains can be infected with RSV has opened the way to a detailed investigation of RSV immunity. We report here the finding of major histocompatibility complex-restricted, RSV-specific memory cytotoxic T cells in the spleens of BALB/c and C57BL mice after intranasal infection; these T cells recognize the Long, A2, and 8/60 (human) strains of RSV. Both K and D locus major histocompatibility complex alleles can restrict the cytotoxic response; however, in the two haplotypes tested, Dd is a low-responder allele and Kb is a nonresponder allele for RSV. UV-inactivated RSV (when given intraperitoneally) can prime mice for development of cytotoxic T cell memory, restimulate cytotoxic T cell cultures in vitro, and form a target for the cytotoxic cells.     

Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

Background

CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.

Methods

Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.

Results

Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.

Conclusions

We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.     

Thioredoxin suppresses airway hyperresponsiveness and airway inflammation in asthma

Thioredoxin (TRX) is a 12-kDa redox (reduction/oxidation)-active protein that has a highly conserved site (-Cys-Gly-Pro-Cys-) and scavenges reactive oxygen species. Here we examined whether exogenously administered TRX modulated airway hyperresponsiveness (AHR) and airway inflammation in a mouse asthma model. Increased AHR to inhaled acetylcholine and airway inflammation accompanied by eosinophilia were observed in OVA-sensitized mice. Administration of wild-type but not 32S/35S mutant TRX strongly suppressed AHR and airway inflammation, and upregulated expression of mRNA of several cytokines (e.g., IL-1alpha, IL-1beta, IL-1 receptor antagonist, and IL-18) in the lungs of OVA-sensitized mice. In contrast, TRX treatment at the time of OVA sensitization did not improve AHR or airway inflammation in OVA-sensitized mice. Thus, TRX inhibited the asthmatic response after sensitization, but did not prevent sensitization itself. TRX and redox-active protein may have clinical benefits in patients with asthma.     

呼吸道合胞病毒感染促进豚鼠产生气道高反应性及其机制研究

目的：通过检测气道反应性和M2受体功能,研究呼吸道合胞病毒（RSV）感染与哮喘发病的关系及机制。方法：34只豚鼠随机分为4组：Hep-2滴鼻＋生理盐水雾化（Hep-2／NS,A）组,RSV滴鼻＋生理盐水雾化（RSV／NS,B）组,Hep-2滴鼻＋鸡卵蛋白（OVA）雾化（Hep-2／OVA,C）组和RSV滴鼻＋OVA雾化（RSV／OVA,D）组,其中A和B纽各9只,C和D组各8只,以A组为对照组。21d通过电刺激迷走神经检测各组气道反应性和M2受体功能,行嗜酸性粒细胞计数以及病理学观察。结果：B组气道内压力（mmH2O）与A组无明显差异（P〉0．05）,给予匹罗卡品,IP下降幅度高于A组,但差别无显著性（P〉0．05）。C组IP明显高于A且（P〈0．05）,且给予匹罗卡品,IP下降幅度明显低于A组,差别有显著性（P〈0．05）。D组IP明显高于C组（P〈0．05）,给予匹罗卡品后IP下降幅度明显低于C组（P〈0．05）。结论：RSV感染可促进过敏原引起的M2R功能障碍,从而促进AHR发生。     

Prostaglandin modulation of airway inflammation and hyperresponsiveness in mice sensitized without adjuvant

As adjuvant during sensitization may cause unspecific immune reactions, the aim of the present study was to define the role of cyclooxygenase (COX) activity on airway inflammation and airway hyperresponsiveness (AHR) in an adjuvant-free allergic mouse model.Administration of diclofenac and indomethacin (non-selective COX inhibitors), FR122047 (COX-1 inhibitor) and lumiracoxib (selective COX-2 inhibitor) enhanced AHR. Only diclofenac and lumiracoxib reduced the inflammatory cell content of bronchoalveolar lavage (BAL). Moreover, levels of prostaglandins in BAL were reduced by indomethacin and FR122047 but were unaffected by lumiracoxib. However, compared with antigen controls, none of the COX inhibitors displayed major effects on the production of cytokines, smooth muscle mass, number of goblet cells and eosinophils, or collagen deposition in the airways.These data in mice sensitized without adjuvant support the fact that COX products have a general bronchoprotective role in allergic airway inflammation. Furthermore, the data suggest that COX-1 activity predominantly generates prostanoids in BAL, whereas COX-2 activity is associated with the accumulation of inflammatory cells in BAL. This study further supports that AHR on the one hand, and the inflammatory response and generation of prostanoids on the other, are dissociated and, at least in part, uncoupled events.     

Breast-feeding protects against respiratory syncytial virus infections.

Eight out of 115 infants admitted to hospital with respiratory syncytial (RS) virus infection had been breast-fed compared with 46 out of 167 controls; this difference was statistically significant. Twenty-one specimens of human colostrum were examined, and all contained RS virus neutralising activity. Specific IgA and IgG were detected in 18 specimens, whereas IgM was detected in none. The titre of IgA antibody was usually higher and correlated more closely to the titre of neutralising activity than that of IgG. Infants inhale milk feeds and regurgitate them through the nose, and the IgA collecting in the respiratory tract might protect against severe respiratory infection. Alternatively, if severe RS virus illness is a sign of hypersensitivity to the virus breast-feeding might protect the infant from an early sensitising infection.