LPS aggravates lung inflammation induced by RSV by promoting the ERK-MMP-12 signaling pathway in mice.

BACKGROUND: RSV can lead to persistent airway inflammation and airway hyperresponsiveness (AHR), and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. Lipopolysaccharide (LPS) is also implicated in the onset and exacerbation of asthma. However, whether inhalation of LPS can boost airway inflammation induced by RSV is not clear. In this study, we utilized an LPS- and RSV-superinfected mouse model to explore underlying pathogenesis. METHODS: Mice were infected with RSV on day 0 and inoculated with LPS from day 35 to day 41, samples were collected on day 42. Inflammatory cells, lung histopathology and AHR were measured. Cytokines were detected by ELISA and ERK, JNK, p38 was determined by western blot. MMP408, PD98059, SP600125 and SB203580 were used to inhibit MMP-12, ERK, JNK and p38 respectively. RESULTS: LPS exposure superimposed on RSV-infected lungs could lead to more vigorous cellular influx, lung structures damage, augmented AHR and higher MMP-12 levels. Inhibition of MMP-12 or ERK signaling pathway in vivo both diminished LPS-driven airway inflammation and AHR. CONCLUSIONS: Exposure to LPS in RSV-infected mice is associated with enhanced increases in ERK-MMP-12 expression that translates into increased lung inflammation and AHR. These findings contribute novel information to the field investigating the onset of post-RSV bronchiolitis recurrent wheezing as a result of LPS exposure.

Qingfei oral liquid alleviates airway hyperresponsiveness and mucus hypersecretion via TRPV1 signaling in RSV-infected asthmatic mice.

Pediatric asthma is exacerbated by Respiratory Syncytial Virus (RSV) infection, and Transient Receptor Potential Vanilloid 1 (TRPV1) promotes production of inflammatory cytokines and mucus hypersecretion in the pathology of this disease. Our previous research revealed that Qingfei oral liquid (QF) inhibited airway inflammation and mucus hypersecretion in RSV-infected asthmatic mice models and that this may be associated with the TRPV1-regulation of NF-κB and Mucin 5AC (MUC5AC) expression, but the exact mechanism is unknown. In the present study, LC-MS was used for analyzing the chemicals in QF, ovalbumin (OVA)-induced asthmatic mice inhaled RSV three consecutive times to create an RSV-infected asthmatic model. We found treatment from QF alleviated airway hyperresponsiveness (AHR) and reduced congestion, edema, and infiltration of inflammatory cells into pulmonary tissues. Additionally, QF was found to decrease expression of NF-κB and its downstream inflammatory cytokines IL-1ß, IL-4, IL-5, and IL-13, as well as a decrease in MUC5AC and pro-inflammatory cytokines in PKC via a reduction in Protein Kinase C-dependent signaling. These findings suggest that QF can alleviate AHR and mucus hypersecretion caused by RSV infection in asthmatic mice, and its mechanism may be associated with the regulation of the TRPV1 signaling pathway.

Respiratory syncytial virus infection and bronchial hyperreactivity in children up to two years of age in correlation with atopy.

BACKGROUND/AIM: Bronchiolitis in early childhood caused by respiratory syncytial virus (RSV) is considered to be important risk factor of the recurrent wheezing and asthma development. The aim of this study was to examine the frequency of RSV infection and atopy in children up to two years of age and to determine their correlation with bronchial hyperreactivity. METHODS: The study included 175 children aged 5-24 months. The presence of RSV infection was identified by serum levels of IgA and IgG determined by ELISA. Bronchial hyperreactivity (BHR) has been defined as the existence of chronic bronchial disease and/or three or more previous suspected diagnosis of acute bronchial disease. Atopy was confirmed by detection of the specific serum IgE using quantitative multitest Phadiatop infant (cut off 0.35 kUA/L). RESULTS: The children with atopy were more frequently infected with RSV (43.3%) than those without atopy (22.8%; p = 0.02). The higher frequency of RSV infection was found in children with BHR in comparison with those without it but only in the group who also had atopy (77.8% vs. 28.6%, p = 0.018). In the female children, BHR and RSV infection were associated in 62.5% of cases, regardless the atopy. In the male children with atopy, RSV infection was associated with BHR in 83.3% of the cases, while in those without atopy, RSV infection with BHR was found in only 17.4% of the cases. CONCLUSION: Children up to two years of age with atopy are more frequently infected with RSV (43.3%) than nonatopic children. Every third child with atopy develops BHR and 77.8% of them also have RSV infection. Atopic children are at higher risk for development of BHR when infected with RSV also.

Matrix Metalloproteinase 9 Exerts Antiviral Activity against Respiratory Syncytial Virus.

Increased lung levels of matrix metalloproteinase 9 (MMP9) are frequently observed during respiratory syncytial virus (RSV) infection and elevated MMP9 concentrations are associated with severe disease. However little is known of the functional role of MMP9 during lung infection with RSV. To determine whether MMP9 exerted direct antiviral potential, active MMP9 was incubated with RSV, which showed that MMP9 directly prevented RSV infectivity to airway epithelial cells. Using knockout mice the effect of the loss of Mmp9 expression was examined during RSV infection to demonstrate MMP9's role in viral clearance and disease progression. Seven days following RSV infection, Mmp9-/- mice displayed substantial weight loss, increased RSV-induced airway hyperresponsiveness (AHR) and reduced clearance of RSV from the lungs compared to wild type mice. Although total bronchoalveolar lavage fluid (BALF) cell counts were similar in both groups, neutrophil recruitment to the lungs during RSV infection was significantly reduced in Mmp9-/- mice. Reduced neutrophil recruitment coincided with diminished RANTES, IL-1ß, SCF, G-CSF expression and p38 phosphorylation. Induction of p38 signaling was required for RANTES and G-CSF expression during RSV infection in airway epithelial cells. Therefore, MMP9 in RSV lung infection significantly enhances neutrophil recruitment, cytokine production and viral clearance while reducing AHR.

CCL7 and IRF-7 Mediate Hallmark Inflammatory and IFN Responses following Rhinovirus 1B Infection.

Rhinovirus (RV) infections are common and have the potential to exacerbate asthma. We have determined the lung transcriptome in RV strain 1B-infected naive BALB/c mice (nonallergic) and identified CCL7 and IFN regulatory factor (IRF)-7 among the most upregulated mRNA transcripts in the lung. To investigate their roles we employed anti-CCL7 Abs and an IRF-7-targeting small interfering RNA in vivo. Neutralizing CCL7 or inhibiting IRF-7 limited neutrophil and macrophage influx and IFN responses in nonallergic mice. Neutralizing CCL7 also reduced activation of NF-κB p65 and p50 subunits, as well as airway hyperreactivity (AHR) in nonallergic mice. However, neither NF-κB subunit activation nor AHR was abolished with infection of allergic mice after neutralizing CCL7, despite a reduction in the number of neutrophils, macrophages, and eosinophils. IRF-7 small interfering RNA primarily suppressed IFN-α and IFN-ß levels during infection of allergic mice. Our data highlight a pivotal role of CCL7 and IRF-7 in RV-induced inflammation and IFN responses and link NF-κB signaling to the development of AHR.

MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice.

BACKGROUND: Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection. METHODS: Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression. RESULTS: RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice. CONCLUSIONS: MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.

Interleukin-1ß mediates virus-induced m2 muscarinic receptor dysfunction and airway hyperreactivity.

Respiratory viral infections are associated with the majority of asthma attacks. Inhibitory M2 receptors on parasympathetic nerves, which normally limit acetylcholine (ACh) release, are dysfunctional after respiratory viral infection. Because IL-1ß is up-regulated during respiratory viral infections, we investigated whether IL-1ß mediates M2 receptor dysfunction during parainfluenza virus infection. Virus-infected guinea pigs were pretreated with the IL-1ß antagonist anakinra. In the absence of anakinra, viral infection increased bronchoconstriction in response to vagal stimulation but not to intravenous ACh, and neuronal M2 muscarinic receptors were dysfunctional. Pretreatment with anakinra prevented virus-induced increased bronchoconstriction and M2 receptor dysfunction. Anakinra did not change smooth muscle M3 muscarinic receptor response to ACh, lung viral loads, or blood and bronchoalveolar lavage leukocyte populations. Respiratory virus infection decreased M2 receptor mRNA expression in parasympathetic ganglia extracted from infected animals, and this was prevented by blocking IL-1ß or TNF-α. Treatment of SK-N-SH neuroblastoma cells or primary cultures of guinea pig parasympathetic neurons with IL-1ß directly decreased M2 receptor mRNA, and this was not synergistic with TNF-α treatment. Treating guinea pig trachea segment with TNF-α or IL-1ß in vitro increased tracheal contractions in response to activation of airway nerves by electrical field stimulation. Blocking IL-1ß during TNF-α treatment prevented this hyperresponsiveness. These data show that virus-induced hyperreactivity and M2 dysfunction involves IL-1ß and TNF-α, likely in sequence with TNF-α causing production of IL-1ß.

Human parainfluenza type 3 virus impairs the efficacy of glucocorticoids to limit allergy-induced pulmonary inflammation in guinea-pigs.

Viral exacerbations of allergen-induced pulmonary inflammation in pre-clinical models reportedly reduce the efficacy of glucocorticoids to limit pulmonary inflammation and airways hyper-responsiveness to inhaled spasmogens. However, exacerbations of airway obstruction induced by allergen challenge have not yet been studied. hPIV-3 (human parainfluenza type 3 virus) inoculation of guinea-pigs increased inflammatory cell counts in BAL (bronchoalveolar lavage) fluid and caused hyper-responsiveness to inhaled histamine. Both responses were abolished by treatment with either dexamethasone (20 mg/kg of body weight, subcutaneous, once a day) or fluticasone propionate (a 0.5 mg/ml solution aerosolized and inhaled over 15 min, twice a day). In ovalbumin-sensitized guinea-pigs, allergen (ovalbumin) challenge caused two phases of airway obstruction [measured as changes in sGaw (specific airways conductance) using whole body plethysmography]: an immediate phase lasting between 4 and 6 h and a late phase at about 7 h. The late phase, airway hyper-responsiveness to histamine and inflammatory cell counts in BAL were all significantly reduced by either glucocorticoid. Inoculation of guinea-pigs sensitized to ovalbumin with hPIV-3 transformed the allergen-induced airway obstruction from two transient phases into a single sustained response lasting up to 12 h. This exacerbated airway obstruction and airway hyper-responsiveness to histamine were unaffected by treatment with either glucocorticoid whereas inflammatory cell counts in BAL were only partially inhibited. Virus- or allergen-induced pulmonary inflammation, individually, are glucocorticoid-sensitive, but in combination generate a phenotype where glucocorticoid efficacy is impaired. This suggests that during respiratory virus infection, glucocorticoids might be less effective in limiting pulmonary inflammation associated with asthma.

Dual role of interleukin-10 in the regulation of respiratory syncitial virus (RSV)-induced lung inflammation.

RSV lower respiratory tract infections (LRTI) are among the most common diseases necessitating hospital admission in children. In addition to causing acute respiratory failure, RSV infections are associated with sequelae such as secondary bacterial infections and reactive airway disease. One characteristic host response observed in severe RSV-induced LRTI and/or subsequent development of asthma is increased expression of interleukin (IL)-10. However, contradictory results have been reported regarding whether IL-10 inhibits asthmatic responses or intensifies the disease. We aimed to reconcile these discordant observations by elucidating the role of IL-10 in regulating the host response to RSV LRTI. In this study, we used a lung-specific, inducible IL-10 over-expression (OE) transgenic mouse model to address this question. Our results showed that the presence of IL-10 at the time of RSV infection not only attenuated acute inflammatory process (i.e. 24 h post-infection), but also late inflammatory changes [characterized by T helper type 2 (Th2) cytokine and chemokine expression]. While this result appears contradictory to some clinical observations where elevated IL-10 levels are observed in asthmatic patients, we also found that delaying IL-10 OE until the late immune response to RSV infection, additive effects rather than inhibitory effects were observed. Importantly, in non-infected, IL-10 OE mice, IL-10 OE alone induced up-regulation of Th2 cytokine (IL-13 and IL-5) and Th2-related chemokine [monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand 3 (CCL3) and regulated upon activation normal T cell expressed and secreted (RANTES)] expression. We identified a subset of CD11b(+)CD11c(+)CD49b(+)F4/80(-)Gr-1(-) myeloid cells as a prinicipal source of IL-10-induced IL-13 production. Therefore, the augmented pathological responses observed in our 'delayed' IL-10 over-expression model could be attributed to IL-10 OE alone. Taken together, our study indicated dual roles of IL-10 on RSV-induced lung inflammation which appear to depend upon the timing of when elevated IL-10 is expressed in the lung.

Toll-like receptor-2/6 and Toll-like receptor-9 agonists suppress viral replication but not airway hyperreactivity in guinea pigs.

Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam2CSK4 and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non-M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus.

Neural-endocrine mechanisms of respiratory syncytial virus-associated asthma in a rat model.

We examined the underlying neural-endocrine mechanisms of asthma associated with respiratory syncytial virus infection. Thirty Sprague-Dawley rats were randomly divided into control group, respiratory syncytial virus (RSV) group, and anti-nerve growth factor (NGF) IgG group. An RSV infection model was established by nasal drip once a week. In the anti-NGF antibody intervention group, each rat was given an intraperitoneal injection of anti-NGF IgG 3 h before RSV infection. Optical microscopy and transmission electron microscopy were used to observe the structural changes in adrenal medulla cells. Changes in adrenaline and norepinephrine in serum were detected by ELISA. NGF expression was assayed by immunohistochemistry. Expression differences in synaptophysin mRNA were detected by RT-PCR. Transmission electron microscopy displayed widened adrenal medulla intercellular spaces, reduced chromaffin particle concentration, and increased mitochondria in the RSV infection group. At the same time, NGF expression was increased in the RSV infection group significantly. In addition, the adrenaline concentration was significantly decreased compared with the control and anti-NGF antibody groups. Synaptophysin mRNA expression was significantly increased in the RSV infection and anti-NGF antibody groups. However, compared with the RSV infection group, synaptophysin mRNA expression was significantly decreased in the anti-NGF antibody group. We conclude that RSV infection could induce adrenal medulla cell differentiation to nerve cells by over-expression of NGF, resulting in the decreased endocrine function found in asthma progression.

TNF-alpha is a key mediator in the development of Th2 cell response to inhaled allergens induced by a viral PAMP double-stranded RNA.

BACKGROUND: Viral pathogen-associated molecular patterns, such as dsRNA, disrupt airway tolerance to inhaled allergens. Specifically, the Th2 and Th17 cell responses are induced by low-dose dsRNA and the Th1-dominant response by high-dose dsRNA. OBJECTIVE: In this model, we evaluate the role of TNF-α in the development of adaptive immune dysfunction to inhaled allergens induced by airway sensitization with dsRNA-containing allergens. METHODS: A virus-associated asthma mouse model was generated via simultaneous airway administration of ovalbumin (OVA) and low (0.1 µg) or high (10 µg) doses of polyinosine-polycytidylic acid (poly[I:C]). The effect of TNF-α on Th2 airway inflammation was evaluated using TNF-α-deficient mice and recombinant TNF-α. RESULTS: TNF-α production was enhanced by airway exposure to low and high doses of poly[I:C]. After airway sensitization with OVA plus low-dose poly[I:C], TNF-α-deficient mice exhibited less OVA-induced airway inflammation than did wild-type (WT) mice. However, this did not occur upon sensitization with high-dose poly[I:C]. In terms of T-cell response, the production of IL-4 from lung T cells after OVA challenge was enhanced by airway sensitization with OVA plus low-dose poly[I:C] in WT mice, and this phenotype was inhibited by the absence of TNF-α. Moreover, the Th2 cell response induced by sensitization with OVA plus low-dose poly[I:C], which was abolished in TNF-α-deficient mice, was restored in these mice upon addition of recombinant TNF-α. CONCLUSION: The results of this study suggest that TNF-α produced by airway exposure to low-dose dsRNA is a key mediator in the development of Th2 cell response to inhaled allergens.

Neonatal bronchial hyperresponsiveness precedes acute severe viral bronchiolitis in infants.

BACKGROUND: Respiratory syncytial virus and other respiratory tract viruses lead to common colds in most infants, whereas a minority develop acute severe bronchiolitis often requiring hospitalization. We hypothesized that such an excessive response to respiratory tract viral infection is caused by host factors reflected in pre-existing increased bronchial responsiveness. OBJECTIVE: We sought to compare bronchial responsiveness and lung function in 1-month-old neonates who later develop acute severe bronchiolitis with those who do not. METHODS: We measured infant lung function (n=402) and bronchial responsiveness to methacholine (n=363) using the raised-volume rapid thoracoabdominal compression technique before any respiratory symptoms in 1-month-old neonates from the Copenhagen Prospective Study of Asthma in Childhood birth cohort born to mothers with asthma. The children were prospectively monitored for respiratory symptoms and given a diagnosis of acute severe bronchiolitis according to a fixed algorithm. RESULTS: Thirty-four (8.5%) infants had acute severe bronchiolitis before 2 years of age, 21 (62%) were hospitalized, and 23 (67%) of the cases were associated with respiratory syncytial virus. Children who later had acute severe bronchiolitis irrespective of viral species had a 2.5-fold increased responsiveness to methacholine (provocative dose of methacholine producing a 15% decrease in transcutaneous oxygen pressure [PD(15)]) at age 1 month compared with control subjects (median PD(15) in cases vs control subjects, 0.13 vs 0.33 µmol; P=.01), whereas differences in baseline airflow were not significant for forced expiratory volume at 0.5 seconds (mean z score for cases vs control subjects, -0.18 vs -0.01; P=.36) and forced expiratory flow at 50% of forced vital capacity (mean z score for cases vs control subjects, -0.37 vs -0.09; P=.13). CONCLUSION: Bronchial hyperresponsiveness in at-risk neonates precedes acute severe bronchiolitis in response to infections with respiratory tract virus.

Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines.

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T(H)2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺iNKT cells are present in the thymic CD44⁺/⁻ NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺iNKT cells produce T(H)2 (IL-13), T(H)9 (IL-9 and IL-10), and T(H)17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T(H)17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.

HIV and asthma, is there an association?

OBJECTIVE: To evaluate whether asthma and airway hyper-responsiveness are associated with HIV infection. METHODS: We reviewed the literature on HIV-associated pulmonary diseases, pulmonary symptoms, and immune changes which may play a role in asthma. The information was analyzed comparing the pre-HAART era to the post-HAART era data. RESULTS: HIV-seropositive individuals commonly experience respiratory complaints yet it is unclear if the frequency of these complaints have changed with the initiation of HAART. Changes in pulmonary function testing and serum IgE are seen with HIV infection even in the post-HAART era. An increased prevalence of asthma among HIV-seropositive children treated with HAART has been reported. CONCLUSION: The spectrum of HIV-associated pulmonary disease has changed with the introduction of HAART. Current data is limited to determine if asthma and airway hyper-responsiveness are more common among HIV-seropositive individuals treated with HAART.

Th17 cytokines are critical for respiratory syncytial virus-associated airway hyperreponsiveness through regulation by complement C3a and tachykinins.

Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. In mice, acute RSV infection causes airway hyperresponsiveness (AHR), inflammation, and mucus hypersecretion. Infected cells induce complement activation, producing the anaphylatoxin C3a. In this paper, we show RSV-infected wild-type mice produce Th17 cytokines, a response not previously associated with viral infections. Mice deficient in the C3aR fail to develop AHR following acute RSV infection, and production of Th17 cytokines was significantly attenuated. Tachykinin production also has been implicated in RSV pathophysiology, and tachykinin receptor-null mice were similarly protected from developing AHR. These animals were also deficient in production of Th17 cytokines. Tachykinin release was absent in mice deficient in C3aR, whereas C3a levels were unchanged in tachykinin receptor-null animals. Thus, our data reveal a crucial sequence following acute RSV infection where initial C3a production causes tachykinin release, followed by activation of the IL-17A pathway. Deficiency of either receptor affords protection from AHR, identifying two potential therapeutic targets.

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness.

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.

Role of TNF-α in virus-induced airway hyperresponsiveness and neuronal M2 muscarinic receptor dysfunction.

BACKGROUND AND PURPOSE: Infections with respiratory viruses induce exacerbations of asthma, increase acetylcholine release and potentiate vagally mediated bronchoconstriction by blocking inhibitory M2 muscarinic receptors on parasympathetic neurons. Here we test whether virus-induced M2 receptor dysfunction and airway hyperresponsiveness are tumour necrosis factor-alpha (TNF-α) dependent. EXPERIMENTAL APPROACH: Guinea pigs were pretreated with etanercept or phosphate-buffered saline 24 h before intranasal infection with parainfluenza. Four days later, pulmonary inflation pressure, heart rate and blood pressure were measured. M2 receptor function was assessed by the potentiation by gallamine (an M2 receptor antagonist) of bronchoconstriction caused by electrical stimulation of the vagus nerves and measured as increased pulmonary inflation pressure. Human airway epithelial cells were infected with influenza and TNF-α concentration in supernatant was measured before supernatant was applied to human neuroblastoma cells. M2 receptor expression in these neuroblastoma cells was measured by qRT-PCR. KEY RESULTS: Influenza-infected animals were hyperresponsive to vagal stimulation but not to intravenous ACh. Gallamine did not potentiate vagally induced bronchoconstriction in virus-infected animals, indicating M2 receptor dysfunction. Etanercept prevented virus-induced airway hyperresponsiveness and M2 receptor dysfunction, without changing lung viral titres. Etanercept caused a non-significant decrease in total cells, macrophages and neutrophils in bronchoalveolar lavage. Influenza infection significantly increased TNF-α release from isolated epithelial cells, sufficient to decrease M2 receptors in neuroblastoma cells. This ability of supernatants from infected epithelial cells to inhibit M2 receptor expression was blocked by etanercept. CONCLUSIONS AND IMPLICATIONS: TNF-α is a key mediator of virus-induced M2 muscarinic receptor dysfunction and airway hyperresponsiveness.

Identification of the Mhc region as an asthma susceptibility locus in recombinant congenic mice.

Mouse models of allergic asthma are characterized by airway hyperreactivity (AHR), Th2-driven eosinophilic airway inflammation, high allergen-specific IgE (anti-OVA IgE) levels in serum, and airway remodeling. Because asthma susceptibility has a strong genetic component, we aimed to identify new asthma susceptibility genes in the mouse by analyzing the asthma phenotypes of the Leishmania major resistant (lmr) recombinant congenic (RC) strains. The lmr RC strains are derived from C57BL/6 and BALB/c intercrosses and carry congenic loci on chromosome 17 (lmr1) and 9 (lmr2) in both backgrounds. Whereas the lmr2 locus on chromosome 9 contributes to a small background-specific effect on anti-OVA IgE and AHR, the lmr1 locus on chromosome 17 mediates a strong effect on Th2-driven eosinophilic airway inflammation and background-specific effects on anti-OVA IgE and AHR. The lmr1 locus contains almost 600 polymorphic genes. To narrow down this number of candidate genes, we performed genome-wide transcriptional profiling on lung tissue from C.lmr1 RC mice and BALB/c control mice. We identified a small number of differentially expressed genes located within the congenic fragment, including a number of Mhc genes, polymorphic between BALB/c and C57Bl/6. The analysis of asthma phenotypes in the C.B10-H2b RC strain, carrying the C57Bl/6 haplotype of the Mhc locus in a BALB/c genetic background, reveals a strikingly similar asthma phenotype compared with C.lmr1, indicating that the differentially expressed genes located within the C.B10-H2b congenic fragment are the most likely candidate genes to contribute to the reduced asthma phenotypes associated with the C57Bl/6 allele of lmr1.

CXCR2 is required for neutrophilic airway inflammation and hyperresponsiveness in a mouse model of human rhinovirus infection.

Human rhinovirus (RV) infection is responsible for the majority of virus-induced asthma exacerbations. Using a mouse model of human RV infection, we sought to determine the requirement of CXCR2, the receptor for ELR-positive CXC chemokines, for RV-induced airway neutrophilia and hyperresponsiveness. Wild-type and CXCR2(-/-) mice were inoculated intranasally with RV1B or sham HeLa cell supernatant. Following RV1B infection, CXCR2(-/-) mice showed reduced airway and lung neutrophils and cholinergic responsiveness compared with wild-type mice. Similar results were obtained in mice treated with neutralizing Ab to Ly6G, a neutrophil-depleting Ab. Lungs from RV-infected, CXCR2(-/-) mice showed significantly reduced production of TNF-alpha, MIP-2/CXCL2, and KC/CXCL1 and lower expression of MUC5B compared with RV-treated wild-type mice. The requirement of TNF-alpha for RV1B-induced airway responses was tested using TNFR1(-/-) mice. TNFR1(-/-) animals displayed reduced airway responsiveness to RV1B, even when exogenous MIP-2 was added to the airways. We conclude that CXCR2 is required for RV-induced neutrophilic airway inflammation and that neutrophil TNF-alpha release is required for airway hyperresponsiveness.