??T cells suppress inflammation and disease during rhinovirus-induced asthma exacerbations.
ABSTRACT: Most asthma exacerbations are triggered by virus infections, the majority being caused by human rhinoviruses (RV). In mouse models, ??T cells have been previously demonstrated to influence allergen-driven airways hyper-reactivity (AHR) and can have antiviral activity, implicating them as prime candidates in the pathogenesis of asthma exacerbations. To explore this, we have used human and mouse models of experimental RV-induced asthma exacerbations to examine ??T-cell responses and determine their role in the immune response and associated airways disease. In humans, airway ??T-cell numbers were increased in asthmatic vs. healthy control subjects during experimental infection. Airway and blood ??T-cell numbers were associated with increased airways obstruction and AHR. Airway ??T-cell number was also positively correlated with bronchoalveolar lavage (BAL) virus load and BAL eosinophils and lymphocytes during RV infection. Consistent with our observations of RV-induced asthma exacerbations in humans, infection of mice with allergic airways inflammation increased lung ??T-cell number and activation. Inhibiting ??T-cell responses using anti-??TCR (anti-??T-cell receptor) antibody treatment in the mouse asthma exacerbation model increased AHR and airway T helper type 2 cell recruitment and eosinophilia, providing evidence that ??T cells are negative regulators of airways inflammation and disease in RV-induced asthma exacerbations.
Project description:Rhinovirus (RV) exposure has been implicated in childhood development of wheeze evoking asthma and exacerbations of underlying airways disease. Studies such as the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC) and Childhood Origins of ASThma (COAST) have identified RV as a pathogen inducing severe respiratory disease. RVs also modulate airway hyperresponsiveness (AHR), a key characteristic of such diseases. Although potential factors underlying mechanisms by which RV induces AHR have been postulated, the precise mechanisms of AHR following RV exposure remain elusive.A challenge to RV-related research stems from inadequate models for study. While human models raise ethical concerns and are relatively difficult in terms of subject recruitment, murine models are limited by susceptibility of infection to the relatively uncommon minor group (RV-B) serotypes, strains that are generally associated with infrequent clinical respiratory virus infections. Although a transgenic mouse strain that has been developed has enhanced susceptibility for infection with the common major group (RV-A) serotypes, few studies have focused on RV in the context of allergic airways disease rather than understanding RV-induced AHR. Recently, the receptor for the virulent RV-C CDHR3, was identified, but a dearth of studies have examined RV-C-induced effects in humans.Currently, the mechanisms by which RV infections modulate airway smooth muscle (ASM) shortening or excitation-contraction coupling remain elusive. Further, only one study has investigated the effects of RV on bronchodilatory mechanisms, with only speculation as to mechanisms underlying RV-mediated modulation of bronchoconstriction.
Project description:Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-gamma. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.
Project description: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.
Project description:<h4>Background</h4>Exacerbations of asthma are linked to significant decline in lung function and are often poorly controlled by corticosteroid treatment. Clinical investigations indicate that viral and bacterial infections play crucial roles in the onset of steroid-resistant inflammation and airways hyperresponsiveness (AHR) that are hallmark features of exacerbations. We have previously shown that interferon ? (IFN?) and lipopolysaccharide (LPS) cooperatively activate pulmonary macrophages and induce steroid-resistant airway inflammation and AHR in mouse models. Furthermore, we have established a mouse model of respiratory syncytial virus (RSV)-induced exacerbation of asthma, which exhibits macrophage-dependent, steroid-resistant lung disease. Emerging evidence has demonstrated a key role for bromo- and extra-terminal (BET) proteins in the regulation of inflammatory gene expression in macrophages. We hypothesised that BET proteins may be involved in the regulation of AHR and airway inflammation in our steroid-resistant exacerbation models.<h4>Methodology/principal findings</h4>We investigated the effects of a BET inhibitor (I-BET-762) on the development of steroid-resistant AHR and airway inflammation in two mouse models. I-BET-762 administration decreased macrophage and neutrophil infiltration into the airways, and suppressed key inflammatory cytokines in both models. I-BET treatment also suppressed key inflammatory cytokines linked to the development of steroid-resistant inflammation such as monocyte chemoattractant protein 1 (MCP-1), keratinocyte-derived protein chemokine (KC), IFN?, and interleukin 27 (IL-27). Attenuation of inflammation was associated with suppression of AHR.<h4>Conclusions/significance</h4>Our results suggest that BET proteins play an important role in the regulation of steroid-resistant exacerbations of airway inflammation and AHR. BET proteins may be potential targets for the development of future therapies to treat steroid-resistant inflammatory components of asthma.
Project description:Surface major histocompatibility complex class I-related chain (MIC) A and B molecules are increased by IL-15 and have a role in the activation of natural killer group 2 member D-positive natural killer and CD8 T cells. MICA and MICB also exist in soluble forms (sMICA and sMICB). Rhinoviruses (RVs) are the major cause of asthma exacerbations, and IL-15 levels are decreased in the airways of subjects with asthma. The role of MIC molecules in immune responses in the lung has not been studied. Here, we determine the relationship between MICA and MICB and RV infection in vitro in respiratory epithelial cells and in vivo in healthy subjects and subjects with asthma.Surface MICA and MICB, as well as sMICA and sMICB, in respiratory epithelial cells were measured in vitro in response to RV infection and exposure to IL-15. Levels of sMICA and sMICB in serum, sputum, and BAL were measured and correlated with blood and bronchoalveolar immune cells in healthy subjects and subjects with asthma before and during RV infection.RV increased MICA and MICB in vitro in epithelial cells. Exogenous IL-15 upregulated sMICB levels in RV-infected epithelial cells. Levels of sMICB molecules in serum were increased in healthy subjects compared with subjects with stable asthma. Following RV infection, airway levels of sMIC are upregulated, and there are positive correlations between sputum MICB levels and the percentage of bronchoalveolar natural killer cells in healthy subjects but not subjects with asthma.RV infection induces MIC molecules in respiratory epithelial cells in vitro and in vivo. Induction of MICB molecules is impaired in subjects with asthma, suggesting these molecules may have a role in the antiviral immune response to RV infections.
Project description:Rhinovirus (RV) infection is involved in acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). RV primarily infects upper and lower airway epithelium. Immunoproteasomes (IP) are proteolytic machineries with multiple functions including the regulation of MHC class I antigen processing during viral infection. However, the role of IP in RV infection has not been explored. We sought to investigate the expression and function of IP during airway RV infection. Primary human tracheobronchial epithelial (HTBE) cells were cultured at air-liquid interface (ALI) and treated with RV16, RV1B, or interferon (IFN)-? in the absence or presence of an IP inhibitor (ONX-0914). IP gene (i.e. LMP2) deficient mouse tracheal epithelial cells (mTECs) were cultured for the mechanistic studies. LMP2-deficient mouse model was used to define the in vivo role of IP in RV infection. IP subunits LMP2 and LMP7, antiviral genes MX1 and OAS1 and viral load were measured. Both RV16 and RV1B significantly increased the expression of LMP2 and LMP7 mRNA and proteins, and IFN-? mRNA in HTBE cells. ONX-0914 down-regulated MX1 and OAS1, and increased RV16 load in HTBE cells. LMP2-deficient mTECs showed a significant increase in RV1B load compared with the wild-type (WT) cells. LMP2-deficient (compared with WT) mice increased viral load and neutrophils in bronchoalveolar lavage (BAL) fluid after 24 h of RV1B infection. Mechanistically, IFN-? induction by RV infection contributed to LMP2 and LMP7 up-regulation in HTBE cells. Our data suggest that IP are induced during airway RV infection, which in turn may serve as an antiviral and anti-inflammatory mechanism.
Project description:Background:Asthma exacerbations are associated with the recruitment of neutrophils to the lungs. These cells release proteases and mediators, many of which act at G protein-coupled receptors (GPCRs) that couple via Gq to promote bronchoconstriction and inflammation. Common asthma therapeutics up-regulate expression of the regulator of G protein signalling (RGS), RGS2. As RGS2 reduces signaling from Gq-coupled GPCRs, we have defined role(s) for this GTPase-activating protein in an acute neutrophilic model of lung inflammation. Methods:Wild type and Rgs2 -/- C57Bl6 mice were exposed to nebulized lipopolysaccharide (LPS). Lung function (respiratory system resistance and compliance) was measured using a SCIREQ flexivent small animal ventilator. Lung inflammation was assessed by histochemistry, cell counting and by cytokine and chemokine expression in bronchoalveolar lavage (BAL) fluid. Results:Lipopolysaccharide inhalation induced transient airways hyperreactivity (AHR) and neutrophilic lung inflammation. While AHR and inflammation was greatest 3 h post-LPS exposure, BAL neutrophils persisted for 24 h. At 3 h post-LPS inhalation, multiple inflammatory cytokines (CSF2, CSF3, IL6, TNF) and chemokines (CCL3, CCL4, CXCL1, CXCL2) were highly expressed in the BAL fluid, prior to declining by 24 h. Compared to wild type counterparts, Rgs2 -/- mice developed significantly greater airflow resistance in response to inhaled methacholine (MCh) at 3 h post-LPS exposure. At 24 h post-LPS exposure, when lung function was recovering in the wild type animals, MCh-induced resistance was increased, and compliance decreased, in Rgs2 -/- mice. Thus, Rgs2 -/- mice show AHR and stiffer lungs 24 h post-LPS exposure. Histological markers of inflammation, total and differential cell counts, and major cytokine and chemokine expression in BAL fluid were similar between wild type and Rgs2 -/- mice. However, 3 and 24 h post-LPS exposure, IL12B expression was significantly elevated in BAL fluid from Rgs2 -/- mice compared to wild type animals. Conclusions:While Rgs2 is bronchoprotective in acute neutrophilic inflammation, no clear anti-inflammatory effect was apparent. Nevertheless, elevated IL12B expression in Rgs2 -/- animals raises the possibility that RGS2 could dampen Th1 responses. These findings indicate that up-regulation of RGS2, as occurs in response to inhaled corticosteroids and long-acting ?2-adrenoceptor agonists, may be beneficial in acute neutrophilic exacerbations of airway disease, including asthma.
Project description:The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if Tollip regulates the airway inflammatory response to RV infection in vivo under IL-13 and IL-33 treatment. Following IL-13, IL-33, and RV treatment, Tollip-deficient (vs. -sufficient) HTBE cells produced excessive IL-8, accompanied by decreased sST2 production but increased IRAK1 activation. IL-8 production following IL-13/IL-33/RV exposure was markedly attenuated in IRAK1-deficient HTBE cells, as well as in Tollip KO HTBE cells treated with an IRAK1 inhibitor or a recombinant sST2 protein. Tollip KO (vs. wild-type) mice developed exaggerated airway neutrophilic responses to RV in the context of IL-13 and IL-33 treatment. Collectively, these data demonstrate that Tollip restricts excessive IL-8 production in type 2 cytokine-exposed human airways during RV infection by promoting sST2 production and inhibiting IRAK1 activation. sST2 and IRAK1 may be therapeutic targets for attenuating excessive neutrophilic airway inflammation in asthma, especially during RV infection.
Project description:BACKGROUND:Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV) to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome) and examine the changes during rhinovirus (RV) infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood. METHODS:Nasal airway secretions were obtained from children (?3 yrs. old) during PCR-confirmed RV infections (n = 10) and age-matched controls (n = 10). Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC) differentiated at air-liquid interface (ALI). Bioinformatics tools were used to determine the unified (nasal and bronchial) signature airway secretory miRNAome and changes during RV infection in children. RESULTS:Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV. CONCLUSIONS:Comparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway secretion of EVs containing miR-155, which is predicted in silico to regulate antiviral immunity. Further characterization of the airway secretory microRNAome during health and disease may lead to completely new strategies to treat and monitor respiratory conditions in all ages.
Project description:BACKGROUND:Macrophages (M?) can be M1/M2 polarized by Th1/2 signals, respectively. M2-like M? are thought to be important in asthma pathogenesis, and M1-like in anti-infective immunity, however their roles in virus-induced asthma exacerbations are unknown. Our objectives were (i) to assess polarised M? phenotype responses to rhinovirus (RV) infection in vitro and (ii) to assess M? phenotypes in healthy subjects and people with asthma before and during experimental RV infection in vivo. METHODS:We investigated characteristics of polarized/unpolarized human monocyte-derived M? (MDM, from 3-6 independent donors) in vitro and evaluated frequencies of M1/M2-like bronchoalveolar lavage (BAL) M? in experimental RV-induced asthma exacerbation in 7 healthy controls and 17 (at baseline) and 18 (at day 4 post infection) people with asthma. FINDINGS:We observed in vitro: M1-like but not M2-like or unpolarized MDM are potent producers of type I and III interferons in response to RV infection (P<0.0001), and M1-like are more resistant to RV infection (P<0.05); compared to M1-like, M2-like MDM constitutively produced higher levels of CCL22/MDC (P = 0.007) and CCL17/TARC (P<0.0001); RV-infected M1-like MDM were characterized as CD14+CD80+CD197+ (P = 0.002 vs M2-like, P<0.0001 vs unpolarized MDM). In vivo we found reduced percentages of M1-like CD14+CD80+CD197+ BAL M? in asthma during experimental RV16 infection compared to baseline (P = 0.024). INTERPRETATION:Human M1-like BAL M? are likely important contributors to anti-viral immunity and their numbers are reduced in patients with allergic asthma during RV-induced asthma exacerbations. This mechanism may be one explanation why RV-triggered clinical and pathologic outcomes are more severe in allergic patients than in healthy subjects. FUNDING:ERC FP7 Advanced grant 233015, MRC Centre Grant G1000758, Asthma UK grant 08-048, NIHR Biomedical Research Centre funding scheme, NIHR BRC Centre grant P26095, the Predicta FP7 Collaborative Project grant 260895, RSF grant 19-15-00272, Megagrant No 14.W03.31.0024.