Modification of the inflammatory response to allergen challenge after exposure to bacterial lipopolysaccharide

The potential role of respiratory infections in altering the development of atopy and asthma is complex. Infections have been suggested to be effective in preventing the induction of T-helper 2-polarized allergen-specific immunity in early life, but also to exacerbate asthma in older, sensitized individuals. The mechanism(s) underlying these effects are poorly defined. The aim of this work was to determine the influence of lipopolysaccharide (LPS) exposure on the development of sensitization to allergen and the response to allergen challenge in vivo. Piebald-Virol-Glaxo rats were exposed to a single aerosol of LPS 1 d before or 1, 2, 4, 6, 8, or 10 d after sensitization with ovalbumin (OVA). On Day 11 animals were exposed to 1% OVA and responses to allergen were measured 24 h later, monitoring inflammatory cell influx and microvascular leakage into bronchoalveolar lavage (BAL) fluid as well as pulmonary responses to methacholine using the forced oscillation technique. Histologic analysis was included to complement the BAL results. Single aerosol exposure to LPS 1 d before and up to 4 d after intraperitoneal injection of OVA protected against the development of OVA-specific immunoglobulin (Ig) E. LPS exposure 6, 8, or 10 d after sensitization further exacerbated the OVA-induced cellular influx, resulting in neutrophilia and increased Evans Blue dye leakage with no effect on serum IgE levels. In addition, LPS abolished the OVA-induced hyperresponsiveness in sensitized animals when given 18 h after OVA challenge. This study demonstrates that exposure to LPS can modify the development of allergic inflammation in vivo by two independent mechanisms. Exposure early in the sensitization process, up to Day 6 after exposure to allergen, prevented allergen sensitization. Exposure to LPS after allergen challenge in sensitized animals abolished the hyperresponsiveness and modified the inflammatory cell influx characteristic of late-phase response to allergen

Muscarinic blockade of methacholine induced airway and parenchymal lung responses in anaesthetised rats

Background-It has previously been shown that M-1, cholinergic receptors are involved in the parenchymal response to inhaled methacholine in puppies using the M-1, selective antagonist pirenzepine. Although M-3, receptors are responsible for acetylcholine induced bronchoconstriction in isolated rat lung, the role of M-1, receptors has not been determined in the rat in vivo

IFN-λ and IgE-mediated allergic disease: A potential future role?

Reduced early microbial exposure has become a leading candidate to explain the rise in allergic disease, and research has focused on studying the interaction between the developing immune system and the microbial environment. However, despite intense interest, the pathways that lead to dysregulation of the immune system in allergic disease are still poorly understood. The newly described type III IFN-λ molecules were initially shown to exhibit antiviral activity, but these molecules are also likely to have an important role to play in the immune-epithelial interface, given their immunomodulatory functions and restricted receptor expression to immune and epithelial cells. Previous studies on the role of IFN-λ in allergic disease have been limited to allergic asthma. More recently, a genetic variation flanking IL28B encoding IFN-λ3 has been associated with allergic disease. Here, we examine this family and suggest how IFN-λ may be an important player in allergic disease

TLR4 Polymorphisms mediate impaired responses to respiratory Syncytial virus and lipopolysaccharide

Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the 299Gly and 399Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence (299Asp/399Thr), or the 299Gly or 399Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing 299Gly or 399Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-κB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-α. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus. The TLR4 gene which encodes the receptor recognizing bacterial LPS is highly polymorphic. Two cosegregating missense polymorphisms have been identified in the TLR4 gene at minor allele frequencies between 8 and 10% in Caucasian populations (1), which result, respectively, in aspartic acid to glycine substitution at position 299 (Asp299Gly) and threonine to isoleucine substitution at position 399 (Thr399Ile) in the receptor protein. These polymorphisms have been linked with blunted airway (2) and systemic inflammatory responses (3) to inhaled LPS in adults and attenuated LPS-induced responses in primary airway epithelial cells (2). Moreover, they have also been associated with increased risk for severe respiratory syncytial virus (RSV)3 bronchiolitis (4, 5, 6) in previously healthy infants. In this regard, studies in mice have shown that TLR4 recognizes not only bacterial but also viral motifs, including the F protein of RSV (7) and, moreover, TLR4-deficient mice infected with RSV show impaired pulmonary cellular responses and delayed viral clearance (7, 8). The primary target for RSV in humans is airway respiratory epithelial cells (9) and the first link of defense against the virus involves epithelial production of a plethora of cytokines, chemokines, and immunomodulatory mediators (9, 10). Although RSV infects >50% of infants during their first year of life, only a small percentage (1–3%) of RSV-infected infants develop infections of sufficient severity to require hospitalization (11). It has been reported (12) that the ability of individual infants to increase TLR4 expression on blood monocytes during acute RSV bronchiolitis is inversely proportional to the degree of ensuing hypoxia, suggesting that the ability to up-regulate TLR4 is closely linked to disease severity. In addition, RSV has also been shown to up-regulate TLR4 in airway epithelial cells in vitro (13) and we have previously reported similar TLR4 up-regulation in the upper airways of infected infants (14). It has also recently been reported that these common TLR4 mutations are associated with an increased risk of severe RSV bronchiolitis (6) and increased risk for hospitalization (4) in previously healthy infants. However, the mechanisms by which TLR4 polymorphisms might impair host defense against RSV are currently unknown. In this present study, we have addressed this issue by comparing the in vitro response to RSV and LPS in bronchial epithelial cells transfected with constructs containing each allele of the TLR4 Asp299Gly (299Asp or 299Gly) and Thr399Ile (399Thr or 399Ile) polymorphisms, measuring markers of cellular immunity. We further extended the studies to investigate the effects of these TLR4 polymorphisms on responses of PBMC to these stimuli

Supplementary Material for: Epigenetic Regulation in Early Childhood: A Miniaturized and Validated Method to Assess Histone Acetylation

<b><i>Introduction:</i></b> Chronic inflammatory diseases including allergies and asthma are the result of complex interactions between genes and environmental factors. Epigenetic mechanisms comprise a set of biochemical reactions that regulate gene expression. In order to understand the cause-effect relationship between environmental exposures and disease development, methods capable of assessing epigenetic regulation (also) in large cohorts are needed. <b><i>Methods:</i></b> For this purpose, we developed and evaluated a miniaturized chromatin immunoprecipitation (ChIP) assay allowing for a cost-effective assessment of histone acetylation of candidate genes in a quantitative fashion. This method was then applied to assess H3 and H4 histone acetylation changes in cord blood (CB) samples from an established cohort of Australian children exposed in the fetal period to either very low or very high levels of maternal folate. <b><i>Results:</i></b> Our ChIP assay was validated for a minimum requirement of 1 × 10⁵ target cells (e.g. CD4+ T cells). Very high levels of maternal folate were significantly associated with increased H3/H4 acetylation at <i>GATA3</i> and/or <i>IL9</i> promoter regions in CD4+ T cells in CB. <b><i>Conclusion:</i></b> We developed a ChIP method allowing reliable assessment of H3/H4 acetylation using 1 × 10⁵ cells only. Practical application of this assay demonstrated an association between high maternal folate exposure and increased histone acetylation, corresponding to a more transcriptionally permissive chromatin status in the promoter regions of some Th2-related genes