Junbae Jee

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

BackgroundThe Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.MethodsExpression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.ResultsWe found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.ConclusionsCFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.

Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant.

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.

IKKβ in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites

Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKβ) in IECs (IKKβΔIEC), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKβΔIEC mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKβΔIEC mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKβΔIEC mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKβΔIEC mice, but did not affect IgA Ab responses. In summary, we show that IKKβ in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.

Neutrophils negatively regulate induction of mucosal IgA responses after sublingual immunization.

Induction of mucosal immunoglobulin-A (IgA) capable of providing a first line of defense against bacterial and viral pathogens remains a major goal of needle-free vaccines given via mucosal routes. Innate immune cells are known to play a central role in induction of IgA responses by mucosal vaccines, but the relative contribution of myeloid cell subsets to these responses has not firmly been established. Using an in vivo model of sublingual vaccination with Bacillus anthracis edema toxin (EdTx) as adjuvant, we examined the role of myeloid cell subsets for mucosal secretory IgA responses. Sublingual immunization of wild-type mice resulted in a transient increase of neutrophils in sublingual tissues and cervical lymph nodes. These mice later developed Ag-specific serum IgG responses, but not serum or mucosal IgA. Interestingly, EdTx failed to increase neutrophils in sublingual tissues and cervical lymph nodes of IKKβΔMye mice, and these mice developed IgA responses. Partial depletion of neutrophils before immunization of wild-type mice allowed the development of both mucosal and serum IgA responses. Finally, co-culture of B cells with neutrophils from either wild-type or IKKβΔMye mice suppressed secretion of IgA, but not IgM or IgG. These results identify a new role for neutrophils as negative regulators of IgA responses.

Routes of Allergic Sensitization and Myeloid Cell IKKβ Differentially Regulate Antibody Responses and Allergic Airway Inflammation in Male and Female Mice

Gender influences the incidence and/or the severity of several diseases and evidence suggests a higher rate of allergy and asthma among women. Most experimental models of allergy use mice sensitized via the parenteral route despite the fact that the mucosal tissues of the gastrointestinal and respiratory tracts are major sites of allergic sensitization and/or allergic responses. We analyzed allergen-specific Ab responses in mice sensitized either by gavage or intraperitoneal injection of ovalbumin together with cholera toxin as adjuvant, as well as allergic inflammation and lung functions following subsequent nasal challenge with the allergen. Female mice sensitized intraperitoneally exhibited higher levels of serum IgE than their male counterparts. After nasal allergen challenge, these female mice expressed higher Th2 responses and associated inflammation in the lung than males. On the other hand, male and female mice sensitized orally developed the same levels of allergen-specific Ab responses and similar levels of lung inflammation after allergen challenge. Interestingly, the difference in allergen-specific Ab responses between male and female mice sensitized by the intraperitoneal route was abolished in IKKβΔMye mice, which lack IKKβ in myeloid cells. In summary, the oral or systemic route of allergic sensitization and IKKβ signaling in myeloid cells regulate how the gender influences allergen-specific responses and lung allergic inflammation.