Thomas R. Businga

Intestinal helminths protect in a murine model of asthma.

Underdeveloped nations are relatively protected from the worldwide asthma epidemic; the hygiene hypothesis suggests this is due to suppression of Th2-mediated inflammation by increased exposure to pathogens and their products. Although microbial exposures can promote Th2-suppressing Th1 responses, even Th2-skewing infections, such as helminths, appear to suppress atopy, suggesting an alternate explanation for these observations. To investigate whether induction of regulatory responses by helminths may counter allergic inflammation, we examined the effects of helminth infection in a murine model of atopic asthma. We chose Heligosomoides polygyrus, a gastrointestinal nematode, as the experimental helminth; this worm does not enter the lung in its life cycle. We found that H. polygyrus infection suppressed allergen-induced airway eosinophilia, bronchial hyperreactivity, and in vitro allergen-recall Th2 responses in an IL-10-dependent manner; total and OVA-specific IgE, however, were increased by worm infection. Finally, helminth-infected mice were protected against eosinophilic inflammation induced by adoptive transfer of OVA-stimulated CD4+ cells, and transfer of cells from helminth-infected/OVA-exposed mice suppressed OVA-induced eosinophilic inflammation, suggesting a role for regulatory cells. Increased CD4+CD25+Foxp3+ cells were found in thoracic lymph nodes of helminth-infected/OVA-exposed mice. Helminthic colonization appears to protect against asthma and atopic disorders; the regulatory cytokine, IL-10, may be a critical player.

CpG oligodeoxynucleotides do not require TH1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma

BACKGROUND Oligodeoxynucleotides (ODNs) containing the dinucleotide CpG in a specific sequence context (CpG-ODNs) have the ability to prevent the development of eosinophilic airway inflammation and bronchial hyperreactivity in a murine model of asthma. We have previously demonstrated that CpG-ODNs stimulate expression of the T(H1)-inducing cytokines IFN-gamma and IL-12 in a murine model of asthma and that this stimulation is associated with the protection against asthmatic inflammation. OBJECTIVE The purpose of this study was to examine whether the protection conferred by CpG-ODNs in a schistosome egg-egg antigen murine model of asthma is dependent on the induction of IFN-gamma, IL-12, or both. METHODS C57BL/6 mice were sensitized to schistosome eggs in the presence or absence of CpG-ODNs or control ODNs and then stimulated with soluble egg antigen in the airway. The protection offered by CpG-ODNs in these mice was compared with the protection induced by CpG-ODNs in IL-12 and IFN-gamma knockout mice and in mice treated with anticytokine blocking antibodies. Double-knockout mice (IL-12/IFN-gamma) were also generated and used in these studies. Determinations included airway eosinophilic inflammation and bronchial hyperreactivity to inhaled methacholine. RESULTS We found that CpG-ODNs confer protection against both airway eosinophilia and bronchial hyperreactivity in the absence of IFN-gamma or IL-12 or in the presence of both cytokines together. However, in the absence of either IL-12 or IFN-gamma, mice require 10 times as much CpG-ODNs to be protected against the induction of airway eosinophilia. The T(H2) cytokines IL-4 and IL-5 were reduced in all of the CpG-treated mice, although less in the absence of IL-12 and IFN-gamma. CONCLUSION These data indicate that CpG-ODNs prevent the generation of T(H2)-like immune responses by multiple mechanisms, which involve, but do not require, IL-12 and IFN-gamma. A direct suppressive effect of CpG-ODNs on T(H2) responses is suggested by their reduction in IFN-gamma and IL-12 knockout mice.

Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia.

Mutations in a group of genes that contribute to ciliary function cause Bardet–Biedl syndrome (BBS). Most studies of BBS have focused on primary, sensory cilia. Here, we asked whether loss of BBS proteins would also affect motile cilia lining the respiratory tract. We found that BBS genes were expressed in human airway epithelia, and BBS2 and BBS4 localized to cellular structures associated with motile cilia. Although BBS proteins were not required for ciliogenesis, their loss caused structural defects in a fraction of cilia covering mouse airway epithelia. The most common abnormality was bulges filled with vesicles near the tips of cilia. We discovered this same misshapen appearance in airway cilia from Bbs1, Bbs2, Bbs4, and Bbs6 mutant mice. The structural abnormalities were accompanied by functional defects; ciliary beat frequency was reduced in Bbs mutant mice. Previous reports suggested BBS might increase the incidence of asthma. However, compared with wild-type controls, neither airway hyperresponsiveness nor inflammation increased in Bbs2−/− or Bbs4−/− mice immunized with ovalbumin. Instead, these animals were partially protected from airway hyperresponsiveness. These results emphasize the role of BBS proteins in both the structure and function of motile cilia. They also invite additional scrutiny of motile cilia dysfunction in patients with this disease.

Immunomodulatory Effects of CpG Oligodeoxynucleotides on Established Th2 Responses

ABSTRACT CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-γ) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-γ, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8+ T lymphocytes. Although IFN-γ plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-γ was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-γ, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-γ was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-γ and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.

Modulation of murine allergic rhinosinusitis by CpG oligodeoxynucleotides

Background Allergic rhinosinusitis is characterized by eosinophilic inflammation of the upper airway, which is induced by TH‐2 cytokines. CpG oligodeoxynucleotides (ODN) are known to induce TH‐1 and to suppress TH‐2 cytokines in a variety of settings, including murine models of asthma.

Anti-Inflammatory Effects of the Neurotransmitter Agonist Honokiol in a Mouse Model of Allergic Asthma

Chronic airway inflammation is a hallmark of asthma, an immune-based disease with great societal impact. Honokiol (HNK), a phenolic neurotransmitter receptor (γ-aminobutyric acid type A) agonist purified from magnolia, has anti-inflammatory properties, including stabilization of inflammation in experimentally induced arthritis. The present study tested the prediction that HNK could inhibit the chronic inflammatory component of allergic asthma. C57BL/6 mice sensitized to and challenged with OVA had increased airway hyperresponsiveness to methacholine challenge and eosinophilia compared with naive controls. HNK-treated mice showed a reduction in airway hyperresponsiveness as well as a significant decrease in lung eosinophilia. Histopathology studies revealed a marked drop in lung inflammation, goblet cell hyperplasia, and collagen deposition with HNK treatment. Ag recall responses from HNK-treated mice showed decreased proinflammatory cytokines in response to OVA, including TNF-α–, IL-6–, Th1-, and Th17-type cytokines, despite an increase in Th2-type cytokines. Regulatory cytokines IL-10 and TGF-β were also increased. Assessment of lung homogenates revealed a similar pattern of cytokines, with a noted increase in the number of FoxP3+ cells in the lung. HNK was able to alter B and T lymphocyte cytokine secretion in a γ-aminobutyric acid type A-dependent manner. These results indicate that symptoms and pathology of asthma can be alleviated even in the presence of increased Th2 cytokines and that neurotransmitter agonists such as HNK have promise as a novel class of anti-inflammatory agents in the treatment of chronic asthma.

Oral administration of CpG-ODNs suppresses antigen-induced asthma in mice

Oligodeoxynucleotides containing CpG motifs (CpG‐ODNs) can protect against eosinophilic airway inflammation in asthma. Previously we have found that parenteral or mucosal administration of CpG‐ODNs is effective in preventing (as well as reversing established) disease. In this study, we examined the effect of oral CpG‐ODNs on the development of immune tolerance. Using an ovalbumin (OVA)‐induced murine model of asthma, we found that CpG‐ODNs, administered orally around the time of sensitization, prevented eosinophilic airway inflammation in a dose‐dependent manner. Although oral co‐administration of CpG‐ODNs with OVA (known to induce tolerance) did not significantly change the inhibition of OVA‐induced airway eosinophilia, it did modulate OVA‐specific immunoglobulin responses: oral administration of OVA alone suppressed OVA‐specific IgG1 production, but only mice that received CpG‐ODNs demonstrated enhanced levels of OVA‐specific IgG2c. Finally, we examined whether oral administration of CpG‐ODNs, alone or with OVA, could reverse established eosinophilic airway inflammation. Again, neither OVA nor CpG‐ODNs alone modulated established eosinophilic airway inflammation, but a combination of the OVA and CpG‐ODNs successfully desensitized the mice. This desensitization was associated with suppression of OVA‐specific IgE and enhancement of OVA‐specific IgG2c production. These findings provide the first indication that oral administration of CpG‐ODNs is effective in preventing and reversing antigen‐induced eosinophilic airway inflammation. CpG‐ODNs may be useful as a component of oral immunotherapy to promote tolerance in established asthma.

CaMKII Is Essential for the Proasthmatic Effects of Oxidation

Ca2+/calmodulin-dependent protein kinase (CaMKII) transduces oxidative stress into asthma-related diseases. A Breath of Fresh Air for Asthma Patients Reactive oxygen species (ROS) have a bad reputation, and rightly so. They’ve been implicated in contributing to a wide swath of diseases, including coronary heart disease, cancer, and asthma. Indeed, asthma is an increasing public health burden—affecting 8.5% of the population in the United States alone. Now Sanders et al. find that oxidative activation of the Ca2+/calmodulin-dependent protein kinase (ox-CaMKII) may respond to ROS in lung epithelium and contribute to asthma pathogenesis. The authors observed that asthma patients have enhanced activation of ox-CaMKII in bronchial epithelium, which increases in response to inhaled antigen. Then they looked in two different mouse models of allergic asthma to examine the mechanistic connection. They found that blocking CaMKII either genetically or with a small-molecule inhibitor could alleviate ROS-mediated asthma progression. These data suggest that blocking CaMKII could be a new therapeutic strategy for asthma patients. Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca2+/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl− current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.

Safety assessment of inhaled xylitol in mice and healthy volunteers

BackgroundXylitol is a 5-carbon sugar that can lower the airway surface salt concentration, thus enhancing innate immunity. We tested the safety and tolerability of aerosolized iso-osmotic xylitol in mice and human volunteers.MethodsThis was a prospective cohort study of C57Bl/6 mice in an animal laboratory and healthy human volunteers at the clinical research center of a university hospital. Mice underwent a baseline methacholine challenge, exposure to either aerosolized saline or xylitol (5% solution) for 150 minutes and then a follow-up methacholine challenge. The saline and xylitol exposures were repeated after eosinophilic airway inflammation was induced by sensitization and inhalational challenge to ovalbumin. Normal human volunteers underwent exposures to aerosolized saline (10 ml) and xylitol, with spirometry performed at baseline and after inhalation of 1, 5, and 10 ml. Serum osmolarity and electrolytes were measured at baseline and after the last exposure. A respiratory symptom questionnaire was administered at baseline, after the last exposure, and five days after exposure. In another group of normal volunteers, bronchoalveolar lavage (BAL) was done 20 minutes and 3 hours after aerosolized xylitol exposure for levels of inflammatory markers.ResultsIn naïve mice, methacholine responsiveness was unchanged after exposures to xylitol compared to inhaled saline (p = 0.49). There was no significant increase in Penh in antigen-challenged mice after xylitol exposure (p = 0.38). There was no change in airway cellular response after xylitol exposure in naïve and antigen-challenged mice. In normal volunteers, there was no change in FEV1 after xylitol exposures compared with baseline as well as normal saline exposure (p = 0.19). Safety laboratory values were also unchanged. The only adverse effect reported was stuffy nose by half of the subjects during the 10 ml xylitol exposure, which promptly resolved after exposure completion. BAL cytokine levels were below the detection limits after xylitol exposure in normal volunteers.ConclusionsInhalation of aerosolized iso-osmotic xylitol was well-tolerated by naïve and atopic mice, and by healthy human volunteers.

Tim-1 regulates Th2 responses in an airway hypersensitivity model

T‐cell immunoglobulin mucin‐1 (Tim‐1) is a transmembrane protein postulated to be a key regulator of Th2‐type immune responses. This hypothesis is based in part upon genetic studies associating Tim‐1 polymorphisms in mice with a bias toward airway hyperrespon‐siveness (AHR) and the development of Th2‐type CD4+ T cells. Tim‐1 expressed by Th2 CD4+ T cells has been proposed to function as a co‐stimulatory molecule. Tim‐1 is also expressed by B cells, macrophages, and dendritic cells, but its role in responses by these cell types has not been firmly established. Here, we generated Tim‐1‐deficient mice to determine the role of Tim‐1 in a murine model of allergic airway disease that depends on the development and function of Th2 effector cells and results in the generation of AHR. We found antigen‐driven recruitment of inflammatory cells into airways is increased in Tim‐1‐deficient mice relative to WT mice. In addition, we observed increased antigen‐specific cytokine production by splenocytes from antigen‐sensitized Tim‐1‐deficient mice relative to those from controls. These data support the conclusion that Tim‐1 functions in pathways that suppress recruitment of inflammatory cells into the airways and the generation or activity of CD4+ T cells.