Lungs Of Allergic Mice Research Articles

TXA2 attenuates allergic lung inflammation through regulation of Th2, Th9, and Treg differentiation.

In lung, thromboxane A2 (TXA2) activates the TP receptor to induce proinflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased approximately 2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naive CD4+ T cell differentiation to Th9 cells and IL-9 production were inhibited dose-dependently by TXA2 in vitro. TP receptor-deficient mice had an approximately 2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared with wild-type mice. Naive CD4+ T cells from TP-deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared with CD4+ T cells from wild-type mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, proinflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.

Aerosol Inhalation of Heat-Killed Clostridium butyricum CGMCC0313-1 Alleviates Allergic Airway Inflammation in Mice.

Epidemiological studies have shown that exposure to beneficial microorganisms can reduce the risk of asthma, but the clinical use of live probiotics is controversial due to the risk of infection. As heat-killed probiotics can also exhibit immunomodulatory activity, this study is aimed at investigating whether heat-killed Clostridium butyricum (HKCB) CGMCC0313-1 could reduce allergic airway inflammation in an ovalbumin-induced mouse model. Mice received aerosol inhalation of HKCB, oral administration of HKCB, or oral administration of live Clostridium butyricum (CB) during sensitization. Bronchoalveolar lavage fluid cell number, histology, and levels of the cytokines interferon-gamma and IL-4, the autophagy-related proteins LC3B, Beclin1, and p62, and members of the nuclear factor kappa B (NF-κB)/NLRP3 inflammasome signaling pathway were examined. Our results demonstrated that aerosol inhalation of HKCB, oral HKCB administration, and oral live CB administration alleviated allergic airway inflammation and mucus secretion in allergic mice. Aerosol inhalation of HKCB was the most effective method; it restored the Th1/Th2 balance, ameliorated autophagy, and inhibited the NF-κB/NLRP3 inflammasome signaling pathway in the lungs of allergic mice. Thus, aerosol inhalation of HKCB could be a promising strategy for the prevention or treatment of asthma.

Intranasal delivery of allergen in a nanoemulsion adjuvant inhibits allergen-specific reactions in mouse models of allergic airway disease.

Atopic diseases are an increasing problem that involve both immediate hypersensitivity reactions mediated by IgE and unique cellular inflammation. Many forms of specific immunotherapy involve the administration of allergen to suppress allergic immune responses but are focused on IgE-mediated reactions. In contrast, the effect of allergen-specific immunotherapy on allergic inflammation is complex, not entirely consistent and not well understood. We have previously demonstrated the ability of allergen administered in a nanoemulsion (NE) mucosal adjuvant to suppress IgE-mediated allergic responses and protect from allergen challenge in murine food allergy models. This activity was associated with decreases in allergen-specific IL-10 and reductions in allergic cytokines and increases in regulatory T cells. Here, we extend these studies to using 2 distinct models, the ovalbumin (OVA) and cockroach (CRA) models of allergic airway disease, which are based predominantly on allergic inflammation. Acute or chronic allergic airway disease was induced in mice using ovalbumin and cockroach allergen models. Mice received three therapeutic immunizations with allergen in NE, and reactivity to airway challenge was determined. Therapeutic immunization with cockroach or OVA allergen in NE markedly reduced pathology after airway challenge. The 2 models demonstrated protection from allergen challenge-induced pathology that was associated with suppression of Th2-polarized immune responses in the lung. In addition, the reduction in ILC2 numbers in the lungs of allergic mice along with reduction in epithelial cell alarmins, IL-25 and IL-33, suggests an overall change in the lung immune environment induced by the NE immunization protocol. These results demonstrate that suppression of allergic airway inflammation and bronchial hyper-reactivity can be achieved using allergen-specific immunotherapy without significant reductions in allergen-specific IgE and suggest that ILC2 cells may be critical targets for this activity.

Lung Regulatory T Cells Express Adiponectin Receptor 1: Modulation by Obesity and Airway Allergic Inflammation.

Regulatory T cells (Tregs) decrease in the adipose tissue upon weight gain, contributing to persistent low-grade inflammation in obesity. We previously showed that adipose tissue Tregs express the adiponectin receptor 1 (AdipoR1); however, the expression in lung Tregs is still unknown. Here, we aimed to determine whether Helios+ and Helios− Treg subsets expressed AdipoR1 in the lungs of obese mice and whether different obesity grades affected the expression upon allergic lung inflammation. For diet-induced obesity (DIO), mice were fed a high-fat diet (HFD) for up to 15 weeks (overweight), 21 weeks (obesity), and 26 weeks (morbid obesity). Overweight and morbidly obese mice were sensitized and challenged with ovalbumin (OVA) to induce allergic lung inflammation. The AdipoR1 expression was reduced significantly in the lung Helios+ and Helios− Tregs of obese mice compared with lean mice. Airway allergic inflammation showed reduced AdipoR1 expression in lung Foxp3+ Tregs. Obesity significantly exacerbated the eosinophilic airway inflammation and reduced the number of Helios+ Tregs in lung and adipose tissue in the obesity-associated asthma model. Upon further weight gain, AdipoR1-expressing Tregs in the lungs of allergic mice were increased, whereas AdipoR1-expressing Tregs in adipose tissue were reduced. These data suggest that obesity-associated adipose tissue inflammation may exacerbate allergic inflammation by downregulating the AdipoR1+ Tregs in the lungs.

Defense mechanisms during respiratory infections in hosts with underlying asthma

Abstract Influenza can often be complicated by superinfections by bacteria such as Streptococcus pneumoniae. While these opportunistic bacterial pathogens do co-infect during seasonal influenza epidemics, the relationship is exaggerated during pandemic influenza virus infections as documented during all four previous influenza pandemics. Approximately 29–55% of hospitalized patients with Swine Flu with severe disease had a bacterial co-infection; however, asthmatics that were hospitalized during this influenza pandemic were less likely to have co-infections. We built a mouse model that recapitulated these intriguing epidemiologic findings that underlying asthma protected the host from severe morbidity and mortality associated with IAV and S. pneumoniae co-infection. These mice had significantly reduced bacterial load in the lungs, increased eosinophils and macrophages in the airways, but reduced tissue inflammation and scarring compared to non-allergic co-infected mice. We hypothesized that TH2-biased programming of eosinophils within the lungs of allergic mice heightened their immune responses to invading respiratory pathogens. We found that allergen sensitization and IAV exposure activated eosinophils marked by upregulation of CD69, ICAM-1, and IL-5Rα, maintained their viability, and enhanced bacterial uptake through receptor-mediated phagocytosis. Activated eosinophils prevented IAV-induced cytopathology on respiratory epithelia and were able to inhibit the growth and kill S. pneumoniae without undergoing cytolysis. Our data suggest a novel pathway by which eosinophils in hosts with asthma may protect the host from severe diseases induced by respiratory pathogens by inhibiting viral-bacterial synergy.

Schistosoma japonicum peptide SJMHE1 suppresses airway inflammation of allergic asthma in mice.

Helminths and their products can shape immune responses by modulating immune cells, which are dysfunctional in inflammatory diseases such as asthma. We previously identified SJMHE1, a small molecule peptide from the HSP60 protein of Schistosoma japonicum. SJMHE1 can inhibit delayed‐type hypersensitivity and collagen‐induced arthritis in mice. In the present study, we evaluated this peptide's potential intervention effect and mechanism on ovalbumin‐induced asthma in mice. SJMHE1 treatment suppressed airway inflammation in allergic mice, decreased the infiltrating inflammatory cells in the lungs and bronchoalveolar lavage fluid, modulated the production of pro‐inflammatory and anti‐inflammatory cytokines in the splenocytes and lungs of allergic mice, reduced the percentage of Th2 cells and increased the proportion of Th1 and regulatory T cells (Tregs). At the same time, Foxp3 and T‐bet expression increased, and GATA3 and RORγt decreased in the lungs of allergic mice. We proved that SJMHE1 can interrupt the development of asthma by diminishing airway inflammation in mice. The down‐regulation of Th2 response and the up‐regulation of Th1 and Tregs response may contribute to the protection induced by SJMHE1 in allergic mice. SJMHE1 can serve as a novel therapy for asthma and other allergic or inflammatory diseases.

Conjugated bile acids attenuate allergen-induced airway inflammation and hyperresponsiveness by inhibiting UPR transducers.

Conjugated bile acids (CBAs), such as tauroursodeoxycholic acid (TUDCA), are known to resolve the inflammatory and unfolded protein response (UPR) in inflammatory diseases, such as asthma. Whether CBAs exert their beneficial effects on allergic airway responses via 1 arm or several arms of the UPR, or alternatively through the signaling pathways for conserved bile acid receptor, remains largely unknown. We used a house dust mite-induced (HDM-induced) murine model of asthma to evaluate and compare the effects of 5 CBAs and 1 unconjugated bile acid in attenuating allergen-induced UPR and airway responses. Expression of UPR-associated transcripts was assessed in airway brushings from human patients with asthma and healthy subjects. Here we show that CBAs, such as alanyl β-muricholic acid (AβM) and TUDCA, significantly decreased inflammatory, immune, and cytokine responses; mucus metaplasia; and airway hyperresponsiveness, as compared with other CBAs in a model of allergic airway disease. CBAs predominantly bind to activating transcription factor 6α (ATF6α) compared with the other canonical transducers of the UPR, subsequently decreasing allergen-induced UPR activation and resolving allergic airway disease, without significant activation of the bile acid receptors. TUDCA and AβM also attenuated other HDM-induced ER stress markers in the lungs of allergic mice. Quantitative mRNA analysis of airway epithelial brushings from human subjects demonstrated that several ATF6α-related transcripts were significantly upregulated in patients with asthma compared with healthy subjects. Collectively, these results demonstrate that CBA-based therapy potently inhibits the allergen-induced UPR and allergic airway disease in mice via preferential binding of the canonical transducer of the UPR, ATF6α. These results potentially suggest a novel avenue to treat allergic asthma using select CBAs.

MTOR-Mediated Autophagy Is Involved in the Protective Effect of Ketamine on Allergic Airway Inflammation.

Unresolved inflammation underpins the pathogenesis of allergic airway diseases, such as asthma. Ketamine, accepted as a promising therapy for resistant asthma, has been demonstrated to attenuate allergic airway inflammation. However, the anti-inflammatory mechanism by ketamine in this setting is largely unknown. We aimed to investigate whether autophagy was involved in the protective effect of ketamine on allergic airway inflammation. Female C57BL/6 mice were sensitized to ovalbumin (OVA) and treated with ketamine at 25, 50, or 100 mg/kg prior to OVA challenge. In this model, the pulmonary morphological findings and airway inflammation were significantly inhibited at 50 mg/kg but not at 25 or 100 mg/kg. Moreover, 50 mg/kg ketamine abrogated the increased concentrations of inflammatory cytokines in bronchoalveolar lavage fluid (BALF) of allergic mice, as well as activated the expression of phosphorylated mammalian target of rapamycin (p-MTOR) and inhibited autophagy in allergic mice. To confirm whether the effect of 50 mg/kg ketamine on asthma was mediated by inhibiting autophagy, rapamycin was administered to mice sensitized to OVA and exposed to 50 mg/kg ketamine. All of the effect of 50 mg/kg ketamine was reversed by rapamycin treatment, including increased p-MTOR and decreased autophagy. Taken together, the present study demonstrates that 50 mg/kg ketamine inhibits allergic airway inflammation by suppressed autophagy, and this effect is mediated by the activation of MTOR in the lungs of allergic mice.

Steroid sparing effects of doxofylline

Glucocorticosteroids are widely used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). However, there are growing concerns about the side effect profile of this class of drug, particularly an increased risk of pneumonia. Over the last two decades there have been many attempts to find drugs to allow a reduction of glucocorticosteroids, including xanthines such as theophylline. Use of xanthines has been shown to lead to a reduction in the requirement for glucocorticosteroids, although xanthines also have a narrow therapeutic window limiting their wider use. Doxofylline is another xanthine that has been shown to be of clinical benefit in patients with asthma or COPD, but to have a wider therapeutic window than theophylline. In the present study we have demonstrated that doxofylline produces a clear steroid sparing effect in both an allergic and a non-allergic model of lung inflammation. Thus, we have shown that concomitant treatment with a low dose of doxofylline and a low dose of the glucocorticosteroid dexamethasone (that alone had no effect) significantly reduced both allergen-induced eosinophil infiltration into the lungs of allergic mice, and lipopolysaccharide (LPS)-induced neutrophil infiltration into the lung, equivalent to a higher dose of each drug. Our results suggest that doxofylline demonstrates significant anti-inflammatory activity in the lung which can result in significant steroid sparing activity.

Nanoparticle conjugation enhances the immunomodulatory effects of intranasally delivered CpG in house dust mite-allergic mice.

An emerging strategy in preventing and treating airway allergy consists of modulating the immune response induced against allergens in the lungs. CpG oligodeoxynucleotides have been investigated in airway allergy studies, but even if promising, efficacy requires further substantiation. We investigated the effect of pulmonary delivery of nanoparticle (NP)-conjugated CpG on lung immunity and found that NP-CpG led to enhanced recruitment of activated dendritic cells and to Th1 immunity compared to free CpG. We then evaluated if pulmonary delivery of NP-CpG could prevent and treat house dust mite-induced allergy by modulating immunity directly in lungs. When CpG was administered as immunomodulatory therapy prior to allergen sensitization, we found that NP-CpG significantly reduced eosinophilia, IgE levels, mucus production and Th2 cytokines, while free CpG had only a moderate effect on these parameters. In a therapeutic setting where CpG was administered after allergen sensitization, we found that although both free CpG and NP-CpG reduced eosinophilia and IgE levels to the same extent, NP conjugation of CpG significantly enhanced reduction of Th2 cytokines in lungs of allergic mice. Taken together, these data highlight benefits of NP conjugation and the relevance of NP-CpG as allergen-free therapy to modulate lung immunity and treat airway allergy.

Abstract 5178: Chitinase-3-like-1 (CHI3L1) expressed during allergic pulmonary inflammation alters lung microenvironment accelerating breast cancer metastasis

Abstract Metastasis is the primary cause of mortality in women diagnosed with breast cancer. Metastasis to the lungs is greater in patients with pulmonary inflammatory illnesses, such as asthma. However, it is largely unknown how pulmonary inflammation impacts mammary tumor growth. Our laboratory has established a novel breast cancer model in which mice are induced with allergic pulmonary inflammation prior to mammary tumor implantation. In this study, we are determining how preexisting allergen-induced inflammation changes the pulmonary microenvironment to exacerbate tumor metastasis. Our studies have shown that pre-existing pulmonary inflammation increases the growth and metastasis of breast cancer cells. In the lungs of allergic tumor bearing mice, we found that sustained tissue injury resulting from pulmonary inflammation promoted infiltration of immune cells, collagen production, and enlargement of smooth muscle. In addition, we found high levels of a glycoprotein known as chitinase-3-like-1 protein (CHI3L1) in circulation and in lung tissue. We and others have reported that CHI3L1 is correlated with poor prognosis and decreased survival in breast cancer patients. In addition, we determined that myeloid derived cells from the lungs of allergic mice produced higher amounts of CHI3L1 compared to controls. Increases in CHI3L1 expression have been correlated with adverse effects on lung architecture through an increased deposition of collagen, smooth muscle actin and extracellular matrix proteins. Thus, we hypothesize that CHI3L1 expression in the lungs of mice with allergic pulmonary inflammation may alter the lung architecture and accelerate breast cancer metastasis. We assessed myeloid cell populations in the lungs of allergic mice and found higher levels of monocytes CD11b+Ly6C+Ly6G-, granulocytes CD11b+Ly6G+Ly6C-, and macrophages CD11b+F4/80+Ly6G-. These subpopulations were further increased in 4T1 tumor bearers with allergic pulmonary inflammation. Additionally, we show that CHI3L1 knockout tumor bearers with allergic pulmonary inflammation had decreased myeloid cells, dampened inflammation in the lungs, and a significant reduction in tumor volume and metastasis compared to controls. These changes may be driving the establishment of a premetastatic milieu in the lungs and aiding in the continued support of metastatic foci. Understanding the role of allergen induced CHI3L1 in tumor bearers and its effects on the pulmonary microenvironment could result in targeted therapies for breast cancer. Citation Format: Nathalia Gazaniga, Camilla S. Castro, Stephania Libreros, Ramon Garcia-Areas, Vijaya Iragavarapu-Charyulu. Chitinase-3-like-1 (CHI3L1) expressed during allergic pulmonary inflammation alters lung microenvironment accelerating breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2015-5178

Abstract 2072: Chitinase-3-like-1 protein expression associated with pulmonary inflammation accelerates metastasis to the lung

Abstract Disseminated metastasis accounts for majority of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3-like-1 protein (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. We found that there are elevated levels of CHI3L1 in plasma of mammary tumor-bearing mice. However, the biological and physiological functions of CHI3L1 are still unclear. We previously found that CHI3L1 has an inhibitory role on the expression of interferon-gamma, and in up-regulation of pro-inflammatory mediators such MCP-1, IL-8 and MMP-9 all of which contribute towards tumor growth and metastasis. In determining the cause of high levels of CHI3L1 in plasma, we found that in addition to tumor cells, splenic macrophages and more interestingly pulmonary macrophages from mammary tumor bearers secrete high levels of CHI3L1. Additionally, allergic mice also expressed higher levels of CHI3L1 in circulation and lung tissue. More specifically, CD11b+Ly6C+ cells from the lungs of allergic mice produced higher amounts of CHI3L1 compared to control mice. We have previously established that pulmonary myeloid cells through their production of CHI3L1 set up the proper microenvironment for growth of incoming mammary tumor cells. Thus, we hyphothesize that CHI3L1 levels in the lungs of mice with allergic pulmonary inflammation could accelerate metatstasis. To understand the role of CHI3L1 in accelerating pulmonary metastasis, pulmonary inflammation was induced by sensitization with ragweed allergen. Allergic mice implanted with either 4T1 or 67NR mammary tumors had a 5-fold increase in tumor volume, and formation of metastatic foci in their lungs compared to control mammary tumor bearers. Further, allergic mice showed accelerated tumor growth and shorter survival. In contrast, CHI3L1 null mice showed decreased tumor volume and metastasis, and increased survival compared to control mice. In vivo administration of chitin (β-(1-4)-poly-N-acetyl D-glucosamine) microparticles, the ligand for CHI3L1, to control and allergic mammary tumor bearers resulted in a significant reduction in pulmonary metastasis and tumor growth. These studies suggest that CHI3L1 plays a role in tumor progression and that chitin microparticles can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful target for treatment of breast cancer. Citation Format: Stephania Libreros, Ramon Garcia-Areas, Vijaya Iragavarapu-Charyulu. Chitinase-3-like-1 protein expression associated with pulmonary inflammation accelerates metastasis to the lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2072. doi:10.1158/1538-7445.AM2014-2072

Infection with respiratory syncytial virus influences FasL-mediated apoptosis of pulmonary γδ T cells in a murine model of allergen sensitization

Background: It has been reported that adoptive transfer of γδ T cells increases the cellular infiltration, especially eosinophils, in the lungs of allergic mice, suggesting that γδ T cells may play a proinflammatory role in allergic airway inflammation. Respiratory syncytial virus (RSV) infection can decrease the number of Th2-type γδ T cells. However, the underlying mechanisms remain unknown. Methods: BALB/c mice were inoculated intranasally with RSV before or after sensitization to OVA. The amounts of Th1/Th2 cytokines as well as the levels of specific antibodies were determined by ELISA. The apoptotic death of pulmonary γδ T cells was analyzed by flow cytometry. Results: Adoptive transfer of γδ T cells increased the production of Th2 cytokines in the lungs and allergy-related antibodies in the serum, further confirming that γδ T cells act as pro-inflammatory cells or a promoter for the development of allergic asthma. RSV infection before sensitization to OVA enhanced apoptotic death of pulmonary γδ T cells. The percentage and absolute number of FasL-expressing γδ T cells in the lungs of allergic mice were elicited significantly by prior RSV infection. Blocking FasL with monoclonal antibody diminished apoptotic death of γδ T cells, suggesting that FasL is important for RSV-induced apoptosis of pulmonary γδ T cells. Conclusions: This work provides evidence that RSV infection suppresses the subsequent development of OVA-induced allergic responses partly by enhancing FasL-mediated apoptosis of pulmonary γδ T cells.

Expression of VPAC1 in a murine model of allergic asthma

Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter of the inhibitory non-adrenergic non-cholinergic nervous system and influences the mammalian airway function in various ways. Hence known for bronchodilatory, immunomodulatory and mucus secretion modulating effects by interacting with the VIP receptors VPAC1 and VPAC2, it is discussed to be a promising target for pharmaceutical intervention in common diseases such as COPD and bronchial asthma. Here we examined the expression and transcriptional regulation of VPAC1 in the lungs of allergic mice using an ovalbumin (OVA) -induced model of allergic asthma. Mice were sensitized to OVA and challenged with an OVA aerosol. In parallel a control group was sham sensitized with saline. VPAC1 expression was examined using RT-PCR and real time-PCR studies were performed to quantify gene transcription. VPAC1 mRNA expression was detected in all samples of OVA-sensitized and challenged animals and control tissues. Further realtime analysis did not show significant differences at the transcriptional level.Although the present studies did not indicate a major transcriptional regulation of VPAC1 in states of allergic airway inflammation, immunomodulatory effects of VPAC1 might still be present due to regulations at the translational level.

P33 Effects of hydrogen sulfide on apoptosis protein and antioxidant enzymes in the lungs from allergic mice

Oxidative stress plays an important role in the pathogenesis of airway allergic. In allergic asthma, activated eosinophils and macrophages produce large amounts of reactive oxygen species (ROS) that may contribute to tissue injury. ROS were also involved in apoptotic pathway which includes enhanced ROS production by eosinophils. On the other hand, antioxidants agents can prevent spontaneous eosinophil apoptosis involving a major role for oxidative stress in eosinophil apoptosis. Apoptosis pathways can be initiated by diverse stimuli but all of them converge into common mechanisms involving cleavage of specific aspartic acids (caspases), as caspases 3, 6, 7, 8 and 9. Activation of these caspases results in activation of executioner caspases, such as caspase-3, that go ahead into cell apoptosis. Caspase-3 has been shown to be an important effector caspase in eosinophils following mitochondrial activation involving the Bcl-2 family protein Bax [1] . Our previous study showed that the pre-treatment of OVA-sensitized mice with hydrogen sulfide (H2S)-donor, NaHS, resulted in significant reduction of eosinophil migration to the lungs and increase in superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) antioxidant enzyme activities [2] . We, herein, investigated the effect of H2S on the expression of apoptotic proteins caspase-3 and Bax and on the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the lungs of allergic mice. BALB/c mice were subcutaneously sensitized with ovalbumin (OVA) and, 7 days later, were intranasally challenged with OVA twice-daily for 2 consecutive days. Half of the challenged mice were treated with NaHS or with the irreversible inhibitor of CSE enzyme, propargylglicine (PAG), 30 min before the OVA challenge. All the mice were killed 48 h after the first challenge; the lungs were removed and homogenized in buffer phosphate containing a protease inhibitor cocktail. Caspase-3 and Bax protein expression were assessed by Western blotting and GSH and GSSG levels by HPLC. Results demonstrated that the treatment of allergic mice with NaHS increased the GSH levels into lungs, but GSSG was not affected. NaHS-treatment also produced significant reduction of caspase-3 expression and this effect was reverted by treatment of allergic mice with PAG. The expression of protein Bax was modified neither by NaHS nor by PAG treatments. Our results suggest that the H2S could prevent allergic lungs damage through antioxidant and antiapoptosis effects. As the GSH is considered one of the most important antioxidant agents against free radicals, the decrease in caspase-3 expression can be a consequence of this enzyme activity in the lung of allergic mice.

P12 Effects of hydrogen sulfide on apoptosis protein and antioxidant enzymes in the lungs from allergic mice

Oxidative stress plays an important role in the pathogenesis of airway allergic. In allergic asthma, activated eosinophils and macrophages produce large amounts of reactive oxygen species (ROS) that may contribute to tissue injury. ROS were also involved in apoptotic pathway which includes enhanced ROS production by eosinophils. On the other hand, antioxidants agents can prevent spontaneous eosinophil apoptosis involving a major role for oxidative stress in eosinophil apoptosis. Apoptosis pathways can be initiated by diverse stimuli but all of them converge into common mechanisms involving cleavage of specific aspartic acids (caspases), as caspases 3, 6, 7, 8 and 9. Activation of these caspases results in activation of executioner caspases, such as caspase-3, that go ahead into cell apoptosis. Caspase-3 has been shown to be an important effector caspase in eosinophils following mitochondrial activation involving the Bcl-2 family protein Bax [1] . Our previous study showed that the pre-treatment of OVA-sensitized mice with hydrogen sulfide (H 2 S)-donor, NaHS, resulted in significant reduction of eosinophil migration to the lungs and increase in superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) antioxidant enzyme activities [2] . We, herein, investigated the effect of H 2 S on the expression of apoptotic proteins caspase-3 and Bax and on the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the lungs of allergic mice. BALB/c mice were subcutaneously sensitized with ovalbumin (OVA) and, 7 days later, were intranasally challenged with OVA twice-daily for 2 consecutive days. Half of the challenged mice were treated with NaHS or with the irreversible inhibitor of CSE enzyme, propargylglicine (PAG), 30 min before the OVA challenge. All the mice were killed 48 h after the first challenge; the lungs were removed and homogenized in buffer phosphate containing a protease inhibitor cocktail. Caspase-3 and Bax protein expression were assessed by Western blotting and GSH and GSSG levels by HPLC. Results demonstrated that the treatment of allergic mice with NaHS increased the GSH levels into lungs, but GSSG was not affected. NaHS-treatment also produced significant reduction of caspase-3 expression and this effect was reverted by treatment of allergic mice with PAG. The expression of protein Bax was modified neither by NaHS nor by PAG treatments. Our results suggest that the H 2 S could prevent allergic lungs damage through antioxidant and antiapoptosis effects. As the GSH is considered one of the most important antioxidant agents against free radicals, the decrease in caspase-3 expression can be a consequence of this enzyme activity in the lung of allergic mice.

Thioperamide induces CD4+ CD25+ Foxp3+ regulatory T lymphocytes in the lung mucosa of allergic mice through its action on dendritic cells

Background:Histamine is an important mediator in the development of allergic reactions. The biological effects of histamine are mediated through four histaminergic receptors. In recent years, an important role has been assigned to the proinflammatory functions of histamine regarding the H4 receptor. Previously, we have demonstrated that injection of immature dendritic cells treated with histamine into allergic mice promotes an increase in CD8+ Tc2 lymphocytes, which are involved in the worsening of allergy symptoms during the chronic phase of the disease. The aim of this study was to evaluate the role of the H3/H4 receptor antagonist, thioperamide, in allergy.Methods:Ovalbumin-allergized mice and nonallergized mice were injected with phosphate-buffered saline, dendritic cells, or thioperamide-treated dendritic cells. After treatment, the lungs of the mice were obtained and analyzed for changes in the populations of dendritic cells and T lymphocytes, as well as the expression of H and H4 receptors in mononuclear lung cells.Results:We found an increase in regulatory T cells in the lungs of allergic mice intratracheally injected with dendritic cells which had their H3/H4 receptors blocked with thioperamide. We also found an increase in the production of interleukin-10 by dendritic cells of the lung. Finally, we observed a decrease in serum levels of specific anti-IgE and a reduction of eosinophils in bronchoalveolar lavage from allergic mice.Conclusion:Thioperamide induces a significant improvement in symptoms of allergic reaction perhaps via induction of regulatory T lymphocytes. These findings may become relevant in the understanding of type 1 hypersensivity reactions.

Role of the Mac-1 and VLA-4 integrins, and concomitant Th2-cytokine production, in nitric oxide modulated eosinophil migration from bone marrow to lungs in allergic mice

Although numerous studies demonstrate the participation of nitric oxide (NO) in various inflammatory diseases, the precise function of NO in allergic asthma remains unclear. We investigated whether iNOS inhibition could interfere with the kinetics of VLA-4 and Mac-1 expression and adhesion properties of bone marrow and peripheral blood eosinophils of sensitized mice after antigen exposure. Treatment of allergic mice with 1400W (iNOS inhibitor) increased the adhesion of bone marrow eosinophils to ICAM-1, but not blood eosinophils, at 24h and 48h after OVA-challenge. Conversely, adhesion of blood eosinophils from 1400W-treated mice to VCAM-1 diminished at 24h and was almost completely blocked at 48h. 1400W did not induce any change in the adhesion of bone marrow eosinophils to VCAM-1, at 24h, but cells collected 48h after challenge showed significantly lower adherence. Flow cytometry demonstrated that 1400W resulted in a significantly increased Mac-1 expression on bone marrow eosinophils at 24h, as compared to control mice. However, at 24h, 1400W significantly decreased Mac-1 and VLA-4 expressions on blood eosinophils. At 48h, the expressions of both Mac-1 and VLA-4 returned to previous levels. Results show a temporal effect of iNOS upon Mac-1 expression and function, the chief adhesion molecule involved in the eosinophil efflux from the bone marrow at 24h. In contrast, Mac-1 and VLA-4 were involved in eosinophil mobilization from blood to lungs at 48h after antigen challenge. Data suggest an important role of the Mac-1 and VLA-4 in the iNOS-modulated migration of eosinophils to the lungs of allergic mice.

Histamine‐treated dendritic cells improve recruitment of type 2 CD8 T cells in the lungs of allergic mice

Histamine controls the function of dendritic cells (DCs). It appears to be required for the normal development of DCs. It also induces the chemotaxis of immature DCs and promotes the differentiation of CD4(+) T cells into cells with a T helper type 2 (Th2) profile. Moreover, we have recently shown that histamine stimulates both the uptake and the cross-presentation of antigens by DCs, supporting the theory that histamine promotes activation of CD8(+) T cells during the development of allergic pathologies. Here, we investigated whether the course of an allergic response, in a well-defined model of ovalbumin (OVA)-induced allergic airway inflammation, could be modulated by intratracheal injection of OVA-pulsed DCs previously treated with histamine (DCHISs). Compared with control DCs, DCHISs induced: (i) greater recruitment of CD8(+) T cells in the lung, (ii) greater stimulation of the production of interleukin (IL)-5 by lung CD8(+) T cells, and (iii) increased recruitment of CD11c/CD8 double-positive DCs in the lungs of allergic mice. Moreover, mice treated with DCHISs showed increased levels of serum-specific immunoglobulin E (IgE) antibodies directed to OVA, and a higher proportion of eosinophils in bronchoalveolar lavage (BAL) compared with mice treated with OVA-pulsed control DCs. Our results support the notion that histamine, by acting on DCs, increases the severity of allergic processes.

Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration

Previous studies in our laboratory have shown that platelets are essential for the migration of eosinophils into the lungs of allergic mice, and that this is dependent on the functional expression of platelet P-selectin. We sought to investigate whether the same is true for nonallergic, acute inflammatory stimuli administered to distinct anatomic compartments. Neutrophil trafficking was induced in two models, namely zymosan-induced peritonitis and LPS-induced lung inflammation, and the platelet dependence of these responses investigated utilizing mice rendered thrombocytopenic. The relative contribution of selectins was also investigated. The results presented herein clearly show that platelet depletion (>90%) significantly inhibits neutrophil recruitment in both models. In addition, we show that P-selectin glycoprotein ligand-1, but not P-selectin, is essential for neutrophil recruitment in mice in vivo, thus suggesting the existence of different regulatory mechanisms for the recruitment of leukocyte subsets in response to allergic and nonallergic stimuli. Further studies in human blood demonstrate that low-dose prothrombotic and pro-inflammatory stimuli (CCL17 or CCL22) synergize to induce platelet and neutrophil activation, as well as the formation of platelet-neutrophil conjugates. We conclude that adhesion between platelets and neutrophils in vivo is an important event in acute inflammatory responses. Targeting this interaction may be a successful strategy for inflammatory conditions where current therapy fails to provide adequate treatment.