白介素17单克隆抗体对变应性鼻炎小鼠气道炎症的作用

目的 探讨白介素17单克隆抗体（IL-17mAb）的不同给予剂量及方式在变应性鼻炎小鼠气道炎症中的作用。方法 将48只小鼠采用随机数字表法分为A、 B、 C、 D、 E、 F组,每组8只。分别于第0、 7、 14 d将20 μg卵清蛋白（OVA）加2 mg铝佐剂腹腔注射处理A、C、D、E及F组小鼠,间隔7 d,第22天开始进行鼻腔激发,每天每侧鼻孔各给予OVA 10 μl（共500 μg）滴鼻,连续7 d。A、C、D、E组小鼠于每次OVA鼻腔激发前1 h分别给予生理盐水、100 ng IL-17mAb、500 ng IL-17mAb、5 μg IL-17mAb滴鼻,F组小鼠于每次OVA鼻腔激发前4 h给予5 μg IL-17mAb腹腔注射,B组小鼠于相同时间点给予等量生理盐水腹腔注射及滴鼻。所有小鼠于最后1次激发后评估鼻部症状学变化,Diff-Quik染色观察鼻腔灌洗液（NLF）中嗜酸性粒细胞浸润情况,ELISA方法检测血清及NLF中IL-6、IL-10水平,鼻黏膜组织行甲苯胺蓝染色观察肥大细胞。结果 ４周末A组所有小鼠症状学评分均＞5分,提示造模成功。F组小鼠的挠鼻及喷嚏次数均少于A组（P＜0.05）;F组小鼠NLF中嗜酸性粒细胞数、血清IL-6水平低于A组,血清及NLF中IL-10水平均高于A组（P＜0.05）;E组小鼠血清中IL-10水平高于A组（P＜0.05）;A组小鼠鼻黏膜组织中肥大细胞数多于B组,统计学意义显著（P＜0.01）;F组小鼠鼻黏膜组织中肥大细胞数少于A组,但差异无统计学意义（P＞0.05）;F组小鼠鼻黏膜组织中肥大细胞数与B组比较,差异无统计学意义（P＞0.05）。结论 高剂量的（5 μg）IL-17mAb腹腔注射处于激发阶段的变应性鼻炎小鼠促使小鼠变应性鼻炎症状明显减轻,鼻腔灌洗液嗜酸性粒细胞减少。促使变应性鼻炎小鼠血清中IL-6表达降低,血清中及鼻腔灌洗液中IL-10表达升高,因此推测这些细胞因子的变化可能抑制Th17/促进Treg的分化,进而对变态反应产生抑制作用。     

Role of Eosinophil Granulocytes in Allergic Airway Inflammation Endotypes

Eosinophil granulocytes are intriguing members of the innate immunity system that have been considered important defenders during parasitic diseases as well as culprits during allergy‐associated inflammatory diseases. Novel studies have, however, found new homoeostasis‐maintaining roles for the cell. Recent clinical trials blocking different Th2 cytokines have uncovered that asthma is heterogeneous entity and forms different characteristic endotypes. Although eosinophils are present in allergic asthma with early onset, the cells may not be essential for the pathology. The cells are, however, likely disease causing in asthma with a late onset, which is often associated with chronic rhinosinusitis. Assessment of eosinophilia, fraction exhaled nitric oxide (FeNO) and periostin are markers that have emerged useful in assessing and monitoring asthma severity and endotype. Current scientific knowledge suggests that eosinophils are recruited by the inflammatory environment, activated by the innate interleukin (IL)‐33 and prevented from apoptosis by both lymphocytes and innate immune cells such as type two innate immune cells. Eosinophils contain four specific granule proteins that exhibit an array of toxic and immune‐modulatory activates. The granule proteins can be released by different mechanisms. Additionally, eosinophils contain a number of inflammatory cytokines and lipid mediators as well as radical oxygen species that might contribute to the disease both by the recruitment of other cells and the direct damage to supporting cells, leading to exacerbations and tissue fibrosis. This review aimed to outline current knowledge how eosinophils are recruited, activated and mediate damage to tissues and therapies used to control the cells.     

Interleukin-9 Is Required for Allergic Airway Inflammation Mediated by the Cytokine TSLP

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Highlights? TSLP induces IL-9 from human and mouse Th9 cells ? TSLP enhances Th9-mediated allergic lung inflammation ? Blocking IL-9 attenuates TSLP-induced lung inflammation     

Modulation of Allergic Airway Inflammation by the Oral Pathogen Porphyromonas gingivalis

Accumulating evidence suggests that bacteria associated with periodontal disease may exert systemic immunomodulatory effects. Although the improvement in oral hygiene practices in recent decades correlates with the increased incidence of asthma in developed nations, it is not known whether diseases of the respiratory system might be influenced by the presence of oral pathogens. The present study sought to determine whether subcutaneous infection with the anaerobic oral pathogen Porphyromonas gingivalis exerts a regulatory effect on allergic airway inflammation. BALB/c mice sensitized and subsequently challenged with ovalbumin exhibited airway hyperresponsiveness to methacholine aerosol and increased airway inflammatory cell influx and Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13) content relative to those in nonallergic controls. Airway inflammatory cell and cytokine contents were significantly reduced by establishment of a subcutaneous infection with P. gingivalis prior to allergen sensitization, whereas serum levels of ovalbumin-specific IgE and airway responsiveness were not altered. Conversely, subcutaneous infection initiated after allergen sensitization did not alter inflammatory end points but did reduce airway responsiveness in spite of increased serum IgE levels. These data provide the first direct evidence of a regulatory effect of an oral pathogen on allergic airway inflammation and responsiveness. Furthermore, a temporal importance of the establishment of infection relative to allergen sensitization is demonstrated for allergic outcomes.A causative relationship between decreased microbial exposure and infection in recent decades and the concurrent increase in asthma prevalence in developed countries has been suggested and is thought to be attributable, at least in part, to a phenomenon known as the hygiene hypothesis (30). Originally put forth by Strachan (32), the hypothesis proposes that increased cleanliness of modern industrialized societies has resulted in decreased exposure to bacterial, viral, and other immunomodulatory organisms and their products, particularly in early life, and that this has in turn resulted in a loss of potentially protective effects of these exposures on the development of allergic diseases. Accumulating clinical and experimental evidence largely supports the hygiene hypothesis as it relates to asthma, although a consensus has not been reached. As reviewed recently (31), a variety of infections of a viral, bacterial, and parasitic nature influence the host immune response, such that regulation of the Th1-Th2 balance is modified to promote Th1 responses and impede Th2 responses, thereby reducing Th2-mediated allergic outcomes. However, this is likely a simplistic view of the effects of infections on immune system development and responses, and other factors, including host genetic makeup and timing of exposures to the infective agent relative to allergen exposure, undoubtedly contribute to the overall allergic phenotype.In addition to the influence of environmental exposure to microbes, the potential regulation of allergic diseases by the microflora of the host is receiving increased attention. Evidence suggests that the composition of the gastrointestinal microflora differs between individuals with and without allergy (reviewed in reference 25), and disruption of the normal gut microflora by antibiotic administration leads to allergic airway responses following allergen challenge in mice not previously sensitized to the allergen (24). Moreover, although similar benefits have not yet been demonstrated in humans, the oral administration of probiotic bacteria was recently shown to decrease allergic airway inflammation in mice (8, 9).As in the gut, the microenvironment of the oral cavity is complex and comprises hundreds of bacterial species. Porphyromonas gingivalis, a Gram-negative opportunistic periodontal pathogen, can initiate periodontal lesions in nonhuman primates when introduced into the periodontal microbiota (15) and is a major etiological agent in severe forms of periodontal disease such as chronic periodontitis (21). Interest in chronic oral infections and their potential role in adverse systemic health effects has been heightened by observations of positive associations between serum concentrations of antibodies to oral pathogens such as P. gingivalis and the incidence of cardiovascular diseases and renal dysfunction (3, 19, 28, 29). Recently, however, an inverse relationship between serum concentrations of antibodies to P. gingivalis and the prevalence of asthma, wheeze, and hay fever was observed in a representative sample of the population of the United States (2). Furthermore, a significant inverse association between periodontitis and the incidences of hay fever and allergy to house dust mites was reported for a northeast German population, with a borderline significant inverse association between periodontitis and asthma also observed (10). While limitations of these observational studies include potential recall bias pertaining to asthma symptoms and the inability to directly assess cause and effect, these findings nonetheless suggest a potential protective effect of infection with oral pathogens such as P. gingivalis on asthma pathogenesis.In order to examine the influence of oral pathogens on the development of allergic airway disease under controlled experimental conditions, the present study sought to determine whether infection with P. gingivalis modified allergic outcomes in a murine model of asthma. To accomplish this, a subcutaneous chamber model was employed wherein mice were subjected to a local infection with live P. gingivalis either before or after sensitization to allergen, and the effects of this infection on subsequent responses to allergen challenge were assessed. The results indicate that P. gingivalis infection exerts a modulatory effect on allergic airway responses and that this effect is dependent on the timing of infection relative to allergic sensitization.     

Hesperidin Suppresses Ovalbumin-Induced Airway Inflammation in a Mouse Allergic Asthma Model

Hesperidin, a flavanone glycoside comprised of the flavanone hesperetin and the disaccharide rutinose, is a plentiful and inexpensive by-product of citrus cultivation. It has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, and anticarcinogenic properties. In this study, we attempt to determine whether hesperidin inhibits inflammatory mediators in the mouse allergic asthma model. Mice were sensitized and challenged by ovalbumin (OVA) to induce chronic airway inflammation and airway remodeling. The administration of hesperidin significantly decreased the number of infiltrating inflammatory cells and Th2 cytokines in bronchoalveolar lavage (BAL) fluid compared with the OVA-induced group of mice. In addition, hesperidin reduced OVA-specific IgE levels in serum. Hesperidin markedly alleviated the OVA-induced airway hyperresponsiveness (AHR) to inhaled methacholine. Based on lung histopathological studies using hematoxylin and eosin and alcian blue-periodic acid-Schiff staining, hesperidin inhibited inflammatory cell infiltration and mucus hypersecretion compared with the OVA-induced group of mice. These findings provide new insight into the immunopharmacological role of hesperidin in terms of its effects in a murine model of asthma.     

Angiogenesis in Mice with Chronic Airway Inflammation : Strain-Dependent Differences

Chronic inflammation is associated with blood vessel proliferation and enlargement and changes in vessel phenotype. We sought to determine whether these changes represent different types of angiogenesis and whether they are stimulus dependent. Chronic airway inflammation, produced by infection with Mycoplasma pulmonis, was compared in strains of mice known to be resistant (C57BL/6) or susceptible (C3H). Tracheal vascularity, assessed in whole mounts after Lycopersicon esculentum lectin staining, increased in both strains at 1, 2, 4, and 8 weeks after infection, but the type of vascular remodeling was different. The number of vessels doubled in tracheas of C57BL/6 mice, with corresponding increases of capillaries and venules. In contrast, neither the number nor the length of vessels changed in C3H mice. Instead, vessel diameter and endothelial cell number doubled, and the proportion of venules doubled with a corresponding decrease of capillaries. Although the infection had no effect on baseline plasma leakage, in both strains it potentiated the leakage produced by substance P. We conclude that the same stimulus can result in blood vessel proliferation or enlargement, depending on the host response. Endothelial cells proliferate in both cases, but in one case new capillaries form whereas in the other capillaries convert to venules.     

Distinct Requirements for Interleukin-6 in Airway Inflammation Induced by Diesel Exhaust in Mice

To clarify the possible role of interleukin-6 in aggravation of inflammatory responses in diesel exhaust-exposed mice, we compared the infiltration of inflammatory cells and the production of chemokines between interleukin-6-deficient and wild-type mice following 0, 1.0, or 3.0 mg diesel particles/m³ diesel exhaust inhalation for 4 weeks. Exposure to diesel exhaust significantly increased the number of inflammatory cells and the amount of CCL17 and CXCL3 in bronchoalveolar lavage fluids from wild-type mice, but not in interleukin-6-deficient mice. These findings suggest that interleukin-6 plays a critical role in airway inflammatory responses induced by diesel exhaust inhalation.     

The Iron Chelator and Anticancer Agent Dp44mT Relieves Allergic Inflammation in Mice With Allergic Rhinitis

Our previous study showed that an iron chelator and anticancer agent Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) has an antiinflammatory effect in human mast cells. However, antiinflammatory effect of Dp44mT remains unclear in animal models. In this study, we assessed whether administration of Dp44mT could relieve clinical symptoms of ovalbumin (OVA)-induced allergic rhinitis (AR) mice. After administration of Dp44mT, number of rubs was significantly decreased, and levels of histamine and IgE were suppressed in serum of AR mice. Also, serum levels of interleukin (IL)-1β, thymic stromal lymphopoietin (TSLP), and tumor necrosis factor (TNF)-α increased by OVA challenge were significantly lowered by administration of Dp44mT. T helper type 1 (Th1) cytokine interferon-γ level was significantly increased by administration of Dp44mT, whereas Th2 cytokines such as IL-4, IL-5, and IL-13 were significantly reduced by administration of Dp44mT. In intranasal tissues of AR mice, levels of IL-1β, TSLP, TNF-α, and IL-6 and activities and protein levels of caspase-1 were significantly reduced by administration of Dp44mT. Interestingly, administration of Dp44mT reduced number of infiltrated eosinophils and mast cells through the inhibition of macrophage inflammatory protein-2 and intercellular adhesion molecule-1 in intranasal tissues of AR mice. In conclusion, these results indicate that Dp44mT also has potential antiinflammatory effects in vivo as well as in vitro.     

Effects of Diet-Induced Mild Obesity on Airway Hyperreactivity and Lung Inflammation in Mice

Purpose

Obesity has been suggested to be linked to asthma. However, it is not yet known whether obesity directly leads to airway hyperreactivity (AHR) or obesity-induced airway inflammation associated with asthma. We investigated obesity-related changes in adipokines, AHR, and lung inflammation in a murine model of asthma and obesity.

Materials and Methods

We developed mouse models of chronic asthma via ovalbumin (OVA)-challenge and of obesity by feeding a high-fat diet, and then performed the methacholine bronchial provocation test, and real-time PCR for leptin, leptin receptor, adiponectin, adiponectin receptor (adipor1 and 2), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) β, and tumor necrosis factor (TNF) α in lung tissue. We also measured cell counts in bronchoalveolar lavage fluid.

Results

Both obese and lean mice chronically exposed to OVA developed eosinophilic lung inflammation and AHR to methacholine. However, obese mice without OVA challenge did not develop AHR or eosinophilic inflammation in lung tissue. In obese mice, lung mRNA expressions of leptin, leptin receptor, VEGF, TGF, and TNF were enhanced, and adipor1 and 2 expressions were decreased compared to mice in the control group. On the other hand, there were no differences between obese mice with or without OVA challenge.

Conclusion

Diet-induced mild obesity may not augment AHR or eosinophilic lung inflammation in asthma.     

Small Interfering RNA Targeting T-cell Ig Mucin-3 Decreases Allergic Airway Inflammation and Hyperresponsiveness

Since CD4+ T cells play a pivotal role in the development of airway inflammation and hyperresponsiveness, targeting activated CD4+ T cell subsets and increasing the cells with regulatory function would be a logical therapeutic approach. We showed that this outcome can be achieved by local therapy with Tim-3, which is a negative regulator of CD4+ T cells. Tim-3 expression was up-regulated by ovalbumin (OVA) induction. Attenuating Tim-3 expression by RNA interference suppressed allergen-induced immune responses. Intranasal application of Tim-3 shRNA diminished airway inflammation and hyperresponsiveness. Multiple mechanisms were involved in the inhibitory effects, including regulation the imbalance of Th1/Th17 and increasing Treg cell expression. Our results indicate that the Tim-3 pathway is highly involved in the regulation of asthma. Targeting Tim-3 by siRNA may hold therapeutic potential in preventing the development of allergic asthma.     

Receptor Interacting Protein 2 (RIP2) Is Dispensable for OVA-Induced Airway Inflammation in Mice

Purpose

Asthma is a pulmonary chronic inflammatory disease characterized by airway obstruction and hyperresponsiveness. Pattern recognition receptors are known to play a key role in the development of allergic diseases as well as host defenses against microbial infection. Receptor interacting protein 2 (RIP2), a serine/threonine kinase, is an adaptor molecule of NOD1 and NOD2, and genetic variation in this receptor is known to be associated with the severity of allergic asthma in children. In this study, we examined the role of RIP2 in the development of allergic airway inflammation in a mouse model.

Methods

Airway inflammation was induced in mice through intranasal administration of ovalbumin (OVA) after 2 intraperitoneal immunizations with OVA. Lung inflammation and mucus hypersecretion were examined histologically and total cell infiltration in bronchoalveolar (BAL) fluids was determined. Levels of the Th2-related cytokines, IL-5 and IL-13, in lung extracts were measured by ELISA. Serum antigen-specific IgE and IgG1 levels were also assessed.

Results

OVA-induced lung inflammation and mucus hypersecretion were not different between WT and RIP2-deficient mice. The IL-5 and IL-13 levels in the bronchoalveolar (BAL) fluids were also not impaired in RIP2-deficient mice compared to WT mice. Moreover, RIP2 deficiency did not affect serum OVA-specific IgG1 and IgE levels.

Conclusions

Our results suggest that RIP2 is not associated with the development of allergic airway inflammation.     

Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation

Immunological cross talk between mucosal tissues such as the intestine and the lung is poorly defined during homeostasis and disease. Here, we show that a low-dose infection with the intestinally restricted helminth parasite Trichuris muris results in the production of Th1 cell-dependent gamma interferon (IFN-γ) and myeloid cell-derived interleukin-10 (IL-10) in the lung without causing overt airway pathology. This cross-mucosal immune response in the lung inhibits the development of papain-induced allergic airway inflammation, an innate cell-mediated type 2 airway inflammatory disease. Thus, we identify convergent and nonredundant roles of adaptive and innate immunity in mediating cross-mucosal suppression of type 2 airway inflammation during low-dose helminth-induced intestinal inflammation. These results provide further insight in identifying novel intersecting immune pathways elicited by gut-to-lung mucosal cross talk.     

An Interaction of LPS and RSV Infection in Augmenting the AHR and Airway Inflammation in Mice

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection (LRTI) in children under 5 years of age, especially infants with severe bronchiolitis. Our preliminary clinical experiments showed that bacterial colonization was commonly observed in children with virus-induced wheezing, particularly in those with recurrent wheezing, suggesting that bacterial colonization with an accompanying viral infection may contribute to disease severity. In most cases, RSV-infected infants were colonized with pathogenic bacteria (mainly Gram-negative bacteria). LPS is the main component of Gram-negative bacteria and acts as a ligand for Toll-like receptor 4 (TLR4). Relevant studies have reported that the TLR family is crucial in mediating the link between viral components and immunologic responses to infection. Of note, TLR4 activation has been associated with disease severity during RSV infection. In the present study, we identified that LPS aggravated RSV-induced AHR and airway inflammation in BALB/c mice using an RSV coinfection model. We found that the airway inflammatory cells and cytokines present in BALF and TRIF in lung tissue play a role in inducing AHR and airway inflammation upon RSV and bacteria coinfection, which might occur through the TRIF-MMP-9-neutrophil-MMP-9 signalling pathway. These results may aid in the development of novel treatments and improve vaccine design.     

KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice

Although natural killer T cells (NKT cells) are altered in obese asthmatic mice, their function remains completely unclear. To further explore the potential mechanism of NKT cells in airway inflammation of obesity-associated asthma, we examined the effects of α-galactosylceramide (KRN7000) on airway inflammation in obese asthmatic mice. Male C57BL/6J mice were divided into five groups: (1) control; (2) asthma; (3) A + KRN, asthma with KRN7000; (4) obese asthma; and (5) OA + KRN, obese asthma with KRN7000. Cytometric bead array (CBA) was used to detect interleukin-4 (IL-4), IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the serum. Flow cytometry was used to detect NKT cells and CD69⁺ NKT cells. Airway inflammation was observed in pathological sections, and calmodulin (CaM) expression was observed by immunohistochemistry in lung tissues. Airway inflammation in the obese asthma group was more severe than that of the asthma group. Airway inflammation of the OA + KRN group was reduced more than that of the A + KRN group. CD69+ NKT cells were only significantly reduced in the OA + KRN group. The levels of serum IFN-γ and TNF-α increased more in the OA + KRN group than in the A + KRN group. CaM is widely expressed in the cytoplasm of the lung tissues and was sharply decreased in the OA + KRN group. KRN7000 can significantly reduce airway inflammation in obesity-associated asthma by regulating NKT cell cytokine secretion and intracellular calcium. These results may contribute to the development of novel therapeutic approaches.