Small Airway Impairment and Bronchial Hyperresponsiveness in Asthma Onset

Purpose

Our study tried to find a relationship between baseline FEF_25-75% and airway hyperresponsiveness (AHR) and whether a greater FEF_25-75% impairment may be a marker of a more severe hyperresponsiveness in subjects with normal FEV1 and FEV1/FVC and suggestive asthma symptoms. Besides, we tried to asses a FEF_25-75% cut-off value to identify hyper-reactive subjects.

Methods

4,172 subjects (2,042 M; mean age: 38.3±14.9; mean FEV1 % predicted: 100.5±12.7 and FEV1/FVC: 85.4±6.8) were examined after performing a methacholine (Mch) test. All subjects reported a symptom onset within 3 years before the test. Subjects with PD20<400 or >400 µg were arbitrarily considered affected by moderate/severe and borderline AHR, respectively.

Results

PD20 values were 213 (IQR:86-557), 340 (IQR:157-872) and 433 (IQR:196-1032) µg in subjects with baseline FEF_25-75≤50%, FEF_25-75 between 50 and 70% and FEF_25-75>70% respectively (P<0.0001). Only in moderate/severe hyper-reactive subjects (excluded borderlines), PD20 was lower in the FEF_25-75≤50% subgroup than in the 1 with FEF_25-75>70%. The hyperreactive subjects percentage, was higher in those with FEF_25-75≤50% and lower in those with FEF_25-75>70% (P<0.0001). FEF_25-75<50% (compared to FEF_25-75>70%) was a higher AHR risk factor, especially in subjects with moderate/severe AHR (OR: 2.18 [IQR:1.41-3.37]; P<0.0001). Thresholds yielding the highest combined sensitivity/specificity for FEF_25-75% were 75.19 (area under curve [AUC]: 0.653) and 74.95 (AUC:0.688) in subjects with PD20<2,400 and <400 µg respectively. FEV1, FVC, and FEV1/FVC measured in subjects with different FEF_25-75≤50%, FEF_25-75>50 and ≤70% or FEF_25-75>70% levels were similar both in normoreactive and hyperreactive subjects.

Conclusions

At asthma onset, reduced baseline FEF_25-75 values with normal FEV1 and FEV1/FVC may predict AHR. Detectable predictive cut-off values do not exist because even normoreactive subjects can show lower FEF_25-75 values. Furthermore, a greater FEF_25-75 reduction may be associated to a more severe AHR, suggesting a possible FEF_25-75 role in the management of asthma when FEV1 and FEV1/FVC are normal.     

Anisakis pegreffii-Induced Airway Hyperresponsiveness Is Mediated by Gamma Interferon in the Absence of Interleukin-4 Receptor Alpha Responsiveness

Infection with the fish parasite Anisakis following exposure to contaminated fish can lead to allergic reactions in humans. The present study examined the immunological mechanisms underlying the development of allergic airway inflammation in mice after different routes of sensitization to Anisakis. Wild-type and interleukin-4 receptor alpha (IL-4Rα)-deficient BALB/c mice were sensitized intraperitoneally with live or heat-killed Anisakis larvae or by intranasal administration of an Anisakis extract and were subsequently challenged intranasally with an Anisakis extract. Both routes of sensitization induced IL-4Rα-dependent allergic airway responses, whereas allergen-specific antibody responses developed only when mice were sensitized intraperitoneally. Intranasal sensitization induced airway hyperresponsiveness (AHR) in wild-type mice only, showing that AHR was IL-4/IL-13 dependent. Unexpectedly, infection with Anisakis larvae induced AHR in both wild-type and IL-4Rα-deficient mice. IL-4Rα-independent AHR was mediated by gamma interferon (IFN-γ), as evidenced by the fact that in vivo neutralization of IFN-γ abrogated AHR. Together, these results demonstrate that both infection with larvae and inhalational exposure to Anisakis proteins are potent routes of allergic sensitization to Anisakis, explaining food- and work-related allergies in humans. Importantly for diagnosis, allergic airway inflammation can be independent of detectable Anisakis-specific antibodies. Moreover, depending on the route of sensitization, AHR can be induced either by IL-4/IL-13 or by IFN-γ.While it is believed that many parasitic worms protect against allergy, the fish parasite Anisakis can induce acute gastroallergic or anaphylactic reactions in a subset of infected patients (4). Anisakis spp. are nematode parasites of marine mammals with larval stages that pass through several intermediate hosts. The infectious-stage larvae (L₃) are found worldwide in sea fish or cephalopods and can be accidentally ingested by humans (32). If ingested live due to consumption of raw or undercooked fish, Anisakis L₃ are able to parasitize humans, causing the zoonotic disease known as anisakiasis. This is usually an acute and transient infection, with the larvae dying within a few weeks, since the host environment does not permit development into adult worms (6). Within hours of being ingested, Anisakis L₃ penetrate the mucosal layers of the gastrointestinal tract, causing direct tissue damage that may lead to abdominal pain, nausea, and/or diarrhea. Furthermore, some patients develop an immunoglobulin E (IgE)-mediated “gastroallergic anisakiasis,” which presents with clinical manifestations ranging from urticaria to allergic reactions and life-threatening anaphylactic shock (5, 13, 14). To date, nine allergens from Anisakis simplex, some of which are cross-reactive to other seafood allergens, have been characterized on a molecular level (28). The importance of the problem is demonstrated by studies that have found Anisakis to be a leading cause of food allergy in Spain (2) and have found a higher prevalence of sensitization to Anisakis than to seafood among almost 5 million Japanese (24). Anisakis is also an important cause of occupational allergy in fish-processing workers, as shown in a recent epidemiological study by our group, including 578 workers from fish-processing factories in South Africa, in which Anisakis sensitization was associated with dermatitis and nonspecific bronchial hyperreactivity (37). Because sensitization to Anisakis was associated with increased fish consumption, we investigated the underlying immune mechanisms by infecting mice with Anisakis L₃ and subsequently challenging them orally with an Anisakis crude antigen extract. This induced striking allergic reactions, including airway inflammation and lung mucus hypersecretion, which were associated with T-helper 2 (Th2)/type 2 responses (37). Furthermore, mice exposed epicutaneously to Anisakis proteins developed protein contact dermatitis (36). Local skin pathology was interleukin-13 (IL-13) dependent, as evidenced by the fact that it was abolished in IL-13- and IL-4 receptor alpha (IL-4Rα)-deficient mice, whereas IL-4 was important for systemic allergic sensitization (36). Together, these studies show that sensitization by Anisakis infection and subsequent oral challenge with an Anisakis extract can cause allergic airway disease, while epicutaneous exposure to Anisakis proteins can lead to dermatitis, explaining the observations of human prevalence studies.However, it is still unclear whether live infection is needed for the development of allergic airway reactions or whether exposure to nematode-derived protein is sufficient (4). This is an important clinical question in view of the fact that several case and prevalence studies, including ours, indicate that sensitization by inhalation of Anisakis proteins might be an important cause of work-related allergies (1, 3, 7, 37, 39, 40). Aerosolized food allergens cause as much as 10% of asthma in the occupational environment (41), and Anisakis allergens aerosolized during fish cleaning, cooking, or fish meal production may therefore pose a risk for workers.In this study we aimed to determine whether sensitization through Anisakis infection is essential for the induction of allergic airway disease or if exposure to larval proteins or heat-killed larvae is sufficient to induce allergic airway reactions. In addition, we investigated the effects of IL-4/IL-13 signaling by using mice deficient in IL-4Rα, an important receptor chain in allergic airway disease through which both IL-4 and IL-13 signal (8). Wild-type and IL-4Rα^−/− mice were either sensitized intraperitoneally (i.p.) with live or heat-killed Anisakis L₃ or sensitized intranasally to an Anisakis extract and were subsequently challenged by intranasal administration of an Anisakis extract in order to mimic aerosolized exposure. All sensitization protocols induced symptoms of allergic airway disease, but allergen-specific antibody responses were present only when mice were sensitized intraperitoneally by live or heat-killed larvae. Interestingly, while the development of airway hyperresponsiveness (AHR) was dependent on IL-4Rα responsiveness when mice were sensitized intranasally, mice sensitized with live or heat-killed Anisakis L₃ developed AHR independently of IL-4Rα responsiveness. This IL-4Rα-independent AHR was shown to be dependent on gamma interferon (IFN-γ).     

Eosinophilic Airway Inflammation and Airway Hyperresponsiveness According to Aeroallergen Sensitization Pattern in Patients With Lower Airway Symptoms

Purpose

Sensitization to specific allergens may be important in the development of allergic airway inflammation and airway hyperresponsiveness (AHR). We evaluated the effect of specific aeroallergen sensitization on eosinophilic airway inflammation and AHR.

Methods

We reviewed retrospectively the clinical data of subjects who underwent skin prick tests to aeroallergens, induced sputum analysis, and methacholine bronchial provocation tests to evaluate lower airway symptoms as well as analyzed the associations between the pattern of aeroallergen sensitization and sputum eosinophilia or AHR.

Results

Of the 1,202 subjects be enrolled, 534 (44.4%) were sensitized to at least one aeroallergen in skin tests. AHR was demonstrated in 23.5% and sputum eosinophilia in 38.8%. Sputum eosinophilia was significantly associated with sensitization to perennial allergens (OR, 1.9; 95% CI, 1.4-2.5), house dust mite (OR, 1.7; 95% CI, 1.3-2.3), dog (OR, 1.9; 95% CI, 1.1-3.3), and cat (OR, 2.1; 95% CI, 1.4-3.4). AHR was associated with sensitization to perennial allergens (OR, 2.7; 95% CI, 2.0-3.7), house dust mite (OR, 2.2; 95% CI, 1.6 3.2), Alternaria (OR, 2.3; 95% CI, 1.2-4.7), and cat (OR, 2.7; 95% CI, 1.7-4.3). Sensitization to more perennial allergens increased the risk for sputum eosinophilia and AHR. There was no relationship with individual seasonal allergens.

Conclusion

The development of airway eosinophilic inflammation and AHR in an adult Korean population was associated with sensitization to perennial allergens rather than seasonal allergens.     

Protective Role for Interleukin-5 during Chronic Toxoplasma gondii Infection

To investigate the role of interleukin-5 (IL-5) during Toxoplasma gondii infection, IL-5 knockout (KO) mice and C57BL/6 control mice were infected intraperitoneally with ME49 cysts and the course of infection was monitored. The mortality rate during chronic infection was significantly greater in IL-5-deficient animals, and consistent with this finding, the KO mice harbored a greater number of brain cysts and tachyzoites than did their wild-type counterparts. Although the IL-5 KO animals did not succumb until late during infection, increased susceptibility, as measured by accelerated weight loss, was detectable during the acute stages of infection. The amounts of total immunoglobulin (Ig), IgM, and IgG2b were comparable in both strains, while the amount of IgG1 was much smaller in IL-5 KO mice. Spleen cell production of IL-12 in response to T. gondii antigen was approximately threefold lower in the KO strain, and this decrease correlated with a selective loss of B lymphocytes during culture. A link between the presence of B cells and augmented IL-12 production was established by the finding that after removal of B cells with monoclonal antibody and complement, wild-type- and KO-derived cells produced equivalent levels of IL-12 in response to T. gondii antigen. These results demonstrate a protective role of IL-5 against T. gondii infection and suggest that IL-5 may play a role in the production of IL-12.     

Dectin-1 is Inducible and Plays an Essential Role for Mycobacteria-Induced Innate Immune Responses in Airway Epithelial Cells

Introduction  

Airway epithelial cells are the first cells to be challenged upon contact with mycobacteria. In response, they express pattern-recognition receptors that play fundamental roles as sentinels and mediators of pulmonary innate immunity. The c-type lectin Dectin-1 is expressed predominantly on the surface of myeloid lineage cells. In this study, we examined the induction, regulation, and functions of Dectin-1 in pulmonary epithelial cells.     

Interleukin-20 Promotes Airway Remodeling in Asthma

Previous studies have demonstrated that interleukin-20 (IL-20) is a pro-inflammatory cytokine, and it has been implicated in psoriasis, lupus nephritis, rheumatoid arthritis, atherosclerosis, and ulcerative colitis. Little is known about the effects of IL-20 in airway remodeling in asthma. The aim of our study was to demonstrate the function of IL-20 in airway remodeling in asthma. To identify the expression of IL-20 and its receptor, IL-20R1/IL-20R2, in the airway epithelium in bronchial tissues, bronchial biopsy specimens were collected from patients and mice with asthma and healthy subjects and stained with specific antibodies. To characterize the effects of IL-20 in asthmatic airway remodeling, we silenced and stimulated IL-20 in cell lines isolated from mice by shRNA and recombinant protein approaches, respectively, and detected the expression of α-SMA and FN-1 by Western blot analysis. First, overexpression of IL-20 and its receptor, IL-20R1/IL-20R2, was detected in the airway epithelium collected from patients and mice with asthma. Second, IL-20 increased the expression of fibronectin-1 and α-SMA, and silencing of IL-20 in mouse lung epithelial (MLE)-12 cells decreased the expression of fibronectin-1 and α-SMA. IL-20 may be a critical cytokine in airway remodeling in asthma. This study indicates that targeting IL-20 and/or its receptors may be a new therapeutic strategy for 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.     

哮喘患者血浆类胰蛋白酶、血嗜酸粒细胞和气道高反应性相互关系

为探讨肥大细胞、嗜酸粒细胞和气道高反应性相互关系 ,本文检测了 36例哮喘患者 (哮喘组 )及 2 9例健康人 (对照组 )血浆中激活肥大细胞的特征性标志物类胰蛋白酶值、血嗜酸粒细胞 (EOS )值及乙酰甲胆碱的支气管激发试验 (PC2 0 )值 ,并对三者关系进行分析比较 ,结果 :(1)哮喘组血浆类胰蛋白酶增高率为 36 11% (13/ 36)和EOS增高率为 38 89% (14/ 36 ) ,明显高于对照组 (P分别 <0 0 0 5 ) ;(2 )哮喘组PC2 0 值 <8g/L者占 81 82 % (2 7/ 33) ,显著低于对照组 (P <0 0 0 5 ) ;(3)哮喘EOS增高组的PC2 0 值为 1 2 1g/L ,明显低于EOS正常组的 5 11g/L (P <0 0 1) ;(4 )哮喘类胰蛋白酶增高组中有 61 5 3% (8/13)EOS值增高 ,明显高于类胰蛋白酶正常组的 2 6 0 9% (6/ 2 3) (P <0 0 5 )。综上所述 ,哮喘患者呈气道高反应 ,血EOS和类胰蛋白酶值增高 ;EOS与气道高反应性有关 ;类胰蛋白酶与EOS增高有关 ,但未直接与气道高反应性相关     

An Essential Role for Gamma Interferon in Innate Resistance to Shigella flexneri Infection

Shigella spp. are the major cause of bacillary dysentery worldwide. To identify immune effectors associated with protection of the naive host during infection, the susceptibility to pulmonary Shigella infection of each of various mouse strains that have a targeted deletion in a specific aspect of the immune system was evaluated. Our results demonstrate that mice deficient in gamma interferon are 5 orders of magnitude more susceptible to Shigella than are wild-type mice, whereas mice deficient in B and T lymphocytes or in T lymphocytes alone exhibit no difference in susceptibility. Significantly lower numbers of shigellae were recovered from immunocompetent compared with gamma-interferon-deficient mice after infection. While immunocompetent mice were able to clear a sublethal Shigella inoculum by day 5 postinfection, progressively increasing numbers of shigellae were cultured from the lungs of gamma interferon-deficient mice over the same period. Histopathology of the lungs from immunocompetent mice infected with a sublethal Shigella inoculum showed mild inflammatory changes, whereas the lungs from gamma interferon-deficient mice demonstrated progressively worsening acute bronchiolitis with ulceration. Further, the time to death in gamma interferon-deficient mice correlates inversely with the size of the Shigella inoculum. To identify the cellular source of gamma interferon, we infected SCID mice, T-cell-receptor-deficient mice, beige mice (a mouse strain deficient in natural killer [NK] cell activity), and mice depleted of NK cells using anti-asialo-GM₁. Each NK cell-deficient mouse strain exhibited a 10-fold-greater susceptibility to Shigella infection than immunocompetent mice. To test the protective effects of gamma interferon in vitro, survival of intracellular Shigella was examined in primary macrophages from wild-type mice, primary macrophages from gamma interferon-deficient mice, a macrophage cell line, and a fibroblast cell line. Following activation with gamma interferon, each cell type eradicated intracellular Shigella, while nonactivated macrophages fostered Shigella replication and nonactivated fibroblast cells fostered both Shigella replication and intercellular spread. Taken together, these data establish that NK cell-mediated gamma interferon is essential to resistance following primary Shigella infection.     

Rhinitis Patients With Sputum Eosinophilia Show Decreased Lung Function in the Absence of Airway Hyperresponsiveness

Purpose

Sputum eosinophilia is observed frequently in patients with rhinitis. Sputum eosinophilia in patients with non-asthmatic allergic rhinitis has been suggested to be related to nonspecific airway hyperresponsiveness (AHR). However, the clinical significance of sputum eosinophilia in patients with non-asthmatic rhinitis without AHR has not been determined. We conducted a retrospective study examining the influence of sputum eosinophilia in patients with non-asthmatic rhinitis without AHR on pulmonary function and expression of fibrosis-related mediators.

Methods

Eighty-nine patients with moderate-to-severe perennial rhinitis without AHR were included. All underwent lung function tests (forced expiratory volume in 1 second [FEV1] and forced vital capacity [FVC]), skin tests to inhalant allergens, methacholine bronchial challenge tests, and hypertonic saline-induced sputum to determine eosinophil counts. Sputum mRNA levels for transforming growth factor-β (TGF-β), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were also examined. Patients were divided into two groups according to the presence of sputum eosinophilia (≥3%, eosinophilia-positive [EP] and <3%, eosinophilia-negative [EN] groups).

Results

FEV1 was significantly lower (P=0.04) and FEV1/FVC tended to be lower (P=0.1) in the EP group than in the EN group. In sputum analyses, the MMP-9 mRNA level (P=0.005) and the ratio of MMP-9 to TIMP-1 expression (P=0.01) were significantly higher in the EP group than in the EN group. There was no significant difference in TGF-β mRNA expression between the two groups.

Conclusions

Sputum eosinophilia in patients with moderate-to-severe perennial rhinitis without AHR influenced FEV1 and the expression pattern of fibrosis-related mediators.     

Dehydroepiandrosterone Suppresses Eosinophil Infiltration and Airway Hyperresponsiveness via Modulation of Chemokines and Th2 Cytokines in Ovalbumin-Sensitized Mice

In this study, we evaluated the anti-inflammatory response and the mechanism by which dehydroepiandrosterone modulates immunity in ovalbumin-sensitized asthmatic mice. Female BALB/c mice were sensitized and challenged with ovalbumin and then treated with oral administration of dehydroepiandrosterone on days 21 to 27. The results showed dehydroepiandrosterone could suppress airway hyperresponsiveness and decrease eosinophil infiltration of the lungs in ovalbumin-sensitized mice. Moreover, dehydroepiandrosterone inhibited chemokines, including CCL11/eotaxin-1 and CCL24/eotaxin-2, and Th2-associated cytokine levels in bronchoalveolar lavage fluid. After the inflammatory human bronchial epithelial cell line BEAS-2B was treated with dehydroepiandrosterone, levels of proinflammatory cytokines and chemokines were inhibited, including IL-6, IL-8, CCL11, and CCL24. We suggested that dehydroepiandrosterone inhibited inflammation in bronchial epithelial cells as indicated by the suppression of Th2-associated cytokines and chemokines. Dehydroepiandrosterone also suppressed eosinophil migration and infiltration into the lung to improve the symptoms of asthma in ovalbumin-sensitized mice.     

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.