Heme oxygenase‐1 inhibits basophil maturation and activation but promotes its apoptosis in T helper type 2‐mediated allergic airway inflammation

The anti‐inflammatory role of heme oxygenase‐1 (HO‐1) has been studied extensively in many disease models including asthma. Many cell types are anti‐inflammatory targets of HO‐1, such as dendritic cells and regulatory T cells. In contrast to previous reports that HO‐1 had limited effects on basophils, which participate in T helper type 2 immune responses and antigen‐induced allergic airway inflammation, we demonstrated in this study, for the first time, that the up‐regulation of HO‐1 significantly suppressed the maturation of mouse basophils, decreased the expression of CD40, CD80, MHC‐II and activation marker CD200R on basophils, blocked DQ‐ovalbumin uptake and promoted basophil apoptosis both in vitro and in vivo, leading to the inhibition of T helper type 2 polarization. These effects of HO‐1 were mimicked by exogenous carbon monoxide, which is one of the catalytic products of HO‐1. Furthermore, adoptive transfer of HO‐1‐modified basophils reduced ovalbumin‐induced allergic airway inflammation. The above effects of HO‐1 can be reversed by the HO‐1 inhibitor Sn‐protoporphyrin IX. Moreover, conditional depletion of basophils accompanying hemin treatment further attenuated airway inflammation compared with the hemin group, indicating that the protective role of HO‐1 may involve multiple immune cells. Collectively, our findings demonstrated that HO‐1 exerted its anti‐inflammatory function through suppression of basophil maturation and activation, but promotion of basophil apoptosis, providing a possible novel therapeutic target in allergic asthma.     

Differential control of CD4+ T‐cell subsets by the PD‐1/PD‐L1 axis in a mouse model of allergic asthma

Studies examining the role of PD‐1 family members in allergic asthma have yielded conflicting results. Using a mouse model of allergic asthma, we demonstrate that blockade of PD‐1/PD‐L1 has distinct influences on different CD4⁺ T‐cell subsets. PD‐1/PD‐L1 blockade enhances airway hyperreactivity (AHR), not by altering the magnitude of the underlying Th2‐type immune response, but by allowing the development of a concomitant Th17‐type immune response. Supporting differential CD4⁺ T‐cell responsiveness to PD‐1‐mediated inhibition, naïve PD‐1^?/? mice displayed elevated Th1 and Th17 levels, but diminished Th2 cytokine levels, and ligation of PD‐1 in WT cells limited cytokine production by in vitro polarized Th1 and Th17 cells, but slightly enhanced cytokine production by in vitro polarized Th2 cells. Furthermore, PD‐1 ligation enhanced Th2 cytokine production by naïve T cells cultured under nonpolarizing conditions. These data demonstrate that different CD4⁺ T‐cell subsets respond differentially to PD‐1 ligation and may explain some of the variable results observed in control of allergic asthma by the PD‐1 family members. As the PD‐1/PD‐L1 axis limits asthma severity by constraining Th17 cell activity, this suggests that severe allergic asthma may be associated with a defective PD‐1/PD‐L1 regulatory axis in some individuals.     

Cytotoxic T lymphocyte antigen 4 immunoglobulin modified dendritic cells attenuate allergic airway inflammation and hyperresponsiveness by regulating the development of T helper type 1 (Th1)/Th2 and Th2/regulatory T cell subsets in a murine model of asthma

T helper type 2 (Th2) and regulatory T cells (T(reg) ) have been postulated to have critical roles in the pathogenesis of allergic asthma. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified dendritic cells (DC-CTLA4Ig) have the potential to reduce Th2 cells and induce T(reg) cells. In the present study, we evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice in an experimental model of asthma. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA for 7 days. Just prior to the first challenge, DC-CTLA4Ig, DCs or DCs infected with DC-green fluorescent protein (GFP) were injected intravenously into mice. The administration of DC-CTLA4Ig reduced airway hyperresponsiveness, relieved asthmatic airway inflammation and decreased the numbers of esosinophils in the BALF in OVA-sensitized/challenged mice. In addition, DC-CTLA4Ig altered the balance of Th1/Th2 cytokine production in the lungs with increased interferon (IFN)-γ levels and decreased interleukin (IL)-4 levels, decreased the percentage of Th2 and increased both the percentage of Th1 and T(reg) cells in the lungs of OVA-sensitized/challenged mice. This research demonstrates that DC-CTL4Ig reduces airway hyperresponsiveness effectively and prevents airway inflammation in OVA-sensitized/challenged mice, which is due most probably to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in the local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/T(reg) cells.     

Bacillus‐derived poly‐γ‐glutamic acid attenuates allergic airway inflammation through a Toll‐like receptor‐4‐dependent pathway in a murine model of asthma

Background Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly‐γ‐glutamic acid (γ‐PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ‐PGA promoted Th1 cell development in a manner dependent on antigen‐presenting cells, but inhibited Th2 cell development. Objective To investigate the effect of γ‐PGA on dendritic cells (DCs), and its potential for treating Th2‐mediated allergic asthma. Methods Wild‐type, Toll‐like receptor (TLR)‐2 deficient, and TLR‐4‐defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ‐PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ‐PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. Results γ‐PGA selectively signalled conventional DCs to activate NF‐κB and mitogen‐activated protein kinase, leading to the up‐regulation of CD86, CD40, and IL‐12, but not IL‐10 and IL‐6. These effects of γ‐PGA were dependent on TLR‐4 and independent of TLR‐2. Importantly, the intranasal administration of γ‐PGA to OVA‐sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ‐PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen‐specific restimulation. These anti‐asthmatic effects of γ‐PGA were also abolished in TLR‐4‐defective mice. Conclusions and Clinical Relevance Our data indicate that γ‐PGA activates DCs to favour Th1 cell induction through a TLR‐4‐dependent pathway and alleviates pathologic symptoms in a Th2‐biased asthmatic model. These findings highlight the potential of γ‐PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role. Cite this as: K. Lee, S.‐H. Kim, H. J. Yoon, D. J. Paik, J. M. Kim and J. Youn, Clinical & Experimental Allergy, 2011 (41) 1143–1156.     

Gene expression profiling by microarray analysis reveals an important role for caspase‐1 in dengue virus‐induced p53‐mediated apoptosis

Recently, a dengue virus‐induced apoptosis p53‐ and mitochondria‐mediated were reported in human and animal cells. To understand further the underlying mechanisms, a p53‐deficient cell line, H1299, and a p53‐knockin cell line, H273, were infected with dengue type 1 virus and the cellular gene expression profiles at the mRNA level were analyzed by affymetrix array analysis. The results showed 183 genes with at least twofold increase at mRNA expression level in dengue virus‐infected cells. Among the 183 genes, 68 genes were up‐regulated in both H1299 and H273 cells and 78 genes were found to be up‐regulated in only H273 cells. Eleven selected genes, mainly involved in IFN‐pathway, cell cycle, signal transduction, and ubiquitin–proteasome pathways were confirmed using qualitative and quantitative PCR. Interestingly, an approximately 32‐fold increase in caspase‐1 expression was observed in the p53‐knockin cell line, H273. Gene silencing of caspase‐1 or inhibition of caspase‐1 activity led to reduction in dengue virus‐induced apoptosis with minimal effect on virus replication. J. Med. Virol. 81:1069–1081, 2009. © 2009 Wiley‐Liss, Inc.     

Induction of glucocorticoid receptor‐β expression in epithelial cells of asthmatic airways by T‐helper type 17 cytokines

Background Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor‐β (GR‐β) in many cell types. T‐helper type 17 (Th17) cytokine (IL‐17A and F) expressions increase in mild and in difficult‐to‐treat asthma. We hypothesize that IL‐17A and F cytokines alone or in combination, induce the expression of GR‐β in bronchial epithelial cells. Objectives To confirm the expression of the GR‐β and IL‐17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL‐17A and F on the expression of GR‐β in bronchial epithelial cells obtained from normal subjects and asthmatic patients. Methods The expression of IL‐17A and F, GR‐α and GR‐β was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q‐RT‐PCR. Immunohistochemistry for IL‐17 and GR‐β was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL‐6 in response to IL‐17A and F and dexamethasone was determined by Q‐RT‐PCR using primary airway epithelial cells from normal and asthmatic subjects. Results We detected significantly higher levels of IL‐17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR‐α expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL‐17F and GR‐β in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR‐β in response to IL‐17A and F in the cells from asthmatics (P0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL‐17‐induced IL‐6 expression in cells from normal individuals but not in those from asthmatics (P0.05). Conclusion Evidence of an increased GR‐β expression in epithelial cells following IL‐17 stimulation suggests a possible role for Th17‐associated cytokines in the mechanism of steroid hypo‐responsiveness in asthmatic subjects. Cite this as: A. Vazquez‐Tello, A. Semlali, J. Chakir, J. G. Martin, D. Y. Leung, D. H. Eidelman and Q. Hamid, Clinical & Experimental Allergy, 2010 (40) 1312–1322.     

Micro‐RNA‐155‐mediated control of heme oxygenase 1 (HO‐1) is required for restoring adaptively tolerant CD4+ T‐cell function in rodents

T cells chronically stimulated by a persistent antigen often become dysfunctional and lose effector functions and proliferative capacity. To identify the importance of micro‐RNA‐155 (miR‐155) in this phenomenon, we analyzed mouse miR‐155‐deficient CD4⁺ T cells in a model where the chronic exposure to a systemic antigen led to T‐cell functional unresponsiveness. We found that miR‐155 was required for restoring function of T cells after programmed death receptor 1 blockade. Heme oxygenase 1 (HO‐1) was identified as a specific target of miR‐155 and inhibition of HO‐1 activity restored the expansion and tissue migration capacity of miR‐155^?/? CD4⁺ T cells. Moreover, miR‐155‐mediated control of HO‐1 expression in CD4⁺ T cells was shown to sustain in vivo antigen‐specific expansion and IL‐2 production. Thus, our data identify HO‐1 regulation as a mechanism by which miR‐155 promotes T‐cell‐driven inflammation.     

T‐helper 1 and T‐helper 2 adjuvants induce distinct differences in the magnitude,quality and kinetics of the early inflammatory response at the site of injection

Vaccine adjuvants activate the innate immune system and thus influence subsequent adaptive T‐cell responses. However, little is known about the initial immune mechanisms preceding the adjuvant‐induced differentiation of T‐helper (Th) cells. The effect of a T‐helper 1 (Th1) adjuvant, dimethyldioctadecylammonium liposomes with monophosphoryl lipid‐A (DDA/MPL), and a T‐helper 2 adjuvant, aluminium hydroxide [Al(OH)₃], on early, innate chemotactic signals and inflammatory cell influx at the site of injection was therefore investigated. Injection of the adjuvants into the peritoneal cavity of mice demonstrated distinct differences in the magnitude, quality and kinetics of the response. The inflammatory response to DDA/MPL was prominent, inducing high local levels of pro‐inflammatory cytokines, chemokines and a pronounced inflammatory exudate consisting of neutrophils, monocytes/macrophages and activated natural killer cells. This was in contrast to the response induced by Al(OH)₃, which, although sharing some of the early chemokine signals, was more moderate and consisted almost exclusively of neutrophils and eosinophils. Notably, Al(OH)₃ specifically induced the release of a significant amount of interleukin (IL)‐5, whereas DDA/MPL induced high amounts of tumour necrosis factor‐α (TNF‐α), IL‐1α and IL‐6. Finally, a microarray analysis confirmed that the effect of DDA/MPL was broader with more than five times as many genes being specifically up‐regulated after injection of DDA/MPL compared with Al(OH)₃. Thus, the adjuvants induced qualitatively distinct local inflammatory signals early after injection.     

Larval Echinococcus multilocularis infection reduces dextran sulphate sodium‐induced colitis in mice by attenuating T helper type 1/type 17‐mediated immune reactions

The tumour‐like growth of larval Echinococcus multilocularis tissue (causing alveolar echinococcosis, AE) is directly linked to the nature/orientation of the periparasitic host immune‐mediated processes. Parasite‐mediated immune suppression is a hallmark triggering infection outcome in both chronic human and murine AE. So far, little is known about secondary systemic immune effects of this pathogen on other concomitant diseases, e.g. endogenous gut inflammation. We examined the influence of E. multilocularis infection on murine dextran sodium sulphate (DSS) ‐induced colitis. At 3 months after E. multilocularis infection (chronic stage), the mice were challenged with 3% DSS in the drinking water for 5 days plus subsequently with tap water (alone) for another 4 days. After necropsy, fixed tissues/organs were sectioned and stained with haematoxylin & eosin for assessing inflammatory reactions. Cytokine levels were measured by flow cytometry and quantitative RT‐PCR. Colitis severity was assessed (by board‐certified veterinary pathologists) regarding (i) colon length, (ii) weight loss and (iii) a semi‐quantitative score of morphological changes. The histopathological analysis of the colon showed a significant reduction of DSS‐induced gut inflammation by concomitant E. multilocularis infection, which correlated with down‐regulation of T helper type 1 (Th1)/Th17 T‐cell responses in the colon tissue. Echinococcus multilocularis infection markedly reduced the severity of DSS‐induced gut inflammation upon down‐regulation of Th1/Th17 cytokine expression and attenuation of CD11b⁺ cell activation. In conclusion, E. multilocularis infection remarkably reduces DSS‐induced colitis in mice by attenuating Th1/Th17‐mediated immune reactions.     

Basophils as a primary inducer of the T helper type 2 immunity in ovalbumin‐induced allergic airway inflammation

Antigen‐induced allergic airway inflammation is mediated by T helper type 2 (Th2) cells and their cytokines, but the mechanism that initiates the Th2 immunity is not fully understood. Recent studies show that basophils play important roles in initiating Th2 immunity in some inflammatory models. Here we explored the role of basophils in ovalbumin (OVA) ‐induced airway allergic inflammation in BALB/c mice. We found that OVA sensitization and challenge resulted in a significant increase in the amount of basophils in blood and lung, along with the up‐regulation of activation marker of CD200R. However, depletion of basophils with MAR‐1 or Ba103 antibody attenuated airway inflammation, represented by the significantly decreased amount of the Th2 subset in spleen and draining lymph nodes, interlukin‐4 level in lung and OVA‐special immunoglobulin E (sIgE) levels in serum. On the other hand, adoptive transfer of basophils from OVA‐challenged lung tissue to naive BALB/c mice provoked the Th2 immune response. In addition, pulmonary basophils from OVA‐challenged mice were able to uptake DQ‐OVA and express MHC class II molecules and CD40 in vivo, as well as to release interleukin‐4 following stimulation by IgE–antigen complexes and promote Th2 polarization in vitro. These findings demonstrate that basophils may participate in Th2 immune responses in antigen‐induced allergic airway inflammation and that they do so through facilitating antigen presentation and providing interleukin‐4.     

Neurotransmitter signalling via NMDA receptors leads to decreased T helper type 1‐like and enhanced T helper type 2‐like immune balance in humans

Given the pivotal roles that CD4⁺ T cell imbalance plays in human immune disorders, much interest centres on better understanding influences that regulate human helper T‐cell subset dominance in vivo. Here, using primary CD4⁺ T cells and short‐term T helper type 1 (Th1) and Th2‐like lines, we investigated roles and mechanisms by which neurotransmitter receptors may influence human type 1 versus type 2 immunity. We hypothesized that N‐methyl‐d ‐aspartate receptors (NMDA‐R), which play key roles in memory and learning, can also regulate human CD4⁺ T cell function through induction of excitotoxicity. Fresh primary CD4⁺ T cells from healthy donors express functional NMDA‐R that are strongly up‐regulated upon T cell receptor (TCR) mediated activation. Synthetic and physiological NMDA‐R agonists elicited Ca²⁺ flux and led to marked inhibition of type 1 but not type 2 or interleukin‐10 cytokine responses. Among CD4⁺ lines, NMDA and quinolinic acid preferentially reduced cytokine production, Ca²⁺ flux, proliferation and survival of Th1‐like cells through increased induction of cell death whereas Th2‐like cells were largely spared. Collectively, the findings demonstrate that (i) NMDA‐R is rapidly up‐regulated upon CD4⁺ T cell activation in humans and (ii) Th1 versus Th2 cell functions such as proliferation, cytokine production and cell survival are differentially affected by NMDA‐R agonists. Differential cytokine production and proliferative capacity of Th1 versus Th2 cells is attributable in part to increased physiological cell death among fully committed Th1 versus Th2 cells, leading to increased Th2‐like dominance. Hence, excitotoxicity, beyond its roles in neuronal plasticity, may contribute to ongoing modulation of human T cell responses.