Glucocorticoid-induced TNFR family-related protein (GITR)-mediated activation of JNK was shown to regulate the suppressive activity of CD4(+)CD25(+) naturally occurring T regulatory cells (nTregs) in wild-type (WT) hosts. In this study, CD4(+)CD25(+) T cells were shown to be capable of becoming pathogenic effector cells in sensitized and challenged CD8(-/-) recipient mice. Only GITR-expressing CD4(+)CD25(+) T cells, but neither GITR knocked-in CD4(+)CD25(-) T cells nor GITR-silenced CD4(+)CD25(+) T cells, enhanced development of lung allergic responses. Inhibition of JNK in WT nTregs or nTregs from GITR(-/-)and JNK2(-/-) mice failed to enhance lung allergic responses in sensitized and challenged CD8(-/-) recipient mice. The failure to enhance responses was associated with increased bronchoalveolar lavage fluid levels of IL-10 and TGF-? and decreased levels of IL-5, IL-6, and IL-13. In contrast, nTregs from JNK1(-/-) mice, similar to WT nTregs, were fully effective in enhancing responses. Thus, GITR stimulation of nTregs and signaling through JNK2, but not JNK1, triggered the loss of regulatory function while concomitantly gaining pathogenic CD4(+) T effector cell function responsible for exacerbating asthma-like immunopathology.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illnesses in infants worldwide. Both RSV-G and RSV-F glycoproteins play pathogenic roles during infection with RSV. The objective of this study was to compare the effects of anti-RSV-G and anti-RSV-F monoclonal antibodies (mAbs) on airway hyperresponsiveness (AHR) and inflammation after primary or secondary RSV infection in mice. In the primary infection model, mice were infected with RSV at 6 weeks of age. Anti-RSV-G or anti-RSV-F mAbs were administered 24 hours before infection or Day +2 postinfection. In a secondary infection model, mice were infected (primary) with RSV at 1 week (neonate) and reinfected (secondary) 5 weeks later. Anti-RSV-G and anti-RSV-F mAbs were administered 24 hours before the primary infection. Both mAbs had comparable effects in preventing airway responses after primary RSV infection. When given 2 days after infection, anti-RSV-G-treated mice showed significantly decreased AHR and airway inflammation, which persisted in anti-RSV-F-treated mice. In the reinfection model, anti-RSV-G but not anti-RSV-F administered during primary RSV infection in neonates resulted in decreased AHR, eosinophilia, and IL-13 but increased levels of IFN-? in bronchoalveolar lavage on reinfection. These results support the use of anti-RSV-G in the prevention and treatment of RSV-induced disease.
Ozone pollution is associated with adverse effects on respiratory health in adults and children but its effects on the neonatal lung remain unknown. This study was carried out to define the effect of acute ozone exposure on the neonatal lung and to profile the transcriptome response. Newborn mice were exposed to ozone or filtered air for 3 h. Total RNA was isolated from lung tissues at 6 and 24 h after exposure and was subjected to microarray gene expression analysis. Compared to filtered air-exposed littermates, ozone-exposed newborn mice developed a small but significant neutrophilic airway response associated with increased CXCL1 and CXCL5 expression in the lung. Transcriptome analysis indicated that 455 genes were down-regulated and 166 genes were up-regulated by at least 1.5 fold at 6 h post-ozone exposure (t-test, p < 0.05). At 24 h, 543 genes were down-regulated and 323 genes were up-regulated in the lungs of ozone-exposed, compared to filtered air-exposed, newborn mice (t-test, p < 0.05). After controlling for false discovery rate, 50 genes were identified as significantly down-regulated and only a few (RORC, GRP, VREB3, and CYP2B6) were up-regulated at 24 h post-ozone exposure (q < 0.05). Gene ontology enrichment analysis revealed that cell cycle-associated functions including cell division/proliferation were the most impacted pathways, which were negatively regulated by ozone exposure, an adverse effect that was associated with reduced bromo-deoxyuridine incorporation. These results demonstrate that acute ozone exposure alters cell proliferation in the developing neonatal lung through a global suppression of cell cycle function.
Human rhinovirus (HRV) is the most common viral etiology in acute exacerbations of asthma. However, the exact mechanisms underlying HRV infection in allergic airways are poorly understood. IL-13 increases interleukin-1 receptor associated kinase M (IRAK-M) and subsequently inhibits airway innate immunity against bacteria. However, the role of IRAK-M in lung HRV infection remains unclear. Here, we provide the first evidence that IRAK-M over-expression promotes lung epithelial HRV-16 replication and autophagy, but inhibits HRV-16-induced IFN-? and IFN-?1 expression. Inhibiting autophagy reduces HRV-16 replication. Exogenous IFN-? and IFN-?1 inhibit autophagy and HRV-16 replication. Our data indicate the enhancing effect of IRAK-M on epithelial HRV-16 infection, which is partly through the autophagic pathway. Impaired anti-viral interferon production may serve as a direct or an indirect (e.g., autophagy) mechanism of enhanced HRV-16 infection by IRAK-M over-expression. Targeting autophagic pathway or administrating anti-viral interferons may prevent or attenuate viral (e.g., HRV-16) infections in allergic airways.
Although national asthma guidelines help organize standards for asthma care, current asthma management is still primarily symptom based. Recent reports provide insights on how to improve asthma management through steps to better understand the natural history of asthma, individualize asthma care, reduce asthma exacerbations, manage inner city asthma, and some potential new ways to use available medications to improve asthma control. Despite many significant gains in managing asthma, we must now find improved strategies to prevent asthma exacerbations, alter the natural history of the disease, and to reduce health disparities in asthma care. Perhaps new directions in personalized medicine including a systems biology approach, along with improved health care access and communication will lead to better methods to alleviate the burden of asthma. This review will discuss the benefits and limitations of the current approach to asthma management, new studies that could impact new directions in asthma management, and new insights related to mechanisms of asthma and allergic airways inflammation that could eventually lead to improved asthma control.
Naturally occurring CD4(+)CD25(+)Foxp3(+) T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-?. nTregs from CD8(-/-) mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-?, and high levels of IL-6. Administration of anti-IL-6 or anti-IL-6R to wild-type recipients prior to transfer of CD8(-/-) nTregs restored suppression. nTregs from IL-6(-/-) mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8(+) cells. Transfer of nTregs from CD8(-/-) donors reconstituted with CD8(+) T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8-T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.
Invariant NKT cells (iNKT cells) play a pivotal role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation. However, it is unclear what role they play in the initiation (sensitization) phase as opposed to the effector (challenge) phase. The role of iNKT cells during sensitization was examined by determining the response of mice to intratracheal transfer of OVA-pulsed or OVA-alpha-galactosylceramide (OVA/alphaGalCer)-pulsed bone marrow-derived dendritic cells (BMDCs) prior to allergen challenge. Wild-type (WT) recipients of OVA-BMDCs developed AHR, increased airway eosinophilia, and increased levels of Th2 cytokines in bronchoalveolar lavage fluid, whereas recipients of OVA/alphaGalCer BMDCs failed to do so. In contrast, transfer of these same OVA/alphaGalCer BMDCs into IFN-gamma-deficient (IFN-gamma(-/-)) mice enhanced the development of these lung allergic responses, which was reversed by exogenous IFN-gamma treatment following OVA-BMDC transfer. Further, Jalpha18-deficient recipients, which lack iNKT cells, developed the full spectrum of lung allergic responses following reconstitution with highly purified WT liver or spleen iNKT cells and transfer of OVA-BMDCs, whereas reconstituted recipients of OVA/alphaGalCer BMDCs failed to do so. Transfer of iNKT cells from IFN-gamma(-/-) mice restored the development of these responses in Jalpha18-deficient recipients following OVA-BMDC transfer; the responses were enhanced following OVA/alphaGalCer BMDC transfer. iNKT cells from these IFN-gamma(-/-) mice produced higher levels of IL-13 in vitro compared with WT iNKT cells. These data identify IFN-gamma as playing a critical role in dictating the consequences of iNKT cell activation in the initiation phase of the development of AHR and airway inflammation.
Respiratory syncytial virus (RSV) bronchiolitis in infants may be followed by the development of asthma-like symptoms. Age at first infection dictates consequences upon reinfection. Reinfection of mice initially exposed as neonates to RSV enhanced development of airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus hyperproduction. RSV lower respiratory tract disease is associated with activation of the leukotriene pathway.
Respiratory syncytial virus (RSV) is the most common cause of hospitalization for respiratory tract infection in young children. It is also a significant cause of morbidity and mortality in elderly individuals and in persons with asthma and chronic obstructive pulmonary disease. Currently, no reliable vaccine or simple RSV antiviral therapy is available. Recently, we determined that the minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), could markedly attenuate inflammatory responses induced by lipopolysaccharide through direct interactions with the Toll-like receptor 4 (TLR4) interacting proteins CD14 and MD-2. CD14 and TLR4 have been implicated in the host response to RSV. Treatment of bronchial epithelial cells with POPG significantly inhibited interleukin-6 and -8 production, as well as the cytopathic effects induced by RSV. The phospholipid bound RSV with high affinity and inhibited viral attachment to HEp2 cells. POPG blocked viral plaque formation in vitro by 4 log units, and markedly suppressed the expansion of plaques from cells preinfected with the virus. Administration of POPG to mice, concomitant with viral infection, almost completely eliminated the recovery of virus from the lungs at 3 and 5 days after infection, and abrogated IFN-gamma (IFN-gamma) production and the enhanced expression of surfactant protein D (SP-D). These findings demonstrate an important approach to prevention and treatment of RSV infections using exogenous administration of a specific surfactant phospholipid.
Naturally occurring Foxp3(+)CD4(+)CD25(+) T cells isolated from lungs of naive mice regulate lung allergic airway hyperresponsiveness, inflammation, levels of Th2 cytokines, and mucus production. OVA-specific (alphabetaTCR(+)) CD4(+)CD25(+) T cells suppressed ragweed-induced airway hyperresponsiveness and inflammation as did anti-TCR-treated OVA-specific CD4(+)CD25(+) T cells, suggesting that Ag-specificity was not required for expression of regulatory activities. Suppression was associated with increased levels of IL-10 and TGF-beta; decreased levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid; and reduced recruitment and activation of CD8(+) T cells in the airways. Following intratracheal administration, OVA-specific CD4(+)CD25(+) T cells were identified in both the airway lumens and lung parenchyma, and in some instances in close proximity to host CD8(+) T cells. These results demonstrate that the regulatory activities of naturally occurring Foxp3(+)CD4(+)CD25(+) T cells on lung allergic responses are Ag-nonspecific and thus, independent of Ag-specific recognition.
Suppressing the abnormalities associated with asthma has been difficult to accomplish using immunotherapy or vaccination once the disease is established. The effector cells necessary for effective immunization/vaccination and immunotherapy of asthma are also not well understood. Therefore, we vaccinated allergen (OVA)-sensitized mice to determine whether therapeutic immunization could suppress airway hyperresponsiveness (AHR) and inflammation and to identify key immune effector cells and cytokines. Mice were immunized with a vaccine comprised of Ag and cationic liposome-DNA complexes (CLDC), a vaccine which has previously been shown to elicit strong CD4(+) and CD8(+) T cell responses and activation of Th1 immunity. We showed that immunization with the OVA-CLDC vaccine significantly suppressed AHR, eosinophilia, goblet cell metaplasia, and Th2 cytokine production. In contrast, immunization with CLDC alone suppressed eosinophilia and Th2 cytokine production, but failed to suppress AHR and goblet cell changes. Using adoptive transfer experiments, we found that suppression of AHR was mediated by Ag-specific CD8(+) T cells and was dependent on IFN-gamma production by the transferred T cells. Thus, we conclude that generation of strong, allergen-specific CD8(+) T cell responses by immunization may be capable of suppressing AHR and allergic airway inflammation, even in previously sensitized and challenged mice.
Previous studies have shown that leukotriene B4 (LTB4), a proinflammatory lipid mediator, is linked to the development of airway hyperresponsiveness through the accumulation of IL-13-producing CD8+ T cells, which express a high affinity receptor for LTB4, BLT1 (Miyahara et al., Am J Respir Crit Care Med 2005;172:161-167; J Immunol 2005;174:4979-4984). By using leukotriene A4 hydrolase-deficient (LTA4H-/-) mice, which fail to synthesize LTB4, we determined the role of this lipid mediator in allergen-induced airway responses. Two approaches were used. In the first, LTA4H-/- mice and wild-type (LTA4H+/+) mice were systemically sensitized and challenged via the airways to ovalbumin. In the second, mice were passively sensitized with anti-ovalbumin IgE and exposed to ovalbumin via the airways. Mast cells were generated from bone marrow of LTA4H+/+ mice or LTA4H-/- mice. After active sensitization and challenge, LTA4H-/- mice showed significantly lower airway hyperresponsiveness compared with LTA4H+/+ mice, and eosinophil numbers and IL-13 levels in the bronchoalveoloar lavage of LTA4H-/- mice were also significantly lower. LTA4H-/- mice also showed decreased airway reactivity after passive sensitization and challenge. After LTA4H+/+ mast cell transfer, LTA4H-/- mice showed increased airway reactivity after passive sensitization and challenge, but not after systemic sensitization and challenge. These data confirm the important role for LTB4 in the development of altered airway responses and suggest that LTB4 secretion from mast cells is critical to eliciting increased airway reactivity after passive sensitization with allergen-specific IgE.
gammadelta T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of gammadelta T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total gammadelta T cells or specific subsets of gammadelta T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-delta(-/-) mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of gammadelta T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vgamma1+, but not of Vgamma4+ T cells, reduced O3-induced AHR, and transfer of total gammadelta T cells or Vgamma1+ T cells to TCR-delta(-/-) mice restored AHR. After transfer of Vgamma1+ cells to TCR-delta(-/-) mice, restoration of AHR after O3 exposure was blocked by anti-TNF-alpha. However, AHR could be restored in TCR-delta(-/-)mice by transfer of gammadelta T cells from TNF-alpha-deficient mice, indicating that another cell type was the source of TNF-alpha. These results demonstrate that TNF-alpha and activation of Vgamma1+ gammadelta T cells are required for the development of AHR after O3 exposure.
Dendritic cells (DCs) are considered to be the most efficient antigen-presenting cells. Intratracheal administration of allergen-pulsed bone marrow-derived dendritic cells (BMDCs) before allergen challenge induces airway hyperresponsiveness (AHR) and inflammation. Ovalbumin (OVA)-pulsed BMDCs from wild-type (WT) mice were transferred into naive WT, CD4(-/-), CD8(-/-), or IL-13(-/-) mice. Two days (short protocol) or 10 days (long protocol) after BMDC transfer, mice were challenged with 1% OVA for 3 days and assayed 2 days later. Transfer of OVA-primed BMDCs into BALB/c or C57BL/6 mice elicited AHR in both protocols. Airway eosinophilia, Th2 cytokines, or goblet cell metaplasia were increased in the long but not short protocol. Lung T cells from both protocols produced Th2 cytokines in response to OVA in vitro. Carboxyfluorescein diacetate succinimidylester-labeled BMDCs were observed in bronchoalveolar lavage (BAL) fluid and lung parenchyma at early time points, and were detected in draining lymph nodes 48 hours after transfer. CD8(-/-) mice developed AHR comparable to WT mice in the short protocol, but decreased levels of AHR, airway eosinophilia, Th2 cytokines in BAL fluid, and goblet cell metaplasia compared with WT mice in the long protocol. CD4(-/-) or IL-13(-/-) mice did not develop AHR or airway inflammation in either protocol. These data suggest that allergen-pulsed BMDCs initiate development of AHR that is dependent initially on CD4(+) T cells, and at later time periods on CD8+ T cells and IL-13. Thus, the timing of allergen challenge after transfer of allergen-pulsed BMDC affects the development of AHR and airway inflammation.
Ligation of the leukotriene B(4) (LTB(4)) receptor 1 on effector memory CD8(+) T cells by LTB(4) is important for the recruitment of CD8(+) T cells into the airways, which appears central to the induction of airway hyperresponsiveness (AHR) and allergic inflammation. Phosphorylation of extracellular signal-regulated kinase (ERK) is important in activation and cytokine production from many cell types.
Recent studies revealed a critical role for thymic stromal lymphopoietin (TSLP) released from epithelial cells and OX40 ligand (OX40L) expressed on dendritic cells (DCs) in T(H)2 priming and polarization.
Naturally occurring Foxp3(+)CD4(+)CD25(+) T regulatory cell (nTreg)-mediated suppression of lung allergic responses is abrogated following ligation of glucocorticoid-induced tumor necrosis receptor (GITR) family-related protein. In vitro stimulation of nTregs with GITR ligand increased phosphorylation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated protein kinase (ERK) or p38 MAPK. SP600125, a known JNK inhibitor, prevented GITR-mediated phosphorylation of JNK. Activation of JNK was associated with increases in the upstream mitogen-activated protein kinase kinase 7 (MKK7) and the downstream transcription factor NF-??. Phosphorylated c-Jun (p-c-Jun), indicative of the activation of JNK, was detected in the immunoprecipitates of nTregs from wild-type but not JNK- or GITR-deficient mice. Treatment with an inhibitor of JNK phosphorylation resulted in complete reversal of all GITR-induced changes in nTreg phenotype and function, with full restoration of suppression of in vivo lung allergic responses and in vitro proliferation of activated CD4(+)CD25(-) T cells. Thus, regulation of JNK phosphorylation plays a central role in T regulatory cell function with therapeutic implications for the treatment of asthma and autoimmune diseases.
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