Jennifer L. Ather

Serum Amyloid A Activates the NLRP3 Inflammasome and Promotes Th17 Allergic Asthma in Mice

IL-1β is a cytokine critical to several inflammatory diseases in which pathogenic Th17 responses are implicated. Activation of the NLRP3 inflammasome by microbial and environmental stimuli can enable the caspase-1–dependent processing and secretion of IL-1β. The acute-phase protein serum amyloid A (SAA) is highly induced during inflammatory responses, wherein it participates in systemic modulation of innate and adaptive immune responses. Elevated levels of IL-1β, SAA, and IL-17 are present in subjects with severe allergic asthma, yet the mechanistic relationship among these mediators has yet to be identified. In this study, we demonstrate that Saa3 is expressed in the lungs of mice exposed to several mixed Th2/Th17-polarizing allergic sensitization regimens. SAA instillation into the lungs elicits robust TLR2-, MyD88-, and IL-1–dependent pulmonary neutrophilic inflammation. Furthermore, SAA drives production of IL-1α, IL-1β, IL-6, IL-23, and PGE2, causes dendritic cell (DC) maturation, and requires TLR2, MyD88, and the NLRP3 inflammasome for secretion of IL-1β by DCs and macrophages. CD4+ T cells polyclonally stimulated in the presence of conditioned media from SAA-exposed DCs produced IL-17, and the capacity of polyclonally stimulated splenocytes to secrete IL-17 is dependent upon IL-1, TLR2, and the NLRP3 inflammasome. Additionally, in a model of allergic airway inflammation, administration of SAA to the lungs functions as an adjuvant to sensitize mice to inhaled OVA, resulting in leukocyte influx after Ag challenge and a predominance of IL-17 production from restimulated splenocytes that is dependent upon IL-1R signaling.

Nuclear Factor-κB Activation in Airway Epithelium Induces Inflammation and Hyperresponsiveness

RATIONALE Nuclear factor (NF)-kappaB is a prominent proinflammatory transcription factor that plays a critical role in allergic airway disease. Previous studies demonstrated that inhibition of NF-kappaB in airway epithelium causes attenuation of allergic inflammation. OBJECTIVES We sought to determine if selective activation of NF-kappaB within the airway epithelium in the absence of other agonists is sufficient to cause allergic airway disease. METHODS A transgenic mouse expressing a doxycycline (Dox)-inducible, constitutively active (CA) version of inhibitor of kappaB (IkappaB) kinase-beta (IKKbeta) under transcriptional control of the rat CC10 promoter, was generated. MEASUREMENTS AND MAIN RESULTS After administration of Dox, expression of the CA-IKKbeta transgene induced the nuclear translocation of RelA in airway epithelium. IKKbeta-triggered activation of NF-kappaB led to an increased content of neutrophils and lymphocytes, and concomitant production of proinflammatory mediators, responses that were not observed in transgenic mice not receiving Dox, or in transgene-negative littermate control animals fed Dox. Unexpectedly, expression of the IKKbeta transgene in airway epithelium was sufficient to cause airway hyperresponsiveness and smooth muscle thickening in absence of an antigen sensitization and challenge regimen, the presence of eosinophils, or the induction of mucus metaplasia. CONCLUSIONS These findings demonstrate that selective activation NF-kappaB in airway epithelium is sufficient to induce airway hyperresponsiveness and smooth muscle thickening, which are both critical features of allergic airway disease.

Airway epithelial NF-κB activation promotes allergic sensitization to an innocuous inhaled antigen.

Activation of NF-κB in airway epithelium is observed in allergic asthma and is induced by inhalation of numerous infectious and reactive substances. Many of the substances that activate NF-κB in the airway epithelium are also capable of acting as adjuvants to elicit antigen-specific sensitization to concomitantly inhaled protein, thereby circumventing the inherent bias of the lung to promote tolerance to innocuous antigens. We have used a transgenic mouse inducibly expressing a constitutively active mutant of the inhibitor of nuclear factor κB (IκB) kinase β ((CA)IKKβ) that activates NF-κB only in nonciliated airway epithelial cells to test whether activation of this intracellular signaling pathway in this specific cell type is sufficient to establish a pulmonary environment permissive to the development of allergic sensitization to inhaled protein. When airway epithelial (CA)IKKβ was transiently expressed in antigen-naive mice only during initial inhalation of ovalbumin, the mice became allergically sensitized to the antigen. As a consequence, subsequent inhalation of ovalbumin alone led to an allergic asthma-like response that included airway hyperresponsiveness to methacholine, eosinophilia, mucus expression, elevated serum levels of antigen-specific IgE and IgG1, and splenic CD4(+) T cells that secreted T helper type 2 and type 17 cytokines in response to in vitro antigen restimulation. Furthermore, CD11c(+) cells in the mediastinal lymph nodes (MLN) of (CA)IKKβ-expressing mice displayed significantly elevated levels of activation markers. These data implicate airway epithelial NF-κB activation as a critical modulator of the adaptive immune response to inhaled antigens via the secretion of soluble mediators that affect the capacity of CD11c(+) cells to undergo maturation and promote antigen-specific allergic responses.

Mitochondria-targeted drugs enhance Nlrp3 inflammasome-dependent IL-1β secretion in association with alterations in cellular redox and energy status

The Nlrp3 inflammasome is activated in response to an array of environmental and endogenous molecules leading to caspase-1-dependent IL-1β processing and secretion by myeloid cells. Several identified Nlrp3 inflammasome activators also trigger reactive oxygen species (ROS) production. However, the initial concept that NADPH oxidases are the primary source of ROS production during inflammasome activation is becoming less accepted. Therefore, the importance of mitochondria-derived ROS has been recently explored. In this study, we explore the impact of mitochondria dysfunction and ROS production on Nlrp3 inflammasome stimulation and IL-1β secretion induced by serum amyloid A (SAA) in primary mouse peritoneal macrophages. To induce mitochondrial dysfunction, we utilized antimycin A, which blocks electron flow at complex III, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial oxidative phosphorylation uncoupler. We also utilized a superoxide dismutase mimetic, MnTBAP, which targets the mitochondria, as well as the broad-spectrum antioxidants DPI (diphenyleneiodonium chloride) and ebselen. Our findings demonstrate that SAA alone induces mitochondrial ROS in a time-dependent manner. We observed that MnTBAP and ebselen blocked IL-1β secretion caused by SAA only when added before stimulation, and DPI augmented IL-1β secretion. Surprisingly, these effects were not directly related to intracellular or mitochondrial ROS levels. We also found that mitochondria-targeted drugs increased IL-1β secretion regardless of their impact on mitochondrial function and ROS levels, suggesting that mitochondrial ROS-dependent and -independent mechanisms play a role in the Nlrp3 inflammasome/IL-1β secretion axis in SAA-stimulated cells. Finally, we found that FCCP significantly sustained the association of the Nlrp3 inflammasome complex, which could explain the most robust effect among the drugs tested in enhancing IL-1β secretion in SAA-treated cells. Overall, our data suggest that the Nlrp3 inflammasome/IL-1β secretion axis is a very highly regulated inflammatory pathway that is susceptible not only to changes in mitochondrial or intracellular ROS, but also to changes in overall mitochondrial function.

Interleukin-1 Receptor and Caspase-1 Are Required for the Th17 Response in Nitrogen Dioxide–Promoted Allergic Airway Disease

Nitrogen dioxide (NO2) is an environmental pollutant and endogenously generated oxidant associated with the development, severity, and exacerbation of asthma. NO2 exposure is capable of allergically sensitizing mice to the innocuous inhaled antigen ovalbumin (OVA), promoting neutrophil and eosinophil recruitment, and a mixed Th2/Th17 response upon antigen challenge that is reminiscent of severe asthma. However, the identity of IL-17A-producing cells and the mechanisms governing their ontogeny in NO2-promoted allergic airway disease remain unstudied. We measured the kinetics of lung inflammation after antigen challenge in NO2-promoted allergic airway disease, including inflammatory cells in bronchoalveolar lavage and antigen-specific IL-17A production from the lung. We determined that IL-17A(+) cells were predominately CD4(+)T cell receptor (TCR)β(+) Th17 cells, and that a functional IL-1 receptor was required for Th17, but not Th2, cytokine production after in vitro antigen restimulation of lung cells. The absence of natural killer T cells, γδ T cells, or the inflammasome scaffold nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain (Nlrp)3 did not affect the development of NO2-promoted allergic inflammation or IL-17A production. Similarly, neutrophil depletion or the neutralization of IL-1α during sensitization exerted no effect on these parameters. However, the absence of caspase-1 significantly reduced IL-17A production from lung cells without affecting Th2 cytokines or lung inflammation. Finally, the intranasal administration of IL-1β and the inhalation of antigen promoted allergic sensitization that was reflected by neutrophilic airway inflammation and IL-17A production from CD4(+)TCRβ(+) Th17 cells subsequent to antigen challenge. These data implicate a role for caspase-1 and IL-1β in the IL-1 receptor-dependent Th17 response manifest in NO2-promoted allergic airway disease.

Airway Epithelial Indoleamine 2,3-Dioxygenase Inhibits CD4+ T Cells during Aspergillus fumigatus Antigen Exposure

Indoleamine 2,3-dioxygenase (IDO) suppresses the functions of CD4(+) T cells through its ability to metabolize the essential amino acid tryptophan. Although the activity of IDO is required for the immunosuppression of allergic airway disease by the Toll-Like-Receptor 9 (TLR9) agonist, oligonucleotides comprised of cytosine and guanine nucleotides linked by phosphodiester bonds (CpG) DNA, it is unclear whether IDO expression by resident lung epithelial cells is sufficient to elicit these effects. Therefore, we created a transgenic mouse inducibly overexpressing IDO within nonciliated airway epithelial cells. Upon inhalation of formalin-fixed Aspergillus fumigatus hyphal antigens, the overexpression of IDO from airway epithelial cells of these mice reduced the number of CD4(+) T cells within the inflamed lung and impaired the capacity of antigen-specific splenic CD4(+) effector T cells to secrete the cytokines IL-4, IL-5, IL-13, and IFN-γ. Despite these effects, allergic airway disease pathology was largely unaffected in mice expressing IDO in airway epithelium. In support of the concept that dendritic cells are the major cell type contributing to the IDO-inducing effects of CpG DNA, mice expressing TLR9 only in the airway epithelium did not augment IDO expression subsequent to the administration of CpG DNA. Furthermore, the systemic depletion of CD11c(+) cells rendered mice incapable of CpG DNA-induced IDO expression. Our results demonstrate that an overexpression of IDO within the airway epithelium represents a novel mechanism by which the number of CD4(+) T cells recruited to the lung and their capacity to produce cytokines can be diminished in a model of allergic airway disease, and these results also highlight the critical role of dendritic cells in the antiasthmatic effects of IDO induction by CpG DNA.

Epithelial, dendritic, and CD4+ T cell regulation of and by reactive oxygen and nitrogen species in allergic sensitization ☆

BACKGROUND While many of the contributing cell types and mediators of allergic asthma are known, less well understood are the factors that induce allergy in the first place. Amongst the mediators speculated to affect initial allergen sensitization and the development of pathogenic allergic responses to innocuous inhaled antigens and allergens are exogenously or endogenously generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). SCOPE OF REVIEW The interactions between ROS/RNS, dendritic cells (DCs), and CD4(+) T cells, as well as their modulation by lung epithelium, are of critical importance for the genesis of allergies that later manifest in allergic asthma. Therefore, this review will primarily focus on the initiation of pulmonary allergies and the role that ROS/RNS may play in the steps therein, using examples from our own work on the roles of NO(2) exposure and airway epithelial NF-κB activation. MAJOR CONCLUSIONS Endogenously generated ROS/RNS and those encountered from environmental sources interact with epithelium, DCs, and CD4(+) T cells to orchestrate allergic sensitization through modulation of the activities of each of these cell types, which quantitiatively and qualitatively dictate the degree and type of the allergic asthma phenotype. GENERAL SIGNIFICANCE Knowledge of the effects of ROS/RNS at the molecular and cellular levels has the potential to provide powerful insight into the balance between inhalational tolerance (the typical immunologic response to an innocuous inhaled antigen) and allergy, as well as to potentially provide mechanistic targets for the prevention and treatment of asthma.

NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigen-specific CD4+ T cell polarization

BackgroundNitrogen dioxide (NO2) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO2 is also produced endogenously in the lung during acute inflammatory responses. NO2 can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c+ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c+ cells in NO2-promoted allergic sensitization.MethodsWe systemically depleted CD11c+ cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO2 followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c+ cells from wildtype mice were studied after exposure to NO2 and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production.ResultsTransient depletion of CD11c+ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c+ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO2 exposure. By 48 hours, CD11c+MHCII+ DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c+CD11b- and CD11c+CD11b+ pulmonary cells exposed to NO2in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647+ CD11c+MHCII+ DCs present in MLN from NO2-exposed mice by 48 hours. Co-cultures of ova-specific CD4+ T cells from naïve mice and CD11c+ pulmonary cells from NO2-exposed mice produced IL-1, IL-12p70, and IL-6 in vitro and augmented antigen-induced IL-5 production.ConclusionsCD11c+ cells are critical for NO2-promoted allergic sensitization. NO2 exposure causes pulmonary CD11c+ cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.

Distinct Functions of Airway Epithelial Nuclear Factor-κB Activity Regulate Nitrogen Dioxide–Induced Acute Lung Injury

Reactive oxidants such as nitrogen dioxide (NO(2)) injure the pulmonary epithelium, causing airway damage and inflammation. We previously demonstrated that nuclear factor-κ B (NF-κB) activation within airway epithelial cells occurs in response to NO(2) inhalation, and is critical for lipopolysaccharide-induced or antigen-induced inflammatory responses. Here, we investigated whether manipulation of NF-κB activity in lung epithelium affected severe lung injuries induced by NO(2) inhalation. Wild-type C57BL/6J, CC10-IκBα(SR) transgenic mice with repressed airway epithelial NF-κB function, or transgenic mice expressing a doxycycline-inducible, constitutively active I κ B kinase β (CC10-rTet-(CA)IKKβ) with augmented NF-κB function in airway epithelium, were exposed to toxic levels of 25 ppm or 50 ppm NO(2) for 6 hours a day for 1 or 3 days. In wild-type mice, NO(2) caused the activation of NF-κB in airway epithelium after 6 hours, and after 3 days resulted in severe acute lung injury, characterized by neutrophilia, peribronchiolar lesions, and increased protein, lactate dehydrogenase, and inflammatory cytokines. Compared with wild-type mice, neutrophilic inflammation and elastase activity, lung injury, and several proinflammatory cytokines were significantly suppressed in CC10-IκBα(SR) mice exposed to 25 or 50 ppm NO(2). Paradoxically, CC10-rTet-(CA)IKKβ mice that received doxycycline showed no further increase in NO(2)-induced lung injury compared with wild-type mice exposed to NO(2), instead displaying significant reductions in histologic parameters of lung injury, despite elevations in several proinflammatory cytokines. These intriguing findings demonstrate distinct functions of airway epithelial NF-κB activities in oxidant-induced severe acute lung injury, and suggest that although airway epithelial NF-κB activities modulate NO(2)-induced pulmonary inflammation, additional NF-κB-regulated functions confer partial protection from lung injury.

Airway epithelial NF-κB activation promotes the ability to overcome inhalational antigen tolerance.

Inhalational antigen tolerance typically protects against the development of allergic airway disease but may be overcome to induce allergic sensitization preceding the development of asthma.