The finding that murine and simian cells have differential susceptibility to diphtheria toxin (DTx) led to the development of genetically engineered mouse strains that express the simian or human diphtheria toxin receptor (DTR) under the control of various mouse gene promoters. Injection of DTx into DTR engineered mice allows for rapid and transient depletion of various cell populations. There are several advantages to this approach over global knockout mice, including normal mouse development and temporal control over when cell depletion occurs. As a result, many DTR engineered mouse strains have been developed, resulting in significant insights into the cell biology of various disease states. We used Foxp3DTR mice to attempt local depletion of Foxp3+ cells in the lung in a model of tolerance breakdown. Intratracheal administration of DTx resulted in robust depletion of lung Foxp3+ cells. However, DTx administration was accompanied by significant local inflammation, even in control C57Bl/6 mice. These data suggest that DTx administration to non-transgenic mice is not always an immunologically inert event, and proper controls must be used to assess various DTx-mediated depletion regimens.
diphtheria toxin; lung; regulatory T cells; tolerance
Recent published studies have highlighted the complexity of the immune response to allergens, and the various asthma phenotypes that arise as a result. While the interplay of regulatory and effector immune cells responding to allergen would seem to dictate the nature of the asthmatic response, little is known as to how tolerance versus reactivity to allergen occurs in the lung. The vast majority of mouse models study allergen encounter in naïve animals, and therefore exclude the possibility that previous encounters with allergen may influence future sensitization. To address this, we studied sensitization to the model allergen OVA in mice in the context of pre-existing tolerance to OVA. Allergen sensitization by either systemic administration of OVA with aluminum hydroxide or mucosal administration of OVA with low-dose lipopolysaccharide (LPS) was suppressed in tolerized animals. However, higher doses of LPS induced a mixed Th2 and Th17 response to OVA in both naïve and tolerized mice. Interestingly, tolerized mice had more pronounced Th17 type inflammation than naïve mice receiving the same sensitization, suggesting pre-existing tolerance altered the inflammatory phenotype. These data show that a pre-existing tolerogenic immune response to allergen can impact subsequent sensitization in the lung. These findings have potential significance in understanding late-onset disease in severe asthmatics.
Negative regulation of innate immune responses is essential in order to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration and activation, and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G-protein coupled receptors, the function of which in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. Here we show that DC from lpa2−/− mice are hyperactive compared to their wild-type counterparts, and are also less susceptible to inhibition by different LPA species. In transient transfection assays, we found that lpa2-overexpression inhibits NF-κB-driven gene transcription. Using an adoptive transfer approach, we found that allergen-pulsed lpa2−/− DC induced substantially more lung inflammation than wild-type DC after inhaled allergen challenge. Finally, lpa2−/− mice develop greater allergen-driven lung inflammation than their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits dendritic cell activation and allergic airway inflammation.
Emerging evidence indicates that airway epithelial barrier function is compromised in asthma, a disease characterized by Th2-skewed immune response against inhaled allergens, but the mechanisms involved are not well understood. The purpose of this study was to investigate the effects of Th2-type cytokines on airway epithelial barrier function. 16HBE14o- human bronchial epithelial cells monolayers were grown on collagen coated Transwell inserts. The basolateral or apical surfaces of airway epithelia were exposed to human interleukin-4 (IL-4), IL-13, IL-25, IL-33, thymic stromal lymphopoietin (TSLP) alone or in combination at various concentrations and time points. We analyzed epithelial apical junctional complex (AJC) function by measuring transepithelial electrical resistance (TEER) and permeability to FITC-conjugated dextran over time. We analyzed AJC structure using immunofluorescence with antibodies directed against key junctional components including occludin, ZO-1, β-catenin and E-cadherin. Transepithelial resistance was significantly decreased after both basolateral and apical exposure to IL-4. Permeability to 3 kDa dextran was also increased in IL-4-exposed cells. Similar results were obtained with IL-13, but none of the innate type 2 cytokines examined (TSLP, IL-25 or IL-33) significantly affected barrier function. IL-4 and IL-13-induced barrier dysfunction was accompanied by reduced expression of membrane AJC components but not by induction of claudin- 2. Enhanced permeability caused by IL-4 was not affected by wortmannin, an inhibitor of PI3 kinase signaling, but was attenuated by a broad spectrum inhibitor of janus associated kinases. Our study indicates that IL-4 and IL-13 have disruptive effect on airway epithelial barrier function. Th2-cytokine induced epithelial barrier dysfunction may contribute to airway inflammation in allergic asthma.
IL-13; IL-25; IL-33; IL-4; Janus associated kinase (JAK); TSLP; Th2 cytokines; adherens junction; airway epithelial cell; allergy; apical junctional complex; asthma; barrier dysfunction; tight junction
Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response.
We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro.
16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy.
PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, β-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction.
PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization.
Asthma; polyI:C; Toll-like receptor 3; epithelial permeability; protein kinase C; tight junctions; adherens junctions
Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1–6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2−/− CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.
Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2−/− DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2−/− DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2+/+ DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2+/+ DCs, coincubation of Nrf2−/− DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.
Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4+ T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O2) and hypoxic (1% O2) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4+ T-cell cytokines, especially IFN-γ. The enhancing effects of hypoxia on IFN-γ secretion were independent of mouse strain, and were also unaffected using CD4+ T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1α. Using T cells from IFN-γ receptor–deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-γ expression were not due to effects on IFN-γ consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45–related factor 2 attenuated the enhancing effect of hypoxia on IFN-γ secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4+ T cells.
hypoxia; gene regulation; CD4+ T cells; effector cytokine; IFN-γ
There is growing evidence that hypoxia-inducible transcription factors are involved in the pathophysiology of asthma. Hypoxia-inducible factor-1α (HIF-1α) in particular controls the expression of many hypoxia regulated genes, but whether HIF-1α directly contributes to allergen-driven immune responses is not known.
Partially HIF-1α-deficient mice (HIF-1α+/−) or wild-type littermate controls were used in all experiments. Spleen CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and cytokine secretion was measured in vitro. Mice were sensitized by intraperitoneal injection of ovalbumin (Ova) plus alum, and then challenged by intranasal Ova followed by bronchoalveolar lavage (BAL) and isolation of spleen cells. BAL cells were counted and the differential determined using cytospin, and splenocytes were incubated with Ova to measure recall cytokine production.
Interferon-γ secretion was significantly higher in anti-CD3 plus anti-CD28 stimulated CD4+ T cells obtained from HIF-1α+/− mice compared to wild-type controls. HIF-1α+/− mice were protected from lung eosinophilia 72 h after allergen challenge, in association with enhanced secretion of interferon-γ in recall responses of splenocytes.
HIF-1α contributes to allergic immune responses and lung eosinophilia in a mouse model of asthma.
Gene regulation; Transcription Factors; Hypoxia; Asthma mouse model
Inflammation-associated lung injury is a major cause of morbidity and mortality for patients in intensive care units. Although the cellular and molecular events that initiate lung inflammation are now well understood, the mechanisms that promote its resolution remain poorly defined. In this issue of the JCI, D’Alessio et al. show in a mouse model that recovery from acute lung injury is not simply a passive process, but involves Tregs in an active resolution program (see the related article beginning on page 2898).
Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.
Bisphenol A (BPA) is a high–production volume chemical classified as an environmental estrogen and used primarily in the plastics industry. BPA’s increased usage correlates with rising BPA levels in people and a corresponding increase in the incidence of asthma. Due to limited studies, the contribution of maternal BPA exposure to allergic asthma pathogenesis is unclear. Using two established mouse models of allergic asthma, we examined whether developmental exposure to BPA alters hallmarks of allergic lung inflammation in adult offspring. Pregnant C57BL/6 dams were gavaged with 0, 0.5, 5, 50, or 500 μg BPA/kg/day from gestational day 6 until postnatal day 21. To induce allergic inflammation, adult offspring were mucosally sensitized with inhaled ovalbumin containing low-dose lipopolysaccharide or ip sensitized using ovalbumin with alum followed by ovalbumin aerosol challenge. In the mucosal sensitization model, female offspring that were maternally exposed to ≥ 50 μg BPA/kg/day displayed enhanced airway lymphocytic and lung inflammation, compared with offspring of control dams. Peritoneally sensitized, female offspring exposed to ≤ 50 μg BPA/kg/day presented dampened lung eosinophilia, compared with vehicle controls. Male offspring did not exhibit these differences in either sensitization model. Our data demonstrate that maternal exposure to BPA has subtle and qualitatively different effects on allergic inflammation, which are critically dependent upon route of allergen sensitization and sex. However, these subtle, yet persistent changes due to developmental exposure to BPA did not lead to significant differences in overall airway responsiveness, suggesting that early life exposure to BPA does not exacerbate allergic inflammation into adulthood.
asthma; plastics; developmental exposure; developmental basis of adult disease
Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus (S. aureus) skin infections. S. aureus is sensed by many pattern recognition receptors including toll-like receptor (TLR) 2. We hypothesized that an effective innate immune response will include skin barrier repair and that this response is impaired in AD subjects. S. aureus-derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, CLDN1, CLDN23, occludin and ZO-1 in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape-stripping. Tlr2−/− mice had a delayed and incomplete barrier recovery following tape-stripping. AD subjects had reduced epidermal TLR2 expression as compared to nonatopic (NA) subjects, which inversely correlated (r= 0.654, P= 0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may play a role in their incompetent skin barrier.
atopic dermatitis; eczema herpeticum; claudin-1
Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.
Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.
Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.
Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.
Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.
nuclear factor-κB; α-smooth muscle actin; idiopathic pulmonary fibrosis
Cytokine expression patterns of T cells can be regulated by pre-commitment to stable effector phenotypes, further modification of moderately stable phenotypes, and quantitative changes in cytokine production in response to acute signals. We showed previously that the epidermal growth factor family member Amphiregulin is expressed by T cell receptor-activated mouse CD4 T cells, particularly Th2 cells, and helps eliminate helminth infection. Here we report a detailed analysis of the regulation of Amphiregulin expression by human T cell subsets. Signaling through the T cell receptor induced Amphiregulin expression by most or all T cell subsets in human peripheral blood, including naive and memory CD4 and CD8 T cells, Th1 and Th2 in vitro T cell lines, and subsets of memory CD4 T cells expressing several different chemokine receptors and cytokines. In these different T cell types, Amphiregulin synthesis was inhibited by an antagonist of protein kinase A, a downstream component of the cAMP signaling pathway, and enhanced by ligands that increased cAMP or directly activated protein kinase A. Prostaglandin E2 and adenosine, natural ligands that stimulate adenylyl cyclase activity, also enhanced Amphiregulin synthesis while reducing synthesis of most other cytokines. Thus, in contrast to mouse T cells, Amphiregulin synthesis by human T cells is regulated more by acute signals than pre-commitment of T cells to a particular cytokine pattern. This may be appropriate for a cytokine more involved in repair than attack functions during most inflammatory responses.
Atopic dermatitis (AD) is characterized by dry skin and a hyperreactive immune response to allergens, two cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJ) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway.
We evaluated the expression/function of the TJ protein, claudin-1 in epithelium from AD and nonatopic (NA) subjects and screened two American populations for SNPs in CLDN1.
Expression profiles of nonlesional epithelium from extrinsic AD, NA and psoriasis subjects were generated using Illumina’s BeadChips. Dysregulated intercellular proteins were validated by tissue staining and qPCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed using a knockdown approach in primary human keratinocytes (PHK). Twenty seven haplotype-tagging SNPs in CLDN1 were screened in two independent AD populations.
We observed strikingly reduced expression of the TJ proteins claudin-1 and -23 only in AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with Th2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro, we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging single nucleotide polymorphisms revealed associations with AD in two North American populations.
Taken together, these data suggest that an impaired epidermal TJ is a novel feature of skin barrier dysfunction and immune dysregulation observed in AD, and that CLDN1 may be a new susceptibility gene in this disease.
atopic dermatitis; claudin-1; tight junctions
The biological effects of acute particulate air pollution exposure in host innate immunity remain obscure and have relied largely on in vitro models. We hypothesized that single acute exposure to ambient or engineered particulate matter (PM) in the absence of other secondary stimuli would activate lung dendritic cells (DC) in vivo and provide information on the early immunological events of PM exposure and DC activation in a mouse model naïve to prior PM exposure. Activation of purified lung DC was studied following oropharyngeal instillation of ambient particulate matter (APM). We compared the effects of APM exposure with that of diesel-enriched PM (DEP), carbon black particles (CBP) and silver nanoparticles (AgP). We found that PM species induced variable cellular infiltration in the lungs and only APM exposure induced eosinophilic infiltration. Both APM and DEP activated pulmonary DC and promoted a Th2-type cytokine response from naïve CD4+ T cells ex vivo. Cultures of primary peribronchial lymph node cells from mice exposed to APM and DEP also displayed a Th2-type immune response ex vivo. We conclude that exposure of the lower airway to various PM species induces differential immunological responses and immunomodulation of DC subsets. Environmental APM and DEP activated DC in vivo and provoked a Th2 response ex vivo. By contrast, CBP and AgP induced altered lung tissue barrier integrity but failed to stimulate CD4+ T cells as effectively. Our work suggests that respirable pollutants activate the innate immune response with enhanced DC activation, pulmonary inflammation and Th2-immune responsiveness.
Innate immunity; Allergic immunity; Dendritic cell; Lung; Inflammation; Immunotoxicology; Toxicology; Particulate matter; Nanoparticles
Amphiregulin, a member of the Epidermal Growth Factor family, is expressed by activated mouse Th2 cells. Amphiregulin produced by mouse hematopoietic cells contributes to the elimination of a nematode infection by a Type 2 effector response.
To identify the human peripheral blood cell population expressing amphiregulin.
Amphiregulin-expressing cells were identified by flow cytometry of cell surface markers and histological staining. Histamine and amphiregulin in supernatants were measured by enzyme immunoassay. Quantitative real-time PCR was used to measure mRNA expression.
Stimulation of human peripheral blood mononuclear cells by anti-CD3 + anti-CD28 antibodies induced expression of amphiregulin mRNA and protein by a non-T cell population. The amphiregulin-producing cells were basophils, as judged by morphology and expression of CD203c and CD123 (IL-3 receptor alpha chain). Activated mouse basophils also produced amphiregulin. Amphiregulin expression by basophils in response to anti-TCR stimulation required IL-3 produced by T cells, and IL-3 alone induced high levels of amphiregulin expression by purified basophils. Amphiregulin was expressed at much higher levels when human basophils were stimulated by IL-3 than by IgE cross-linking, whereas the opposite was true for IL-4 expression and histamine release. Heparin-binding Epidermal Growth Factor-like growth factor was also expressed by IL-3-stimulated human basophils. PBMC from asthmatic human subjects contained significantly higher numbers of basophils able to produce amphiregulin, compared to allergic or non-allergic controls.
IL-3 can induce basophils to express high levels of amphiregulin, which may contribute to tissue remodeling during type 2 immune responses such as asthma.
Basophils; amphiregulin; IL-3
Oxidative stress plays an important role in immune regulation and dendritic cell (DC) maturation. Recent studies indicate that allergens, including ragweed extract (RWE), possess prooxidant activities, but how RWE interacts with DCs is not well understood. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a key transcription factor that regulates constitutive and coordinated induction of a battery of antioxidant genes. We hypothesized that RWE would activate DCs and that this response would be augmented in the absence of Nrf2. We generated bone marrow–derived DCs (BM-DCs) and isolated lung DCs from Nrf2+/+ and Nrf2−/− mice and studied the effects of RWE on DCs in vitro. Under resting conditions, Nrf2−/− BM-DCs exhibited constitutively greater levels of inflammatory cytokines and costimulatory molecules than Nrf2+/+ BM-DCs. Exposure to RWE impaired endocytic activity, significantly induced oxidative stress, and enhanced the expression of CD80, CD86, and MHCII in Nrf2−/− BM-DCs when compared with Nrf2+/+ BM-DC, in association with reduced expression of Nrf2-regulated antioxidant genes. RWE significantly induced the secretion of inflammatory cytokines IL-6 and TNF-α in BM-DCs and lung DCs from Nrf2−/− mice than Nrf2+/+ mice and significantly inhibited the secretion of IL-12 in Nrf2+/+ BM-DCs and IL-18 in Nrf2+/+ and Nrf2−/− BM-DCs. The stimulatory effects of RWE on DC activation were inhibited to varying degrees by the antioxidant N-acetyl cysteine. Our findings indicate that a defect in Nrf2-mediated signaling mechanisms alters the response of DCs to a common environmental allergen, which may contribute to the susceptibility to allergic diseases.
Nrf2; dendritic cells; ragweed extract; antioxidant genes; oxidative stress
Immune responses can be compartmentalized into innate versus adaptive components. This relatively recent dichotomy positioned the innate immune system at the interface between the host and the external environment and provided a new conceptual framework with which to view allergic diseases, including asthma. Airway epithelial cells and dendritic cells are key components of the innate immune system in the nose and lung and are now known to be intimately involved in allergen recognition and in modulating allergic immune responses. Here we review current thinking about how these two key cell types sense and respond to inhaled allergens, and emphasize how an understanding of “allergic innate immunity” can translate into new thinking about mechanisms of allergen sensitization and potentially lead to new therapeutic targets.
Innate immunity; Allergy; Asthma; Dendritic cells
Dendritic cells (DC) are potent professional antigen-presenting cells that drive primary immune responses to infections or other agonists perceived as ‘dangerous’. Muc1 is the only cell surface mucin or MUC gene product that is expressed in DC. Unlike other members of this glycoprotein family, Muc1 possesses a unique cytosolic region capable of signal transduction and attenuating toll-like receptor (TLR) activation. The expression and function of Muc1 has been intensively investigated on epithelial and tumor cells, but relatively little is known about its function on DC. We hypothesized that Muc1 would influence in vitro generation and primary DC activation in response to the TLR4 and TLR5 ligands lipopolysaccharide and flagellin. Compared with Muc1+/+ DC, we found that Muc1−/− DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF-α and VEGF), and better stimulation of allogeneic naïve CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1−/− DC also induced greater secretion of TNF-α and IFN-γ compared to similarly activated Muc1+/+ DC. Taken together, our results indicate that deletion of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC.
Inflammation; Dendritic cells; Muc1; Toll-like receptor; Innate immunity; Immunomodulation; Host defence; Cytokines
Epidemiologic studies have associated exposure to airborne particulate matter (PM) with exacerbations of asthma. It is unknown how different sources of PM affect innate immunity. We sought to determine how car- and diesel exhaust–derived PM affects dendritic cell (DC) activation. DC development was modeled using CD34+ hematopoietic progenitors. Airborne PM was collected from exhaust plenums of Fort McHenry Tunnel providing car-enriched particles (CEP) and diesel-enriched particles (DEP). DC were stimulated for 48 hours with CEP, DEP, CD40-ligand, or lipopolysaccharide. DC activation was assessed by flow cytometry, enzyme-linked immunosorbent assay, and standard culture techniques. DEP increased uptake of fluorescein isothiocyanate–dextran (a model antigen) by DC. Diesel particles enhanced cell-surface expression of co-stimulatory molecules (e.g., CD40 [P < 0.01] and MHC class II [P < 0.01]). By contrast, CEP poorly affected antigen uptake and expression of cell surface molecules, and did not greatly affect cytokine secretion by DC. However, DEP increased production of TNF, IL-6, and IFN-γ (P < 0.01), IL-12 (P < 0.05), and vascular endothelial growth factor (P < 0.001). In co-stimulation assays of PM-exposed DC and alloreactive CD4+ T cells, both CEP and DEP directed a Th2-like pattern of cytokine production (e.g., enhanced IL-13 and IL-18 and suppressed IFN-γ production). CD4+ T cells were not functionally activated on exposure to either DEP or CEP. Car- and diesel-enriched particles exert a differential effect on DC activation. Our data support the hypothesis that DEP (and to a lesser extent CEP) regulate important functional aspects of human DC, supporting an adjuvant role for this material.
asthma; allergy; innate immunity; Toll-like receptors; pollution