Steve N. Georas

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.

Tight junction defects in patients with atopic dermatitis.

BACKGROUND Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. OBJECTIVE We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). METHODS Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illuminas BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. RESULTS We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 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 SNPs revealed associations with AD in 2 North American populations. CONCLUSION Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

AMP-activated kinase regulates cytoplasmic HuR.

ABSTRACT While transport of RNA-binding protein HuR from nucleus to cytoplasm is emerging as a key regulatory step for HuR function, the mechanisms underlying this process remain poorly understood. Here, we report that the AMP-activated kinase (AMPK), an enzyme involved in responding to metabolic stresses, potently regulates the levels of cytoplasmic HuR. Inhibition of AMPK, accomplished either through cell treatment or by adenovirus infection to express dominant-negative AMPK, was found to increase the level of HuR in the cytoplasm and to enhance the binding of HuR to p21, cyclin B1, and cyclin A mRNA transcripts and elevate their expression and half-lives. Conversely, AMPK activation, achieved by means including infection to express constitutively active AMPK, resulted in reduced cytoplasmic HuR; decreased levels and half-lives of mRNAs encoding p21, cyclin A, and cyclin B1; and diminished HuR association with the corresponding transcripts. We therefore propose a novel function for AMPK as a regulator of cytoplasmic HuR levels, which in turn influences the mRNA-stabilizing function of HuR and the expression of HuR target transcripts.

T-helper cell type-2 regulation in allergic disease

Substantial experimental evidence now supports the notion that allergic diseases are characterised by a skewing of the immune system towards a T-helper cell type-2 (Th2) phenotype. Studies using both human and mouse model systems have provided key evidence for the role that Th2 cytokines play in driving many of the hallmarks of allergic inflammation. Furthermore, the signalling pathways by which Th2 cytokines exert their effects on airway target cells are rapidly being elucidated, and antagonists of the Th2 pathway are under active development. In this review, the current knowledge of the role of T-helper cell type-2 cells in asthma is summarised, focusing on how and where T-helper cell type-2 cells differentiate from naïve precursors. The signalling molecules and transcription factors involved in T-helper cell type-2 differentiation will be reviewed in detail, in an attempt to translate studies using genetically modified mice into meaningful insights about asthma and other allergic diseases.

Disruption of the Transcription Factor Nrf2 Promotes Pro-Oxidative Dendritic Cells That Stimulate Th2-Like Immunoresponsiveness upon Activation by Ambient Particulate Matter

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.

Biology and genetics of atopic disease

Several immunological disorders including allergic rhinitis, bronchial asthma, atopic dermatitis, food allergies, urticaria, nonhereditary angioedema, systemic anaphylaxis, and allergic conjunctivitis are associated with a positive family history, and share a positive response in the Prausnitz-Kuster (wheal and flare) reaction. Studies have shown that 20-30% of the population has a strong genetic predisposition for this condition, termed atopy, whose hallmark is a greatly elevated serum IgE concentration. A great deal is known about the cellular interactions that mediate the sensitization, immediate and late-phase reactions that follow encounters with allergen, as well as about the cell surface and signaling events that result in mediator release from inflammatory cells. Less is known of the genes that confer genetic predisposition for atopy; however, a worldwide effort to identify atopy genes is making significant progress.

Cutaneous Injection of Human Subjects with Macrophage Inflammatory Protein-1α Induces Significant Recruitment of Neutrophils and Monocytes

Macrophage inflammatory protein (MIP-1α), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1α have been well documented, the in vivo biological activities of MIP-1α in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1α and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10–24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1α stimulation. These studies substantiate the biological effects of MIP-1α on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1α to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.

Future Research Directions in Asthma. An NHLBI Working Group Report

Asthma is a common chronic disease without cure. Our understanding of asthma onset, pathobiology, classification, and management has evolved substantially over the past decade; however, significant asthma-related morbidity and excess healthcare use and costs persist. To address this important clinical condition, the NHLBI convened a group of extramural investigators for an Asthma Research Strategic Planning workshop on September 18-19, 2014, to accelerate discoveries and their translation to patients. The workshop focused on (1) in utero and early-life origins of asthma, (2) the use of phenotypes and endotypes to classify disease, (3) defining disease modification, (4) disease management, and (5) implementation research. This report summarizes the workshop and produces recommendations to guide future research in asthma.

Transcriptional regulation of lysophosphatidic acid-induced interleukin-8 expression and secretion by p38 MAPK and JNK in human bronchial epithelial cells

HBEpCs (human bronchial epithelial cells) contribute to airway inflammation by secreting a variety of cytokines and chemokines in response to allergens, pathogens, viruses and environmental toxins and pollutants. The potent neutrophil chemoattractant, IL-8 (interleukin-8), is a major cytokine secreted by HBEpCs. We have recently demonstrated that LPA (lysophosphatidic acid) stimulated IL-8 production in HBEpCs via protein kinase C delta dependent signal transduction. However, mechanisms of IL-8 expression and secretion are complex and involve multiple protein kinases and transcriptional factors. The present study was undertaken to investigate MAPK (mitogen-activated protein kinase) signalling in the transcriptional regulation of IL-8 expression and secretion in HBEpCs. Exposure of HBEpCs to LPA (1 microM) enhanced expression and secretion of IL-8 by 5-8-fold and stimulated threonine/tyrosine phosphorylation of ERK (extracellular-signal-regulated kinase), p38 MAPK and JNK (c-Jun N-terminal kinase). The LPA-induced secretion of IL-8 was blocked by the p38 MAPK inhibitor SB203580, by p38 MAPK siRNA (small interfering RNA), and by the JNK inhibitor JNK(i) II, but not by the MEK (MAPK/ERK kinase) inhibitor, PD98059. LPA enhanced the transcriptional activity of the IL-8 gene; that effect relied on activation of the transcriptional factors NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein-1). Furthermore, SB203580 attenuated LPA-dependent phosphorylation of IkappaB (inhibitory kappaB), NF-kappaB and phospho-p38 translocation to the nucleus, NF-kappaB transcription and IL-8 promoter-mediated luciferase reporter activity, without affecting the JNK pathway and AP-1 transcription. Similarly, JNK(i) II only blocked LPA-mediated phosphorylation of JNK and c-Jun, AP-1 transcription and IL-8 promoter-mediated luciferase reporter activity, without blocking p38 MAPK-dependent NF-kappaB transcription. Additionally, siRNA for LPA(1-3) receptors partially blocked LPA-induced IL-8 production and activation of MAPKs. The LPA1 and LPA3 receptors, as compared with LPA2, were most efficient in transducing LPA-mediated IL-8 production. These results show an independent role for p38 MAPK and JNK in LPA-induced IL-8 expression and secretion via NF-kappaB and AP-1 transcription respectively in HBEpCs.

Activation of Epidermal Toll-Like Receptor 2 Enhances Tight Junction Function: Implications for Atopic Dermatitis and Skin Barrier Repair

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.