Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways.

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.

Identification of inflammatory markers suitable for non-invasive, repeated measurement studies in biobehavioral research: A feasibility study.

INTRODUCTION: Studying the role of the immune system in the interaction between mental and physical health is challenging. To study individuals with an intensive, longitudinal study design that requires repetitive sampling in their daily life, non-invasive sampling techniques are a necessity. Urine can be collected in a non-invasive way, but this may be demanding for participants and little is known about fluctuation of inflammatory markers in urine over time. The aim of this study was to investigate the feasibility of non-invasive sampling, and to explore intra-individual differences in inflammatory markers in urine. MATERIALS & METHODS: Ten healthy individuals collected 24-hour urine for 63 consecutive days. In a pilot analysis, 39 inflammatory markers were examined for detectability in urine, stability over time and under storage conditions, and daily fluctuations. Multiplex analyses were used to quantify levels of eight selected markers: C-reactive protein (CRP), Fractalkine, Interleukin-1 receptor-antagonist (IL-1RA), interferon-α (IFNα), interferon-γ (IFNγ), Interferon gamma-induced protein 10 (IP10), Macrophage inflammatory protein-1ß (MIP-1ß), and Vascular Endothelial Growth Factor (VEGF). Cross-correlations were calculated between the overnight and 24-hour samples were calculated, to examine whether 24-hour urine could be replaced by the overnight portion for better feasibility. We examined intra- and interindividual differences in the levels of inflammatory markers in urine and the fluctuations thereof. RESULTS: This study showed that levels of selected inflammatory markers can be detected in urine. Cross-correlation analyses showed that correlations between levels of inflammatory markers in the night portion and the 24-hour urine sample varied widely between individuals. In addition, analyses of time series revealed striking inter- and intra-individual variation in levels of inflammatory markers and their fluctuations. CONCLUSION: We show that the assessment of urinary inflammatory markers is feasible in an intensive day-to-day study in healthy individuals. However, 24-hour urine cannot be replaced by an overnight portion to alleviate the protocol burden. Levels of inflammatory markers show substantial variation between and within persons.

Characterization of a lung epithelium specific E-cadherin knock-out model: Implications for obstructive lung pathology.

The airway epithelium regulates responses to aeroallergens, acting as a physical and immunological barrier. In asthma, epithelial barrier function and the expression of adherens junction protein E-cadherin is compromised, but it is unknown whether this is cause or consequence of the disease. We hypothesized that airway epithelial loss of E-cadherin is a critical step in the development of manifestations of asthma. We generated a transgenic mouse model with conditional loss of E-cadherin in lung epithelial cells at birth and onwards. We observed normal lung development at the time of birth in mice lacking E-cadherin in the lung epithelium. However, E-cadherin deficiency led to progressive epithelial damage in mice growing into adulthood, as evidenced by airway epithelial denudation, decreased zonula occludens (ZO)-1 expression, loss of ciliated cells, and enlarged alveolar spaces. In addition, spontaneous goblet cell metaplasia with mucus production was observed. These epithelial changes were accompanied by elevated levels of the epithelial-derived chemokine CCL17, infiltration of eosinophils and dendritic cells, and mucus production. In conclusion, loss of E-cadherin induces features in the lung reminiscent of those observed in asthma, indicating that the disruption of E-cadherin-mediated cell-cell contacts may play a key role in the development of asthma manifestations.

Serum periostin does not reflect type 2-driven inflammation in COPD.

Although Th2 driven inflammation is present in COPD, it is not clearly elucidated which COPD patients are affected. Since periostin is associated with Th2 driven inflammation and inhaled corticosteroid (ICS)-response in asthma, it could function as a biomarker in COPD. The aim of this study was to analyze if serum periostin is elevated in COPD compared to healthy controls, if it is affected by smoking status, if it is linked to inflammatory cell counts in blood, sputum and endobronchial biopsies, and if periostin can predict ICS-response in COPD patients.Serum periostin levels were measured using Elecsys Periostin immunoassay. Correlations between periostin and inflammatory cell count in blood, sputum and endobronchial biopsies were analyzed. Additionally, the correlation between serum periostin levels and treatment responsiveness after 6 and 30 months was assessed using i.e. ΔFEV1% predicted, ΔCCQ score and ΔRV/TLC ratio. Forty-five COPD smokers, 25 COPD past-smokers, 22 healthy smokers and 23 healthy never-smokers were included. Linear regression analysis of serum periostin showed positive correlations age (B = 0.02, 95%CI 0.01-0.03) and FEV1% predicted (B = 0.01, 95%CI 0.01-0.02) in healthy smokers, but not in COPD patients In conclusion, COPD -smokers and -past-smokers have significantly higher periostin levels compared to healthy smokers, yet periostin is not suitable as a biomarker for Th2-driven inflammation or ICS-responsiveness in COPD.

Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce long-term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as ß-glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment. AIMS: Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)-driven mouse model of allergic asthma. MATERIALS AND METHODS: HDM-sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen-induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue. RESULTS: ESR measurement shows suppression of early allergic response in HDM-SCIT- and DerP1/2-SCIT-treated mice. Both HDM-SCIT and DerP1/2-SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2-SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2-SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL-33, CCL17 and eotaxin levels in lung tissue. DISCUSSION: Taken together, these data show that purified DerP1/2-SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM-induced activation of lung structural cells including airway epithelium. CONCLUSIONS: We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.

Pim1 kinase activity preserves airway epithelial integrity upon house dust mite exposure.

Most patients with allergic asthma are sensitized to house dust mite (HDM). The allergenicity of HDM largely depends on disruption of the integrity and proinflammatory activation of the airway epithelium. In this study, we hypothesized that Pim1 kinase activity attenuates HDM-induced asthma by preserving airway epithelial integrity. The effects of Pim1 kinase activity on barrier function and release of the proinflammatory mediators IL-1α and CCL20 were studied in vitro in 16HBE and primary bronchial epithelial cells (PBECs). Pim1-proficient and -deficient mice were exposed to a HDM-driven model of allergic asthma, and airway hyperresponsiveness (AHR) was measured upon methacholine challenge. Airway inflammation and proinflammatory mediators in lung tissue and BAL fluid were determined. We observed that inhibition of Pim1 kinase prolongs the HDM-induced loss of barrier function in 16HBE cells and sensitizes PBECs to HDM-induced barrier dysfunction. Additionally, inhibition of Pim1 kinase increased the HDM-induced proinflammatory activity of 16HBE cells as measured by IL-1α secretion. In line herewith, HDM exposure induced an enhanced production of the proinflammatory chemokines CCL17 and CCL20 in Pim1-deficient mice compared with wild-type controls. While we observed a marked increase in eosinophilic and neutrophilic granulocytes as well as mucus cell metaplasia and AHR to methacholine in mice exposed to HDM, these parameters were independent of Pim1 kinase activity. In contrast, levels of the Th2-cytokines IL-5 and IL-10 were significantly augmented in HDM-treated Pim1-deficient mice. Taken together, our study shows that Pim1 kinase activity maintains airway epithelial integrity and protects against HDM-induced proinflammatory activation of the airway epithelium.

Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation.

Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC.

Airway epithelial barrier function regulates the pathogenesis of allergic asthma.

The integrity of the airway epithelium in patients with asthma is often disrupted, with loss of epithelial cell-cell contacts. Airway epithelial barrier dysfunction may have important implications for asthma, because structural epithelial barrier function is tightly interwoven with the ability of the epithelium to regulate the immune system. We propose that changes at the airway epithelial barrier play a central role in the sensitisation to allergens and pathogenesis of allergic asthma. Many of the recently identified susceptibility genes for asthma are expressed in airway epithelium. However, the exact mechanisms by which the expression of epithelial susceptibility genes translates into a functionally altered response to aeroallergens in asthma are still unknown. In this review, we will focus on the role of airway epithelial barrier function in the susceptibility to develop allergic asthma and discuss therapeutic strategies aimed at the epithelial barrier.

DAMPs activating innate and adaptive immune responses in COPD.

Chronic obstructive pulmonary disease (COPD), a progressive lung disease characterized by sustained neutrophilic airway inflammation, is caused by chronic exposure to noxious stimuli, e.g., cigarette smoke. This chronic exposure can induce immunogenic cell death of structural airway cells, inducing the release of damage-associated molecular patterns (DAMPs). Levels of several DAMPs, including S100 proteins, defensins, and high-mobility group box-1 (HMGB1), are increased in extracellular lung fluids of COPD patients. As DAMPs can attract and activate immune cells upon binding to pattern recognition receptors, we propose that their release may contribute to neutrophilic airway inflammation. In this review, we discuss the novel role of DAMPs in COPD pathogenesis. Relevant DAMPs are categorized based on their subcellular origin, i.e. cytoplasm, endoplasmic reticulum, nucleus, and mitochondria. Furthermore, their potential role in the pathophysiology of COPD will be discussed.

Basophil Activation Test in the diagnosis and monitoring of mastocytosis patients with wasp venom allergy.

Background: There is need for an accurate diagnostic test in mastocytosis patients with wasp venom allergy (WVA) and monitoring of these patients during immunotherapy (IT). In this study, we aimed to evaluate sensitivity and specificity of the Basophil Activation Test (BAT) as a diagnostic and monitoring test in patients with mastocytosis and WVA. Methods: Seventeen patients with mastocytosis and WVA and 6 mastocytosis patients without WVA were included. BAT was performed before the start of IT (1st visit) and at 6 weeks (2nd visit) and 1 year (3rd visit), after reaching the maintenance dose. Of 17 patients included, 11 complerted the 3rd visit.In mastocytosis patients with WVA, dose-dependent wasp-venom induced upregulation of CD63 and CD203c expression on basophils was observed compared to mastocytosis patients without WVA. Serum specific IgE, IgG4 and tryptase levels were measured in all patients. Results: BAT had a sensitivity of 87% and specificity of 100% in diagnosing WVA in mastocytosis patients. Basophil allergen threshold sensitivity with respect to CD63 and CD203c was significantly decreased in the second visit compared to the first visit and increased significantly in the third visit compared to the second visit. Specific IgE levels increased significantly in the 2nd visit compared to first and decreased significantly in the third visti compared to the second. Specific IgG4 levels rose significantly in the 2nd visit compared to the 1st and on the 3rd visit compared to the 2nd . Tryptase levels did not change significantly during the study. Conclusions: BAT can represent a diagnostic test in allergic patients with mastocytosis and these patients are better to be monitored for a longer period during IT. © 2013 Clinical Cytometry Society.

House dust mite-induced calcium signaling instigates epithelial barrier dysfunction and CCL20 production.

BACKGROUND: House dust mite (HDM) affects the immunological and physical barrier function of airway epithelium, leading to allergic sensitization, airway remodeling, and eosinophilic inflammation in mouse models, although the mechanisms are still largely unknown. OBJECTIVE: Given the implications for adenosine triphosphate (ATP)-dependent Ca(2+) signaling in allergic sensitization in mice, we sought to determine the role of intracellular Ca(2+) concentration ([Ca(2+)](i)) in HDM-induced barrier dysfunction and pro-inflammatory activity of bronchial epithelium. METHODS: We investigated the effect of HDM on accumulation of [Ca(2+)](i) levels, barrier function, and CCL20 release in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from healthy subjects and asthma patients. Involvement of ATP-dependent activation of purinergic receptors and downstream Ca(2+) influx was studied, using the ATP hydrolyzing agent apyrase, the purinergic receptor agonist PPADS, the calcium chelator BAPTA-AM, and calpain inhibitors. RESULTS: Asthma PBECs were more susceptible to HDM-induced barrier dysfunction, CCL20 secretion, and Ca(2+) influx than healthy PBECs. Furthermore, we show that the HDM-induced increase in CCL20 in PBECs and 16HBE cells and the HDM-induced barrier dysfunction in 16HBE cells are dependent on [Ca(2+)](i) accumulation. Additionally, we demonstrate that [Ca(2+)](i) accumulation is initiated partly through the activation of purinergic receptors, which contributes to HDM-induced epithelial barrier dysfunction by disruption of cell-cell contacts, but not CCL20 secretion. CONCLUSION: Our data show for the first time that Ca(2+) signaling plays a crucial role in barrier dysfunction and the pro-inflammatory response of bronchial epithelium upon HDM exposure and may thus have important implications for the development of allergic asthma.

The composition of house dust mite is critical for mucosal barrier dysfunction and allergic sensitisation.

BACKGROUND: House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation. OBJECTIVES: The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo. METHODS: Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model. RESULTS: The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity. CONCLUSIONS: This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

Iron administration reduces airway hyperreactivity and eosinophilia in a mouse model of allergic asthma.

The prevalence of allergic diseases has increased dramatically during the last four decades and is paralleled by a striking increase in iron intake by infants in affluent societies. Several studies have suggested a link between increased iron intake and the marked increase in prevalence of allergic diseases. We hypothesized that the increased iron intake by infants offers an explanation for the increased prevalence of allergic disease in industrialized societies during the past four decades. A well-established mouse model of ovalbumin (OVA)-driven allergic asthma was used to test the effects of differences in iron intake and systemic iron levels on the manifestations of allergic asthma. Surprisingly, iron supplementation resulted in a significant decrease in airway eosinophilia, while systemic iron injections lead to a significant suppression of both allergen-induced airway eosinophilia and hyperreactivity compared to placebo. In contrast, mice fed on an iron-deprived diet did not show any difference in developing experimentally induced allergic asthma when compared to those fed on an iron-sufficient control diet. In contrast to our hypothesis, airway manifestations of allergic asthma are suppressed by both increased levels of iron intake and systemic iron administrations in the mouse model.

Flow cytometric analysis of cytokine expression in short-term allergen-stimulated T cells mirrors the phenotype of proliferating T cells in long-term cultures.

BACKGROUND: Allergen-specific T(H) cells play an important role in IgE-mediated disorders as allergies. Since this T(H) cell-population only accounts for a small percentage of T(H) cells, they are difficult to phenotype without prior selection or expansion. METHODS: Grass-pollen-specific T(H) cell profiles were evaluated in 5 allergic and 4 non-allergic individuals using three different approaches: CD154 expression on ex vivo grass-pollen-activated PBMCs (i); CFSE-dilution in grass-pollen-restimulated PBMCs (ii) and T cell lines enriched for allergen-specific T cells (iii). RESULTS: Relatively low numbers of allergen-specific T(H) cells were detected using CD154 expression, limiting the power to detect phenotypic differences between allergic and non-allergic individuals. In contrast, higher frequencies of proliferating T(H) cells were detected by loss-of-CFSE intensity in PBMCs and TCLs after grass-pollen-stimulation, resulting in the detection of significantly more IL-4 producing T(H) cells in allergic vs non-allergic individuals. In addition, higher numbers of IFNγ producing T(H) cells were detected in long-term cultures compared to the CD154 expressing T(H) cells. CONCLUSION: To detect allergen-specific T(H) cells for a common allergen as grass-pollen, expansion is not absolutely necessary, although within 8-day grass-pollen cultures, higher numbers of proliferating T(H) cells resulted in increased statistical power to detect phenotypic differences. However, this approach also detects more bystander activated T(H) cells. TCLs resulted in comparable percentages of cytokine expressing T cells as 8-day cultures. Therefore enrichment can be necessary for detection of T(H) cells specific for a single allergen or allergen-derived peptides, but is dispensable for the detection and phenotyping of allergen-specific T(H) cells using crude extracts.

Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis.

BACKGROUND: Anaphylaxis to insect venom (Hymenoptera) is most severe in patients with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene expression between patients with indolent systemic mastocytosis (ISM) and a history of insect venom anaphylaxis (IVA) compared to those patients without a history of anaphylaxis, and to determine the predictive use of gene expression profiling. METHODS: Whole-genome gene expression analysis was performed in peripheral blood cells. RESULTS: Twenty-two adults with ISM were included: 12 with a history of IVA and 10 without a history of anaphylaxis of any kind. Significant differences in single gene expression corrected for multiple testing were found for 104 transcripts (P < 0.05). Gene ontology analysis revealed that the differentially expressed genes were involved in pathways responsible for the development of cancer and focal and cell adhesion suggesting that the expression of genes related to the differentiation state of cells is higher in patients with a history of anaphylaxis. Based on the gene expression profiles, a naïve Bayes prediction model was built identifying patients with IVA. CONCLUSIONS: In ISM, gene expression profiles are different between patients with a history of IVA and those without. These findings might reflect a more pronounced mast cells dysfunction in patients without a history of anaphylaxis. Gene expression profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in patients with ISM. Prospective studies are needed to substantiate any conclusions.

Frat oncoproteins act at the crossroad of canonical and noncanonical Wnt-signaling pathways.

Wnt-signal transduction is critical for development and tissue homeostasis in a wide range of animal species and is frequently deregulated in human cancers. Members of the Frat/GBP family of glycogen synthase kinase 3beta (Gsk3b)-binding oncoproteins are recognized as potent activators of the Wnt/beta-catenin pathway in vertebrates. Here, we reveal a novel, Gsk3b-independent function of Frat converging on the activation of JNK and AP-1. Both these have been used as readouts for the noncanonical Frizzled/PCP pathway, which controls polarized cell movements and the establishment of tissue polarity. We find that Frat synergizes with Diversin, the mammalian homolog of the Drosophila PCP protein diego, in the activation of JNK/AP-1 signaling. Importantly, Frat mutants deficient for binding to Gsk3b retain oncogenic activity in vivo, suggesting that Wnt/beta-catenin-independent events contribute to Frat-induced malignant transformation. The observed activities of Frat are reminiscent of the dual function of Dishevelled in the Wnt/beta-catenin and Frizzled/PCP pathways and suggest that Frat may also function to bridge canonical and noncanonical Wnt pathways.

Enforced expression of GATA-3 during T cell development inhibits maturation of CD8 single-positive cells and induces thymic lymphoma in transgenic mice.

The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4(+)CD8(+) double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRalphabeta up-regulation and CD69 expression. LacZ expression remained high ( approximately 80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, approximately 50% of the mice developed thymic lymphoblastoid tumors that were CD4(+)CD8(+/low) and mostly CD3(+). In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69(low)HSA(low) CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8(+) T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.

Enforced expression of GATA-3 in transgenic mice inhibits Th1 differentiation and induces the formation of a T1/ST2-expressing Th2-committed T cell compartment in vivo.

The transcription factor GATA-3 is essential for early T cell development and differentiation of naive CD4(+) T cells into Th2 effector cells. To study the function of GATA-3 during T cell-mediated immune responses in vivo, we investigated CD2-GATA3-transgenic mice in which GATA-3 expression is driven by the CD2 locus control region. Both in the CD4(+) and the CD8(+) T cell population the proportion of cells exhibiting a CD44(high)CD45RB(low)CD62L(low) Ag-experienced phenotype was increased. In CD2-GATA3-transgenic mice, large fractions of peripheral CD4(+) T cells expressed the IL-1 receptor family member T1/ST2, indicative of advanced Th2 commitment. Upon in vitro T cell stimulation, the ability to produce IL-2 and IFN-gamma was decreased. Moreover, CD4(+) T cells manifested rapid secretion of the Th2 cytokines IL-4, IL-5, and IL-10, reminiscent of Th2 memory cells. In contrast to wild-type CD4(+) cells, which lost GATA-3 expression when cultured under Th1-polarizing conditions, CD2-GATA3-transgenic CD4(+) cells maintained expression of GATA-3 protein. Under Th1 conditions, cellular proliferation of CD2-GATA3-transgenic CD4(+) cells was severely hampered, IFN-gamma production was decreased and Th2 cytokine production was increased. Enforced GATA-3 expression inhibited Th1-mediated in vivo responses, such as Ag-specific IgG2a production or a delayed-type hypersensitivity response to keyhole limpet hemocyanin. Collectively, these observations indicate that enforced GATA-3 expression selectively inhibits Th1 differentiation and induces Th2 differentiation. The increased functional capacity to secrete Th2 cytokines, along with the increased expression of surface markers for Ag-experienced Th2-committed cells, would argue for a role of GATA-3 in Th2 memory formation.

Regulation of helper T cell responses to staphylococcal superantigens.

Staphylococcal superantigens (sAgs) including toxic shock syndrome toxin-1 (TSST-1) and related enterotoxins are exoproteins with unique immunobiological properties. They bind to major histocompatibility complex (MHC) class II molecules of antigen-presenting cells outside the peptide groove, and induce massive proliferation of T cells bearing specific V beta determinants. This tri-molecular interaction leads to uncontrolled release of various proinflammatory cytokines especially interferon-gamma (IFN-gamma) and tumor necrosis factor-a (TNF-alpha), the key cytokines causing sAg-mediated shock. The effector T cells involved in this hyper-immune response are predominantly of the T helper-1 (Th1) phenotype. There is also some evidence that polarization to a Th2 response with the production of classical anti-inflammatory cytokines (such as interleukins IL-4 and IL-6) also occurs. Moreover, the emergence of a novel regulatory T cell (Tr1) subset, producing mainly IL-10 but little or no IL-2 and IL-4, has recently been described following repeated sAg stimulation. In this review, the current knowledge regarding the regulation of T helper cell subsets in response to staphylococcal sAgs is critically evaluated, and the role of various cytokines which directly influence T cell differentiation and polarization is summarized. Particular emphasis is directed towards pro-inflammatory as well as anti-inflammatory and regulatory effector functions during toxic shock. Based on this review, we propose that a delayed production of IL-10 by Tr1 cells may be the most prominent driving force in the down-regulation of the Th1 hyper-immune response, and the critical determinant for the eventual recovery of the host.

Expression of the transcription factor GATA-3 is required for the development of the earliest T cell progenitors and correlates with stages of cellular proliferation in the thymus.

GATA-3 is a zinc-finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA-3 expression during T cell development in vivo in the mouse, the GATA-3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA-3+ cells throughout T cell development in the thymus, the proportions of GATA-3+ cells varied considerably between the distinct differentiation stages. The two periods of TCR alpha and beta gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA-3+ cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA-3+ cells. In addition, we generated chimeric mice by injection of GATA-3-deficient, lacZ-expressing ES cells into wild-type blastocysts. In this in vivo competition analysis, no contribution of GATA-3-deficient cells to the T cell lineage was detected, not even in the earliest CD44+CD25- double-negative (CD4-CD8-) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA-3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.