Kia Joo Puan

Allergic airway diseases in a tropical urban environment are driven by dominant mono‐specific sensitization against house dust mites

Southeast Asian populations are increasingly affected by allergic airway diseases. Etiology and specific causes, however, are still unknown. The aim of this study is therefore to identify allergens and risk factors for the high prevalence of allergic airway disease in the tropical urban environment.

Genetic analysis of an allergic rhinitis cohort reveals an intercellular epistasis between FAM134B and CD39

BackgroundExtracellular ATP is a pro-inflammatory molecule released by damaged cells. Regulatory T cells (Treg) can suppress inflammation by hydrolysing this molecule via ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also termed as CD39. Multiple studies have reported differences in CD39+ Treg percentages in diseases such as multiple sclerosis, Hepatitis B and HIV-1. In addition, CD39 polymorphisms have been implicated in immune-phenotypes such as susceptibility to inflammatory bowel disease and AIDS progression. However none of the studies published so far has linked disease-associated variants with differences in CD39 Treg surface expression. This study aims at identifying variants affecting CD39 expression on Treg and at evaluating their association with allergic rhinitis, a disease characterized by a strong Treg involvement.MethodsCohorts consisting of individuals of different ethnicities were employed to identify any association of CD39 variants to surface expression. Significant variant(s) were tested for disease association in a published GWAS cohort by one-locus and two-locus genetic analyses based on logistic models. Further functional characterization was performed using existing microarray data and quantitative RT-PCR on sorted cells.ResultsOur study shows that rs7071836, a promoter SNP in the CD39 gene region, affects the cell surface expression on Treg cells but not on other CD39+ leukocyte subsets. Epistasis analysis revealed that, in conjunction with a SNP upstream of the FAM134B gene (rs257174), it increased the risk of allergic rhinitis (P = 1.98 × 10-6). As a promoter SNP, rs257174 controlled the expression of the gene in monocytes but, notably, not in Treg cells. Whole blood transcriptome data of three large cohorts indicated an inverse relation in the expression of the two proteins. While this observation was in line with the epistasis data, it also implied that a functional link must exist. Exposure of monocytes to extracellular ATP resulted in an up-regulation of FAM134B gene expression, suggesting that extracellular ATP released from damaged cells represents the connection for the biological interaction of CD39 on Treg cells with FAM134B on monocytes.ConclusionsThe interplay between promoter SNPs of CD39 and FAM134B results in an intercellular epistasis which influences the risk of a complex inflammatory disease.

Type I IFNs and IL-18 Regulate the Antiviral Response of Primary Human γδ T Cells against Dendritic Cells Infected with Dengue Virus

Little is known about the cellular mechanisms of innate immunity against dengue virus (DV) infection. Specifically, the γδ T cell response to DV has not been characterized in detail. In this article, we demonstrate that markers of activation, proliferation, and degranulation are upregulated on γδ T cells in PBMC isolated from individuals with acute dengue fever. Primary γδ T cells responded rapidly in vitro to autologous DV-infected dendritic cells by secreting IFN-γ and upregulating CD107a. The anti-DV IFN-γ response is regulated by type I IFN and IL-18 in a TCR-independent manner, and IFN-γ secreting γδ T cells predominantly expressed IL-18Rα. Antagonizing the ATP-dependent P2X7 receptor pathway of inflammasome activation significantly inhibited the anti-DV IFN-γ response of γδ T cells. Overnight priming with IL-18 produced effector γδ T cells with significantly increased ability to lyse autologous DV-infected dendritic cells. Monocytes were identified as accessory cells that augmented the anti-DV IFN-γ response of γδ T cells. Lack of monocytes in culture is associated with lower IL-18 levels in culture supernatant and diminished production of IFN-γ by γδ T cells, whereas addition of exogenous IL-18 restored the IFN-γ response of γδ T cells in monocyte-depleted cocultures with DV-infected DC. Our results indicate that primary γδ T cells contribute to the immune response during DV infection by providing an early source of IFN-γ, as well as by killing DV-infected cells, and suggest that monocytes participate as accessory cells that sense DV infection and amplify the cellular immune response against this virus in an IL-18–dependent manner.

γ/δ T cell subsets in human aging using the classical α/β T cell model

Aging is associated with an increased susceptibility to infections and diseases. It has also been associated with reduced functionality and altered distribution of immune cells, especially T cells. Whereas classical α/β T cells, especially CD8+ T cells, were shown to be highly susceptible to aging, the effects of viral persistent stimulations on the fate of γ/δ T cells are much less documented. Healthy, elderly individuals of Chinese ethnical background were recruited under the aegis of SLAS‐II. In this observational study, γ/δ T cell populations were characterized by flow cytometry and compared with the α/β CD4+ and CD8+ T cells in elderly and young controls. In our study, we identified a reduced frequency of γ/δ T cells but not α/β T cells with aging. The classical markers of α/β T cell aging, including CD28, CD27, and CD57, did not prove significant for γ/δ T cells. The extreme range of expression of these markers in γ/δ T cells was responsible for the lack of relationship between γ/δ T cell subsets, CD4/CD8 ratio, and anti‐CMV titers that was significant for α/β T cells and, especially, CD8+ T cells. Although markers of aging for γ/δ T cells are not clearly identified, our data collectively suggest that the presence of CD27 γ/δ T cells is associated with markers of α/β T cell aging.

Immune modulation of inflammatory conditions: regulatory T cells for treatment of GvHD

The immune system is a highly balanced network of different cell types. This balance is disturbed in the setting of organ or stem cell transplantation, which can lead to graft rejection or “Graft versus host disease” (GvHD). Conventional pharmacological treatment by broad immune suppression is restricted by dose-limiting side effects. A novel strategy for prevention and control is cell therapy. This applies particularly to GvHD. A number of phase I trials have already been launched. The most appropriate cell type appears to be the regulatory T (Treg) cell as it is a natural “suppressor” of the immune system. Treg cells are able to inhibit various effector cells including CD4+ and CD8+ T cells, the main drivers of GvHD. Like other T cells, also Treg cells can be divided into naïve and memory-type cells. We have previously identified effector/memory Treg cells (TREM), the regulatory counterparts of CD4+ effector/memory T cells (TEM). TREM may be particularly suited to inhibit proinflammatory reactions in peripheral tissues as they express the chemokine receptor CCR6, a feature they share with proinflammatory Th17 cells. As specific marker, they also express CD39 but lack the expression of CD49d and CD127. We could show that a simple depletion of CD49d and CD127 expressing cells yields a population of “untouched” Treg cells that is highly pure and largely consist of highly suppressive TREM cells. Mouse models have confirmed the efficacy of Treg cells in controlling GvHD but the translation has been lagging. First clinical trials suggesting safety of adoptive Treg transfer increase the need for methods that allow obtaining clinical-grade Treg cells in sufficient amounts. The new approach may therefore provide a promising new alternative to facilitate a simple access to these cells.

Genome-wide analysis of the genetic regulation of gene expression in human neutrophils

Neutrophils are an abundant immune cell type involved in both antimicrobial defence and autoimmunity. The regulation of their gene expression, however, is still largely unknown. Here we report an eQTL study on isolated neutrophils from 114 healthy individuals of Chinese ethnicity, identifying 21,210 eQTLs on 832 unique genes. Unsupervised clustering analysis of these eQTLs confirms their role in inflammatory responses and immunological diseases but also indicates strong involvement in dermatological pathologies. One of the strongest eQTL identified (rs2058660) is also the tagSNP of a linkage block reported to affect leprosy and Crohns disease in opposite directions. In a functional study, we can link the C allele with low expression of the β-chain of IL18-receptor (IL18RAP). In neutrophils, this results in a reduced responsiveness to IL-18, detected both on the RNA and protein level. Thus, the polymorphic regulation of human neutrophils can impact beneficial as well as pathological inflammatory responses.

Differential IL-1β secretion by monocyte subsets is regulated by Hsp27 through modulating mRNA stability.

Monocytes play a central role in regulating inflammation in response to infection or injury, and during auto-inflammatory diseases. Human blood contains classical, intermediate and non-classical monocyte subsets that each express characteristic patterns of cell surface CD16 and CD14; each subset also has specific functional properties, but the mechanisms underlying many of their distinctive features are undefined. Of particular interest is how monocyte subsets regulate secretion of the apical pro-inflammatory cytokine IL-1β, which is central to the initiation of immune responses but is also implicated in the pathology of various auto-immune/auto-inflammatory conditions. Here we show that primary human non-classical monocytes, exposed to LPS or LPS + BzATP (3’-O-(4-benzoyl)benzyl-ATP, a P2X7R agonist), produce approx. 80% less IL-1β than intermediate or classical monocytes. Despite their low CD14 expression, LPS-sensing, caspase-1 activation and P2X7R activity were comparable in non-classical monocytes to other subsets: their diminished ability to produce IL-1β instead arose from 50% increased IL-1β mRNA decay rates, mediated by Hsp27. These findings identify the Hsp27 pathway as a novel therapeutic target for the management of conditions featuring dysregulated IL-1β production, and represent an advancement in understanding of both physiological inflammatory responses and the pathogenesis of inflammatory diseases involving monocyte-derived IL-1β.

Large-scale Isolation of Highly Pure "Untouched" Regulatory T Cells in a GMP Environment for Adoptive Cell Therapy.

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate “untouched” human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of “untouched” Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.

A functional SNP associated with atopic dermatitis controls cell type-specific methylation of the VSTM1 gene locus

BackgroundExpression quantitative trait loci (eQTL) databases represent a valuable resource to link disease-associated SNPs to specific candidate genes whose gene expression is significantly modulated by the SNP under investigation. We previously identified signal inhibitory receptor on leukocytes-1 (SIRL-1) as a powerful regulator of human innate immune cell function. While it is constitutively high expressed on neutrophils, on monocytes the SIRL-1 surface expression varies strongly between individuals. The underlying mechanism of regulation, its genetic control as well as potential clinical implications had not been explored yet.MethodsWhole blood eQTL data of a Chinese cohort was used to identify SNPs regulating the expression of VSTM1, the gene encoding SIRL-1. The genotype effect was validated by flow cytometry (cell surface expression), correlated with electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and bisulfite sequencing (C-methylation) and its functional impact studied the inhibition of reactive oxygen species (ROS).ResultsWe found a significant association of a single CpG-SNP, rs612529T/C, located in the promoter of VSTM1. Through flow cytometry analysis we confirmed that primarily in the monocytes the protein level of SIRL-1 is strongly associated with genotype of this SNP. In monocytes, the T allele of this SNP facilitates binding of the transcription factors YY1 and PU.1, of which the latter has been recently shown to act as docking site for modifiers of DNA methylation. In line with this notion rs612529T associates with a complete demethylation of the VSTM1 promoter correlating with the allele-specific upregulation of SIRL-1 expression. In monocytes, this upregulation strongly impacts the IgA-induced production of ROS by these cells. Through targeted association analysis we found a significant Meta P value of 1.14 × 10–6 for rs612529 for association to atopic dermatitis (AD).ConclusionLow expression of SIRL-1 on monocytes is associated with an increased risk for the manifestation of an inflammatory skin disease. It thus underlines the role of both the cell subset and this inhibitory immune receptor in maintaining immune homeostasis in the skin. Notably, the genetic regulation is achieved by a single CpG-SNP, which controls the overall methylation state of the promoter gene segment.

Systematic characterization of basophil anergy

Cohort studies indicated that in certain individuals the basophils do not respond toward allergens due to a desensitization of their Fc epsilon receptor pathway. Cause and functional role as well as the implications on allergic reactions, however, are not clear yet.