FUT6 deficiency compromises basophil function by selectively abrogating their sialyl-Lewis x expression.

Sialyl-Lewis x (sLex, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLex expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLex+ and sLex- basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts.

Resistin expression in human monocytes is controlled by two linked promoter SNPs mediating NFKB p50/p50 binding and C-methylation.

Resistin is a key cytokine associated with metabolic and inflammatory diseases. Especially in East Asian populations, the expression levels are strongly influenced by genetic polymorphisms. Mechanisms and functional implications of this genetic control are still unknown. By employing reporter assays, EMSA, inhibition studies, bisulphite sequencing, ChIP-Seq and gene-editing we show that the p50/p50 homodimer known to act as repressor for a number of pro-inflammatory genes plays a central role in the genetic regulation of resistin in monocytes along with promoter methylation. In the common RETN haplotype p50/p50 constitutively dampens the expression by binding to the promoter. In an Asian haplotype variant however this interaction is disrupted by the A allele of rs3219175. The SNP is in very close linkage to rs34861192, a CpG SNP, located 280 bp upstream which provides an allele-specific C-methylation site. rs34861192 is located in a 100 bp region found to be methylated in the common but not in the Asian haplotype, resulting in the latter having a higher basal expression, which also associates with elevated histone acetylation (H3K27ac). Genotype associations within cohort data of 200 East Asian individuals revealed significant associations between this haplotype and the plasma levels of factors such as TGF-b, S100B, sRAGE and IL-8 as well as with myeloid DC counts. Thus, the common RETN haplotype is tightly regulated by the epigenetic mechanism linked to p50/p50-binding. This control is lost in the Asian haplotype, which may have evolved to balance the antagonistic RETN effects on pathogen protection vs. metabolic and inflammatory disease induction.

A Co-culture Model of PBMC and Stem Cell Derived Human Nasal Epithelium Reveals Rapid Activation of NK and Innate T Cells Upon Influenza A Virus Infection of the Nasal Epithelium.

Background: We established an in vitro co-culture model involving H3N2-infection of human nasal epithelium with peripheral blood mononuclear cells (PBMC) to investigate their cross-talk during early H3N2 infection. Methods: Nasal epithelium was differentiated from human nasal epithelial stem/progenitor cells and cultured wtih fresh human PBMC. PBMC and supernatants were harvested after 24 and 48 h of co-culture with H3N2-infected nasal epithelium. We used flow cytometry and Luminex to characterize PBMC subpopulations, their activation and secretion of cytokine and chemokines. Results: H3N2 infection of the nasal epithelium associated with significant increase in interferons (IFN-α, IFN-γ, IL-29), pro-inflammatory cytokines (TNF-α, BDNF, IL-3) and viral-associated chemokines (IP-10, MCP-3, I-TAC, MIG), detectable already after 24 h. This translates into rapid activation of monocytes, NK-cells and innate T-cells (MAIT and γδ T cells), evident with CD38+ and/or CD69+ upregulation. Conclusions: This system may contribute to in vitro mechanistic immunological studies bridging systemic models and possibly enable the development of targeted immunomodulatory therapies.

Analysis of T Cell Subsets in Adult Primary/Idiopathic Minimal Change Disease: A Pilot Study.

AIM: To characterise infiltrating T cells in kidneys and circulating lymphocyte subsets of adult patients with primary/idiopathic minimal change disease. METHODS: In a cohort of 9 adult patients with primary/idiopathic minimal change recruited consecutively at disease onset, we characterized (1) infiltrating immune cells in the kidneys using immunohistochemistry and (2) circulating lymphocyte subsets using flow cytometry. As an exploratory analysis, association of the numbers and percentages of both kidney-infiltrating immune cells and the circulating lymphocyte subsets with kidney outcomes including deterioration of kidney function and proteinuria, as well as time to complete clinical remission up to 48 months of follow-up, was investigated. RESULTS: In the recruited patients with primary/idiopathic minimal change disease, we observed (a) a dominance of infiltrating T helper 17 cells and cytotoxic cells, comprising cytotoxic T cells and natural killer cells, over Foxp3+ Treg cells in the renal interstitium; (b) an increase in the circulating total CD8+ T cells in peripheral blood; and (c) an association of some of these parameters with kidney function and proteinuria. CONCLUSIONS: In primary/idiopathic minimal change disease, a relative numerical dominance of effector over regulatory T cells can be observed in kidney tissue and peripheral blood. However, larger confirmatory studies are necessary.

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

BACKGROUND: Expression 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. METHODS: Whole 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). RESULTS: We 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). CONCLUSION: Low 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.

Differential IL-1ß 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-1ß, 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-1ß 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-1ß instead arose from 50% increased IL-1ß 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-1ß production, and represent an advancement in understanding of both physiological inflammatory responses and the pathogenesis of inflammatory diseases involving monocyte-derived IL-1ß.

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 Crohn's disease in opposite directions. In a functional study, we can link the C allele with low expression of the ß-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.

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.

Are we ready for the use of foxp3(+) regulatory T cells for immunodiagnosis and immunotherapy in kidney transplantation?

The existence of T-cell subsets naturally committed to perform immunoregulation has led to enthusiastic efforts to investigate their role in the immunopathogenesis of transplantation. Being able to modulate alloresponses, regulatory T cells could be used as an immunodiagnostic tool in clinical kidney transplantation. Thus, the measurement of Foxp3 transcripts, the presence of regulatory T cells in kidney biopsies, and the phenotypic characterisation of the T-cell infiltrate could aid in the diagnosis of rejection and the immune monitoring and prediction of outcomes in kidney transplantation. Interestingly, the adoptive transfer of regulatory T cells in animal models has been proven to downmodulate powerful alloresponses, igniting translational research on their potential use as an immunomodulatory therapy. For busy transplant clinicians, the vast amount of information in the literature on regulatory T cells can be overwhelming. This paper aims to highlight the most applicable research findings on the use of regulatory T cells in the immune diagnosis and potential immunomodulatory therapy of kidney transplant patients. However, can we yet rely on differential regulatory T-cell profiles for the identification of rejection or to tailor patient's immunosuppression? Are we ready to administer regulatory T cells as inductive or adjunctive therapy for kidney transplantation?