The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 ...is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.
Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that ...sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.
Allergic rhinitis (AR) is an atopic disease which affects about 600 million people worldwide and results from a complex interplay between genetic and environmental factors. However genetic ...association studies on known candidate genes yielded variable results. The aim of this study is to identify the genetic variants that influence predisposition towards allergic rhinitis in an ethnic Chinese population in Singapore using a genome-wide association study (GWAS) approach. A total of 4461 ethnic Chinese volunteers were recruited in Singapore and classified according to their allergic disease status. The GWAS included a discovery stage comparing 515 atopic cases (including 456 AR cases) and 486 non-allergic non-rhinitis (NANR) controls. The top SNPs were then validated in a replication cohort consisting of a separate 2323 atopic cases (including 676 AR cases) and 511 NANR controls. Two SNPs showed consistent association in both discovery and replication phases; MRPL4 SNP rs8111930 on 19q13.2 (OR = 0.69, P(combined) = 4.46×10(-05)) and BCAP SNP rs505010 on chromosome 10q24.1 (OR = 0.64, P(combined) = 1.10×10(-04)). In addition, we also replicated multiple associations within known candidates regions such as HLA-DQ and NPSR1 locus in the discovery phase. Our study suggests that MRPL4 and BCAP, key components of the HIF-1α and PI3K/Akt signaling pathways respectively, are two novel candidate genes for atopy and allergic rhinitis. Further study on these molecules and their signaling pathways would help in understanding of the pathogenesis of allergic rhinitis and identification of targets for new therapeutic intervention.
SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms ...underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.
The Toll-like receptor proteins are important in host defense and initiation of the innate and adaptive immune responses. A number of studies have identified associations between genetic variation in ...the Toll-like receptor genes and allergic disorders such as asthma and allergic rhinitis. The present study aim to search for genetic variation associated with allergic rhinitis in the Toll-like receptor genes.
A first association analysis genotyped 73 SNPs in 182 cases and 378 controls from a Swedish population. Based on these results an additional 24 SNPs were analyzed in one Swedish population with 352 cases and 709 controls and one Chinese population with 948 cases and 580 controls.
The first association analysis identified 4 allergic rhinitis-associated SNPs in the TLR7-TLR8 gene region. Subsequent analysis of 24 SNPs from this region identified 7 and 5 significant SNPs from the Swedish and Chinese populations, respectively. The corresponding risk-associated haplotypes are significant after Bonferroni correction and are the most common haplotypes in both populations. The associations are primarily detected in females in the Swedish population, whereas it is seen in males in the Chinese population. Further independent support for the involvement of this region in allergic rhinitis was obtained from quantitative skin prick test data generated in both populations.
Haplotypes in the TLR7-TLR8 gene region were associated with allergic rhinitis in one Swedish and one Chinese population. Since this region has earlier been associated with asthma and allergic rhinitis in a Danish linkage study this speaks strongly in favour of this region being truly involved in the development of this disease.
RNA‐sensing toll‐like receptors (TLRs) mediate innate immunity and regulate anti‐viral response. We show here that TLR3 regulates host immunity and the loss of TLR3 aggravates pathology in ...Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infection is markedly increased in human and mouse fibroblasts with defective TLR3 signaling. Up to 100‐fold increase in CHIKV load was observed in Tlr3−/− mice, alongside increased virus dissemination and pro‐inflammatory myeloid cells infiltration. Infection in bone marrow chimeric mice showed that TLR3‐expressing hematopoietic cells are required for effective CHIKV clearance. CHIKV‐specific antibodies from Tlr3−/− mice exhibited significantly lower in vitro neutralization capacity, due to altered virus‐neutralizing epitope specificity. Finally, SNP genotyping analysis of CHIKF patients on TLR3 identified SNP rs6552950 to be associated with disease severity and CHIKV‐specific neutralizing antibody response. These results demonstrate a key role for TLR3‐mediated antibody response to CHIKV infection, virus replication and pathology, providing a basis for future development of immunotherapeutics in vaccine development.
Synopsis
TLR3‐mediated innate response against CHIKV infection modulates the adaptive immune response and TLR3 deficiency results in enhanced viremia and more severe pathology in mice; in CHIKV‐infected patients, TLR3 expression is high and a TLR3 SNP associates with disease severity.
TLR3 regulates host immunity in CHIKV infection and pathology in humans and mice.
A loss of TLR3 was demonstrated to markedly increase virus replication and dissemination that led to more severe joint pathology.
Bone marrow chimeric experiments indicated that TLR3‐expressing hematopoietic cells play a role CHIKV clearance.
The loss of TLR3 impaired neutralizing capacity due to altered virus‐neutralizing epitope specificity.
SNP genotyping analysis on TLR from patients identified SNP rs6552950 as associated with disease severity.
TLR3‐mediated innate response against CHIKV infection modulates the adaptive immune response and TLR3 deficiency results in enhanced viremia and more severe pathology in mice; in CHIKV‐infected patients, TLR3 expression is high and a TLR3 SNP associates with disease severity.
Extracellular 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.
Cohorts 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.
Our 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.
The interplay between promoter SNPs of CD39 and FAM134B results in an intercellular epistasis which influences the risk of a complex inflammatory disease.
γ/δ T cells have a differential susceptibility to immunosenescence compared to α/β T cells, during human aging.
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.
Background Allergic rhinitis (AR) and asthma are common allergic conditions with a shared genetic component to their cause. The 17q12-21 locus includes several genes that have been linked to asthma ...susceptibility, but the role of this locus in AR is unclear. Asthma and AR in adults of Chinese ethnicity in Singapore are predominately caused by sensitization against house dust mites with a nearly complete penetrance of the allergen, which presents a unique opportunity for accurately identifying genetic associations with allergic diseases. Objective We sought to define the functional role of 17q12-21 in patients with AR and allergic asthma. Methods We asked whether single nucleotide polymorphisms (SNPs) in the 17q12-21 locus were associated with AR or asthma in a cohort of 3460 ethnic Chinese subjects residing in Singapore (1435 in the discovery phase and 2025 in the validation phase). Full-blood mRNA gene expression data, plasma IgE levels, and immune cell frequencies in peripheral blood were tested against the tag SNP genotypes. Luciferase assays were used to measure the effect of putative promoter SNPs on expression of the asthma-associated orosomucoid-like 3 gene (ORMDL3). Results Within 17q12-21, only the tag SNP rs8076131 was significantly associated with asthma ( P = 8.53 × 10−10 ; odds ratio, 0.6715), and AR status was independent of SNPs in this region. C-A alleles at rs8076131 resulted in significantly increased ORMDL3 expression in HEK293 cells in vitro relative to T-G alleles. Moreover, subjects with the risk genotype AA exhibited significantly higher total IgE levels and higher blood eosinophil counts than those with the lower-risk genotypes. Conclusion The 17q12-21 locus has a strong genetic association with allergic asthma but not with AR. The polymorphic effect of this locus is attributed to the linkage set tagged by rs8076131, which affects the expression of ORMDL3 , protein phosphatase 1, regulatory inhibitor subunit 1B ( PPP1R1B ), zona pellucida binding protein 2 ( ZPBP2 ), and gasdermin B (GSDMB) and is correlated with high IgE levels and eosinophil counts in subjects bearing the risk genotype.
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