The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G+ granulocytes (Ly6G+Gra), IL-17, and COX2. We show that in the lungs of Mtb-infected wild-type mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G+Gra-resident 4 weeks post-infection onwards. In the genetically susceptible ifng-/- mice, excessive Ly6G+Gra infiltration correlates with severe bacteremia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G+Gra infiltration, associated pathology, and death in ifng-/- mice. Surprisingly, Ly6G+Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or ifng-/- mice. The IL-17-COX2-Ly6G+Gra interplay also operates in WT mice. Inhibiting RORγt, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G+Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCG’s protective efficacy, mainly by targeting the Ly6G+Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by RORγt inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G+Gra axis for TB prevention and therapy.

Macrophages are critical to maintaining intestinal homeostasis and contribute to localized inflammation once dysregulated. Here, we aimed at understanding innate macrophage dysfunction in subjects with chronic idiopathic gut inflammation Crohn’s disease (CD) and chronic infection-related gut inflammation, intestinal tuberculosis (ITB). The study population involved subjects aged 18 years or above, including males and females, who met the inclusion/exclusion criteria (CD: n = 42; ITB: n = 41). RNA-seq analysis of ex vivo Mtb-infected or uninfected monocyte-derived macrophages (MDMs) from CD, ITB and healthy controls was done. The MDMs from a cohort of patients, including CD (n = 42) and ITB (n = 41), were characterized for innate immune responses such as autophagy (CD, n = 15: ITB, n = 09), mitochondrial depolarization (CD, n = 08: ITB, n = 08) and bactericidal capacity (CD, n = 25: ITB, n = 28). Finally, the role of AXL downregulation in impaired autophagy was tested. The MDMs from CD and ITB subjects showed differential regulation of autophagy-associated genes. Functionally, CD MDMs showed significant autophagy impairment compared to ITB MDMs (average MFI-201 in CD vs. 414 in ITB). Both CD and ITB MDMs were permissive to Mtb uptake; however, CD MDMs were more permissive (44

Background: Impaired interferon response and allergic sensitization may contribute to virus -induced wheeze and asthma development in young children. Plasmacytoid dendritic cells (pDCs) play a key role in antiviral immunity as critical producers of type I interferons. pDCs also express the highaffinity IgE receptor through which type I interferon production may be negatively regulated. Whether antiviral function of pDCs is associated with recurrent episodes of wheeze in young children is not well understood. Objective: We sought to evaluate the phenotype and function of circulating pDCs in children with a longitudinally defined wheezing phenotype. Methods: We performed multiparameter flow cytometry on PBMCs from 38 children presenting to the emergency department with an acute episode of respiratory wheeze and 19 controls. RNA sequencing on isolated pDCs from the same individuals was also performed. For each subject, their longitudinal exacerbation phenotype was determined using the Western Australia public hospital database. Results: We observed a significant depletion of circulating pDCs in young children with a persistent phenotype of wheeze. The same individuals also displayed upregulation of the Fc epsilon RI on their pDCs. Based on transcriptomic analysis, pDCs from these individuals did not mount a robust systemic antiviral response as observed in children who displayed a nonrecurrent wheezing phenotype. Conclusions: Our data suggest that circulating pDC phenotype and function are altered in young children with a persistent longitudinal exacerbation phenotype. Expression of high -affinity IgE receptor is increased and their function as major interferon producers is impaired during acute exacerbations of wheeze. (J Allergy Clin Immunol 2024;153:1083-94.)

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

Background : Allergic rhinitis (AR) is strongly associated with a type 2 response, characterized by the cytokines IL-5, IL-4 and IL-13. Several studies have implicated ILC2 and TH2A (CD161+ TH2) but it is not yet entirely clear which subsets are driving the common allergic reactions underlying AR. The objective of this study aims to identify critical pathogenic cell populations associated with AR and to determine their phenotype and functional contribution to disease progression. Methods : We identified integral allergic-specific cell types by transcriptomic sequencing. Whole blood, PBMCs and plasma from a cross-sectional cohort of 216 individuals were analysed by 9-colour flow cytometry and ultra-sensitive cytokine bead arrays using unsupervised clustering algorithms. Clinically active AR cases were further analysed by functional mass cytometry to define phenotype and cytokine secretion (IL-2, IL-3, IL-4, IL-5, IL-9, IL13, IL-17A and IL-22) as well as the expression of the hematopoietic prostaglandin D synthetase (HPGDS). Results : The unbiased analysis revealed that atopy and AR manifestation corelated only with eosinophils, plasma IL-5 and CD161+ TH2 cells. In-depth characterization further revealed that the CD45RB CD161+ TH2 subset were most closely associated with severity. This subset is able to concomitantly secrete multiple cytokines including IL-5, IL-13 and IL-4 and has been previously reported to be associated with other eosinophilic allergies. Conclusion : CD45RB CD161+ TH2 have key roles in driving the allergic response in AR. Neutralizing the CD45RB CD161+ TH2 subset should disrupt the allergic response pathway, thus providing a target for lasting therapeutic interventions.

Tropical urban environments reveal a strong association of CD45RBloCD161+ Th2 subset to allergic rhinitis To the Editor:Allergic airway diseases such as allergic rhinitis (AR) affects more than 400 million individuals worldwide and afflicts substantial health and economic morbidity. [1] AR is strongly associated with a type 2 response, characterized by the cytokines IL-5, IL-4 and IL-13. However, the key drivers behind AR immunopathogenesis remained to be elucidated. This study aims to identify critical pathogenic cell populations associated with AR using the Singapore System Immunology Cohort (SSIC) [2] and a clinician-diagnosed paediatric cohort with active AR manifestation (Supplementary Table 1 ). In both cohorts, the eosinophilic nature of AR was confirmed by higher blood eosinophil numbers (Supplementary Figure 1 ).Whole blood gene expression analysis revealed a total of 23 probes representing 20 unique genes were associated with AR in the SSIC (Table 1A ). To account for ethnicity and environmental influences we validated our findings in BAMSE population-based cohort comprising of Swedish adolescents. Table 1B shows 11 DEGs which was also associated with AR, confirming the transferability of our findings to other populations. For the top DEGs that reached nominal significance in the SSIC we performed an Ingenuity Pathway Analysis (IPA). Supplementary Table 2 revealed important pathways related to hypersensitivity and inflammation and also functional enrichment for eosinophils, basophils and mast cells. In particular, functional activation of Th2 was highlighted as a key pathway for AR pathogenesis. As CRTH2 was reported to be expressed by cell types involved in the eosinophilic response, [3] an unsupervised cluster analysis was performed on the CRTH2+ subset in PBMC of individuals from SSIC (Figure 1 A and B ) to determine CRTH2+ subsets associated with AR. We found that CD161+Th2 subsets in particularly to be strongly associated with AR (Figure 1C and D ) (Supplementary Figure 2 ). Further characterization found that the marker CD45RB to be significantly downregulated on CD161+Th2 cells of AR individuals (Figure 1E ). Low CD45RB expression on T cells is indicative of a mature phenotype. Interestingly, significantly higher circulatory plasma IL-5 levels (Figure 1F) . Furthermore we could also demonstrate AR individuals produced significantly higher IL-5 in an in vitro PMA-stimulation assay (Figure 1G ).While we noted a small population of IL-5 secreting conventional CD161-Th2 (cTh2), IL-5 secretion was significantly elevated in CD161+Th2 cells (Figure 1H ). Strikingly, IL-5 was found to be predominantly secreted by CD45RBlo subset in both cTh2 and CD161+Th2 (Figure 1H and I ). There was also a significant increase in the IL-5 producing CD45RBloCD161+Th2 population from the AR individuals (Figure 1I ). These findings confirm CD45RBloCD161+Th2 as the main producers of IL-5.We further validated our findings in a second paediatric cohort with clinically diagnosed active AR manifestations. To further refine CD161+Th2 subset that is associated with AR, we performed unsupervised PhenoGraph and UMAP clustering on CD161+Th2 (Supplementary Figure 3A and B ). Amongst the UMAP clusters, “cluster 3” was found to be significantly associated with active AR (Supplementary Figure 3C and D ). Deep characterization reveals “cluster 3” to be an IL-5 secreting CD45RBlopopulation, confirming our earlier observation (Supplementary Figure 3E ). Furthermore, this cluster appeared to be a highly differentiated population of mature CD161+Th2 cells with an activated phenotype secreting IL-2, IL-3, IL-4, IL-9 and IL-13 concomitantly (Supplementary Figure 3E and F ). Thus, the severity of eosinophilic airway allergies such as AR seems to be driven by an activated terminally differentiated CD161+Th2 subset that is able to secrete a complex set of inflammatory cytokines.The presence of CD45RBloCD161+Th2 population in both cohorts shows the persistence and pertinence of this population in the pathogenesis of AR. Both cohorts described in this study were collected in Singapore, whereby majority of the individuals are sensitized against HDM. HDM is a perennial allergen in tropical nations such as Singapore, thus T cells in atopic individuals undergo constant stimulation. This could explain the strong association observed between CD45RB expression on CD161+Th2 cells and atopy markers despite the fact that not all subjects demonstrated active AR symptoms during the collection of SSIC cohort. Taken together, our current study unifies the markers previously reported for allergic-specific Th2 subsets and provides clarity for the pathogenic Th2 subset previously reported in different allergic diseases.[4-6] Neutralizing the CD45RBloCD161+Th2 subset should disrupt the allergic response pathway, thus providing a target for lasting therapeutic interventions. Moreover, these cells may also be leveraged as a biomarker for the effectiveness of immunotherapy as well as a potential biomarker of public health surveillance of allergic individuals.

15UBIAir – Clinical & Experimental Lung Centre and CICSUBI Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal 16Allergy and Clinical Immunology, University of Bari Medical School, Bari, Italy 17Institute of Sciences of Food Production, National Research Council (ISPACNR), Bari, Italy 18ISGlobal, Barcelona Institute for Global Health, Barcelona, Spain 19Universitat Pompeu Fabra (UPF), Barcelona, Spain 20CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206loESAM- population (KC1) and a minor CD206hiESAM+ population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have become dominant as the pandemic progresses bear the ORF8 mutation together with multiple spike mutations. A 382-nucleotide deletion (Δ382) in the ORF7b and ORF8 regions has been associated with milder disease phenotype and less systemic inflammation in COVID-19 patients. However, its impact on host immunity against SARS-CoV-2 remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with either wildtype or Δ382 SARS-CoV-2 variant. Interestingly, the immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased adaptive immune response, evidenced by enrichment of genes related to T cell functionality, a more robust SARS-CoV-2-specific T cell immunity, as well as a more rapid antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be upregulated in patients bearing Δ382, and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. This study provides more in-depth insight into the host–pathogen interactions of ORF8 with great promise as a therapeutic target to combat SARS-CoV-2 infection.

Numerous genome-wide association studies, including 1 published in The Journal of Allergy and Clinical Immunology by Ferreria et al, have reported an association between rs2427837 and allergic disease status.1Ferreira M.A.R. Vonk J.M. Baurecht H. Marenholz I. Tian C. Hoffman J.D. et al.Eleven loci with new reproducible genetic associations with allergic disease risk.J Allergy Clin Immunol. 2019; 143: 691-699Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar This single-nucleotide polymorphism (SNP) is located within the first intron of the high-affinity IgE Fc receptor (FCER1A). However, the candidate gene that seems to strongly be correlated with this SNP is OR10J5, which is an olfactory receptor located approximately 250 kb upstream of the polymorphism.1Ferreira M.A.R. Vonk J.M. Baurecht H. Marenholz I. Tian C. Hoffman J.D. et al.Eleven loci with new reproducible genetic associations with allergic disease risk.J Allergy Clin Immunol. 2019; 143: 691-699Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Here, we present evidence to support the idea that rs2427837 is instead correlated with FCER1A mRNA levels in whole blood and protein levels on monocytes and plasmacytoid dendritic cells (pDCs). We first analyzed mRNA data from 31,430 whole blood samples constituting 35 different cohorts that were collected by the eQTLgen Consortium. We consistently detected a strong correlation (P = 5.8E–152) between rs2427837 and FCER1A (Fig 1, A). The expression levels of only 2 other genes were also significantly correlated with this SNP: DARC (P = 4.9E–54) and AIM2 (P = 4.2E–08). Notably, we did not detect a significant correlation with OR10J5 (see Table E1 in this article's Online repository at www.jacionline.org). Furthermore, and consistent with data from the Genotype-Tissue Expression Consortium (https://www.gtexportal.org/home/gene/ENSG00000184155), we did not detect OR10J5 in our blood samples. To identify the cell subsets affected by rs2427837, we analyzed FCER1A protein levels in whole blood subsets by flow cytometry. A previous study reported a correlation between rs2427837 and FCER1A levels at nominal significance in basophils.2Kanada S. Nakano N. Potaczek D.P. Maeda K. Shimokawa N. Niwa Y. et al.Two different transcription factors discriminate the -315C>T polymorphism of the Fc epsilon RI alpha gene: binding of Sp1 to -315C and of a high mobility group-related molecule to -315T.J Immunol. 2008; 180: 8204-8210Crossref PubMed Scopus (43) Google Scholar However, when we analyzed 135 samples from a functional cohort of Singapore Chinese participants, we found no impact of this SNP on FCER1A levels in these cells (Fig 1, B). Rather, we found a strong correlation between rs2427837 and FCER1A protein levels on monocytes (P < .001) but not with other allergy-relevant subsets, namely, eosinophils or neutrophils (Fig 1, C-E). This discrepancy in basophils could be due to cell treatment effects: in the previous study, the cells were treated with lactic acid to strip IgE from the surface receptor. IgE has a stabilizing effect on FCER1A surface expression, which on basophils is further enhanced by the cell type–specific Fc receptor α-chain MS4A2.3Sharma V. Michel S. Gaertner V. Franke A. Vogelberg C. von Berg A. et al.Fine-mapping of IgE-associated loci 1q23, 5q31, and 12q13 using 1000 Genomes Project data.Allergy. 2014; 69: 1077-1084Crossref PubMed Scopus (23) Google Scholar Another recent analysis of FCERӀA levels in PBMC fractions also found no association between FCER1A-positive basophil subsets and atopy or asthma.4Leffler J. Read J.F. Jones A.C. Mok D. Hollams E.M. Laing I.A. et al.Progressive increase of FcepsilonRI expression across several PBMC subsets is associated with atopy and atopic asthma within school-aged children.Pediatr Allergy Immunol. 2019; 30: 646-653PubMed Google Scholar However, the rs2427837 SNP was not considered in this work. We validated our findings in additional samples by using a comprehensive flow cytometry panel, gating out the 4 major FCER1A-expressing immune cell subsets: monocytes, pDCs, myeloid dendritic cells (mDCs), and basophils (see Figs E1 and E2 in this article's Online repository at www.jacionline.org). We found an allele-dependent increase in the FCER1A-positive subset, with the AA genotype conferring the highest frequency (see Fig E2). The impact of this increase was evident only on classical CD16– monocytes, as nonclassical CD16+ monocytes do not express FCER1A and are thus unaffected by the polymorphism (see Fig E2). We then replicated this correlation between FCER1A expression in classical monocytes and the rs2427837 SNP in individuals with AG and GG genotypes, as the AA genotype is rare in East Asians (Fig 2, A). Further analysis in a validation cohort revealed a strong correlation between rs2427837 and FCER1A levels in classical monocytes (P = 6.04E–04) (Fig 2, C). We also observed increased FCER1A mRNA levels in classical monocytes in individuals with the AA genotype compared with in individuals with the AG and GG genotypes when analyzing eQTL data obtained by the DICE project for various immune populations (see Fig E3 in this article's Online repository at www.jacionline.org). Staining with anti-IgE further confirmed that this increase in FCER1A levels also translates into increased IgE surface expression on monocytes (Fig 2, B). Besides classical monocytes, rs2427837 was correlated with FCER1A levels in pDCs. Here, we found that individuals with the G allele expressed high FCER1A levels (Fig 2, D). Although this correlation opposes what we saw with monocytes, it is concordant with the reported effects on stripped basophils.2Kanada S. Nakano N. Potaczek D.P. Maeda K. Shimokawa N. Niwa Y. et al.Two different transcription factors discriminate the -315C>T polymorphism of the Fc epsilon RI alpha gene: binding of Sp1 to -315C and of a high mobility group-related molecule to -315T.J Immunol. 2008; 180: 8204-8210Crossref PubMed Scopus (43) Google Scholar We found no correlation for any other subsets, including untreated basophils and mDCs (Fig 2, D and E and see Fig E4 in this article's Online repository at www.jacionline.org). The rs2427837 SNP has been associated with asthma and total IgE levels in asthmatics, where the major G allele results in higher IgE production and an elevated disease risk. However, the region has multiple SNPs which could have combined effect on the functional consequence of FCER1A.3Sharma V. Michel S. Gaertner V. Franke A. Vogelberg C. von Berg A. et al.Fine-mapping of IgE-associated loci 1q23, 5q31, and 12q13 using 1000 Genomes Project data.Allergy. 2014; 69: 1077-1084Crossref PubMed Scopus (23) Google Scholar Furthermore, when we analyzed whole blood gene expression data we saw a significantly higher level of FCER1A mRNA expression in individuals with a history of asthma from a total of 166 subjects (42 asthma cases and 124 controls) from the Singapore Systems Immunology cohort and a total of 256 subjects (98 asthma cases and 158 controls) from the Barn/Children Allergy/Asthma Milieu Stockholm Epidemiologic birth cohort (P = .045, and P = .0086, respectively). Although basophils have been associated with the clinical manifestations of this SNP, the lack of any detectable genotype effect on untreated cells makes it unlikely that they mediate the genetic effect. Our finding of increased FCER1A levels in pDCs, however, is in line with previous reports showing higher FCER1A levels on pDCs in asthmatic patients.5Foster B. Metcalfe D.D. Prussin C. Human dendritic cell 1 and dendritic cell 2 subsets express FcepsilonRI: correlation with serum IgE and allergic asthma.J Allergy Clinical Immunol. 2003; 112: 1132-1138Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar Various studies have implicated FCER1A and pDC recruitment to the lungs in asthma and allergic airway diseases. In particular, a study in school-aged children found that FCER1A-positive pDC subsets could differentiate children with asthma from atopic children without asthma.4Leffler J. Read J.F. Jones A.C. Mok D. Hollams E.M. Laing I.A. et al.Progressive increase of FcepsilonRI expression across several PBMC subsets is associated with atopy and atopic asthma within school-aged children.Pediatr Allergy Immunol. 2019; 30: 646-653PubMed Google Scholar In addition, pDCs expressing CD123 (IL3-receptor-alpha) are recruited to the lungs during inflammation and migrate to the draining lymph nodes to augment TH2 responses in a mouse model of allergic airway disease.6Chairakaki A.D. Saridaki M.I. Pyrillou K. Mouratis M.A. Koltsida O. Walton R.P. et al.Plasmacytoid dendritic cells drive acute asthma exacerbations.J Allergy Clin Immunol. 2018; 142: 542-556.e12Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar These pDCs exhibit an activated inflammatory phenotype in the nasal mucosa following stimulation with TNF-α: here, they favor a TH2 polarizing profile and can markedly increase in number to trigger airway allergy. Thus, not surprisingly, they are associated with a higher risk of experiencing asthma attacks and a severe form of the disease. Using data from the Genotype-Tissue Expression portal, we also found an eQTL association for rs2427837 in lung tissue for the FCER1A gene (https://www.ncbi.nlm.nih.gov/gene/?term=2205) (see Fig E5 in this article's Online repository at www.jacionline.org). Similar to the effect on pDCs, the AA genotype was correlated with the highest FCER1A mRNA levels. It is noteworthy that functional genotype-dependent FCERIA expression has been previously described for the SNP rs2251746 in basophils and mast cells.7Nishiyama C. Molecular mechanism of allergy-related gene regulation and hematopoietic cell development by transcription factors.Biosci Biotechnol Biochem. 2006; 70: 1-9Crossref PubMed Scopus (20) Google Scholar In monocytes, we found that the AG genotype was associated with increased FCER1A levels and conferred a lower risk of IgE production and allergic disease. This effect could be due to the fact that serum IgE is cleared via FCER1A capture and internalization by monocytes and mDCs.8Alexandra M.G. Nan W. Amy L.P. Prescott G.W. Paul W. Jean-Pierre K. et al.Serum IgE clearance is facilitated by human FcεRI internalization.J Clin Invest. 2014; 124: 1187-1198Crossref PubMed Scopus (56) Google Scholar Importantly, the particularly strong correlation between the AG genotype and IgE surface expression on these cells suggests that the contribution of monocytes to allergy control might be underappreciated. A recent review highlighted the fact that FCER1A engagement in monocytes induces indoleamine 2,3-dioxygenase upregulation, which inhibits T-cell proliferation and enhances regulatory T-cell function by depleting tryptophan.9Maurer M. Altrichter S. Schmetzer O. Scheffel J. Church M.K. Metz M. Immunoglobulin E-mediated autoimmunity.Front Immunol. 2018; 9: 689Crossref PubMed Scopus (49) Google Scholar Moreover, FCER1A cross-linking in monocytes induces IL-10, an anti-inflammatory mediator that might attenuate basophil and dendritic cell activation and monocyte phagocytic activity. Taking the aforesaid data together, we propose a model of FCER1A regulation in monocytes and pDCs that is based on the rs2427837 genotype, which is associated with allergic disease risk.7Nishiyama C. Molecular mechanism of allergy-related gene regulation and hematopoietic cell development by transcription factors.Biosci Biotechnol Biochem. 2006; 70: 1-9Crossref PubMed Scopus (20) Google Scholar (see Fig E6 in this article's Online Repository at www.jacionline.org). Certain limitations to our study should be acknowledged. First, we did not evaluate the correlation between rs2427837 and FCER1A gene expression in patients with allergy versus in healthy controls. Second, the associations that we made between FCER1A gene expression and asthma in the Singapore Systems Immunology cohort and Barn/Children Allergy/Asthma Milieu Stockholm Epidemiologic study cohort derive from whole blood samples, not from individual immune cell subsets. Further studies that combine genetic and immune characterizations of FCER1A in immune cell subsets will help to elucidate the contribution of each factor to asthma and allergic disease. Importantly, we have confirmed the association of the allergy-relevant SNP rs2427837 with FCER1A and believe that targeting FCER1A in monocytes and pDCs might constitute a novel option to reduce allergen-specific responses. The relevant details for the discovery and validation cohorts from the Singapore Chinese cohort have been previously described.E1Puan K.J. Andiappan A.K. Lee B. Kumar D. Lai T.S. Yeo G. et al.Systematic characterization of basophil anergy.Allergy. 2017; 72: 373-384Crossref PubMed Scopus (16) Google Scholar For the discovery cohort, whole blood samples were lysed with lysis buffer and stained with anti–FcεRI-FITC (AER37, eBioscience, San Diego, Calif), anti–IgE-APC (MB10-5C4, Miltenyi Biotec, Bergisch Gladbach, Germany), and anti–CD123-PerCP-Cy5.5 (6H6, eBioscience) antibodies. For the validation cohort, another 4 antibodies were included to identify various immune cell populations: anti-CD14 (61D3, eBioscience), anti-CD16 (3G8, Biolegend, San Diego, Calif), anti-CD1c (L161, Biolegend), and anti–HLA-DR (L243, BD Bioscience, San Jose, Calif). In some experiments, staining was performed by using the same validation antibody panels on PBMCs. A Live/Dead exclusion dye was included to discriminate the viable cells. The samples were analyzed on a BD FACS CantoII or a BD LSRFortessa flow cytometer (BD Bioscience). Data analysis and visualization was performed by using FlowJo software, version 9.9.6 or 10.6.2 (BD Bioscience). The Singapore Chinese cohort genotyping data were obtained from Illumina Infinium chips containing SNPs covering the whole genome. DNA extraction and genotyping were performed as previously described.E2Andiappan A.K. Melchiotti R. Poh T.Y. Nah M. Puan K.J. Vigano E. et al.Genome-wide analysis of the genetic regulation of gene expression in human neutrophils.Nat Commun. 2015; 6: 7971Crossref PubMed Scopus (17) Google Scholar Genotyping data for SNPs in the FCER1A locus (rs2427837 and rs2251746) were extracted, and the correlation with FCER1A protein expression was determined by performing nonparametric tests. eQTL data for the rs2427837 SNP from whole blood and PBMCs were obtained from the eQTLGen Consortium (https://www.eqtlgen.org).E3Westra H.J. Peters M.J. Esko T. Yaghootkar H. Schurmann C. Kettunen J. et al.Systematic identification of trans eQTLs as putative drivers of known disease associations.Nat Genet. 2013; 45: 1238-1243Crossref PubMed Scopus (1048) Google Scholar A meta-analysis of the data from the various cohorts was performed by using the z score; a P value was calculated for a meta P value and combined z score.E4Võsa U. Claringbould A. Westra H.-J. Bonder M.J. Deelen P. Zeng B. et al.Unraveling the polygenic architecture of complex traits using blood eQTL metaanalysis.bioRxiv. 2018; 447367Google Scholar Gene expression plots and eQTL data from lung tissue were downloaded from the Genotype-Tissue Expression Consortium web page. Data for this consortium were published previously.E5Battle A. Brown C.D. Engelhardt B.E. Montgomery S.B. Consortium G. Laboratory DtACCLAWG et al.Genetic effects on gene expression across human tissues.Nature. 2017; 550: 204-213Crossref PubMed Scopus (1317) Google Scholar FCER1A gene expression in whole blood and phenotype information for asthma from the Barn/Children Allergy/Asthma Milieu Stockholm Epidemiologic study cohort were extracted from a previously reported data set.E6Lemonnier N, Melén E, Jiang Y, et al. A novel whole blood gene expression signature for asthma, dermatitis, and rhinitis multimorbidity in children and adolescents [e-pub ahead of print, 2020]. Allergy https://doi.org/10.1111/all.14314. Accessed May 3, 2020.Google Scholar Associations of gene expression with the clinical phenotype were determined by performing nonparametric tests. A P value less than .05 was considered statistically significant. Gene expression data for the Chinese samples were obtained from participants of the Singapore Systems Immunology cohort. This cohort constitutes part of an unpublished study that aimed to identify differentially expressed genes for allergic diseases. RNA was isolated from Tempus blood tubes by using a Qiagen blood kit, after which the RNA quality was determined by using a Bioanalyzer. An Illumina Human HT-12 gene expression chip was used to determine the mRNA levels. The FCER1A mRNA levels extracted for the analysis are presented in this letter. The asthma phenotype was determined from the participants answers to a self-report questionnaire and a positive skin prick test result to the house dust mite allergen. Associations between gene expression and clinical phenotype were determined by performing nonparametric tests. A P value less than .05 was considered statistically significant. FDR, False discovery rate; ID, identifier; Pos, positive. Association statistics are shown. P values and z scores were calculated for the combined meta-analysis of the statistics on all the cohorts described in the eQTLgen Consortium. More details on the analysis are provided in Võsa et al.E4Võsa U. Claringbould A. Westra H.-J. Bonder M.J. Deelen P. Zeng B. et al.Unraveling the polygenic architecture of complex traits using blood eQTL metaanalysis.bioRxiv. 2018; 447367Google Scholar

ABSTRACT Methionine biosynthetic pathway, essential for the growth of Mycobacterium tuberculosis ( Mtb ) in the host, represents an attractive target for the development of novel anti-tuberculars. Here, we have biochemically characterized homoserine acetyl transferase (HSAT viz. MetA) of Mtb , which catalyses the first committed step of methionine and S-adenosylmethionine (SAM) biosynthesis. High-throughput screening of a 2300 compound library resulted in identification of thiram, an anti-fungal organosulfur compound, as the most potent MetA inhibitor. Further analysis of thiram analogs led to the identification of orally bioavailable disulfiram (DIS, an anti-alcoholism FDA approved drug) as a novel inhibitor of MetA. Both thiram and DIS restricted the growth of drug-sensitive and drug-resistant Mtb strains in a bactericidal manner. ThermoFlour assay demonstrated direct binding of DIS with MetA. Metabolomic and transcriptomic studies showed DIS mediated perturbation of methionine and redox homeostasis, respectively, in Mtb . In concordance, the effect of DIS on Mtb growth was partially rescued by supplementation with either L-methionine as well as N-acetyl cysteine, suggesting a multi-target killing mechanism. In Mtb -infected mice, DIS administration restricted bacterial growth, increased efficacy of isoniazid, ameliorated lung pathology, modulated lung immune cell landscape and protective immune response. Taken together, our results demonstrate that DIS can be repurposed for designing an effective anti-tubercular therapy.

Patients with type 2 diabetes (T2D) have a lower risk of Mycobacterium tuberculosis infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence, when being treated with metformin. However, a detailed mechanistic understanding of these protective effects is lacking. Here, we use mass cytometry to show that metformin treatment expands a population of memory-like antigen-inexperienced CD8 + CXCR3 + T cells in naive mice, and in healthy individuals and patients with T2D. Metformin-educated CD8 + T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8 + T cells from Cxcr3 −/− mice do not exhibit this metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhances immunogenicity and protective efficacy against M. tuberculosis challenge. Collectively, these results demonstrate an important function of CD8 + T cells in metformin-derived host metabolic-fitness towards M. tuberculosis infection.

Background and aims: Lipid-rich foam cell macrophages drive atherosclerosis via several mechanisms, including inflammation, lipid uptake, lipid deposition and plaque vulnerability. The atheroma environment shapes mac-rophage function and phenotype; anti-inflammatory macrophages improve plaque stability while pro -in-flammatory macrophages promote rupture. Current evidence suggests a variety of macrophage phenotypes occur in atherosclerotic plaques with local lipids, cytokines, oxidised phospholipids and pathogenic stimuli altering their phenotype. In this study, we addressed differential functioning of macrophage phenotypes via a systematic analysis of in vitro polarised, human monocyte-derived macrophage phenotypes, focussing on molecular events that regulate foam-cell formation. Methods: We examined transcriptomes, protein levels and functionally determined lipid handling and foam cell formation capacity in macrophages polarised with IFN gamma+LPS, IL-4, IL-10, oxPAPC and CXCL4. Results: RNA sequencing of differentially polarised macrophages revealed distinct gene expression changes, with enrichment in atherosclerosis and lipid-associated pathways. Analysis of lipid processing activity showed IL-4 and IL-10 macrophages have higher lipid uptake and foam cell formation activities, while inflammatory and oxPAPC macrophages displayed lower foam cell formation. Inflammatory macrophages showed low lipid uptake, while higher lipid uptake in oxPAPC macrophages was matched by increased lipid efflux capacity. Conclusions: Atherosclerosis-associated macrophage polarisation dramatically affects lipid handling capacity underpinned by major transcriptomic changes and altered protein levels in lipid-handling gene expression. This leads to phenotype-specific differences in LDL uptake, cellular cholesterol levels and cholesterol efflux, in-forming how the plaque environment influences atherosclerosis progression by influencing macrophage phe-notypes.

The impact of ORF8 382-nucleotide deletion (Δ382) on the cellular host immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with wildtype or Δ382 variant of SARS-CoV-2. Interestingly, immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased T cell response, evidenced by enrichment of genes related to T cell functionality that correlated with up-regulation of T cell-associated cytokines, under-expression of neutrophil activation-associated genes, lowered systemic inflammation and effective antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be up-regulated in patients bearing Δ382 and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. These key insights could serve as an important foundation for future human challenge vaccine studies and highlights the prophylactic potential of Δ382 as vaccine candidate.Funding: The study was supported by core and COVID-19 (project number H20/04/g1/006) research grants provided to Singapore Immunology Network by the Biomedical Research Council (BMRC) and A*ccelerate GAP-funded project (ACCL/20-GAP001C20H-E) from the Singapore Ministry of Health's National Medical Research Council. This study was also funded by the National Medical Research Council (NMRC) COVID-19 Research fund (COVID19RF-001). SIgN Immunomonitoring Platform is supported by a BMRC IAF 311006 grant and BMRC transition funds #H16/99/b0/011. ATR is supported by the Singapore International Graduate Award (SINGA), A*STAR. Conflict of Interest: The authors declare no conflict of interest.Ethical Approval: The study design and protocols for the COVID-19 PROTECT study group were evaluated by National Healthcare Group (NHG) Domain Specific Review Board (DSRB) and approved under study number 2012/00917. Collection of healthy donor samples was approved by SingHealth Centralised Institutional Review Board (CIRB) under study number 2017/2806 and NUS IRB 04-140. Written informed consent was obtained from participants in accordance with the Declaration of Helsinki for Human Research.

Background: Immune adaptation with aging is a major of health outcomes. Studies in humans have mainly focus on alpha beta T cells while gamma delta T cells have been neglected despite their role in immunosurveillance. We investigated the impact of aging on gamma delta T cell subsets phenotypes, functions, senescence and their molecular response to stress. Methods: Peripheral blood of young and old donors in Singapore have been used to assess the phenotype, functional capacity, proliferation capacity and gene expression of the various gamma d T cell subsets. Peripheral blood mononuclear cells from apheresis cones and young donors have been used to characterize the telomere length, epigenetics profile and DNA damage response of the various gamma d T cell subsets phenotype. Findings: Our data shows that peripheral V delta 2+ phenotype, functional capacity (cytokines, cytotoxicity, proliferation) and gene expression profile are specific when compared against all other alpha beta and gamma delta T cells in aging. Hallmarks of senescence including telomere length, epigenetic profile and DNA damage response of V delta 2+ also differs against all other alpha beta and gamma delta T cells. Interpretation: Our results highlight the differential impact of lifelong stress on V delta T cells subsets, and highlight possible mechanisms that enable V delta 2+ to be resistant to cellular aging. The new findings reinforce the concept that V delta 2+ have an "innate-like" behavior and are more resilient to the environment as compared to "adaptivelike" V delta 1+ T cells. (c) 2018 The Authors. Published by Elsevier B.V.

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1 flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.

Circulating CCR2+ monocytes are crucial for maintaining the adult tissue-resident F4/80hiMHCIIhi macrophage pool in the intestinal lamina propria. Here we show that a subpopulation of CCR2-independent F4/80hiMHCIIlow macrophages, which are the most abundant F4/80hi cells in neonates, gradually decline in number in adulthood; these macrophages likely represent the fetal contribution to F4/80hi cells. In colon adenomas of ApcMin/+ mice, F4/80hiMHCIIlow macrophages are not only preserved, but become the dominant subpopulation among tumour-resident macrophages during tumour progression. Furthermore, these pro-tumoural F4/80hiMHCIIlow and F4/80hiMHCIIhi macrophages can self-renew in the tumour and maintain their numbers mostly independent from bone marrow contribution. Analyses of colon adenomas indicate that CSF1 may be a key facilitator of macrophage self-renewal. In summary, the tumour microenvironment creates an isolated niche for tissue-resident macrophages that favours macrophage survival and self-renewal.

BackgroundImmune adaptation with aging is a major of health outcomes. Studies in humans have mainly focus on αβ T cells while γδ T cells have been neglected despite their role in immunosurveillance. We investigated the impact of aging on γδ T cell subsets phenotypes, functions, senescence and their molecular response to stress.MethodsPeripheral blood of young and old donors in Singapore have been used to assess the phenotype, functional capacity, proliferation capacity and gene expression of the various γδ T cell subsets. Peripheral blood mononuclear cells from apheresis cones and young donors have been used to characterize the telomere length, epigenetics profile and DNA damage response of the various γδ T cell subsets phenotype.FindingsOur data shows that peripheral Vδ2+ phenotype, functional capacity (cytokines, cytotoxicity, proliferation) and gene expression profile are specific when compared against all other αβ and γδ T cells in aging. Hallmarks of senescence including telomere length, epigenetic profile and DNA damage response of Vδ2+ also differs against all other αβ and γδ T cells.InterpretationOur results highlight the differential impact of lifelong stress on γδ T cells subsets, and highlight possible mechanisms that enable Vδ2+ to be resistant to cellular aging. The new findings reinforce the concept that Vδ2+ have an "innate-like" behavior and are more resilient to the environment as compared to "adaptive-like" Vδ1+ T cells.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

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.