Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

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.

Impaired interferon response in plasmacytoid dendritic cells from children with persistent wheeze.

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 high-affinity 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 FcεRI 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.

Robust Virus-Specific Adaptive Immunity in COVID-19 Patients with SARS-CoV-2 Δ382 Variant Infection.

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.

Functional variants of 17q12-21 are associated with allergic asthma but not allergic rhinitis.

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.

Dendritic cell derived IL-2 inhibits survival of terminally mature cells via an autocrine signaling pathway.

DCs are crucial for sensing pathogens and triggering immune response. Upon activation by pathogen-associated molecular pattern (PAMP) ligands, GM-CSF myeloid DCs (GM-DCs) secrete several cytokines, including IL-2. DC IL-2 has been shown to be important for innate and adaptive immune responses; however, IL-2 importance in DC physiology has never been demonstrated. Here, we show that autocrine IL-2 signaling is functional in murine GM-DCs in an early time window after PAMPs stimulation. IL-2 signaling selectively activates the JAK/STAT5 pathway by assembling holo-receptor complexes at the cell surface. Using the sensitivity of targeted mass spectrometry, we show conclusively that GM-DCs express CD122, the IL-2 receptor ß-chain, at steady state. In myeloid DCs, this cytokine pathway inhibits survival of PAMP-matured GM-DCs which is crucial for maintaining immune tolerance and preventing autoimmunity. Our findings suggest that immune regulation by this novel autocrine signaling pathway can potentially be used in DC immunotherapy.

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis.

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.

Pyrosequencing enhancement for better detection limit and sequencing homopolymers.

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid and real-time sequence determination. Although ample genomic research has been undertaken using pyrosequencing, the requirement of relatively high amount of DNA template and the difficulty in sequencing the homopolymeric regions limit its key advantages in the applications directing towards clinical research. In this study, we demonstrate that pyrosequencing on homopolymeric regions with 10 identical nucleotides can be successfully performed with optimal amount of DNA (0.3125-5 pmol) immobilized on conventional non-porous Sepharose beads. We also validate that by using porous silica beads, the sequencing signal increased 3.5-folds as compared to that produced from same amount of DNA immobilized on solid Sepharose beads. Our results strongly indicate that with optimized quantity of DNA and suitable solid support, the performance of pyrosequencing on homopolymeric regions and its detection limit has been significantly improved.