Biology subjects

Brown, L.

Publications and source records attributed to Brown, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

The human Y and inactive X chromosomes similarly modulate autosomal gene expression

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex-chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors - ZFX and ZFY - encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

genetics↗

bioRxiv2023

A novel ground truth dataset enables robust 3D nuclear instance segmentation in early mouse embryos

For investigations into fate specification and cell rearrangements in time-lapse images of preimplantation embryos, automated 3D instance segmentation of nuclei and subsequent nuclear tracking are invaluable. Often, the images low signal-to-noise ratio and high voxel anisotropy and the nucleis dense packing and variable shapes limit the performance of many segmentation methods, while subsequent tracking of nuclear instances is complicated by low frame rates and sample movements. Supervised machine learning approaches can radically improve segmentation accuracy and enable easier tracking, but they often require large amounts of difficult-to-obtain annotated 3D data. Here we report a novel mouse line expressing near-infrared nuclear reporter H2B-miRFP720. H2B-miRFP720 is the longest wavelength nuclear reporter in mice and can be imaged simultaneously with other reporters with minimal overlap. We then generate a dataset, which we call BlastoSPIM, of 3D microscopy images of H2B-miRFP720-expressing embryos with ground truth for nuclear instance segmentation. Using BlastoSPIM, we benchmark the performance of seven convolutional neural networks and identify Stardist-3D as the most accurate instance segmentation method across preimplantation development. We then construct a complete pipeline for nuclear instance segmentation with our BlastoSPIM-trained Stardist-3D models and lineage tracking from the 8-cell stage to the end of preimplantation development (> 100 nuclei). Finally, we demonstrate BlastoSPIMs usefulness as pre-train data for related problems, both for a different imaging modality and for different model systems. BlastoSPIM, its corresponding Stardist-3D models, and documentation of the full associated analysis pipeline are available at: blastospim.flatironinstitute.org.

developmental biology↗

bioRxiv2023

DeepHeme: A generalizable, bone marrow classifierwith hematopathologist-level performance

Morphology-based classification of cells in the bone marrow aspirate (BMA) is a key step in the diagnosis and management of hematologic malignancies. However, it is time-intensive and must be performed by expert hematopathologists and laboratory professionals. We curated a large, high-quality dataset of 41,595 hematopathologist consensus-annotated single-cell images extracted from BMA whole slide images (WSIs) containing 23 morphologic classes from the clinical archives of the University of California, San Francisco. We trained a convolutional neural network, DeepHeme, to classify images in this dataset, achieving a mean area under the curve (AUC) of 0.99. DeepHeme was then externally validated on WSIs from Memorial Sloan Kettering Cancer Center, with a similar AUC of 0.98, demonstrating robust generalization. When compared to individual hematopathologists from three different top academic medical centers, the algorithm outperformed all three. Finally, DeepHeme reliably identified cell states such as mitosis, paving the way for image-based quantification of mitotic index in a cell-specific manner, which may have important clinical applications.

bioinformatics↗

bioRxiv2022

Toblerone: detecting exon deletion events in cancer using RNA-seq

Cancer is driven by mutations of the genome that can result in the activation of oncogenes or repression of tumour suppressor genes. In acute lymphoblastic leukemia (ALL) focal deletions in IKAROS family zinc finger 1 (IKZF1) result in the loss of zinc-finger DNA-binding domains and a dominant negative isoform that is associated with higher rates of relapse and poorer patient outcomes. Clinically, the presence of IKZF1 deletions informs prognosis and treatment options. In this work we developed a method for detecting exon deletions in genes using RNA-seq with application to IKZF1. We developed a pipeline that first uses a custom transcriptome reference consisting of transcripts with exon deletions. Next, RNA-seq reads are mapped using a pseudoalignment algorithm to identify reads that uniquely support deletions. These are then evaluated for evidence of the deletion with respect to gene expression and other samples. We applied the algorithm, named Toblerone, to a cohort of 99 B-ALL paediatric samples including validated IKZF1 deletions. Furthermore, we developed a graphical desktop app for non-bioinformatics users that can quickly and easily identify and report deletions in IKZF1 from RNA-seq data with informative graphical outputs.

bioinformatics↗

bioRxiv2022

Developing a standardized but extendable framework to increase the findability of infectious disease datasets

Biomedical datasets are increasing in size, stored in many repositories, and face challenges in FAIRness (findability, accessibility, interoperability, reusability). As a Consortium of infectious disease researchers from 15 Centers, we aim to adopt open science practices to promote transparency, encourage reproducibility, and accelerate research advances through data reuse. To improve FAIRness of our datasets and computational tools, we evaluated metadata standards across established biomedical data repositories. The vast majority do not adhere to a single standard, such as Schema.org, which is widely-adopted by generalist repositories. Consequently, datasets in these repositories are not findable in aggregation projects like Google Dataset Search. We alleviated this gap by creating a reusable metadata schema based on Schema.org and catalogued nearly 400 datasets and computational tools we collected. The approach is easily reusable to create schemas interoperable with community standards, but customized to a particular context. Our approach enabled data discovery, increased the reusability of datasets from a large research consortium, and accelerated research. Lastly, we discuss ongoing challenges with FAIRness beyond discoverability.

bioinformatics↗

bioRxiv2022

Structural foundations of potassium selectivity in channelrhodopsins

Kalium channelrhodopsins (KCRs) are light-gated K+ channels recently found in the stramenopile protist Hyphochytrium catenoides. When expressed in neurons, KCRs enable high-precision optical inhibition of spiking (optogenetic silencing). KCRs are capable of discriminating K+ from Na+ without the conventional K+-selectivity filter found in classical K+ channels. The genome of H. catenoides also encodes a third paralog that is more permeable for Na+ than for K+. To identify structural motifs responsible for the unusual K+ selectivity of KCRs, we systematically analyzed a series of chimeras and mutants of this protein. We found that mutations of three critical residues in the paralog convert its Na+ selective channel into a K+ selective one. Our characterization of homologous proteins from other protists (Colponema vietnamica, Cafeteria burkhardae and Chromera velia) and metagenomic samples confirmed the importance of these residues for K+ selectivity. We also show that Trp102 and Asp116, conserved in all three H. catenoides orthologs, are necessary, although not sufficient, for K+ selectivity. Our results provide the foundation for further engineering of KCRs for optogenetic needs. IMPORTANCERecently discovered microbial light-gated ion channels (channelrhodopsins) with a higher permeability for K+ than for Na+ (kalium channelrhodopsins, or KCRs) demonstrate an alternative K+ selectivity mechanism, unrelated to well-characterized "selectivity filters" of voltage- and ligand-gated K+ channels. KCRs can be used for optogenetic inhibition of neuronal firing, and potentially for the development of gene therapies to treat neurological and cardiovascular disorders. In this study we identify structural motifs that determine the K+ selectivity of KCRs that provide the foundation for that provide the foundation for elucidating their K+ selectivity mechanism and for their further engineering as optogenetic tools.

biochemistry↗

bioRxiv2021

Consistent predictors of microbial community composition across scales in grasslands reveal low context-dependency

Environmental circumstances shaping soil microbial communities have been studied extensively, but due to disparate study designs it has been difficult to resolve whether a globally consistent set of predictors exists, or context-dependency prevails. Here, we used a network of 18 grassland sites (11 sampled across regional plant productivity gradients) to examine i) if the same abiotic or biotic factors predict both large- and regional-scale patterns in bacterial and fungal community composition, and ii) if microbial community composition differs consistently with regional plant productivity (low vs high) across different sites. We found that there is high congruence between predictors of microbial community composition across spatial scales; bacteria were predominantly associated with soil properties and fungi with plant community composition. Moreover, there was a microbial community signal that clearly distinguished high and low productivity soils that was shared across worldwide distributed grasslands suggesting that microbial assemblages vary predictably depending on grassland productivity.

ecology↗

bioRxiv2021

SOX transcription factors direct TCF-independent WNT/beta-catenin transcription.

WNT/{beta}-catenin signaling regulates gene expression across numerous biological contexts including development, stem cell homeostasis and tissue regeneration, and dysregulation of this pathway has been implicated in many diseases including cancer. One fundamental question is how distinct WNT target genes are activated in a context-specific manner, given the dogma that most, if not all, WNT/{beta}-catenin responsive transcription is mediated by TCF/LEF transcription factors (TFs) that have similar DNA-binding specificities. Here we show that the SOX family of TFs direct lineage-specific WNT/{beta}-catenin responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and neuromesodermal progenitors (NMPs). Using time-resolved multi-omics analyses, we show that {beta}-catenin association with chromatin is highly dynamic, colocalizing with distinct TCFs and/or SOX TFs at distinct stages of differentiation, indicating both cooperative and competitive modes of genomic interactions. We demonstrate that SOX17 and SOX2 are required to recruit {beta}-catenin to hundreds of lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At a subset of these TCF-independent enhancers, SOX TFs are required to both establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex that includes {beta}-catenin, BCL9, PYGO and transcriptional coactivators to direct SOX/{beta}-catenin-dependent transcription. Given that SOX TFs are expressed in almost every cell type, these results have broad mechanistic implications for the specificity of WNT responses across many developmental and disease contexts.

developmental biology↗

bioRxiv2021

A gene-by-gene mosaic of dosage compensation strategies on the human X chromosome

Researchers have presumed that the "inactive" X chromosome (Xi) has little impact, in trans, on the "active" X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xis. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X-chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes ([≥] 121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X-chromosome genes most likely to drive sex differences in common disease, and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of cis and trans effects differs widely among genes.

genetics↗

bioRxiv2021

Acetylated Chromatin Domains Link Chromosomal Organization to Cell- and Circuit-level Dysfunction in Schizophrenia and Bipolar Disorder

To explore modular organization of chromosomes in schizophrenia (SCZ) and bipolar disorder (BD), we applied population-scale correlational structuring of 739 histone H3-lysine 27 acetylation and H3-lysine 4 trimethylation profiles, generated from the prefrontal cortex (PFC) of 568 cases and controls. Neuronal histone acetylomes and methylomes assembled as thousands of cis-regulatory domains (CRDs), revealing fine-grained, kilo-to megabase scale chromatin organization at higher resolution but firmly integrated into Hi-C chromosomal conformations. Large clusters of domains that were hyperacetylated in disease shared spatial positioning within the nucleus, predominantly regulating PFC projection neuron function and excitatory neurotransmission. Hypoacetylated domains were linked to inhibitory interneuron- and myelination-relevant genes. Chromosomal modular architecture is affected in SCZ and BD, with hyperacetylated domains showing unexpectedly strong convergences defined by cell type, nuclear topography, genetic risk, and active chromatin state across a wide developmental window.

genomics↗

bioRxiv2021

3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

cell biology↗

bioRxiv2020

Reprogramming of the FOXA1 cistrome in treatment-emergent neuroendocrine prostate cancer

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer1,2. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. To investigate the epigenomic basis of this resistance mechanism, we profiled histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identified a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium3,4, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying novel cancer dependencies through epigenomic profiling.

cancer biology↗

bioRxiv2020

Genomic sequencing confirms absence of introgression despite past hybridisation in a critically endangered bird

Genetic swamping resulting from interspecific hybridisation can increase extinction risk for threatened species. The development of high-throughput and reduced-representation genomic sequencing and analyses to generate large numbers of high resolution genomic markers has the potential to reveal introgression previously undetected using small numbers of genetic markers. However, few studies to date have implemented genomic tools to assess the extent of interspecific hybridisation in threatened species. Here we investigate the utility of genome-wide single nucleotide polymorphisms (SNPs) to detect introgression resulting from past interspecific hybridisation in one of the worlds rarest birds. Anthropogenic impacts have resulted in hybridisation and subsequent backcrossing of the critically endangered Aotearoa New Zealand endemic kak[i] (black stilts; Himantopus novaezelandiae) with the non-threatened self-introduced congeneric poaka (Aotearoa New Zealand population of pied stilts, Himantopus himantopus leucocephalus), yet genetic analyses with a limited set of microsatellite markers revealed no evidence of introgression of poaka genetic material in kak[i], excluding one individual. We use genomic data for [~]63% of the wild adult kak[i] population to reassess the extent of introgression resulting from hybridisation between kak[i] and poaka. Consistent with previous genetic analyses, we detected no introgression from poaka into kak[i]. These collective results indicate that, for kak[i], existing microsatellite markers provide a robust, cost-effective approach to detect cryptic hybrids. Further, for well-differentiated species, the use of genomic markers may not be required to detect admixed individuals.

genomics↗

bioRxiv2020

Subtype Heterogeneity and Epigenetic Convergence in Neuroendocrine Prostate Cancer

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyzed cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single cell analysis of human clinical samples exhibit a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

cancer biology↗

bioRxiv2020

Conductance mechanisms of rapidly desensitizing cation channelrhodopsins from cryptophyte algae

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, BCCR sequences match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs, which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption and Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function. IMPORTANCECation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-vis spectroscopy and photoelectrophysiology reveal the desensitization is caused by rapid accumulation of a non-conductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

biochemistry↗

bioRxiv2019

A metabolic pathway for bile acid dehydroxylation by the gut microbiome

The gut microbiota synthesize hundreds of molecules, many of which are known to impact host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at ~500 M and are known to block C. difficile growth1, promote hepatocellular carcinoma2, and modulate host metabolism via the GPCR TGR53. More broadly, DCA, LCA and their derivatives are a major component of the recirculating bile acid pool4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Despite the clear impact of DCA and LCA on host physiology, incomplete knowledge of their biosynthetic genes and a lack of genetic tools in their native producer limit our ability to modulate secondary bile acid levels in the host. Here, we complete the pathway to DCA/LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the 8-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a non-producing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool, and provide a road map for deorphaning and engineering pathways from the microbiome as a critical step toward controlling the metabolic output of the gut microbiota.

microbiology↗

bioRxiv2019

A comparison of pedigree, genetic, and genomic estimates of relatedness for informing pairing decisions in two critically endangered birds.

Conservation management strategies for many highly threatened species include conservation breeding to prevent extinction and enhance recovery. Pairing decisions for these conservation breeding programmes can be informed by pedigree data to minimise relatedness between individuals in an effort to avoid inbreeding, maximise diversity, and maintain evolutionary potential. However, conservation breeding programmes struggle to use this approach when pedigrees are shallow or incomplete. While genetic data (i.e., microsatellites) can be used to estimate relatedness to inform pairing decisions, emerging evidence indicates this approach lacks precision in genetically depauperate species, and more effective estimates will likely be obtained from genomic data (i.e., thousands of genome-wide single nucleotide polymorphisms, or SNPs). Here, we compare relatedness estimates using pedigree-, genetic-, and genomic-based approaches for making pairing decisions in two critically endangered birds endemic to New Zealand: kak[i]/black stilt (Himantopus novaezelandiae) and k[a]k[a]riki karaka/orange-fronted parakeet (Cyanoramphus malherbi). Our findings indicate genetic-based estimates of relatedness are indeed the least precise when assessing known parent-offspring and full sibling relationships. Furthermore, our results show that relatedness estimates and subsequent pairing recommendations using PMx are most similar between pedigree- and genomic-based approaches. These combined results indicate that in lieu of robust pedigrees, SNPs are an effective tool for informing pairing decisions, which has exciting implications for many poorly pedigreed conservation breeding programmes worldwide.

molecular biology↗

bioRxiv2019

Dynamic changes in innate immune and T cell function and composition at the nasal mucosa across the human lifespan

The very young and very old are at increased risk of serious infections, including pneumonia. This may relate to changes in the immune system as young children have limited immunological memory, while immunosenescence, inflammaging and a decreased pool of naive immune cells are described with advanced age. How the immune system changes with age at mucosal surfaces, from where infections frequently develop, is not very clear as access to human tissue samples is limited. Therefore, we aimed to assess the composition and activation state of the immune system at the human mucosa. Here, we profiled nasal immune cells from 207 individuals between 1 to 80 years old using flow cytometry. Neutrophil and monocyte functionality were measured using whole blood assays. Levels of thirty nasal cytokines were measured from nasal lining fluid. Nasopharyngeal colonization by Streptococcus pneumoniae was assessed using classical microbiology and associated with immune responses. We found that young children have a striking paucity of granulocytes at the nasal mucosa compared to adults. In addition, T cell numbers at the nasal mucosa decreased progressively with age and were almost absent in older adults. While nasopharyngeal colonization by Streptococcus pneumoniae was associated with elevated levels of inflammation it had a limited effect on nasal immune composition, including levels of monocytes and neutrophils. These results show that the immune system at the nasal mucosal surface changes drastically with age and provides explanations for the increased susceptibility to infections in young and old age.\n\nSignificance statementHow the immune system changes with age is an intensive area of research, but has been primarily studied in blood. However, blood poorly reflects the immune system at the mucosa, from where infections develop. This manuscript provides a first characterization of how the composition and function of the immune system in the upper respiratory tract changes with age, providing explanations for increased susceptibility to infection in the very young and old. Furthermore, by linking mucosal and systemic measurements with pneumococcal colonization, we observed that reduced monocyte and neutrophil responses associate with the increased burden of pneumococcal colonization in children. This study highlights the need to study the immune system also at other mucosal sites in the context of aging.

immunology↗