Shaping immunity: The influence of natural selection on population immune diversity.

Humans exhibit considerable variability in their immune responses to the same immune challenges. Such variation is widespread and affects individual and population-level susceptibility to infectious diseases and immune disorders. Although the factors influencing immune response diversity are partially understood, what mechanisms lead to the wide range of immune traits in healthy individuals remain largely unexplained. Here, we discuss the role that natural selection has played in driving phenotypic differences in immune responses across populations and present-day susceptibility to immune-related disorders. Further, we touch on future directions in the field of immunogenomics, highlighting the value of expanding this work to human populations globally, the utility of modeling the immune response as a dynamic process, and the importance of considering the potential polygenic nature of natural selection. Identifying loci acted upon by evolution may further pinpoint variants critically involved in disease etiology, and designing studies to capture these effects will enrich our understanding of the genetic contributions to immunity and immune dysregulation.

EpiVar Browser: advanced exploration of epigenomics data under controlled access.

MOTIVATION: Human epigenomic data has been generated by large consortia for thousands of cell types to be used as a reference map of normal and disease chromatin states. Since epigenetic data contains potentially identifiable information, similarly to genetic data, most raw files generated by these consortia are stored in controlled-access databases. It is important to protect identifiable information, but this should not hinder secure sharing of these valuable datasets. RESULTS: Guided by the Framework for responsible sharing of genomic and health-related data from the Global Alliance for Genomics and Health (GA4GH), we have developed an approach and a tool to facilitate the exploration of epigenomics datasets' aggregate results, while filtering out identifiable information. Specifically, the EpiVar Browser allows a user to navigate an epigenetic dataset from a cohort of individuals and enables direct exploration of genotype-chromatin phenotype relationships. Because individual genotypes and epigenetic signal tracks are not directly accessible, and rather aggregated in the portal output, no identifiable data is released, yet the interface allows for dynamic genotype-epigenome interrogation. This approach has the potential to accelerate analyses that would otherwise require a lengthy multi-step approval process and provides a generalizable strategy to facilitate responsible access to sensitive epigenomics data. AVAILABILITY AND IMPLEMENTATION: Online portal: https://computationalgenomics.ca/tools/epivar; EpiVar Browser source code: https://github.com/c3g/epivar-browser; bw-merge-window tool source code: https://github.com/c3g/bw-merge-window.

Epigenetic variation impacts individual differences in the transcriptional response to influenza infection.

Humans display remarkable interindividual variation in their immune response to identical challenges. Yet, our understanding of the genetic and epigenetic factors contributing to such variation remains limited. Here we performed in-depth genetic, epigenetic and transcriptional profiling on primary macrophages derived from individuals of European and African ancestry before and after infection with influenza A virus. We show that baseline epigenetic profiles are strongly predictive of the transcriptional response to influenza A virus across individuals. Quantitative trait locus (QTL) mapping revealed highly coordinated genetic effects on gene regulation, with many cis-acting genetic variants impacting concomitantly gene expression and multiple epigenetic marks. These data reveal that ancestry-associated differences in the epigenetic landscape can be genetically controlled, even more than gene expression. Lastly, among QTL variants that colocalized with immune-disease loci, only 7% were gene expression QTL, while the remaining genetic variants impact epigenetic marks, stressing the importance of considering molecular phenotypes beyond gene expression in disease-focused studies.

DNA methylation-environment interactions in the human genome.

Previously, we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here, we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures-one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

Microbial transmission in the social microbiome and host health and disease.

Although social interactions are known to drive pathogen transmission, the contributions of socially transmissible host-associated mutualists and commensals to host health and disease remain poorly explored. We use the concept of the social microbiome-the microbial metacommunity of a social network of hosts-to analyze the implications of social microbial transmission for host health and disease. We investigate the contributions of socially transmissible microbes to both eco-evolutionary microbiome community processes (colonization resistance, the evolution of virulence, and reactions to ecological disturbance) and microbial transmission-based processes (transmission of microbes with metabolic and immune effects, inter-specific transmission, transmission of antibiotic-resistant microbes, and transmission of viruses). We consider the implications of social microbial transmission for communicable and non-communicable diseases and evaluate the importance of a socially transmissible component underlying canonically non-communicable diseases. The social transmission of mutualists and commensals may play a significant, under-appreciated role in the social determinants of health and may act as a hidden force in social evolution.

Multi-Omic blood analysis reveals differences in innate inflammatory sensitivity between species.

Vertebrates differ greatly in responses to pro-inflammatory agonists such as bacterial lipopolysaccharide (LPS), complicating use of animal models to study human sepsis or inflammatory disorders. We compared transcriptomes of resting and LPS-exposed blood from six LPS-sensitive species (rabbit, pig, sheep, cow, chimpanzee, human) and four LPS-resilient species (mice, rats, baboon, rhesus), as well as plasma proteomes and lipidomes. Unexpectedly, at baseline, sensitive species already had enhanced expression of LPS-responsive genes relative to resilient species. After LPS stimulation, maximally different genes in resilient species included genes that detoxify LPS, diminish bacterial growth, discriminate sepsis from SIRS, and play roles in autophagy and apoptosis. The findings reveal the molecular landscape of species differences in inflammation, and may inform better selection of species for pre-clinical models.

BCG vaccination impacts the epigenetic landscape of progenitor cells in human bone marrow.

While the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90 days after BCG vaccination or placebo administration. We show that BCG vaccination significantly alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occur primarily on the most uncommitted stem cells and are reflective of a persistent myeloid bias. In contrast, BCG-induced changes in chromatin accessibility are most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF)/SP and EGR transcription factors (TFs). These TFs are also activated in the most uncommitted stem cells, indicating that activated TFs, which drive persistent changes in HSC gene expression, likely also drive chromatin dynamics appearing within downstream progenitor cells. This perspective contests the prevailing notion that epigenetic modifications linked to innate immune memory transfer directly from stem cells to their differentiated derivatives. Finally, we show that alterations in gene expression and chromatin accessibility in HSPCs due to BCG vaccination were highly correlated (r>0.8) with the IL-1ß secretion capacity of paired PBMCs upon secondary immune challenge. Overall, our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses.

Mycobacterium tuberculosis infection drives differential responses in the bone marrow hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) play a vital role in the host response to infection through the rapid and robust production of mature immune cells. These HSPC responses can be influenced, directly and indirectly, by pathogens as well. Infection with Mycobacterium tuberculosis (Mtb) can drive lymphopoiesis through modulation of type I interferon (IFN) signaling. We have previously found that the presence of a drug resistance (DR)-conferring mutation in Mtb drives altered host-pathogen interactions and heightened type I IFN production in vitro. But the impacts of this DR mutation on in vivo host responses to Mtb infection, particularly the hematopoietic compartment, remain unexplored. Using a mouse model, we show that, while drug-sensitive Mtb infection induces expansion of HSPC subsets and a skew toward lymphopoiesis, DR Mtb infection fails to induce an expansion of these subsets and an accumulation of mature granulocytes in the bone marrow. Using single-cell RNA sequencing, we show that the HSCs from DR Mtb-infected mice fail to upregulate pathways related to cytokine signaling across all profiled HSC subsets. Collectively, our studies report a novel finding of a chronic infection that fails to induce a potent hematopoietic response that can be further investigated to understand pathogen-host interaction at the level of hematopoiesis.

EpiVar Browser: advanced exploration of epigenomics data under controlled access.

Motivation: Human epigenomic data has been generated by large consortia for thousands of cell types to be used as a reference map of normal and disease chromatin states. Since epigenetic data contains potentially identifiable information, similarly to genetic data, most raw files generated by these consortia are stored in controlled-access databases. It is important to protect identifiable information, but this should not hinder secure sharing of these valuable datasets. Results: Guided by the Framework for responsible sharing of genomic and health-related data from the Global Alliance for Genomics and Health (GA4GH), we have developed a tool to facilitate the exploration of epigenomics datasets' aggregate results, while filtering out identifiable information. Specifically, the EpiVar Browser allows a user to navigate an epigenetic dataset from a cohort of individuals and enables direct exploration of genotype-chromatin phenotype relationships. Because the information about individual genotypes is not accessible and aggregated in the output that is made available, no identifiable data is released, yet the interface allows for dynamic genotype - epigenome interrogation. This approach has the potential to accelerate analyses that would otherwise require a lengthy multi-step approval process and provides a generalisable strategy to facilitate responsible access to sensitive epigenomics data. Availability and implementation: Online portal instance: https://computationalgenomics.ca/tools/epivarSource code: https://github.com/c3g/epivar-browser.

DNA methylation-environment interactions in the human genome.

Previously we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures-one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

Antigen-driven colonic inflammation is associated with development of dysplasia in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an immune-mediated disease of the bile ducts that co-occurs with inflammatory bowel disease (IBD) in almost 90% of cases. Colorectal cancer is a major complication of patients with PSC and IBD, and these patients are at a much greater risk compared to patients with IBD without concomitant PSC. Combining flow cytometry, bulk and single-cell transcriptomics, and T and B cell receptor repertoire analysis of right colon tissue from 65 patients with PSC, 108 patients with IBD and 48 healthy individuals we identified a unique adaptive inflammatory transcriptional signature associated with greater risk and shorter time to dysplasia in patients with PSC. This inflammatory signature is characterized by antigen-driven interleukin-17A (IL-17A)+ forkhead box P3 (FOXP3)+ CD4 T cells that express a pathogenic IL-17 signature, as well as an expansion of IgG-secreting plasma cells. These results suggest that the mechanisms that drive the emergence of dysplasia in PSC and IBD are distinct and provide molecular insights that could guide prevention of colorectal cancer in individuals with PSC.

Universal gut microbial relationships in the gut microbiome of wild baboons.

Ecological relationships between bacteria mediate the services that gut microbiomes provide to their hosts. Knowing the overall direction and strength of these relationships is essential to learn how ecology scales up to affect microbiome assembly, dynamics, and host health. However, whether bacterial relationships are generalizable across hosts or personalized to individual hosts is debated. Here, we apply a robust, multinomial logistic-normal modeling framework to extensive time series data (5534 samples from 56 baboon hosts over 13 years) to infer thousands of correlations in bacterial abundance in individual baboons and test the degree to which bacterial abundance correlations are 'universal'. We also compare these patterns to two human data sets. We find that, most bacterial correlations are weak, negative, and universal across hosts, such that shared correlation patterns dominate over host-specific correlations by almost twofold. Further, taxon pairs that had inconsistent correlation signs (either positive or negative) in different hosts always had weak correlations within hosts. From the host perspective, host pairs with the most similar bacterial correlation patterns also had similar microbiome taxonomic compositions and tended to be genetic relatives. Compared to humans, universality in baboons was similar to that in human infants, and stronger than one data set from human adults. Bacterial families that showed universal correlations in human infants were often universal in baboons. Together, our work contributes new tools for analyzing the universality of bacterial associations across hosts, with implications for microbiome personalization, community assembly, and stability, and for designing microbiome interventions to improve host health.

Communities of bacteria living in the guts of humans and other animals perform essential services for their hosts such as digesting food, degrading toxins, or fighting viruses and other bacteria that cause disease. These services emerge from so-called 'ecological' relationships between different species of bacteria. One species, for example, may break down a molecule in human food into another compound that is, in turn, digested by another species into a small molecule that the human gut can absorb and use. The bacteria involved in such a process may become more or less common together in their host. In other situations, some bacteria may have opposing roles to each other, meaning that if one species becomes more abundant it may reduce the level of the other. However, it is not known if relationships between different bacteria are consistent across hosts (i.e., universal) or unique to each host (personalized). In other words, if a pair of bacteria increase and decrease in abundance together in one host, do they do the same in other hosts? Microbes often swap genes with each other to gain new traits; as each host harbors a distinctive set of gut microbes, it may be possible for microbial relationships to change depending on the bacterial species present in a specific environment. To investigate, Roche et al. studied the bacteria in thousands of samples of feces collected from 56 baboons over a 13-year period. These samples came from a long-term research project in Amboseli, Kenya which has been studying a population of wild baboons continuously since 1971. Roche et al. measured the abundance of hundreds of gut bacteria in the feces to understand the relationships between pairs. This revealed that connections between species were largely universal rather than personalized to each baboon. Furthermore, the pairs of bacteria with the strongest positive or negative associations had the most consistent relationships across the baboons. Microbial relationships that have strong effects on the microbiome's composition might therefore be especially universal. Further analyses measuring gut bacteria in human babies also found that relationships between pairs of bacteria were largely universal. Hence, individual species of bacteria may fill similar ecological roles in each host across humans and other primates, and perhaps also in other mammals. These findings suggest that it may be possible to leverage the ecological relationships between bacteria to develop universal therapies for human diseases associated with gut bacteria, such as inflammatory bowel disease or Clostridium difficile infection.

Genome graphs detect human polymorphisms in active epigenomic state during influenza infection.

Genetic variants, including mobile element insertions (MEIs), are known to impact the epigenome. We hypothesized that genome graphs, which encapsulate genetic diversity, could reveal missing epigenomic signals. To test this, we sequenced the epigenome of monocyte-derived macrophages from 35 ancestrally diverse individuals before and after influenza infection, allowing us to investigate the role of MEIs in immunity. We characterized genetic variants and MEIs using linked reads and built a genome graph. Mapping epigenetic data revealed 2.3%-3% novel peaks for H3K4me1, H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq), and ATAC-seq. Additionally, the use of a genome graph modified some quantitative trait loci estimates and revealed 375 polymorphic MEIs in an active epigenomic state. Among these is an AluYh3 polymorphism whose chromatin state changed after infection and was associated with the expression of TRIM25, a gene that restricts influenza RNA synthesis. Our results demonstrate that graph genomes can reveal regulatory regions that would have been overlooked by other approaches.

Transposable elements are associated with the variable response to influenza infection.

Influenza A virus (IAV) infections are frequent every year and result in a range of disease severity. Here, we wanted to explore the potential contribution of transposable elements (TEs) to the variable human immune response. Transcriptome profiling in monocyte-derived macrophages from 39 individuals following IAV infection revealed significant inter-individual variation in viral load post-infection. Using transposase-accessible chromatin using sequencing (ATAC-seq), we identified a set of TE families with either enhanced or reduced accessibility upon infection. Of the enhanced families, 15 showed high variability between individuals and had distinct epigenetic profiles. Motif analysis showed an association with known immune regulators (e.g., BATFs, FOSs/JUNs, IRFs, STATs, NFkBs, NFYs, and RELs) in stably enriched families and with other factors in variable families, including KRAB-ZNFs. We showed that TEs and host factors regulating TEs were predictive of viral load post-infection. Our findings shed light on the role TEs and KRAB-ZNFs may play in inter-individual variation in immunity.

Reply to Barton et al: signatures of natural selection during the Black Death.

Barton et al.1 raise several statistical concerns regarding our original analyses2 that highlight the challenge of inferring natural selection using ancient genomic data. We show here that these concerns have limited impact on our original conclusions. Specifically, we recover the same signature of enrichment for high FST values at the immune loci relative to putatively neutral sites after switching the allele frequency estimation method to a maximum likelihood approach, filtering to only consider known human variants, and down-sampling our data to the same mean coverage across sites. Furthermore, using permutations, we show that the rs2549794 variant near ERAP2 continues to emerge as the strongest candidate for selection (p = 1.2×10-5), falling below the Bonferroni-corrected significance threshold recommended by Barton et al. Importantly, the evidence for selection on ERAP2 is further supported by functional data demonstrating the impact of the ERAP2 genotype on the immune response to Y. pestis and by epidemiological data from an independent group showing that the putatively selected allele during the Black Death protects against severe respiratory infection in contemporary populations.

Transmission of stimulus-induced epigenetic changes through cell division is coupled to continuous transcription factor activity.

Trained immunity, or innate immune memory, has been attributed to the long-term retention of stimulus-induced histone post-translational modifications (PTMs) following clearance of the initial stimulus. Yet, it remains unknown how this epigenetic memory can persist for months in dividing cells given the lack of any known mechanism for stimulus-induced histone PTMs to be directly copied from parent to daughter strand during DNA replication. Here, using time course RNA-seq, ChIP-seq, and infection assays, we find that trained macrophages are transcriptionally, epigenetically, and functionally re-programmed for at least 14 cell divisions after stimulus washout. However, the epigenetic changes observed after multiple rounds of cell division do not result from the self-sustained propagation of stimulus-induced epigenetic changes through cell division. Instead, long-lasting epigenetic differences between trained and non-trained cells are always coupled with changes in transcription factor (TF) activity, emphasizing the central role played by TFs, and gene expression changes more broadly, in driving the transmission of stimulus-induced epigenetic changes across cell divisions.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology.

There is growing recognition that regionalization of bacterial colonization and immunity along the intestinal tract has an important role in health and disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. This study found that regional epithelial expression of the transcription factor GATA4 controls bacterial colonization and inflammatory tissue immunity in the proximal small intestine by regulating retinol metabolism and luminal IgA. Furthermore, in mice without jejunal GATA4 expression, the commensal segmented filamentous bacteria promoted pathogenic inflammatory immune responses that disrupted barrier function and increased mortality upon Citrobacter rodentium infection. In celiac disease patients, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. Taken together, these results reveal broad impacts of GATA4-regulated intestinal regionalization on bacterial colonization and tissue immunity, highlighting an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease.

Aberrant T-cell exhaustion in severe combined immunodeficiency survivors with poor T-cell reconstitution after transplantation.

BACKGROUND: Severe combined immunodeficiency (SCID) comprises rare inherited disorders of immunity that require definitive treatment through hematopoietic cell transplantation (HCT) or gene therapy for survival. Despite successes of allogeneic HCT, many SCID patients experience incomplete immune reconstitution, persistent T-cell lymphopenia, and poor long-term outcomes. OBJECTIVE: We hypothesized that CD4+ T-cell lymphopenia could be associated with a state of T-cell exhaustion in previously transplanted SCID patients. METHODS: We analyzed markers of exhaustion in blood samples from 61 SCID patients at a median of 10.4 years after HCT. RESULTS: Compared to post-HCT SCID patients with normal CD4+ T-cell counts, those with poor T-cell reconstitution showed lower frequency of naive CD45RA+/CCR7+ T cells, recent thymic emigrants, and TCR excision circles. They also had a restricted TCR repertoire, increased expression of inhibitory receptors (PD-1, 2B4, CD160, BTLA, CTLA-4), and increased activation markers (HLA-DR, perforin) on their total and naive CD8+ T cells, suggesting T-cell exhaustion and aberrant activation, respectively. The exhaustion score of CD8+ T cells was inversely correlated with CD4+ T-cell count, recent thymic emigrants, TCR excision circles, and TCR diversity. Exhaustion scores were higher among recipients of unconditioned HCT, especially when further in time from HCT. Patients with fewer CD4+ T cells showed a transcriptional signature of exhaustion. CONCLUSIONS: Recipients of unconditioned HCT for SCID may develop late post-HCT T-cell exhaustion as a result of diminished production of T-lineage cells. Elevated expression of inhibitory receptors on their T cells may be a biomarker of poor long-term T-cell reconstitution.