Smoking changes adaptive immunity with persistent effects.

Individuals differ widely in their immune responses, with age, sex and genetic factors having major roles in this inherent variability1-6. However, the variables that drive such differences in cytokine secretion-a crucial component of the host response to immune challenges-remain poorly defined. Here we investigated 136 variables and identified smoking, cytomegalovirus latent infection and body mass index as major contributors to variability in cytokine response, with effects of comparable magnitudes with age, sex and genetics. We find that smoking influences both innate and adaptive immune responses. Notably, its effect on innate responses is quickly lost after smoking cessation and is specifically associated with plasma levels of CEACAM6, whereas its effect on adaptive responses persists long after individuals quit smoking and is associated with epigenetic memory. This is supported by the association of the past smoking effect on cytokine responses with DNA methylation at specific signal trans-activators and regulators of metabolism. Our findings identify three novel variables associated with cytokine secretion variability and reveal roles for smoking in the short- and long-term regulation of immune responses. These results have potential clinical implications for the risk of developing infections, cancers or autoimmune diseases.

Immune-response 3'UTR alternative polyadenylation quantitative trait loci contribute to variation in human complex traits and diseases.

Genome-wide association studies (GWASs) have identified thousands of non-coding variants that are associated with human complex traits and diseases. The analysis of such GWAS variants in different contexts and physiological states is essential for deciphering the regulatory mechanisms underlying human disease. Alternative polyadenylation (APA) is a key post-transcriptional modification for most human genes that substantially impacts upon cell behavior. Here, we mapped 9,493 3'-untranslated region APA quantitative trait loci in 18 human immune baseline cell types and 8 stimulation conditions (immune 3'aQTLs). Through the comparison between baseline and stimulation data, we observed the high responsiveness of 3'aQTLs to immune stimulation (response 3'aQTLs). Co-localization and mendelian randomization analyses of immune 3'aQTLs identified 678 genes where 3'aQTL are associated with variation in complex traits, 27.3% of which were derived from response 3'aQTLs. Overall, these analyses reveal the role of immune 3'aQTLs in the determination of complex traits, providing new insights into the regulatory mechanisms underlying disease etiologies.

Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.

The immune factors driving DNA methylation variation in human blood.

Epigenetic changes are required for normal development, yet the nature and respective contribution of factors that drive epigenetic variation in humans remain to be fully characterized. Here, we assessed how the blood DNA methylome of 884 adults is affected by DNA sequence variation, age, sex and 139 factors relating to life habits and immunity. Furthermore, we investigated whether these effects are mediated or not by changes in cellular composition, measured by deep immunophenotyping. We show that DNA methylation differs substantially between naïve and memory T cells, supporting the need for adjustment on these cell-types. By doing so, we find that latent cytomegalovirus infection drives DNA methylation variation and provide further support that the increased dispersion of DNA methylation with aging is due to epigenetic drift. Finally, our results indicate that cellular composition and DNA sequence variation are the strongest predictors of DNA methylation, highlighting critical factors for medical epigenomics studies.

Early IFNß secretion determines variable downstream IL-12p70 responses upon TLR4 activation.

The interleukin-12 (IL-12) family comprises the only heterodimeric cytokines mediating diverse functional effects. We previously reported a striking bimodal IL-12p70 response to lipopolysaccharide (LPS) stimulation in healthy donors. Herein, we demonstrate that interferon ß (IFNß) is a major upstream determinant of IL-12p70 production, which is also associated with numbers and activation of circulating monocytes. Integrative modeling of proteomic, genetic, epigenomic, and cellular data confirms IFNß as key for LPS-induced IL-12p70 and allowed us to compare the relative effects of each of these parameters on variable cytokine responses. Clinical relevance of our findings is supported by reduced IFNß-IL-12p70 responses in patients hospitalized with acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or chronically infected with hepatitis C (HCV). Importantly, these responses are resolved after viral clearance. Our systems immunology approach defines a better understanding of IL-12p70 and IFNß in healthy and infected persons, providing insights into how common genetic and epigenetic variation may impact immune responses to bacterial infection.

Integrative genetic and immune cell analysis of plasma proteins in healthy donors identifies novel associations involving primary immune deficiency genes.

BACKGROUND: Blood plasma proteins play an important role in immune defense against pathogens, including cytokine signaling, the complement system, and the acute-phase response. Recent large-scale studies have reported genetic (i.e., protein quantitative trait loci, pQTLs) and non-genetic factors, such as age and sex, as major determinants to inter-individual variability in immune response variation. However, the contribution of blood-cell composition to plasma protein heterogeneity has not been fully characterized and may act as a mediating factor in association studies. METHODS: Here, we evaluated plasma protein levels from 400 unrelated healthy individuals of western European ancestry, who were stratified by sex and two decades of life (20-29 and 60-69 years), from the Milieu Intérieur cohort. We quantified 229 proteins by Luminex in a clinically certified laboratory and their levels of variation were analyzed together with 5.2 million single-nucleotide polymorphisms. With respect to non-genetic variables, we included 254 lifestyle and biochemical factors, as well as counts of seven circulating immune cell populations measured by hemogram and standardized flow cytometry. RESULTS: Collectively, we found 152 significant associations involving 49 proteins and 20 non-genetic variables. Consistent with previous studies, age and sex showed a global, pervasive impact on plasma protein heterogeneity, while body mass index and other health status variables were among the non-genetic factors with the highest number of associations. After controlling for these covariates, we identified 100 and 12 pQTLs acting in cis and trans, respectively, collectively associated with 87 plasma proteins and including 19 novel genetic associations. Genetic factors explained the largest fraction of the variability of plasma protein levels, as compared to non-genetic factors. In addition, blood-cell fractions, including leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, basophils, and platelets, had a larger contribution to inter-individual variability than age and sex and appeared as confounders of specific genetic associations. Finally, we identified new genetic associations with plasma protein levels of five monogenic Mendelian disease genes including two primary immunodeficiency genes (Ficolin-3 and FAS). CONCLUSIONS: Our study identified novel genetic and non-genetic factors associated to plasma protein levels which may inform health status and disease management.

Single-Cell and Bulk RNA-Sequencing Reveal Differences in Monocyte Susceptibility to Influenza A Virus Infection Between Africans and Europeans.

There is considerable inter-individual and inter-population variability in response to viruses. The potential of monocytes to elicit type-I interferon responses has attracted attention to their role in viral infections. Here, we use single-cell RNA-sequencing to characterize the role of cellular heterogeneity in human variation of monocyte responses to influenza A virus (IAV) exposure. We show widespread inter-individual variability in the percentage of IAV-infected monocytes. Notably, individuals with high cellular susceptibility to IAV are characterized by a lower activation at basal state of an IRF/STAT-induced transcriptional network, which includes antiviral genes such as IFITM3, MX1 and OAS3. Upon IAV challenge, we find that cells escaping viral infection display increased mRNA expression of type-I interferon stimulated genes and decreased expression of ribosomal genes, relative to both infected cells and those never exposed to IAV. We also uncover a stronger resistance of CD16+ monocytes to IAV infection, together with CD16+ -specific mRNA expression of IL6 and TNF in response to IAV. Finally, using flow cytometry and bulk RNA-sequencing across 200 individuals of African and European ancestry, we observe a higher number of CD16+ monocytes and lower susceptibility to IAV infection among monocytes from individuals of African-descent. Based on these data, we hypothesize that higher basal monocyte activation, driven by environmental factors and/or weak-effect genetic variants, underlies the lower cellular susceptibility to IAV infection of individuals of African ancestry relative to those of European ancestry. Further studies are now required to investigate how such cellular differences in IAV susceptibility translate into population differences in clinical outcomes and susceptibility to severe influenza.

Reconstructing 50,000 years of human history from our DNA: lessons from modern genomics.

The advent of high throughput sequencing approaches and ancient DNA techniques have enabled reconstructing the history of human populations at an unprecedented level of resolution. The symposium from the French Academy of Sciences "50,000 ans d'épopée humaine dans notre ADN" has reviewed some of the latest contributions from the fields of genomics, archaeology, and linguistics to our understanding of >300,000 years of human history. DNA has revealed the richness of the human journey, from the deep divergences between human populations in Africa, to the first encounters of Homo Sapiens with other hominins on their way to Eurasia and the peopling of Remote Oceania. The symposium has also emphasized how migrations, cultural practices, and environmental pathogens have contributed to shape the genetic diversity of modern humans, through admixture, genetic drift or genetic adaptation. Finally, special attention was also given to how human behaviours have shaped the genome of other species, through the spreading of microbes and pathogens, as in the case of Yersinia Pestis, or through domestication, as elegantly demonstrated for dogs, horses, and apples. Altogether, this conference illustrated how the complex history of human populations is tightly linked with their contemporary genetic diversity that, in turn, has direct effects on their identity and health.

L'avènement des approches de séquençage à haut débit et des techniques de séquençage de l'ADN ancien ont permis de reconstruire l'histoire des populations humaines à un niveau de résolution sans précédent. Le symposium de l'Académie des sciences « 50 000 ans d'épopée humaine dans notre ADN ¼ a passé en revue certaines des dernières contributions de la génomique, de l'archéologie et de la linguistique à notre compréhension de plus de 300 000 ans d'histoire humaine. L'ADN a révélé la richesse du parcours humain, depuis les premières séparations entre populations humaines en Afrique jusqu'à la rencontre d'Homo sapiens avec d'autres hominidés sur leur chemin vers l'Eurasie et le peuplement de l'Océanie lointaine. Le symposium a également mis l'accent sur la façon dont les migrations, les pratiques culturelles et les agents pathogènes environnementaux ont contribué à façonner la diversité génétique des humains modernes, par le biais du brassage, de la dérive ou de l'adaptation génétique. Enfin, une attention particulière a également été accordée à la manière dont les comportements humains ont façonné le génome d'autres espèces, par la propagation de microbes et d'agents pathogènes, comme dans le cas de Yersinia pestis, ou par la domestication, comme cela a été démontré de manière élégante pour les chiens, les chevaux et les pommes. Dans l'ensemble, cette conférence a illustré comment la diversité génétique contemporaine des populations humaines est étroitement liée à leur histoire complexe et, à son tour, a des effets directs sur leur identité et leur santé.

A genetic variant controls interferon-ß gene expression in human myeloid cells by preventing C/EBP-ß binding on a conserved enhancer.

Interferon ß (IFN-ß) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-ß binding motif, prevents binding of C/EBP-ß, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-ß expression in myeloid cells.

Population variation in miRNAs and isomiRs and their impact on human immunity to infection.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of the immune system, yet their variation and contribution to intra- and inter-population differences in immune responses is poorly characterized. RESULTS: We generate 977 miRNA-sequencing profiles from primary monocytes from individuals of African and European ancestry following activation of three TLR pathways (TLR4, TLR1/2, and TLR7/8) or infection with influenza A virus. We find that immune activation leads to important modifications in the miRNA and isomiR repertoire, particularly in response to viral challenges. These changes are much weaker than those observed for protein-coding genes, suggesting stronger selective constraints on the miRNA response to stimulation. This is supported by the limited genetic control of miRNA expression variability (miR-QTLs) and the lower occurrence of gene-environment interactions, in stark contrast with eQTLs that are largely context-dependent. We also detect marked differences in miRNA expression between populations, which are mostly driven by non-genetic factors. On average, miR-QTLs explain approximately 60% of population differences in expression of their cognate miRNAs and, in some cases, evolve adaptively, as shown in Europeans for a miRNA-rich cluster on chromosome 14. Finally, integrating miRNA and mRNA data from the same individuals, we provide evidence that the canonical model of miRNA-driven transcript degradation has a minor impact on miRNA-mRNA correlations, which are, in our setting, mainly driven by co-transcription. CONCLUSION: Together, our results shed new light onto the factors driving miRNA and isomiR diversity at the population level and constitute a useful resource for evaluating their role in host differences of immunity to infection.

A trans-eQTL network regulates osteoclast multinucleation and bone mass.

Functional characterisation of cell-type-specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans-regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (Pik3cb-/-, Atp8b2+/-, Igsf8-/-, Eml1-/-, Appl2-/-, Deptor-/-) and myeloid-specific Slc40a1 knockout mice displayed abnormal bone phenotypes. We report opposing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast multinucleation and bone mass.

Two common disease-associated TYK2 variants impact exon splicing and TYK2 dosage.

TYK2 belongs to the JAK protein tyrosine kinase family and mediates signaling of numerous antiviral and immunoregulatory cytokines (type I and type III IFNs, IL-10, IL-12, IL-22, IL-23) in immune and non-immune cells. After many years of genetic association studies, TYK2 is recognized as a susceptibility gene for some inflammatory and autoimmune diseases (AID). Seven TYK2 variants have been associated with AIDs in Europeans, and establishing their causality remains challenging. Previous work showed that a protective variant (P1104A) is hypomorphic and also a risk allele for mycobacterial infection. Here, we have studied two AID-associated common TYK2 variants: rs12720270 located in intron 7 and rs2304256, a non-synonymous variant in exon 8 that causes a valine to phenylalanine substitution (c.1084 G > T, Val362Phe). We found that this amino acid substitution does not alter TYK2 expression, catalytic activity or ability to relay signaling in EBV-B cell lines or in reconstituted TYK2-null cells. Based on in silico predictions that these variants may impact splicing of exon 8, we: i) analyzed TYK2 transcripts in genotyped EBV-B cells and in CRISPR/Cas9-edited cells, ii) measured splicing using minigene assays, and iii) performed eQTL (expression quantitative trait locus) analysis of TYK2 transcripts in primary monocytes and whole blood cells. Our results reveal that the two variants promote the inclusion of exon 8, which, we demonstrate, is essential for TYK2 binding to cognate receptors. In addition and in line with GTEx (Genetic Tissue Expression) data, our eQTL results show that rs2304256 mildly enhances TYK2 expression in whole blood. In all, these findings suggest that these TYK2 variants are not neutral but instead have a potential impact in AID.

Author Correction: WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFß1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFß1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Impact and Evolutionary Determinants of Neanderthal Introgression on Transcriptional and Post-Transcriptional Regulation.

Archaic admixture is increasingly recognized as an important source of diversity in modern humans, and Neanderthal haplotypes cover 1%-3% of the genome of present-day Eurasians. Recent work has shown that archaic introgression has contributed to human phenotypic diversity, mostly through the regulation of gene expression. Yet the mechanisms through which archaic variants alter gene expression and the forces driving the introgression landscape at regulatory regions remain elusive. Here, we explored the impact of archaic introgression on transcriptional and post-transcriptional regulation. We focused on promoters and enhancers across 127 different tissues as well as on microRNA (miRNA)-mediated regulation. Although miRNAs themselves harbor few archaic variants, we found that some of these variants may have a strong impact on miRNA-mediated gene regulation. Enhancers were by far the regulatory elements most affected by archaic introgression: up to one-third of the tissues we tested presented significant enrichments. Specifically, we found strong enrichments of archaic variants in adipose-related tissues and primary T cells, even after accounting for various genomic and evolutionary confounders such as recombination rate and background selection. Interestingly, we identified signatures of adaptive introgression at enhancers of some key regulators of adipogenesis, raising the interesting hypothesis of a possible adaptation of early Eurasians to colder climates. Collectively, this study sheds new light on the mechanisms through which archaic admixture has impacted gene regulation in Eurasians and, more generally, increases our understanding of the contribution of Neanderthals to the regulation of acquired immunity and adipose homeostasis in modern humans.

Characterising the genetic basis of immune response variation to identify causal mechanisms underlying disease susceptibility.

Over the last 10 years, genome-wide association studies (GWAS) have identified hundreds of susceptibility loci for autoimmune diseases. However, despite increasing power for the detection of both common and rare coding variants affecting disease susceptibility, a large fraction of disease heritability has remained unexplained. In addition, a majority of the identified loci are located in noncoding regions, and translation of disease-associated loci into new biological insights on the etiology of immune disorders has been lagging. This highlights the need for a better understanding of noncoding variation and new strategies to identify causal genes at disease loci. In this review, I will first detail the molecular basis of gene expression and review the various mechanisms that contribute to alter gene activity at the transcriptional and post-transcriptional level. I will then review the findings from 10 years of functional genomics studies regarding the genetics on gene expression, in particular in the context of infection. Finally, I will discuss the extent to which genetic variants that modulate gene expression at transcriptional and post-transcriptional level contribute to disease susceptibility and present strategies to leverage this information for the identification of causal mechanisms at disease loci in the era of whole genome sequencing.