The Black Death may have influenced the evolution of genes involved in immune responses against pathogens

By Neha MathurReviewed by Danielle Ellis, B.Sc.Oct 24 2022

In a recent study published in Nature, researchers gathered empirical evidence that past pandemics due to infectious pathogens shaped the present-day susceptibility of humans to diseases, such as autoimmune diseases.

Background

The pandemic of plague, commonly called the Black Death, was caused by a bacterium species, Yersinia pestis, which wiped off 30 to 50% of the Afro-Eurasian population to become the single biggest mortality event in human history. Such unprecedented mortality rates indicated that allelic variants of immune genes that mounted an immune response against Y. pestis infection might have undergone strong selection during that time. In all subsequent plague pandemics in Europe, mortality rates reduced significantly, likely due to pathogen evolution, changing cultural practices, or humans' genetic adaption to Y. pestis.

With no recent exposure to the plague, Europeans living through the Black Death seem to represent an immunologically naive population with minimal or no adaptation to Y. pestis. Yet, so far, immune targets of Y. pestis imposed selection during the Black Death have remained elusive. Most evidence of an association between autoimmune disease-related risk alleles and adaptation to past infectious diseases remains indirect because the etiological agents driving this selection remain hidden.

About the study

In the present study, researchers dug ancient genomic data of individuals who died a little before, during, or after the Black Death pandemic in London and Denmark to characterize their genetic variations. The unique sampling design of the study helped the researchers differentiate genetic signatures due to Y. pestis from other selective processes, including other infectious diseases, such as tuberculosis. Additionally, it minimized the degree to which other historical events, such as famine, could have affected the inference of positive selection.

The team visited three London cemeteries to collect deoxyribonucleic acid (DNA) samples of individuals who died before, during, and after the Black Death. In London, they also collected samples of all Black Death victims who died between 1348 and 1349 and were buried in East Smithfield. The radiocarbon, stratigraphy, and historical data verified how these people died. Burials in London were more precisely dated and better geographically controlled than those from Denmark. Next, the team sampled Danish people from five localities across the country. They grouped these people into those that lived in pre-Black Death and post-Black Death times. In this way, the researchers ensured that they identified solely European mitogenomic haplotypes.

The researchers screened 516 samples, of which 318 were from London and 198 from Denmark. They detected human DNA using a modified polymerase chain reaction (PCR) assay for the cellular myelocytomatosis oncogene (c-myc) gene. Further, they identified 360 nuclear loci with adequate endogenous DNA for downstream enrichment and sequencing.

The researchers ensured that deamination and other ancient DNA damage did not manifest as fake genotype calls. So they trimmed four base pairs (bp) from the beginning and end of each sequencing read. The final study dataset comprised 33,110 biallelic variants, with an average coverage of 4.6× reads per site per individual. They filtered out missing genotype calls at over 50% of target sites. Next, the team calculated the minor allele frequency (MAF) per population. Finally, they retained only those sites with a mean MAF of greater than 5% on average across London and Denmark.

Furthermore, the researchers searched within immune genes and genome-wide association study (GWAS) loci for allelic variants that exhibited unexpected changes in frequency between pre- and post-Black Death samples to identify gene variants for which the degree of differentiation (F_ST) was more than expected by coincidence.

First, the team identified 245 common variants with MAF greater than 10%. These allelic variants were also highly differentiated, as indicated by their F_ST, which was >95th percentile. The researchers defined F_ST using neutral sites when comparing pre-and post-Black Death samples from London. Next, they reasoned that alleles conferring increased susceptibility to, or protection from Y. pestis, should increase or decrease in frequency among people who died during the Black Death and sampled post-Black Death, respectively. It narrowed the list of allegedly selected gene loci from 245 to 35. Finally, they investigated whether these loci were as highly differentiated and in the same direction as their London counterparts.

Furthermore, the team investigated whether the four candidate loci identified in the study or genes near them were involved in the transcriptional response to Y. pestis. So they incubated macrophages from 33 samples with heat-killed Y. pestis. Next, they used ribonucleic acid (RNA) sequencing to compare the gene expression profiles of those with that of control samples.

Study findings

Functional data from in vitro infection experiments confirmed that the strongest candidates for positive selection were directly involved in the immune response to Y. pestis. The researchers identified four gene loci strongly differentiated before and after the Black Death outbreak in London. So they calculated the selection coefficient (s) for each of these variants using a Hidden Markov Model (HMM) framework. The same was replicated in the Danish cohort as the strongest candidates of selection. Due to the small sample size, replication power was limited. Thus, some of the 245 highly differentiated loci in London were also likely impacted by natural selection, although they did not survive conservative filtering criteria. Future studies dissecting the evolutionary role played by these variants in the immune response to Y. pestis should use increased sample sizes coupled with additional functional data.

Seven genes within 100 kilobases of four candidate loci showed a transcriptional response to Y. pestis in macrophages, except the leucyl and cysteinyl aminopeptidase (LNPEP) gene. In vivo, toll-like receptor 4 (TLR4) detected Y. pestis by recognizing its lipopolysaccharides (LPS) membrane. To avoid this detection, bacterium deacetylating surface LPS, thereby reducing the binding affinity for TLR4. Toll-like receptor adaptor molecule 2 (TICAM2) helped LPS-bound TLR4 to move into endosomes and activate type I interferon (IFN) responses. It is, therefore, possible that increased TICAM2 expression conferred protection against Y. pestis.

The researchers noted that the endoplasmic reticulum aminopeptidase 2 (ERAP2) locus had two haplotypes (A and B). In macrophages, the decreased ERAP2 expression in individuals harboring the deleterious rs2248374 G allele of haplotype B was coupled with a higher expression of the abridged isoform. Subjects with the advantageous haplotype were more efficient at resisting Y. pestis-induced cell death than subjects with the lethal haplotype. For example, levels of IL-1β, a key proinflammatory cytokine associated with pyroptotic cell death, were threefold lower in individuals homozygous for the beneficial ERAP2 genotype when compared to individuals homozygous for the allegedly lethal one. The findings, however, could not be confirmed in vitro experiments.

Conclusions

The current study used ancient genomic data and functional analyses to gather empirical evidence that Y. pestis infection shaped genetic diversity around some immune loci. Four loci that highly differentiated before and after the Y. pestis-induced Black Death emerged as the strongest candidates for selection. ERAP2 showed the most compelling evidence for selection, with a selection coefficient of 0.4.

This finding suggests that individuals homozygous for the beneficial allele were ~40% more likely to survive the Black Death than those homozygous for the lethal allele. Perhaps the ERAP2 protein increased the presentation of Y. pestis-specific antigens to CD8+ T cells, stimulating a protective immune response.

ERAP2 was transcriptionally responsive to several pathogens, supporting its key role in immune response regulation. Therefore, selection imposed by Y. pestis on ERAP2 affected the immune response to other pathogens and disease traits. For example, the selectively beneficial ERAP2 allele is a known risk factor for Crohn’s disease, and its allelic variation has been associated with other infectious diseases.

Likewise, studies have shown the association of another locus, rs11571319, near cytotoxic T-lymphocyte associated protein 4 (CTLA4), with an increased risk of rheumatoid arthritis and systemic lupus erythematosus. Indeed, retaining the allegedly beneficial allele(s) of some immune genes during the Black Death conferred an increased risk for autoimmune disease in present-day populations.