In southeastern Iberia, one of the most outstanding archaeological entities of the European Bronze Age emerged around 2,200 BCE. This so-called ‘El Argar’ culture, one of the first state-level societies on the European continent, was characterised by large, central hilltop settlements, distinct pottery, specialized weapons and bronze, silver and gold artefacts, alongside an intra-murial burial rite. A new study explores the relation between dynamic shifts at population scale and the major social and political changes of the third and second millennia BCE by analysing the genomes of 136 ancient Iberians, ranging from 3,000 to 1,500 BCE.

Genetic turnover and melting pot

Including published genomes from Iberia, the new study encompasses data from nearly 300 ancient individuals and focuses specifically on the Copper to Bronze Age transition around 2,200 BCE.

“While we knew that the so-called ‘steppe’-related ancestry, which had spread across Europe during the third millennium BCE, eventually reached the northern Iberian Peninsula around 2,400 BCE, we were surprised to see that all prehistoric individuals from the El Argar period carried a portion of this ancestry, while the Chalcolithic individuals did not,” says Max Planck researcher Wolfgang Haak, senior author and principal investigator of the study.

The genomic data reveals some of the processes underlying this genetic shift. While the bulk of the genome shows that Bronze Age individuals are a mix of local Iberian Chalcolithic ancestry and a smaller part of incoming ancestry from the European mainland, the paternally inherited Y chromosome lineages show a complete turnover, linked to the movement of steppe-related ancestry that is also visible in other parts of Europe.

The rich new data from the El Argar sites also show that these two components do not fully account for the genetic make-up of the early Bronze Age societies.

“We also found signals of ancestry that we traced to the central and eastern Mediterranean and western Asia. We cannot say exactly whether these influences arrived at the same time as the steppe-related ancestry, but it shows that it formed an integrative part of the rising El Argar societies, attesting to continued contacts to these regions,” adds Vanessa Villalba-Mouco, postdoctoral researcher at the Max Planck Institute and Instituto de Biología Evolutiva.

Social implications

“Whether the genetic shift was brought about by migrating groups from North and Central Iberia or by climatic deteriorations that affected the eastern Mediterranean around 2,200 BCE is the million-dollar question,” says co-PI and senior author Prof Roberto Risch from the Universitat Autònoma de Barcelona. “It would be foolish to think that it can all be explained by a simple, one-factor model. While the temporal coincidence is striking, it is likely that many factors played a role.”

One of these factors could be pandemics, such as an early form of the Plague, which has been attested to in other regions of Europe around the time. While not found directly among the tested individuals in southern Iberia, it could be a cause or driver for the movement or disappearance of other groups in the region.

“In any case, we can now conclude that the population movement starting in the eastern European steppe zones around 3,000 BCE was not a single migratory event, but required over four centuries to reach the Iberian Peninsula and another 200 years to appear in present-day Murcia and Alicante,” adds Risch.

The archaeological record of the El Argar group shows a clear break with previous Chalcolithic traditions. Burial rites, for example, changed from communal to single and double burials within the building complexes. Elite burials also indicate the formation of strong social hierarchies. Testing for biological relatedness, the researchers found that males are on average more closely related to other people at the site, indicating that the group was likely patrilineally structured. Such a social organization could explain the stark reduction of the Y-lineage diversity.

“We observe similar patterns of social organisation and increasing stratification also in other parts of Early Bronze Age Europe, in fact broadly around the same time and with similar characteristics of early state-like formations. This suggests a structured restart or resetting following some form of crisis or unstable, highly dynamic times,” summarises Haak.

“Our results represent a significant advance in the new era in structural biology in which computation plays a fundamental role,” said Qian Cong, Ph.D., Assistant Professor in the Eugene McDermott Center for Human Growth and Development with a secondary appointment in Biophysics.

Dr. Cong led the study with David Baker, Ph.D., Professor of Biochemistry and Dr. Cong’s postdoctoral mentor at the University of Washington prior to her recruitment to UT Southwestern. The study has four co-lead authors, including UT Southwestern Computational Biologist Jimin Pei, Ph.D.

Proteins often operate in pairs or groups known as complexes to accomplish every task needed to keep an organism alive, Dr. Cong explained. While some of these interactions are well studied, many remain a mystery. Constructing comprehensive interactomes — or descriptions of the complete set of molecular interactions in a cell — would shed light on many fundamental aspects of biology and give researchers a new starting point on developing drugs that encourage or discourage these interactions. Dr. Cong works in the emerging field of interactomics, which combines bioinformatics and biology.

Until recently, a major barrier for constructing an interactome was uncertainty over the structures of many proteins, a problem scientists have been trying to solve for half a century. In 2020 and 2021, a company called DeepMind and Dr. Baker’s lab independently released two AI technologies called AlphaFold (AF) and RoseTTAFold (RF) that use different strategies to predict protein structures based on the sequences of the genes that produce them.

In the current study, Dr. Cong, Dr. Baker, and their colleagues expanded on those AI structure-prediction tools by modeling many yeast protein complexes. Yeast is a common model organism for fundamental biological studies. To find proteins that were likely to interact, the scientists first searched the genomes of related fungi for genes that acquired mutations in a linked fashion. They then used the two AI technologies to determine whether these proteins could be fit together in 3D structures.

Their work identified 1,505 probable protein complexes. Of these, 699 had already been structurally characterized, verifying the utility of their method. However, there was only limited experimental data supporting 700 of the predicted interactions, and another 106 had never been described.

To better understand these poorly characterized or unknown complexes, the University of Washington and UT Southwestern teams worked with colleagues around the world who were already studying these or similar proteins. By combining the 3D models the scientists in the current study had generated with information from collaborators, the teams were able to gain new insights into protein complexes involved in maintenance and processing of genetic information, cellular construction and transport systems, metabolism, DNA repair, and other areas. They also identified roles for proteins whose functions were previously unknown based on their newly identified interactions with other well-characterized proteins.

“The work described in our new paper sets the stage for similar studies of the human interactome and could eventually help in developing new treatments for human disease,” Dr. Cong added.

Dr. Cong noted that the predicted protein complex structures generated in this study are available to download from ModelArchive (https://modelarchive.org/doi/10.5452/ma-bak-cepc). These structures and others generated using this technology in future studies will be a rich source of research questions for years to come, she said.

Dr. Cong is a Southwestern Medical Foundation Scholar in Biomedical Research. Other UTSW researchers who contributed to this study include Jing Zhang and Josep Rizo, Ph.D., who holds the Virginia Lazenby O’Hara Chair in Biochemistry.

Collaborating institutions include: Harvard University, Wayne State University, Cornell University, MRC Laboratory of Molecular Biology, Memorial Sloan Kettering Cancer Center, Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Fred Hutchinson Cancer Research Center, Columbia University, University of Würzburg in Germany, St Jude Children’s Research Hospital, FIRC Institute of Molecular Oncology in Milan, Italy, and the National Research Council, Institute of Molecular Genetics in Rome, Italy.

This work was supported by Southwestern Medical Foundation, the Cancer Prevention and Research Institute of Texas (CPRIT) (RP210041), Amgen, Microsoft, the Washington Research Foundation, Howard Hughes Medical Institute, National Science Foundation (DBI 1937533), National Institutes of Health (R35GM118026, R01CA221858, R35GM136258, R21AI156595), UK Medical Research Council (MRC_UP_1201/10), HHMI Gilliam Fellowship, the Deutsche Forschungsgemeinschaft (KI-562/11-1, KI-562/7-1), AIRC investigator and the European Research Council Consolidator (IG23710 and 682190), Defense Threat Reduction Agency (HDTRA1-21-1-0007), and the National Energy Research Scientific Computing Center.

The work, conducted in mice, reveals that what animals consume initiates the release of a metabolic byproduct from a specific gut microbe that, in turn, modulates the animals’ gut immunity.

The findings, published Nov.10 in Nature, offer a unifying explanation for the complex interplay between diet, gut microbiota, and immune function. They are the result of collaboration among scientists at Harvard Medical School, Brigham and Women’s Hospital, Seoul National University, and Monash University in Australia.

The experiments pinpoint a microbial molecule, the synthesis and release of which are influenced by host diet. That molecule, in turn, stimulates the activation and signaling of a subset of cells known as natural killer (NK) T cells, which are involved in immune regulation and implicated in a range of inflammatory conditions.

While scientists have surmised for a long time that diet plays a role in immune health, the new study elucidates the precise molecular cascade behind this interplay, said study senior author Dennis Kasper, professor of immunology in the Blavatnik Institute at Harvard Medical School.

“We have shown how diet affects the immune system through a microbial mediator in the gut, and this is a really striking example of the diet-microbiota-immunity triad at play,” Kasper said. “What this work really does is provide a step-by-step pathway from beginning to end that explains how and why this triad works and how diet ultimately affects the immune system.”

If confirmed in larger animals and eventually in humans, the findings can help inform the design of small-molecule treatments that enhance both intestinal and overall immunity, the researchers said.

“Gut-resident microbes produce molecules with enormous structural diversity. We used microbial and chemical tools to elucidate how these molecules are synthesized by gut bacteria and how they act in the host gut,” said study first author Sungwhan Oh, a principal investigator at the Center for Experimental Therapeutics and Reperfusion Injury at Brigham and Women’s Hospital and a former postdoctoral fellow in the Kasper lab.”Our findings yield fascinating insights about the microbiome, diet, and immune function and provide interesting clues about how molecules made by our inner neighbors can be used to design therapies.”

In a series of experiments, the team identified the immune-signaling cascade triggered by the metabolic breakdown of dietary amino acids in the mouse gut. This multistep pathway begins with an animal consuming food that contains branched-chain amino acids, so named for the tree branch-like structure of one of their molecular chains. The branched-chain amino acids are then taken up by B. fragilis, a gut-resident microbe, and converted by a specific enzyme into sugar-lipid molecules that also have branched chains. B. fragilis then releases branched-chain molecules that are spotted and picked up by a class of immune-signaling cells known as antigen-presenting cells, which in turn induce NK T cells to exercise their immunoregulatory response through upregulating inflammation-controlling genes and immune-regulatory chemicals.

Notably, the experiments demonstrated that it is the branching of the chain structure that initiates the cascade. Straight-chain versions of the molecule did not yield the same effect. Furthermore, the team found that B. fragilis alters the structure of the sugar-lipid molecules that it metabolizes and renders them better capable of binding to receptors on specific immune cells and initiating a signaling cascade that culminates in downregulating inflammation.

The work also showed that each of three different branched-chain amino acids consumed by mice yielded slightly different structural changes to the bacterial lipid molecules, resulting in different patterns of binding with immune cells.

Study coinvestigator Seung Bum Park, professor of chemistry at Seoul National University, synthesized, and the Harvard team tested, 23 different configurations of the microbe-made immunomodulatory molecule to determine how each one interacts with the immune cells that regulate inflammation.

The Harvard team’s experiments revealed that synthetic, lab-made branched-chain lipid molecules induced NK T cells to release the immune-signaling chemical IL-2, whereas the lab-made straight-chained versions of these molecules did not. Thus activated, the NK T cells, in turn, induced the expression of genes that regulate immunity but not of genes that drive inflammation.

Using a structural biology approach, Jamie Rossjohn, professor of biochemistry and molecular biology at Monash Biomedicine Discovery Institute in Australia, elucidated how the lipid structure engages with and binds to antigen presenting cells — the immune cells that give NK T cells the go-ahead to produce anti-inflammatory chemicals.

In a final step, the researchers treated mice with ulcerative colitis with the branched-chain sugar-lipid molecule. Animals that received treatment with the branched-chain molecule fared much better than untreated animals. They not only gained weight, but when researchers examined the gut cells of the mice under a microscope, they observed that these cells also had minimal signs of colon inflammation.

Taken together, the experiments provide a structural and molecular explanation of previously observed anti-inflammatory effects of this class of sugar-lipids produced by the gut microbe B. fragilis.

“This work offers a great example of transdisciplinary discovery-based research aimed at answering a major question in biomedical sciences, namely, how the immune system can be modulated by the interplay between diet and the microbiota,” Rossjohn said.

In 2014, Kasper and colleagues published a study showing that a sugar-lipid molecule released by B. fragilis had anti-inflammatory effects on the gut and protected mice from colitis, but the scientists did not know how these molecules were made by the microbe, nor the specific structural features of the sugar-lipids that conferred the anti-inflammatory effect. The current study answers this question demonstrating that the sugar-lipid molecules made by this particular organism are branched-chain and it is precisely that branched-chain structure that allows them to bind to immune cells in a way that dampens these cells’ proinflammatory signaling.

“Our new work demonstrates that the branching of the lipid structure induces a very different response — the branching in the structure induces an anti-inflammatory rather than a proinflammatory response,” Kasper said.

The findings offer hope that inflammatory diseases mediated by these NK T cells could one day be treated with inflammation-dampening microbial molecules made in the lab, the researchers said.

The exact function of NK T cells — the immune cells that the microbe-made molecule ultimately activates to control colonic inflammation in mice — is not well-understood, Kasper said. However, given that these cells line the human gastrointestinal tract and the lungs and are also found in the liver and spleen, they likely play a significant role in immune regulation. Previous research points to these cells’ likely involvement in a range of inflammatory conditions, including ulcerative colitis, and to a possible role in airway inflammatory conditions such as asthma.

“We can never isolate enough of these immune-modulatory molecules from bacteria for therapeutic use, but the beauty of this is now we can synthesize them in the lab,” Kasper said. “The idea would be that we’d have a drug that can modulate inflammation in the colon and beyond.”

Co-authors included T. Praveena, Heebum Song, Ji-Sun Yoo, Da-Jung Jung, Deniz Erturk-Hasdemir, Yoon Soo Hwang, ChangWon Lee, Jérôme Le Nours, Hyunsoo Kim, Jesang Lee, and Richard Blumberg.

The work was supported by National Institutes of Health grants K01-DK102771, R01-AT010268, and R01-DK044319; by Department of Defense grant W81XWH-19-1-0625; by Brigham and Women’s Hospital Department of Anesthesiology, Perioperative and Pain Medicine Basic Science Grant; by the National Research Foundation of Korea grants 2014R1A3A2030423 and 2012M3A9C404878; and by the Australian Research Council grant CE140100011 and Australian Research Council Laureate Fellowship and Future Fellowships.

Relevant disclosures

Oh, Blumberg, and Kasper have filed a patent for Bacteroides fragilis α-galactosylceramides(BfaGCs) and related structures. Oh, Park, and Kasper have filed a patent on the functions of BfaGCs and related structures.

These remarkable findings are the result of a multidisciplinary study published in Nature Medicine, led by Dr. Jeff Biernaskie, PhD, professor, Comparative Biology and Experimental Medicine in the Faculty of Veterinary Medicine (UCVM) and Dr. Bryan Yipp, MD, associate professor, Department of Critical Care Medicine, Cumming School of Medicine.

“We found that the males derived benefit from the steroids, and the females, at both the cellular level and at the population level, received limited benefit,” says Yipp, Tier II Canada Research Chair in Pulmonary Immunology, Inflammation and Host Defense. “Currently, it’s possible the mainstay therapy for severe COVID-19 that we’re giving everybody is only benefiting half the population. This is a big problem.”

How do our bodies battle COVID-19 infection?

At the onset of the pandemic, hospitals’ treatments of the severely ill were not yet informed by research into how effective the drugs were under COVID-19 conditions. Steroids were the first identified drugs with proven benefit, but they were only moderately successful at reducing deaths, and exactly what they did was not understood.

In addition, when the study began, no one knew exactly how immune cells would react to COVID-19 infection at a cellular level. Why did some people get really sick while others did not? Why did certain drugs help some but not others?

“To be able to develop new treatments, we wanted to study how different people respond to SARS-CoV2 infection and how different immune responses dictate the severity of their disease,” says Biernaskie, the Calgary Firefighters Burn Treatment Society Chair in Skin Regeneration and Wound Healing.

Yipp and Biernaskie sought to better understand how steroids helped and, at the same time, evaluate why a clinical trial of steroids in COVID-19 showed they only helped some males, but not females.

When Yipp accessed the provincial eCRITICAL database of all ICU admissions during the pandemic, he discovered that the introduction of dexamethasone therapy in Alberta reduced the number of males dying but had no affect on the female population. “That was an unsettling observation.”

Analyzing thousands of immune cells from ICU patients

Blood was collected from both COVID-19 and non-COVID-19 patients who were admitted to Calgary ICUs in severe respiratory distress. Researchers in the Biernaskie lab used cutting-edge single cell RNA sequencing and bioinformatics techniques to simultaneously analyze the functional states of thousands of immune cells from each patient. This allowed them to document cellular behaviours at different stages of the disease (COVID-19 or non-COVID infections) and to measure treatment effects.

“We sampled as many patients as we could, not just at one time point but at a follow-up time point so we could get an idea of the evolution of the disease and the evolution of the immune response,” says Biernaskie.

In most viral infections, proteins called interferons work to clear the virus quickly. But with COVID-19, rather than working fast, “the interferon response trickles along, which actually fuels the fire of inflammation, and then you get worse organ damage,” says Yipp.

“What we found was that specifically in males, we see an exaggerated neutrophil interferon response, that is significantly restrained when a patient is given dexamethasone,” says Biernaskie. “But with females, relative to males, their neutrophil interferon response was much more tempered, so dexamethasone had little effect.”

Find therapies that benefit more people

After identifying the reasons why there’s a sex bias in the way dexamethasone works, Yipp believes that the way forward is for researchers to figure out how to make therapies that benefit more people, or individualized therapies, also known as precision or personalized medicine, so that a blanket approach isn’t being used.

Biernaskie and Yipp credit significant contributions from the trainees and junior scientists involved in the research, including Dr. Nicole Rosin and Sarthak Sinha who spent countless hours managing the project and analyzing the results.

The project was supported by a grant Biernaskie and Yipp received from the Thistledown Foundation and by the Calgary Firefighters Burn Treatment Society, “who enthusiastically supported my request to divert some of the CFBTS Chair funds to support this initiative early on in the pandemic,” Biernaski says.