In a new study, corresponding authors Cheryl Nickerson, Jennifer Barrila and their colleagues demonstrate that under low fluid shear force conditions that simulate those found in microgravity culture during spaceflight, the foodborne pathogen Salmonella infects 3-D models of human intestinal tissue at much higher levels, and induces unique alterations in gene expression.

This study advances previous work by the same team showing that physical forces of fluid shear acting on both the pathogen and host can transform the landscape of infection.

Understanding this subtle interplay of host and pathogen during infection is critical to ensuring astronaut health, particularly on extended space missions. Such research also sheds new light on the still largely mysterious processes of infection on earth, as low fluid shear forces are also found in certain tissues in our bodies that pathogens infect, including the intestinal tract.

While the team has extensively characterized the interaction between conventionally grown shake flask cultures of Salmonella Typhimurium and 3-D intestinal models, this study marks the first time that S. Typhimurium has been grown under the low fluid shear conditions of simulated microgravity and then used to infect a 3-D model of human intestinal epithelium co-cultured with macrophage immune cells, key cell types targeted by Salmonella during infection.

The 3-D co-culture intestinal model used in this study more faithfully replicates the structure and behavior of the same tissue within the human body and is more predictive of responses to infection, as compared with conventional laboratory cell cultures.

Results showed dramatic changes in gene expression of 3-D intestinal cells following infection with both wild-type and mutant S. Typhimurium strains grown under simulated microgravity conditions. Many of these changes occurred in genes known to be intimately involved with S. Typhimurium’s prodigious ability to invade and colonize host cells and escape surveillance and destruction by the host’s immune system.

“A major challenge limiting human exploration of space is the lack of a comprehensive understanding of the impact of space travel on crew health,” Nickerson says. “This challenge will negatively impact both deep space exploration by professional astronauts, as well as civilians participating in the rapidly expanding commercial space market in low Earth orbit. Since microbes accompany humans wherever they travel and are essential for controlling the balance between health and disease, understanding the relationship between spaceflight, immune cell function, and microorganisms will be essential to understand infectious disease risk for humans.”

Nickerson, who co-directed the new study with Jennifer Barrila, is a researcher in the Biodesign Center for Fundamental and Applied Microbiomics and is also a professor with ASU’s School of Life Sciences. The research appears in the current issue of the journal Frontiers in Cellular and Infection Microbiology

Life-altering force

Life on earth has diversified into an almost incomprehensibly vast array of forms, evolving under wildly dissimilar environmental conditions. Yet one parameter has remained constant. Throughout the 3.7-billion-year history of life on earth, all living organisms evolved under, and respond to, the pull of Earth’s gravity.

For more than 20 years, Nickerson has been a pioneer in exploring the effects of the reduced microgravity environment of spaceflight on a range of pathogenic microbes and the impact on interactions with human cells and animals they infect. She and her colleagues have doggedly pursued this research in both land-based and spaceflight settings, the results of which helped lay the foundation for the rapidly growing research field, mechanobiology of infectious disease, the study of how physical forces impact infection and disease outcomes.

Among their important findings is that the low fluid shear conditions associated with the reduced gravity environment of spaceflight and spaceflight analog culture are similar to those encountered by pathogens inside the infected host, and that these conditions can induce unique changes in the ability of pathogenic microbes like Salmonella to aggressively infect host cells and exacerbate disease, a property known as virulence.

The infectious agent explored in the new study, Salmonella Typhimurium, is a bacterial pathogen responsible for gastrointestinal disease in humans and animals. Salmonella is the leading cause of death from food-borne illness in the United States. According to the CDC, Salmonella bacteria cause about 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the United States each year. Foods contaminated by the bacteria are the primary source for most of these illnesses.

Salmonella infection typically causes diarrhea, fever, and stomach cramps, beginning 6 hours to 6 days after infection. Illness from the disease usually lasts 4 to 7 days. In severe cases, hospitalization may be required.

‘Shear’ probability?

Cells in mammalian organisms, including humans, as well as the bacterial cells that infect them, are exposed to extracellular fluid flowing over their outer surfaces. Just as a gentle downstream current will affect the pebbles in the underlying streambed differently than a raging torrent, so the force of fluid gliding over cell surfaces can cause changes to affected cells. This liquid abrasion of cell surfaces is known as fluid shear.

Since spaceflight experiments are rare and access to the space research platform is currently limited, researchers often simulate the low fluid shear conditions that microbes encounter during culture in spaceflight by growing cells in liquid growth media within a device known as a rotating wall vessel bioreactor or RWV. As the cylindrical reactor rotates, cells are maintained in suspension, gently and continuously tumbling in their surrounding culture medium. This process mimics the low fluid shear conditions of microgravity that cells experience during culture in spaceflight.

The team has also shown that this fluid shear level is relevant to conditions that microbial cells encounter in the human intestine and other tissues during infection, triggering changes in gene expression that can help some pathogens better colonize host cells and evade the immune system’s efforts to destroy them.

Portrait of an intruder

The study found significant changes in both gene expression and ability to infect 3-D intestinal models by Salmonella bacteria cultured in the RWV bioreactor. These experiments involved two S. Typhimurium strains, one unaltered or wild type strain and one mutant strain.

The mutant strain was otherwise identical to the wild type but lacked an important protein known as Hfq, a major stress response regulator in Salmonella. In earlier research, Nickerson and her team discovered that Hfq acts as a master regulator of Salmonella’s infection process in both spaceflight and spaceflight analog culture. They later discovered additional pathogens that also use Hfq to regulate their responses to these same conditions.

Unexpectedly, in the current study, the hfq mutant strain was still able to attach, invade into, and survive within 3-D tissue models at levels comparable to the wild type strain. In agreement with this finding, many genes responsible for Salmonella’s ability to colonize human cells, including those associated with cell adherence, motility, and invasion were still activated in the mutant strain under simulated microgravity conditions, despite the removal of Hfq.

From the host perspective, the 3-D intestinal co-culture model responded to Salmonella infection by upregulating genes involved in inflammation, tissue remodeling, and wound healing at higher levels when the bacteria were grown under simulated microgravity conditions prior to use in infection studies. This was observed for both wild type and hfq mutant strains of the pathogen.

Data from this new spaceflight analog study reinforces previous findings from the team’s 2006, 2008 and 2010 Space Shuttle experiments. In particular, the 2010 flight experiment conducted aboard Space Shuttle Discovery, called STL-IMMUNE, used the same wild type strain of S. Typhimurium to infect a 3-D model of human intestinal tissue made from the same epithelial cells used in the new study.

Several commonalities were observed between host cell responses to infection in the new spaceflight analog study and those previously reported when infections took place in true spaceflight during the STL-IMMUNE experiment. These results further reinforce the RWV as a predictive ground-based spaceflight analogue culture system that mimics key aspects of microbial responses to true spaceflight culture.

“During STL-IMMUNE, we discovered that infection of a human 3-D intestinal epithelial model by Salmonella during spaceflight induced key transcriptional and proteomic biosignatures that were consistent with enhanced infection by the pathogen,” Barrila says. “However, due to the technical challenges of performing in-flight infections, we could not quantify whether the bacteria were actually attaching and invading into the tissue at higher levels. The use of the RWV bioreactor as a spaceflight analog culture system in our current study has been a powerful tool which allowed us to explore this experimental question at a deeper level.”

New horizons

Astronauts face a double risk from infectious disease during their missions far from earth. The combined rigors of spaceflight act to weaken their immune systems. At the same time, some pathogens like Salmonella may be triggered by low fluid shear conditions induced by microgravity to become more effective infectious agents.

With longer spaceflight missions in the advanced planning stages and the advent of civilian space travel rapidly emerging, safeguarding space travelers from infectious disease is vital.

Studies like the current one are also helping to pull back the curtain on the infection process, revealing foundational details with broad relevance for the battle against diseases, on Earth and beyond.

The immune system, however, faces far greater challenges in distinguishing self from non-self. Should this complex surveillance network fail to identify a foreign intruder, like a bacterium or virus, the result may be serious, unchecked disease.

Under certain circumstances, however, the immune system can become over-vigilant, identifying our own tissues as foreign and targeting them for destruction, resulting in autoimmune disease. Autoimmune responses are also associated with some cancers.

In a new study, Arizona State University researcher Joshua LaBaer and his colleagues explore components of the immune system known as autoantibodies. While they have been implicated as central players in a range of serious autoimmune diseases, the study observes that autoantibodies are also found in healthy individuals.

This fact may make the diagnostic use of autoantibodies as sentinels of autoimmune disease more challenging, hence the importance of such investigations.

An improved awareness of the pervasiveness and role of autoantibodies in human health and disease may ultimately help in the design of better diagnostics and therapeutics against a range of illnesses.

“Historically, we looked for autoantibodies present only in disease, but we’ve always been intrigued because our healthy controls always had autoantibodies too,” LaBaer says. “So, we decided to see if any of these “healthy autoantibodies” were common in healthy people and sure enough many of them were. Knowing about these will help us avoid confusion in future studies.”

Dr. LaBaer is the executive director of ASU’s Biodesign Institute as well as the director of the Biodesign Virginia G. Piper Center for Personalized Diagnostics.

The research findings appear in the current issue of the journal Cell Reports.

Biological civil war

Autoimmune diseases are a widespread phenomenon, affecting roughly 23 million Americans. Researchers have identified more than 80 autoimmune diseases, including both common ailments like type 1 diabetes, lupus, multiple sclerosis and rheumatoid arthritis, as well as more obscure ailments, which often prove challenging to properly diagnose. Nearly 80% of autoimmune diseases occur in women, for reasons researchers are still trying to puzzle out.

Science has much to learn about the underlying mechanisms responsible for autoimmune reactions. Often such illnesses arise following infections. Two primary components of the so-called adaptive immune system also play important roles in autoimmunity. These are the are white blood cells or lymphocytes, known as T cells and B cells. Lymphocytes are crucial for maintaining health and are essential to survival. These sentries, ceaselessly patrolling the bloodstream, are alerted by the presence of foreign entities known as antigens.

T cells guard against infection from pathogens like bacteria, viruses and fungi. They can also attack and destroy cancerous cells. B cells secrete proteins known as antibodies that either disrupt interactions or target infected cells so that other cells can come in to destroy them. Antibodies operate by binding with pathogens or foreign substances, including toxins, and neutralizing their harmful effects. If an antibody binds with a virus, for example, it may prevent the intruder from entering a normal cell to cause infection. B cells can also recruit other specialized immune cells to migrate to the sites of infected cells and help destroy them.

The immune system’s defensive arsenal is exquisitely sensitive to foreign proteins, peptides, enzyme complexes, RNA and DNA. When these are encountered, B cells can swing into action, producing antibodies directed against these foreign entities.

Mutinous immunity

The immune system, however, faces a formidable challenge. B and T cells must be able to accurately target threats to the body while remaining harmless to host cells and tissues. Immune cells are not born with this knowledge, they learn it over a matter of weeks, receiving training in a kind of biological classroom, where they undergo two rounds of careful screening.

The B and T cells that successfully graduate from their training sessions exhibit two kinds of immune tolerance, central immune tolerance, which develops in the bone marrow and peripheral immune tolerance, which matures in the lymph nodes.

After their training, cells displaying immune tolerance, a kind of non-aggression pact with healthy tissues — are preserved in the body for future use. Immune cells that flunk out of their training and present a risk of autoimmunity are sequestered or destroyed.

Yet sometimes, pathogens or cancers can produce antigens that bear such a close resemblance in terms of sequence or structure to features found in normal tissues in the body that the host tissues are mistaken for disease antigens and targeted by antibodies. Such features are known as self-antigens and the antibodies produced to target them are known as autoantibodies.

The mistaken targeting of self-antigens due to their similarities with disease antigens is known as molecular mimicry and is implicated in many autoimmune disorders, from rheumatoid arthritis to multiple sclerosis.

Scavenger hunt for antibodies

The new study explores common autoantibodies — those occurring in healthy individuals. Though these common autoantibodies don’t appear to cause disease, they nevertheless appear in as many as 40% of the people tested. It is likely that at least some of these common autoantibodies have been mistakenly identified as disease antibodies.

The researchers performed a meta-analysis of 9 datasets. The tool of choice for exploring the common autoantibodies is a device known as a protein microarray. Here, thousands of individual proteins are affixed to a glass slide. When a sample of blood is spread over the microarray, antibodies, (in this case, autoantibodies) bind with specific protein antigens.

The microarrays were subjected to two rounds of screening. In the first round, 182 blood samples from healthy individuals were screened against 7,653 human proteins. In the second round, 90 blood samples were screened against 1,666 human proteins. The experiments identified a total of 77 common autoantibodies.

The blood samples came from healthy individuals of both sexes, ranging in age from infancy to 84 years old. The results showed that the number of autoantibodies increased from birth up to the age of adolescence and then plateaued. Further, the number of autoantibodies detected was the same regardless of sex, a surprising outcome given the large disparity between men and women in the prevalence of autoimmune disease.

Another underlying enigma is why common autoantibodies fail to produce autoimmune disease. Although such antibodies appear to have evaded the screening process leading to immune tolerance, their occurrence in the body remains benign. It is believed that autoimmune pathology requires autoantibodies to bind and form complexes with autoantigens, and this may be blocked in the case of common autoantibodies.

Future research promises to unlock many more secrets concerning the nature of autoantibodies. The current study examined less than half of all human proteins. Most likely, additional common autoantibodies remain to be uncovered.

The study is available online in the journal Clinical Cancer Research.

Synovial sarcoma is rare, with 900 to 1,000 new cases diagnosed annually, and is most typically diagnosed during adolescence and into young and middle adulthood. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine is a major center for the treatment of sarcomas nationwide, as is Siteman Kids at St. Louis Children’s Hospital.

“Synovial sarcoma is responsible for about 10% of all sarcomas, and because sarcoma in general is rare, a lot of these patients — pediatric and adult — travel from all over the country to receive treatment at our specialized Sarcoma Center,” said oncologist Brian A. Van Tine, MD, PhD, a professor of medicine. “If it’s diagnosed early, this cancer can be cured with standard care — surgery, radiation and chemotherapy. But once it spreads, we have no effective curative therapies, so we’re looking for new treatment strategies that take advantage of the genetic quirks of this rare tumor.”

The researchers found that synovial sarcoma is missing an important protein that most tumors rely on to drive their energy metabolism. The absence of this key protein — called malic enzyme 1 (ME1) — forces synovial sarcoma tumors to rely on a different metabolic pathway, which makes it uniquely vulnerable to the inhibition of that alternate pathway. The investigational drug ACXT-3102 interferes with this alternate route. The interference causes volatile waste compounds called reactive oxygen species to build up inside the cancer cells. When enough reactive oxygen species build up inside, the cell dies.

“Because they’re missing ME1, these tumor cells are already crippled in their ability to fight damage from reactive oxygen species,” said Van Tine, who leads the sarcoma program at Siteman. “So, we asked if we could use this broken defense against this cancer. When levels of these compounds skyrocket inside the cells, they die very quickly.”

The drug ACXT-3102 was developed by William G. Hawkins, MD, the Neidorff Family and Robert C. Packman Professor of Surgery, and his team, to treat pancreatic cancer. Because most pancreatic cancers still have ME1, researchers will need to find a second way to attack that tumor type. But because the metabolism of synovial sarcoma is unusual and consistent across patients — the cancer’s defining genetic mistake is present in 90% to 95% of all cases — the researchers suspect that this rare tumor could be treatable with ACXT-3102 alone.

“Synovial sarcoma is caused by a very specific genetic mutation, so it’s a relatively clean cancer, meaning it has a single specific genetic mistake that can be exploited, unlike other cancers that have a complex accumulation of many mutations whose effects are difficult to unravel,” Van Tine said. “Because of this single mutation, it’s harder for synovial sarcoma cells to adapt to an attack on their energy metabolism. Finding a weakness in cancer that we can exploit based on the biology of a rare tumor is really exciting.”

The drug ACXT-3102 was licensed to a Washington University startup company called Accuronix Therapeutics that was co-founded by Hawkins to develop new cancer therapies.

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Materials provided by Washington University School of Medicine. Original written by Julia Evangelou Strait. Note: Content may be edited for style and length.