Zika infection causes developing cranial cells to secrete neurotoxic levels of immune molecules

Infection by the Zika virus causes a population of cells in the cranium of a developing embryo to secrete neurotoxic levels of immune signaling molecules called cytokines, according to a new study by researchers at the Stanford University School of Medicine.

Although the study was conducted solely in cells grown in a laboratory dish, it may provide one possible explanation for why babies born to women infected with the virus can suffer from a birth defect called microcephaly, or abnormally small heads.

“Affected babies have small brains and small skulls,” said assistant professor of medicine Catherine Blish, MD, PhD. “Cells in the cranial neural crest give rise to the bones and cartilage of the skull and face, and they also form an important supportive niche for the developing brain. We wondered if the Zika virus could infect cranial neural crest cells, perhaps giving rise to deficits in skull formation and altered neural development.”

The study was published online Sept. 29 in Cell Host & Microbe. Blish is the senior author. Graduate students Nicholas Bayless and Rachel Greenberg share lead authorship of the study.

A reservoir for the virus

Cranial neural crest cells are cells that arise in humans within about five to six weeks of conception. Although they first appear along what eventually becomes the spinal cord, the neural crest cells migrate over time to affect facial morphology and differentiate into bone, cartilage and connective tissue of the head and face. They also provide critical molecular signals that support nearby developing neurons in the brain. 

Bayless and Greenberg used a technique developed in the laboratory of study co-author Joanna Wysocka, PhD, a professor of chemical and systems biology and of developmental biology, to convert human embryonic stem cells into cranial neural crest precursors in the laboratory. Wysocka’s research focuses on understanding how these cells affect the embryonic development of facial features, including those of humans and chimpanzees.

Recent research has focused on the effect of Zika virus infection of the neural precursor cells that give rise to neurons in the developing brain. But Bayless and Greenberg found that not only can cranial neural crest cells also be infected by the Zika virus, they respond differently than their neighboring neural precursor cells to the infection. Rather than rapidly dying, as the neural precursors do, the cranial neural crest cells act as a reservoir for the virus by allowing it to replicate repeatedly. In addition, they begin to secrete high levels of cytokines, including leukemia inhibitory factor and vascular endothelial growth factor, known to affect neural development.

“The magnitude of altered cytokine secretion caught us by surprise,” said Blish. “These molecules are important for neurogenesis, and the infected cells are secreting them at high levels.”

Abnormally shaped cells

When Bayless and Greenberg incubated neural precursor cells together with infected neural crest cells, the neural precursors appeared abnormal and were more likely to initiate a program of cellular suicide.

They next exposed the neural precursor cells to leukemia inhibitory factor and vascular endothelial growth factor at levels equivalent to those secreted by the infected cells. After three days, they observed an increase in structures associated with cellular migration and growth, as well as a significant increase in the frequency of cell death.

“At least in vitro, these elevated levels of cytokines appear to induce premature differentiation and migration,” said Wysocka. “This abnormal developmental program then leads to cell death.”

The Zika virus, which is spread by the Aedes genus of mosquito, has sprung into the public eye over the past year as it has become increasingly apparent that pregnant women infected with the virus can pass it to the fetus, causing devastating birth defects. Outbreaks of the virus are currently occurring in multiple countries and in three American territories: American Samoa, Puerto Rico and the U.S. Virgin Islands. Local mosquito-borne transmission has also been identified in two areas of Miami, and the World Health Organization has designated the disease a public health emergency of international significance.

“Our study brings attention to the possibility that other infected embryonic cell types in the developing head can influence Zika-associated birth defects, including microcephaly, perhaps through signaling to neighboring cells or by serving as a viral replication reservoir,” said Wysocka. “Formation of the cranial neural crest cells and a cross-talk between brain and craniofacial development occurs during the first three months of human fetal development, which is when epidemiological studies have suggested that Zika infection correlates with poor birth outcomes.”

The researchers said that although the findings are intriguing and merit further study,  their studies were conducted only on cells grown in a laboratory and it is possible that other factors related to Zika infection may affect brain size and outcomes in affected infants.

Tomek Swigut, PhD, a senior scientist in Wysocka’s lab, is also a co-author of the study.

The research was supported by the National Institutes of Health (grants 1DP2AI112193 and DE024430), the Tashia and John Morgridge Faculty Scholar Program from the Stanford Child Health Research Institute, the March of Dimes Birth Defect Foundation, the Howard Hughes Medical Institute, a seed grant from the Stanford Center for Systems Biology and two Ruth L. Kirschstein awards.

Stanford’s Department of Medicine also supported the work.