Article In Brief
Researchers have identified an immune cell in mice that promotes axon regeneration and neuronal survival and could potentially be harnessed as a treatment for brain, spinal cord, and optic nerve damage.
Scientists at the Ohio State University Wexner Medical Center and the University of Michigan have identified an immune cell in mice that promotes axon regeneration and neuronal survival and could potentially be harnessed as a treatment for brain, spinal cord, and optic nerve damage. The findings were published October 26 online in Nature Immunology.
Surprisingly, the cell is a granulocyte that resembles an immature neutrophil. Neutrophils, the most common white blood cell in the human circulation, are the first responders to infection and other threats.
“In the central nervous system, infiltrating mature neutrophils have been shown to cause collateral damage to healthy tissue during the clearance of infection. Therefore it was surprising that this newly discovered immature neutrophil subset had potent neuroprotective and neuro-regenerative properties,” said senior study author Benjamin M. Segal, MD, chair of neurology and director of the Neuroscience Research Institute at Ohio State University.
Monocytes, as opposed to neutrophils, are the immune cell type that has been most closely associated with healing in the periphery in the wake of inflammation, Dr. Segal explained.
The team also showed that a human immune cell line with characteristics of an immature neutrophil can drive the regrowth of severed axons in mice in vivo and neurite outgrowth of explanted human cortical neurons in vitro.
“This research opens up the possibility that damage in the central nervous system might be reversed via manipulation of immune pathways, or by administration of an autologous immune cell therapy,” Dr. Segal said. His lab is now planning to test the beneficial effects of these unusual cells in animal models of multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS).
Dr. Segal, a physician-scientist specializing in neuroinflammatory diseases, including MS, pointed out that while there are more than a dozen drugs that decrease the frequency of MS attacks, there are no drugs that reverse chronic neurological deficits. There is also a lack of medications that prevent or slow neuronal loss in ALS or other neurodegenerative disorders or that restore neurological function in individuals with traumatic brain or spinal cord injury or ischemic reperfusion injury of the optic nerve.
Several decades ago, Larry Benowitz, PhD, a professor of ophthalmology at Harvard Medical School, and his colleagues injected a fungal cell wall extract into the posterior chamber of the eye of mice with optic nerve crush injury, thereby triggering a robust inflammatory response associated with enhanced survival of retinal ganglion cells (RGCs)—the neurons that give rise to the optic nerve—and axon regeneration. The cellular and molecular mechanisms underlying this phenomenon had not been elucidated, compelling Dr. Segal to investigate the model further.
Study Design, Findings
Dr. Segal said that the first step was to identify the inflammatory cell type responsible for the neuroprotective and neuro-regenerative response. His team isolated inflammatory cells from the eyes of mice following intraocular injection of fungal cell wall extract. Neutrophils were the most abundant leukocyte, followed by monocytes. Since the local accumulation of neutrophils was closely associated with RGC rescue and optic nerve regrowth, Dr. Segal and his postdoctoral fellows, Kevin Carbajal, PhD, and Andrew Sas, MD, PhD, questioned whether neutrophils play an active role in the repair process.
“This approach has potential for a number of neurodegenerative diseases in which sustained effective therapeutic options are limited.”
—DR. THOMAS E. LANE
In their next experiment, they attempted to prevent neutrophils from entering the eye following intraocular injection of fungal cell wall extract and optic nerve crush injury. To do so, they administered an anti-serum that blocks CXCR2, a chemokine receptor that guides neutrophils to sites of injury. The scientists speculated that neuronal survival and regeneration might be attenuated by anti-CXCR2 treatment. Instead, they found even more robust regeneration in the mice treated with anti-CXCR2.
Now, they were stumped. They serially analyzed the composition of intraocular infiltrates and found that neutrophil entry into the eye was delayed but not completely suppressed by anti-CXCR2. Furthermore, there was something very different about the neutrophils that had accumulated in the eye. They were relatively immature and expressed markers previously associated with “alternatively-activated” monocytes that were implicated in wound healing. An in-depth analysis of the genes expressed by the eye-infiltrating cells was consistent with an alternatively-activated immature neutrophil, said Dr. Segal.
Next, they isolated the eye-infiltrating immature neutrophils and cultured them with explanted neurons extracted either from the retina or from dorsal root ganglia. The neutrophils directly stimulated both types of neurons to grow neurites, he added.
In order to harvest a sufficient number of pro-regenerative neutrophils for adoptive transfer experiments, they injected fungal wall cell extract into the peritoneal cavity of naïve mice and collected inflammatory cells from the cavity three days later. They found that the peritoneal cells were enriched with immature neutrophils that bore markers similar to those of intraocular pro-regenerative neutrophils.
The researchers then purified these neutrophils and injected them into mice that had just received a crush injury to their optic nerve. They administered another injection three days later. It worked. There was approximately three to four times greater neuronal survival and substantial nerve fiber regrowth in the mice that received the immature neutrophils compared with their counterparts that received vehicle alone or conventional mature neutrophils.
The immature neutrophils were effective in rescuing RGCs and inducing nerve fiber regeneration when their administration was delayed up to 12 hours from the time of nerve crush injury, illustrating their therapeutic potential in a clinically relevant setting.
In order to determine the role of soluble factors, they cultured explanted neurons with conditioned medium from peritoneal immature neutrophils. The conditioned media itself induced neurite outgrowth. The scientists detected growth factors in the neutrophil conditioned media, specifically an abundance of nerve growth factor and insulin-like growth factor-1. There was less neuronal survival and nerve fiber regeneration in the animals when the growth factors were neutralized.
The investigators wanted to know if these healing properties were specific to the eye and optic nerve. So they repeated the studies using a spinal cord injury model and injected fungal cell wall-induced peritoneal neutrophils into the sciatic nerves. Administration of the immature neutrophils drove the regeneration of severed axons within the spinal cord.
Finally, the researchers tested the neuroprotective and pro-regenerative effects of a human cell line, called HL-60, originally derived from promyelocytic leukemia cells, that is widely used as a surrogate of immature neutrophils. They delivered the optic nerve crush injury to immunodeficient mice and then injected the human HL-60 cells into the posterior chamber of the eye. The mice treated with HL-60 cells, but not a control human lymphocyte cell line, experienced robust enhancement of neuronal survival and axon regeneration of nerve fibers.
“The human cells were just as effective as their murine counterparts. They also produced nerve growth factor (but not insulin-like growth factor,)” said Dr. Segal.
“We are encouraged by our results and hope to evaluate their translational potential in the future,” he added.
“These findings are important and novel for a number of reasons,” said Thomas E. Lane, PhD, Chancellor's professor in the department of neurobiology & behavior at the University of California, Irvine. “What Dr. Segal and his colleagues have done is systematically work through experiments to identify this new population of cells and immunologically phenotype them. He showed that adoptive transfer of these neutrophils are therapeutic and can home to the site of injury and promote repair. This approach has potential for a number of neurodegenerative diseases in which sustained effective therapeutic options are limited.”
“The holy grail in neurology is repair of the central nervous system,” added Rhonda Voskuhl, MD, the Jack H. Skirball chair and professor of neurology at UCLA, and director of the UCLA Multiple Sclerosis Program. “These scientists identified a unique subset of neutrophils that have regenerative properties. The hope is to harness these cells and use them to repair injuries of the central nervous system.”
“One caveat is that the work was done in optic nerve and spinal cord traumatic injury models,” said Dr. Voskuhl, who also wrote a commentary on the study in the November 2 online edition of Nature Immunology.
“Can this be extrapolated to brain injury or to neurodegenerative diseases?” she asked. “It is important not to make assumptions that a treatment for spinal cord and optic nerve also works in brain in a one-size-fits-all approach.”
Indeed, Dr. Voskuhl said that she and other scientists have done work showing that cells in the spinal cord and optic nerve can have different responses to injury as compared to brain. “Overall, this is good news of an exciting discovery, and more studies are now warranted,” she said.