Growing evidence suggests the peripheral immune system plays a role in Alzheimer’s disease. Now, researchers led by Gabriela Constantin at the University of Verona, Italy, turn the spotlight on an overlooked peripheral cell: the neutrophil. These short-lived phagocytes are the most abundant white blood cells, and swarm to sites of inflammation, but have not been thought to act in the brain. However, in the July 27 Nature Medicine, Constantin and colleagues reported that neutrophils seep into the brain in both Alzheimer’s disease and in mouse models of AD. In mice, blocking a neutrophil receptor prevented the cells from entering the brain, and reduced amyloid load and microgliosis, as well as giving the animals a boost in long-term memory. The results highlight the importance of peripheral innate immunity in Alzheimer’s, and hint that neutrophils might be one source of tissue damage.
“This is a thought-provoking study that departs from what everybody else is doing,” Tony Wyss-Coray at Stanford University School of Medicine, California, told Alzforum. “One of the most exciting observations is that the immune response seems most prominent at the early stages of disease in the mouse models.” If the findings hold up, they could point to a potential window for anti-inflammatory therapeutic intervention, he added. Other researchers found the data intriguing as well, but cautioned that more studies will be needed to discover if neutrophil infiltration contributes significantly to human disease.
Sneaking Into the Brain. In 5xFAD mice (middle), but not wild-type (left), neutrophils (red) squeeze out of blood vessels (green) and into the brain. Many neutrophils (right) also tarry on blood vessel walls near Aβ deposits (blue). [Courtesy of Nature Medicine, Zenaro et al.]
Several studies report that blood cells such as monocytes and T cells trickle into the AD brain, with monocytes in particular credited with mopping up amyloid deposits (see Apr 2011 news; Aug 2011 news; Apr 2015 conference news). Few researchers have focused on neutrophils, perhaps because these cells live for only hours and would be unlikely to accumulate in tissue. However, one recent study did find neutrophils infiltrating the brain in the 5xFAD mouse (see Baik et al., 2014).
Constantin’s interest grew out of her work studying white blood cell migration in several neuroinflammatory conditions (see Rossi et al., 2011). She was intrigued by the fact that brain endothelial cells in both 5xFAD and 3xTg mice expressed high levels of adhesion factors, suggesting they might capture circulating white blood cells, particularly neutrophils. Joint first authors Elena Zenaro and Enrica Pietronigro found that adhesion molecules were particularly prominent on endothelium near amyloid deposits. Moreover, adding Aβ42 to cultured brain endothelial cells induced expression of cell-surface adhesion factors.
The authors next examined what happened to neutrophils. In brain sections from both mouse models, but not controls, they found the cells loitering outside blood vessels (see image above). They identified them by staining for Ly6G, a cell-surface receptor specific for mouse neutrophils. Accumulation of these cells peaked around the age when cognitive problems began in each model, and occurred in areas with high plaque load and few neurons. This infiltration might have negative consequences, the authors speculated. Neutrophils release many toxic substances, such as reactive oxygen species, cytokines, and neutrophil extracellular traps (NETs), proteins that cage bacteria and damage tissue. The authors detected NETs and the pro-inflammatory cytokine IL-17 around neutrophils in brain tissue, hinting that the phagocytes might be wreaking havoc.
The cells might also injure the blood-brain barrier, the authors suggested. In live imaging experiments, they saw neutrophils crawling along brain blood vessels near amyloid deposits (see image above). Others agreed with this idea. “I find the interaction of the leukocytes at the vasculature as interesting as the extravasation,” Wyss-Coray commented. When white blood cells stick to endothelial cells, they communicate with them through surface receptors, potentially changing the permeability of the endothelium, he noted. Several studies implicate a leaky blood-brain barrier in AD (see May 2014 conference news; Jan 2015 news; Feb 2015 Webinar).
What causes neutrophils to invade the brain? In cell culture experiments, the authors found that synthetic Aβ42 oligomers caused the neutrophil receptor LFA-1 integrin to change shape, assuming a form with high affinity for endothelial adhesion factors. To test the role of this integrin, the authors isolated neutrophils from integrin knockout mice and injected them into 5xFAD animals. The integrin-deficient cells failed to stick to blood vessels, and stayed out of the brain. Likewise, administering an anti-LFA-1 antibody to 5xFAD mice slowed migration of endogenous neutrophils into the brain (see image below).
The authors wondered what effect blocking neutrophil entry would have. They injected anti-Ly6G antibodies into 6-month-old 3xTg mice for four weeks, causing neutrophil numbers to crash. Then they allowed neutrophils to recover for four weeks before testing cognition. While the 6-month-old animals had memory problems, the treated 8-month-olds performed like wild-types in the Y maze and in contextual fear conditioning. The cognitive improvements persisted even six months later, the authors reported. In addition, at eight months the treated mice had about a quarter less microgliosis, half as much insoluble Aβ42, and two-thirds less phosphorylated tau than untreated littermates. Meanwhile, levels of the presynaptic protein synaptotagmin rebounded to normal levels. Treatment with other antibodies targeted to neutrophils, such as anti-LFA-1, produced the same effects. So did crossing 3xTg mice with the integrin knockouts.
“This is the first demonstration that neutrophils are important in [Alzheimer's] disease pathogenesis,” Constantin told Alzforum. The long-term improvements struck her. “We think neutrophils are particularly important in the early phases of disease, during which inflammation mechanisms predominate. Intervening during those early phases could be beneficial.”
Does this happen in human disease? The authors stained postmortem brain sections from 11 AD patients and 11 controls, and saw about 10 times more neutrophils in the brain tissue from patients. The cells accumulated near amyloid plaques, as did NETs. As in mice, neutrophils stuck and spread across the walls of brain blood vessels in AD samples, suggesting they could be injuring the blood-brain barrier. While preliminary, the findings appear consistent with a role in Alzheimer’s disease.
Piet Eikelenboom at Vrije University, Amsterdam, pointed out that systemic inflammation in older people can trigger late-onset dementia (see Fong et al., 2015; Mar 2015 conference news). He wondered if blocking neutrophils during a bout of delirium might lower the risk of developing dementia later. Anti-integrin therapies are in use for some autoimmune diseases, and anti-LFA-1 has been in clinical trials, opening the door for testing this approach in people.
At the same time, several researchers said that more work needs to be done to nail down the mechanisms behind the observed improvement in treated mice. Eikelenboom speculated that neutrophils may have their greatest effect on the blood-brain barrier, rather than on parenchyma. Researchers have delineated two stages to crossing the blood-brain barrier: first, crossing the blood vessel wall; second, crossing the glial membrane formed by astrocyte endfeet to enter the brain parenchyma proper. Many white blood cells never make it past the first stage (see Bechmann et al., 2007). It is not clear from the images in the paper whether neutrophils pass this second barrier, Eikelenboom said.
Moreover, Terrence Town at the University of Southern California, Los Angeles, noted that the total number of infiltrating neutrophils in transgenic mice is quite small. Even among those white blood cells that sneak into brain, only about 2 to 3 percent are neutrophils, according to the authors’ measurements. “Is that sufficient to mediate a biological effect?” Town asked. Instead, changes in microglia might account for the improvements in pathology, he speculated. Microglia can express LFA-1 integrin. Perhaps antibodies against this receptor enter the brain and act directly on these cells to dampen inflammation, he suggested.—Madolyn Bowman Rogers
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Research Models Citations
- Baik SH, Cha MY, Hyun YM, Cho H, Hamza B, Kim DK, Han SH, Choi H, Kim KH, Moon M, Lee J, Kim M, Irimia D, Mook-Jung I. Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model. Neurobiol Aging. 2014 Jun;35(6):1286-92. Epub 2014 Jan 8 PubMed.
- Rossi B, Angiari S, Zenaro E, Budui SL, Constantin G. Vascular inflammation in central nervous system diseases: adhesion receptors controlling leukocyte-endothelial interactions. J Leukoc Biol. 2011 Apr;89(4):539-56. PubMed.
- Fong TG, Davis D, Growdon ME, Albuquerque A, Inouye SK. The interface between delirium and dementia in elderly adults. Lancet Neurol. 2015 Aug;14(8):823-32. Epub 2015 Jun 29 PubMed.
- Bechmann I, Galea I, Perry VH. What is the blood-brain barrier (not)?. Trends Immunol. 2007 Jan;28(1):5-11. Epub 2006 Nov 30 PubMed.
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- Innate Immune Cells Enlisted to Clear Amyloid, Fight Disease
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- DC: Is Alzheimer's Rooted in the Early Life?
- Australia Report: Inflammation
- Zenaro E, Pietronigro E, Della Bianca V, Piacentino G, Marongiu L, Budui S, Turano E, Rossi B, Angiari S, Dusi S, Montresor A, Carlucci T, Nanì S, Tosadori G, Calciano L, Catalucci D, Berton G, Bonetti B, Constantin G. Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Nat Med. 2015 Aug;21(8):880-6. Epub 2015 Jul 27 PubMed.