Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

The innate immune system stimulating cytokine GM-CSF improves learning/memory and interneuron and astrocyte brain pathology in Dp16 Down syndrome mice and improves learning/memory in wild-type mice.

Down syndrome (DS) is characterized by chronic neuroinflammation, peripheral inflammation, astrogliosis, imbalanced excitatory/inhibitory neuronal function, and cognitive deficits in both humans and mouse models. Suppression of inflammation has been proposed as a therapeutic approach to treating DS co-morbidities, including intellectual disability (DS/ID). Conversely, we discovered previously that treatment with the innate immune system stimulating cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which has both pro- and anti-inflammatory activities, improved cognition and reduced brain pathology in a mouse model of Alzheimer's disease (AD), another inflammatory disorder, and improved cognition and reduced biomarkers of brain pathology in a phase II trial of humans with mild-to-moderate AD. To investigate the effects of GM-CSF treatment on DS/ID in the absence of AD, we assessed behavior and brain pathology in 12-14 month-old DS mice (Dp[16]1Yey) and their wild-type (WT) littermates, neither of which develop amyloid, and found that subcutaneous GM-CSF treatment (5 µg/day, five days/week, for five weeks) improved performance in the radial arm water maze in both Dp16 and WT mice compared to placebo. Dp16 mice also showed abnormal astrocyte morphology, increased percent area of GFAP staining in the hippocampus, clustering of astrocytes in the hippocampus, and reduced numbers of calretinin-positive interneurons in the entorhinal cortex and subiculum, and all of these brain pathologies were improved by GM-CSF treatment. These findings suggest that stimulating and/or modulating inflammation and the innate immune system with GM-CSF treatment may enhance cognition in both people with DS/ID and in the typical aging population.

Innate Immune System Activation and Neuroinflammation in Down Syndrome and Neurodegeneration: Therapeutic Targets or Partners?

Innate immune system activation and inflammation are associated with and may contribute to clinical outcomes in people with Down syndrome (DS), neurodegenerative diseases such as Alzheimer's disease (AD), and normal aging. In addition to serving as potential diagnostic biomarkers, innate immune system activation and inflammation may play a contributing or causal role in these conditions, leading to the hypothesis that effective therapies should seek to dampen their effects. However, recent intervention studies with the innate immune system activator granulocyte-macrophage colony-stimulating factor (GM-CSF) in animal models of DS, AD, and normal aging, and in an AD clinical trial suggest that activating the innate immune system and inflammation may instead be therapeutic. We consider evidence that DS, AD, and normal aging are accompanied by innate immune system activation and inflammation and discuss whether and when during the disease process it may be therapeutically beneficial to suppress or promote such activation.

Safety and efficacy of sargramostim (GM-CSF) in the treatment of Alzheimer's disease.

INTRODUCTION: Inflammatory markers have long been observed in the brain, cerebrospinal fluid (CSF), and plasma of Alzheimer's disease (AD) patients, suggesting that inflammation contributes to AD and might be a therapeutic target. However, non-steroidal anti-inflammatory drug trials in AD and mild cognitive impairment (MCI) failed to show benefit. Our previous work seeking to understand why people with the inflammatory disease rheumatoid arthritis are protected from AD found that short-term treatment of transgenic AD mice with the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) led to an increase in activated microglia, a 50% reduction in amyloid load, an increase in synaptic area, and improvement in spatial memory to normal. These results called into question the consensus view that inflammation is solely detrimental in AD. Here, we tested our hypothesis that modulation of the innate immune system might similarly be used to treat AD in humans by investigating the ability of GM-CSF/sargramostim to safely ameliorate AD symptoms/pathology. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in mild-to-moderate AD participants (NCT01409915). Treatments (20 participants/group) occurred 5 days/week for 3 weeks plus two follow-up (FU) visits (FU1 at 45 days and FU2 at 90 days) with neurological, neuropsychological, blood biomarker, and imaging assessments. RESULTS: Sargramostim treatment expectedly changed innate immune system markers, with no drug-related serious adverse events or amyloid-related imaging abnormalities. At end of treatment (EOT), the Mini-Mental State Examination score of the sargramostim group increased compared to baseline (P = .0074) and compared to placebo (P = .0370); the treatment effect persisted at FU1 (P = .0272). Plasma markers of amyloid beta (Aß40 [decreased in AD]) increased 10% (P = .0105); plasma markers of neurodegeneration (total tau and UCH-L1) decreased 24% (P = .0174) and 42% (P = .0019), respectively, after sargramostim treatment compared to placebo. DISCUSSION: The innate immune system is a viable target for therapeutic intervention in AD. An extended treatment trial testing the long-term safety and efficacy of GM-CSF/sargramostim in AD is warranted.

Amylin, Aß42, and Amyloid in Varicella Zoster Virus Vasculopathy Cerebrospinal Fluid and Infected Vascular Cells.

BACKGROUND: Varicella zoster virus (VZV) vasculopathy is characterized by persistent arterial inflammation leading to stroke. Studies show that VZV induces amyloid formation that may aggravate vasculitis. Thus, we determined if VZV central nervous system infection produces amyloid. METHODS: Aß peptides, amylin, and amyloid were measured in cerebrospinal fluid (CSF) from 16 VZV vasculopathy subjects and 36 stroke controls. To determine if infection induced amyloid deposition, mock- and VZV-infected quiescent primary human perineurial cells (qHPNCs), present in vasculature, were analyzed for intracellular amyloidogenic transcripts/proteins and amyloid. Supernatants were assayed for amyloidogenic peptides and ability to induce amyloid formation. To determine amylin's function during infection, amylin was knocked down with small interfering RNA and viral complementary DNA (cDNA) was quantitated. RESULTS: Compared to controls, VZV vasculopathy CSF had increased amyloid that positively correlated with amylin and anti-VZV antibody levels; Aß40 was reduced and Aß42 unchanged. Intracellular amylin, Aß42, and amyloid were seen only in VZV-infected qHPNCs. VZV-infected supernatant formed amyloid fibrils following addition of amyloidogenic peptides. Amylin knockdown decreased viral cDNA. CONCLUSIONS: VZV infection increased levels of amyloidogenic peptides and amyloid in CSF and qHPNCs, indicating that VZV-induced amyloid deposition may contribute to persistent arterial inflammation in VZV vasculopathy. In addition, we identified a novel proviral function of amylin.

Recruiting the innate immune system with GM-CSF to fight viral diseases, including West Nile Virus encephalitis and COVID-19.

As the coronavirus disease 2019 (COVID-19) pandemic grows throughout the world, it is imperative that all approaches to ameliorating its effects be investigated, including repurposing drugs that show promise in other diseases. We have been investigating an approach to multiple disorders that involves recruiting the innate immune system to aid the body's healing and regenerative mechanism(s). In the case of West Nile Virus encephalitis and potentially COVID-19, the proposed intervention to stimulate the innate immune system may give the adaptive immune response the necessary time to develop, finish clearing the virus, and provide future immunity. Furthermore, we have found that GM-CSF-induced recruitment of the innate immune system is also able to reverse brain pathology, neuroinflammation and cognitive deficits in mouse models of Alzheimer's disease and Down syndrome, as well as improving cognition in normal aging and in human patients with cognitive deficits due to chemotherapy, both of which exhibit neuroinflammation. Others have shown that GM-CSF is an effective treatment for both bacterial and viral pneumonias, and their associated inflammation, in animals and that it has successfully treated pneumonia-associated Acute Respiratory Distress Syndrome in humans. These and other data strongly suggest that GM-CSF may be an effective treatment for many viral infections, including COVID-19.

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses.

Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek® ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream® ). High-performance liquid chromatography followed by nano-flow liquid chromatography-mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5-fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre-analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano-flow liquid chromatography-mass spectrometry.

Varicella-Zoster Virus Infection of Primary Human Spinal Astrocytes Produces Intracellular Amylin, Amyloid-ß, and an Amyloidogenic Extracellular Environment.

BACKGROUND: Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid. METHODS: Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-ß [Aß]) and amyloid, as well as extracellular amylin, Aß, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aß42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined. RESULTS: VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aß, and amyloid. No differences in extracellular amylin, Aß40, or Aß42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aß42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aß42 aggregation. CONCLUSIONS: VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression.