Mimicry of Central-Peripheral Immunity in Alzheimer's Disease and Discovery of Neurodegenerative Roles in Neutrophil

Neuroinflammatory roles of central innate immunity in brain parenchyma are well-regarded in the progression of neurodegenerative disorders including Alzheimer's disease (AD), however, the roles of peripheral immunity in central nervous system (CNS) diseases are less clear. Here, we created a microfluidic environment of human AD brains: microglial neuroinflammation induced by soluble amyloid-beta (Abeta), a signature molecule in AD and employed the environment to investigate the roles of neutrophils through the central-peripheral innate immunity crosstalk. We observed that soluble Abeta-activated human microglial cells produced chemoattractants for neutrophils including IL6, IL8, CCL2, CCL3/4, CCL5 and consequently induced reliable recruitment of human neutrophils. Particularly, we validated the discernable chemo-attractive roles of IL6, IL8, and CCL2 for neutrophils by interrupting the recruitment with neutralizing antibodies. Upon recruitment, microglia-neutrophils interaction results in the production of inflammatory mediators such as MIF and IL2, which are known to up-regulate neuroinflammation in AD. We envision that targeting the crosstalk between central-peripheral immune community is a potential strategy to reduce immunological burdens in other neuroinflammatory CNS diseases.


INTRODUCTION
Alzheimer's disease (AD) is the most common form of dementia. It affects more than 35 million people worldwide (1,2). It is characterized by neuropathological features including amyloid-beta (Abeta) plaques, neurofibrillary tangles, and neuroinflammation that lead into synaptic dysfunction and a progressive deterioration of cognitive functions (2)(3)(4)(5). A growing body of evidence suggests that neuroinflammation is the critical hallmark of AD and central innate immune cells (microglia and astrocytes) have the major roles of neuroinflammation in a central nervous system (CNS) (6)(7)(8)(9). Particularly, microglia, a macrophage resident in brains, produce both beneficial neuroinflammatory responses and detrimental neurotoxic effects in the progression of AD (10)(11)(12). Therefore, the regulation of microglial activation has been proposed as a potential therapeutic approach in AD treatment (13).
In contrast to the field's increasing understanding of the neuroinflammatory roles of central innate immunity in AD, comparatively little is known about their interactions with peripheral immunity. Peripheral immune cells including Tcells, B-cells, monocytes, and neutrophils, have been found in the brains of AD human patients and corresponding AD animal models (14)(15)(16)(17). Among them, neutrophils are the body's most abundant and frontline immune cells, respond with both central and peripheral immune effectors during the progression of AD (17). The migration of neutrophils is initiated by chemokine-triggered activation. Neutrophils actively crawl through the endothelium and accumulate in tissues where they released neutrophil extracellular traps (NETs) and IL-17 which are markers of sterile inflammation and toxic cytokines, respectively. Neutrophil depletion or inhibition of trafficking via LFA-1 blockade reduced Alzheimer's diseaselike neuropathology and improved memory in mice showing cognitive dysfunction in transgenic Alzheimer's disease mouse models (18). However, the mechanism study of neutrophil recruitment and induction of neuropathological changes of AD still remains unclear. The challenge of the study is due to, in part, the limited readouts in vivo, the similar profiles of peripheral inflammatory roles compared to the central immunity by microglia and astrocytes, and inconsistent observation and/or variation in AD animal models. Recently, microfluidic organ-onchips demonstrated successfully to provide feasible, regulated, accessible microenvironments to study multicellular interaction and/or multiorgan behaviors in quantitative, reproducible, and large-scaled manners (19).
Here, we present a microfluidic system mimicking centralperipheral innate immunity in AD for the first time. We identified IL6, IL8, CCL2, CCL3, and CCL5 as mediators released by soluble Abeta-stimulated microglia. We validated their contribution to neutrophil recruitment by inhibiting with neutralizing antibodies. Moreover, the recruited neutrophils induce the secretion of inflammatory factors such as MIF and IL2, which may further stimulate the glia cells. Taken together, these studies suggest that central innate immune cells have a critical role in recruiting peripheral immune cells and mitigating AD pathogenesis.

Replicated AD Microenvironment by Using a Chemotactic Microfluidic Platform
We established an in vitro system to study the crosstalk between microglia and neutrophils in the context of AD (Figure 1). The system consists of two compartments: central circular and surrounding annular compartments representing regions of cerebral tissues and blood streams, respectively. The two compartments are connected with microchannels (10 × 5 × 500 µm 3 in width, height, and length) representing a mechanical barrier between two regions, which only chemoattracted neutrophils can penetrate through. The long and thin migration channels serve as mechanical constraints to avoid spontaneous entrance of inactivated neutrophils and activated microglia. Firstly, we cultured human adult microglia cells in the central compartment in addition of soluble Abeta for 3 days to induce the release of microglial inflammatory mediators in the context of AD. Later, we plated freshly isolated human neutrophils on the annular compartment under effective gradients of microglial soluble factors from the central compartment. We tested 200 independent conditions on eight arrays of twenty-five devices in single well plates and measured neutrophil mobility at a single cell resolution in a real-time.

Assessment of Microglial Neuroinflammation Activated by Soluble Abeta
We assessed the status of microglial inflammation by measuring inflammatory cytokines. We found that proinflammatory mediators, CCL2, IL-6, and IL-8 increase significantly by 1.4-, 1.9-, and 2.3-folds, respectively and chemokines, e.g., CCL3/4, CCL5 released from activated microglia only under stimulation of Abeta (Figures 2A,B). Secretion of anti-inflammatory markers, such as IL-1RA, IL-4, IL-10, and TGF-ß were below the limit of detection. These data collectively suggest that soluble Abeta induces microglial proinflammation quantified by using our microfluidic platform.
To identify the microglial activation, Abeta-treated microglial cells were monitored for three days using a time-lapse imaging microscopy. At the early 24 h, microglia remained as branched filopodia in all directions, morphological features typically associated with "resting" microglia ( Figure 2C, upper, −Abeta). At 48 h after cell seeding and Abeta incubation, microglia cells became larger (Figure 2C, lower, +Abeta) and transformed into an amoeboid shape while the length and area of microglial somata increased in all directions (Figures 2C,D). In addition, microglia treated with soluble Abeta showed up-regulation of microglial activation markers such as CD11b and CD68 (Figures 2C,E, Supplementary Figure 1).

Validation of Microfluidic Chemotaxis for Neutrophils
We induced gradients of chemoattractants for neutrophils from the central toward the annular compartments and validated the design of microchannels effective for measuring chemotactic activities of neutrophils. We defined a neutrophil recruitment index, R.I. (Figure 3A, Supplementary Figure 2), representing the fraction of neutrophils recruited to the central compartment by chemoattractants. We assessed the R.I. values under various conditions (fMLP: N-formyl-methionyl-leucylphenalanine, Abeta, and culturing medium) and observed discernable neutrophil recruitment only in addition of fMLP to the central compartment (Figures 3A,B). In this study, we used fMLP for a positive control as fMLP triggers p38 pathway overriding PI(3)K and is certain for effectiveness of neutrophil chemoattraction (20). However, neutrophil recruitment was not discernable with Abeta only and the medium during the observation for 6 h. The results successfully demonstrate that our platform can engage neutrophils with chemical cues actively while avoiding spontaneous entrance of neutrophils with the mechanical barriers. Neutrophil recruitment reached  (1) is added to a central microglial microcompartment which represents a brain parenchyma and stimulates microglia to secrete neutrophil chemoattractants (2). As a result, neutrophils in an annular microcompartment migrate across a mechanical barrier (3) representing the endothelial barrier and release additional proinflammatory factors (4) in the microglial compartment after recruitment.
the peak activity at 18 ± 5 µm/min within the first 6 h (Supplementary Figure 3) and the activity was proportional to the concentration of fMLP, such as 1.6 times faster at 10 nM fMLP compared to 1 nM fMLP ( Figure 3B).
We further analyzed the migratory activities of human neutrophils in a single cellular level. We found that 88.5 ± 3.8% of active neutrophils migrated forwards and 11.5 ± 1.7% migrated backward 10 nM fMLP, whereas 82.2 ± 3.4% migrated forward and 17.8 ± 2.8% backward 1 nM fMLP showing the persistent migration (Figures 3C,D). However, too much fMLP of 1 µM resulted in a 63 % reduction in the total fraction of migrating cells compared to 10 nM (Supplementary Figure 4).

Inducement of Neutrophil Recruitment by A-Beta Activated Microglia
To probe the crosstalk between microglia and neutrophils in the context of AD, we cultured microglia cells in the central compartment at various cell numbers (5,000, 10,000, 20,000 cells per device) with the treatment of soluble Abeta42 (22 nM) for 48 h, and then introduced neutrophils to the surrounding compartment. Microglia cells were pre-labeled with a red membrane dye to monitor their morphogenesis. Microglia were incubated alone for 24 h with reduced 1% FBS PrigrowIII medium without Abeta. Followed by microglial incubation with soluble Abeta, human neutrophil labeled with a green membrane dye were plated on the annular compartment. Neutrophil immediately started to migrate toward the central compartment of microglia of 5,000 with Abeta ( Figure 4A). With the increase of microglia in the central compartment, neutrophil migration became more discernable and the number of recruited neutrophils increases proportional to the microglia, presuming that A-beta activated microglia release neutrophil chemoattractants. The responding neutrophils showed the enhanced polarity and distinct trailing edge toward microglial compartment with Abeta ( Figure 4A, Supplementary Figure 5, Supplementary Videos 1, 2). However, neutrophil migration and the basal cell polarity were not noticeable in the conditions of microglia only, Abeta only, nor culture medium only ( Figure 4B,  Supplementary Video 3).
To confirm neutrophil activation, neutrophils co-cultured with microglial cells with or without Abeta for 5 min, were thoroughly washed away and the numbers of adherent neutrophils were counted. It showed that neutrophils cocultured with Abeta-activated microglia enhanced cellular adhesion to the surface, confirming the neutrophil activation (Supplementary Figure 6). (C) Membrane-staining (red) shows the discernable change in microglial morphology with Abeta stimulation from a branch-ramified shape (resting) to an amoeboid shape (activated). A microglia activation marker of CD11b (green) was upregulated with Abeta stimulation. Both the membrane area (D) and the amounts of expressed CD11b (E) increase in the activated microglia with Abeta. n device = 3, n cell = 150. Scale bars: 30 µm. All parameters are presented as mean ± SEM.

Assessment of Neutrophil Inflammation Activated by Microglial Soluble Factors
To assess the roles of recruited neutrophils in the progression of AD pathology, we investigated neutrophil inflammation. We compared the morphology of neutrophils resting in the annular compartment (Figure 5A left) and activated in the central compartment (Figure 5A right). Microglia-activated neutrophils extended their bodies by 1.8 folds (Figure 5B). In a 37-human cytokine assay, we found two additional cytokines, MIF and IL2 (1.7-fold and 1.67-fold, respectively) from the recruited neutrophils compared to A-beta activated microglia only (Figures 5D,E). The other 5-soluble factors from Abeta-activated microglia remained unchanged after neutrophil recruitment.
Other key proinflammatory and neurotoxic cytokines including interleukin 1β (IL-1β), interleukin 17 and tumor necrosis factor alpha (TNF-alpha) were below the detection limit of the kit used ( Figure 5C). This measurement suggests that the recruitment of neutrophils may exacerbate microglial neuroinflammation activated by Abeta in AD.

DISCUSSION
Microglial cells are cerebral innate immune cells having neuroinflammatory roles for AD pathology. Based on our previous study of neutrophil accumulation on Abeta plaques in AD animal models (17), we hypothesized that neutrophils, the frontline of peripheral immune cells are engaged with neuroinflammation not directly with Abeta but mediated by microglia. Our microscale platforms enabled comprehensive studies of the crosstalk between central and peripheral immunity and overcame significant limitations of previous studies (21)(22)(23). For example, the study of neutrophil involvement in previous models lack control over the complex cellular interactions among multiple types of brain cells and leukocytes (23). Another The effectiveness of microglia-derived chemokines was confirmed by showing the reduced neutrophil accumulation to Abeta-stimulated microglia in addition of neutralizing antibodies for individual chemokines. Statistical significance is denoted by **P < 0.20, ***P < 0.10, ****P < 0.01 n device = 4 (B-E), n device = 3, n neutrophil = 250. Scale bars: 500 µm (upper right), 50 µm (lower pannel). All parameters are presented as mean ± SEM. *not detected.
key feature of our platform is the capability of isolating activated neutrophils from resting neutrophils, which allows selective monitoring the activities of interest in the AD progression (21) and on-chip investigation of drug efficacy, such as, inhibitor treatment against neutrophil recruitment and microglial inflammation (23).
One of the intriguing findings is that several mediators contribute to the robust recruitment of human neutrophils, which is similar to the findings in AD mouse models (24,25). CCL3 has been found to be associated with AD (26,27), reported as expressed by neurons and microglia in post-mortem brains from AD patients (28) and upregulated in experimental models mimicking both amyloid and Tau deposits (29). In addition, a CCL3 polymophism has been associated with AD (30) and CCL3 secretion was found dependent on apoplipoprotein ε4, the greatest genetic risk factor for sporadic AD (31). CCL5 is most commonly involved in AD neurodegenerative processes, which regulates the activities of normal T cells. During AD pathogenesis, an elevated level of the glial CCL5 chemokine is observed in the microcirculatory system of the brain (32). Its elevated levels contribute to the recruitment of immunecompetent cells, which occurs concurrently with increased rates of neuronal deaths (33). In addition to these factors, we identified two proinflammatory-specific proteins released during the crosstalk in between activated microglia and neutrophils. IL2 and MIF are known to enhance human-neutrophil inflammatory activities (25). Especially, MIF (macrophage inhibitor factor), was observed in serial brain sections of transgenic APP AD mice and stained MIF immunolabeling in neutrophils in association with Abeta plaques in the transgenic mouse brain sections (34). In addition, functional studies in murine and human neuronal cell lines revealed that Abeta-induced toxicity could be reversed significantly by a small amount of MIF, showing the beneficial effect in AD mediated by microglia (25). Finally, MIF levels in the brain cytosol and cerebrospinal fluid (CSF) of AD patients showed marked increase compare to and age-matched controls. MIF are regarded as an important proinflammatory mediator by observing in the brain cytosol and cerebrospinal fluid (CSF) of AD patients and affecting tau hyper-phosphorylation, which is found in the late stage of AD (35). IL-2 was found to attenuate Abeta pathology and synaptic impairment in AD mice by engaging astrocytes for clearing Abeta and consequently improving synaptic plasticity (36).

CONCLUSION
We developed a microfluidic platform to explore the crosstalk between microglia and neutrophils in the context of AD and suggested how peripheral immune cells, neutrophils are involved in AD pathology mediated by Abeta-activated microglia to enhance the beneficial neuroinflammation. Our platform will serve as a physiologically relevant in vitro AD brain model for the study of neuroinflammation and a reliable tool for the validation of therapeutic strategy for neuroinflammatory immunotherapy.

Cell Lines, Media, and Reagents
Immortalized human adult microglia cells (SV40-microglia) were created by a company (ABM Inc., Montreal, Canada). The cells were plated onto T25 cell culture flasks (BD Biosciences, San Jose, CA, USA) and maintained in Prigrow III Medium (ABM Inc) supplemented with 10 % FBS (Life Technologies) and 1 % Pen/Strep (Invitrogen) in a CO 2 cell culture incubator.
The cell culture medium was changed every 3 days until cells were confluent.
For neutrophil isolation, human peripheral blood samples from healthy volunteers, aged 18 years and older, were collected by a company (Zen-Bio Inc., Research Triangle Park, NC, USA). Peripheral blood was drawn in a 10-mL tube containing a final concentration of 5 mM EDTA (Vacutainer; Becton Dickinson). Nucleated cells were isolated using a HetaSep gradient, followed by the EasySep Human Neutrophil Enrichment Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's protocol.

Microfluidic Device Fabrication
Negative photoresists, SU-8 5 and SU-8 100 (MicroChem, Newton, MA, USA), were sequentially patterned using standard lithography on a 4" silicon wafer to create a mold for cell migration channels of 5 µm in height and chemokine compartments of 100 µm in height. A mixture of a base and a curing agent with a 10:1 weight ratio (SYLGARD 184 A/B, Dow corning, Midland, MI, USA) was poured onto the SU-8 mold and cured for 1 h at room temperature under vacuum and, subsequently, cured for more than 3 h in an oven at 80 • C. The cured poly dimethyl-siloxane (PDMS) replica was peeled off from the mold and holes were punched for fluid reservoirs. Arrayed holes were also laser-cut (Zing 24, Epilog Laser, Golden, CO, USA) into an acrylic plate of 6 mm in thickness. The machined membrane and the plate were glued together using uncured PDMS and incubated at 80 • C overnight. This assembly was irreversibly bonded first to the PDMS replica using oxygen plasma at 50 mW, 5 cm, for 30 s (PX-250, March Plasma Systems, Petersburg, FL, USA), and later to a glass-bottomed single well plate (P384G-1.5-10872-C, MatTek Corp., Ashland, MA, USA). Immediately after the bonding, 10 µL of poly (l-lysine) solution (PLL, M.W. 70,000-150,000, 1.0 mg/mL, Sigma-Aldrich Co. LLC) was injected into each platform and incubated for 2 h at a room temperature to promote cellular adhesion. PLL-treated surface was rinsed with autoclaved and 0.2 µm filtered water (AM9920, Life Technologies).

Membrane Staining of Microglia
Before the experiment, cells were washed using a serum-free medium and the cell membrane was labeled with red fluorescent dyes (PKH26PCL Red Fluorescent Cell Linker, Sigma-Aldrich). Briefly, after centrifugation (1,200 rpm.g for 5 min), the cells were re-suspended in 1 mL of Diluent C (G8278, Sigma-Aldrich) and immediately mixed with 4 µL of dye solution. The cell/dye mixture was incubated at room temperature for 4 min and periodically mixed by pipetting to achieve a bright, uniform, and reproducible labeling. After the incubation, the staining was stopped by adding an equal volume (1 mL) of 1 % BSA in PBS and incubating for 1 min to remove excess dye. Unbound dye was washed by centrifugation and suspending cells in culture medium (10 6 cells/mL). Ten µL of the cell suspension was injected into each platform and 100 µL of a culturing medium was added into side and central extra wells. The loaded micro-devices were then incubated at 37 • C supplied with 5% CO 2 .

Isolation and Membrane Staining of Neutrophils
Human neutrophils were isolated from fresh human blood samples within 2 h of blood collection by using EasySepHuman Neutrophil Enrichment Kits (STEMCELL Technologies) and following the manufacturer's instructions. After isolation, the neutrophils were washed using 1x PBS, and immediately mixed with 4 µL of dye solution (PKH67 Green Fluorescent Cell Linker, Sigma-Aldrich). The cell/dye mixture was incubated at room temperature for 4 min and periodically mixed by pipetting to achieve a bright, uniform, and reproducible labeling. After the incubation, the staining was stopped by adding an equal volume (1 mL) of 1% BSA in PBS and incubating for 1 min to remove excess dyes. Unbound dyes were washed by centrifugation and suspending cells in culture medium (10 6 cells/mL). Ten µL of the cell suspension was injected into each annular compartment. The loaded microfluidic-device were then incubated at 37 • C supplied with 5% CO 2 in 10 min for the cell attachment and used later in microscope.

Time-Lapse Imaging
To track individual cells at a high throughput, we prepared 24 devices on a single-well plate and time-lapse imaged the entire devices in a large-area mode of 8 × 8 mm 2 with a 15 % stitching by using a fully automated microscope (Ti-E/NIS Elements, Nikon Inc.) integrated with a heated incubating stage (INU-TIZB-F1, Tokai Hit Co., Ltd., Shizuoka, Japan), set at 37.5 • C and 5% CO 2 with moderate humidity. We imaged cells at every 15 min for 6 h in three channels-FITC, Cy5, and a bright fieldwith a 10x objective lens and a perfect focusing system in a phase contrast mode.

Immunostaining
Before immunofluorescent staining, we rinsed the cells twice with DPBS. To fix, cells were incubated in fresh 4% paraformaldehyde aqueous solution (157-4, ElectronMicroscopy Sciences) for more than 15 min at RT followed by rinsing twice with DPBS. To permeabilize, cells were incubated in 0.1% Triton X-100 in PBST (phosphate buffered saline with 0.1% tween R 20) for 15 min at RT. To block, cells were incubated in 3% human serum albumin for overnight in PBST at 4 • C. After incubating with the primary antibody solutions for 24 h at 4 • C, the cells were washed five times. The following antibody (and dilutions) were used: anti-cd11b (1:100, Life Technologies).

Multiple Human Cytokine Array Kit
A human cytokine array kit was purchased from R&D Systems (Catalog # ARY005, Human Cytokine 37-membrane kit array) and utilized following the protocol provided by the manufacturer. Capture antibodies have been spotted in duplicate on nitrocellulose membranes. Cell culture supernatants are mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture is then incubated with the array. Any cytokine/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Streptavidin-Horseradish Peroxidase and chemiluminescent detection reagents are added. A signal is produced, proportional with the amount of cytokine bound. Chemiluminescence is detected in the same manner as a Western blot and the resulting cytokine release profiles were quantified with ImageJ.

Microglial Stimulation by Soluble Abeta
After stabilizing microglia in the central compartment, the culturing medium was replaced by a medium containing Abeta 42 (PAM-4349-v, Amyloid Beta-Protein, Peptides International, Inc.) at 22 nM of 2% FBS and microglia were incubated for 72 h.

Statistical Analysis
Data, expressed as mean ± SEM, were compared using either a two-tailed Student's t-test when comparing two groups/conditions or one-way ANOVA followed by a post hoc test when comparing 3 or more groups/conditions. P < 0.05 was considered significant.

DATA AVAILABILITY
The datasets generated for this study are available on request to the corresponding author.

AUTHOR CONTRIBUTIONS
JP and HC designed, fabricated, and tested devices, designed experiments, performed immunostaining, statistical quantification, generated figures, wrote and edited the manuscript. JP, SB, IM-J, DI, and HC conceived the ideas and edited the manuscript. All authors read and edited the manuscript extensively.

ACKNOWLEDGMENTS
We appreciate Dr. You Jung Kang (UNCC) for critical reviewing our manuscript.