Peripheral Low Level Chronic LPS Injection as a Model of Neutrophil Activation in the Periphery and Brain in Mice

Lipopolysaccharide-induced (LPS) inflammation is used as model to understand the role of inflammation in brain diseases. However, no studies have assessed the ability of peripheral low-level chronic LPS to induce neutrophil activation in the brain. Subclinical levels of LPS were injected intraperitoneally into mice to investigate impacts on neutrophil frequency and activation. Neutrophil activation, as measured by CD11b expression, peaked in the periphery after 4 weeks of weekly injections. Neutrophil frequency and activation increased in the periphery 4–12 hours and 4–8 hours after the fourth and final injection, respectively. Increased levels of G-CSF, TNFa, IL-6, and CXCL2 were observed in the plasma along with increased neutrophil elastase, a marker of neutrophil extracellular traps, peaking 4 hours following the final injection. Neutrophils and neutrophil activation were increased in the brain of LPS injected mice when compared to saline-injected mice 4 hours and 4–8 hours after the final injection, respectively. These results indicate that subclinical levels of peripheral LPS induces neutrophil activation in the periphery and brain. This model of chronic low-level systemic inflammation could be used to understand how neutrophils may act as mediators of the periphery-brain axis of inflammation with age and/or in mouse models of neurodegenerative or neuroinflammatory disease.


Introduction
Neuroin ammation and immune activation are widely accepted as a signi cant contributing factor in the progression and degeneration of patients with Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), and other neurodegenerative diseases [1][2][3] .Moreover, neuroin ammation and immune activation are present before the onset of symptoms 4,5 .An increase in proin ammatory cytokines, such as tumor necrosis factor-alpha (TNFα), and interleukin-1b (IL-1b), and caustic molecules released during in ammation, such as nitric oxide (NO) and myeloperoxidase (MPO), have been associated with a worse prognosis across multiple neurodegenerative diseases 2,6 .Neuroin ammatory markers such as these, along with immune cells, including neutrophils and microglia, are potential therapeutic targets and potential markers for early detection for multiple neurodegenerative diseases.
Multiple studies have correlated the onset and progression of neurodegenerative diseases with proin ammatory factors and immune cell activation and accumulation [7][8][9] .Transcriptomics studies have demonstrated an increase in pro-in ammatory differentially expressed genes (DEGs) in neurodegeneration, and genes involved in neutrophil activation and adhesion have been identi ed in areas with blood brain barrier (BBB) disruption and associated with disease progression 7,[9][10][11] .Neutrophils are of therapeutic interest because they are the most abundant circulating leukocyte and have been shown in both human and mouse models to be associated with a worse prognosis in neurodegenerative diseases [12][13][14] .Further, humanized AD mouse model studies have shown an increase in short-term spatial memory and a decrease in capillary blood ow stalling when targeting neutrophil adhesion or accumulation [14][15][16] .
Neutrophils are essential to ght invaders and clear debris 17 .Neutrophils have three main ways in which they contain pathogens, including phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs).While neutrophils are important in ghting infection and repairing tissues, they can also cause damage to tissues through their release of antimicrobial peptides, enzymes meant to degrade extracellular matrix, and reactive oxygen species [18][19][20] .Neuroin ammation plays a role in neurodegenerative diseases, including both increased in ammatory signaling and dysregulation of resident glial cells in the brain and the in ltration of leukocytes from the periphery to the central nervous system (CNS) 21,22 .Neutrophils are not commonly found in a healthy brain because of their exclusion by the blood brain barrier (BBB) 17,23 .However, the BBB is disrupted in events such as an ischemic stroke [23][24][25] ) and traumatic brain injury 24,25 , in brain diseases such as Alzheimer's Disease (AD) 18,26 and Parkinson's Disease (PD) 27 , and in viral 25,28 , fungal and parasite infection 17 .
Although neutrophils have been documented in the brain in several neuroin ammatory conditions and diseases, such as stroke, multiple sclerosis, and AD, the role of peripheral in ammation in neutrophil in ltration and activation in the brain is unclear 15,18,26,29,30 .
Administration of high levels of lipopolysaccharide (LPS) into the periphery as a model of sepsis results in neutrophil in ltration into the brain [31][32][33] .However, the level of in ammation that is induced in sepsis models is not representative of the chronic low-level in ammation that occurs with age and likely contributes to age-related neurological disease.To date, there are no models of chronic peripheral in ammation and neutrophil brain in ltration and activation.With this pilot study, we demonstrate that low-level chronic in ammatory stimuli (LPS) in the periphery induces neutrophil in ltration and activation in the brain.This model can be used in future studies to investigate the role of neutrophils in neuroin ammation and related brain disorders; how peripheral mediators and genetic risk factors for disease may alter neutrophil responses to these stimuli; and if neutrophils are mechanistic drivers of the subsequent neuroin ammation and neurodegeneration.

Results
Determining the Optimal Duration of Peripheral Neutrophil Activation with Chronic Low-Level LPS LPS is a component of the outer membrane of gram-negative bacteria that binds to toll-like receptor 4 (TLR-4) on immune cells.It is commonly used to induce in ammation in the periphery and brain.However, studies differ in their dose, frequency, and route of administration 3 .Most studies have investigated acute exposure to LPS, administering LPS intraperitoneally (IP) daily for 5-7 days at doses of 0.25-1 mg/kg.Chronic exposure studies have demonstrated microglial and astrocyte activation and memory impairment with similar doses administered IP once or twice weekly for 4-6 weeks 34 .However, no studies investigating chronic LPS-induced neuroin ammation and potential cognitive consequences conducted to date have investigated the induction of neutrophil activation in the periphery or brain.As such, we sought to investigate 0.5 mg/kg of LPS once per week as a potential model of neutrophil in ammation in the brain.This LPS exposure represents the threshold of physiological changes in mice, and can be used to model low-level chronic in ammation, as may occur in persons with microbiome alterations or frequent GI or respiratory infections common in elderly individuals 35 .We examined neutrophil frequency and neutrophil activation, via expression of CD11b, in response to this low level chronic LPS.CD11b is important for neutrophil migration and adhesion and is known to be increased in a TLR-4 dependent manner with LPS stimulation of neutrophils 36 .Importantly, CD11b expression is increased in persons with AD and correlates with disease severity, making it a relevant marker of neutrophil activation in models of neuroin ammation and neurodegeneration 37 .
To determine the optimal duration of chronic LPS injections, male and female mice were IP injected with 0.5 mg/kg of LPS or saline once per week for 8 weeks, and blood was collected at baseline (pre-injection), 12 hours after the 4th injection, and 12 hours after the 8th injection (Fig. 1A).Following injections, neutrophil activation was assessed via ow cytometry.CD11b expression was highest on neutrophils after 4 weeks of injections, suggesting a potential for increased tolerance between 4 weeks and 8 weeks of injections (Fig. 1B).
Based on these data, the next group of mice was injected for 4 weeks to investigate the timing of neutrophil activation in the brain following the nal LPS injection (Fig. 1C).Blood was drawn at baseline and blood and brain tissue were collected 4 hours, 8 hours, or 12 hours after the 4th and nal injection (Fig. 1C).As expected with this level of LPS, there were no signi cant changes in the body weight in either the saline-or LPS-injected mice throughout the experiment nor signi cant differences in body weight between the saline-and LPSinjected groups at any timepoint (Fig. 1D).
Peripheral Neutrophil Activation Following Low-Level Chronic LPS Following 4 weeks of IP LPS injections, blood neutrophil frequency and activation were assessed via ow cytometry (Supplementary Fig. 1).No increases in blood neutrophil frequency or activation were observed in the control mice receiving saline.Neutrophil levels increased 4 hours after the nal LPS injection, with signi cant increases observed 8 and 12 hours following LPS injection (Fig. 2A).
Peripheral cytokines and chemokines with the ability to regulate neutrophil activation, maturation, and recruitment were measured in plasma via a LEGENDPlex to assess the potential role of soluble mediators of neutrophil activation.Of 12 total analytes measured, 5 showed a statistically signi cant change between the LPS and saline groups.G-CSF, TNFa, IL-6, and CXCL2, potential mediators of neutrophil activation, maturation, recruitment, increased 4 hours after LPS injection, with G-CSF remaining elevated zing by 8 hours post LPS administration (Fig. 2B).The anti-in ammatory mediator IL-10 was also increased 4 hours following the nal LPS injection, suggesting a potential compensatory response early following LPS injection 4 .Finally, neutrophil elastase (NE), a marker of neutrophil extracellular trap release 5,6 , was measured via ELISA in plasma.NE was signi cantly increased 4 hours following LPS injection and remained elevated but lower 8 and 12-hours following post LPS 2C) frequency and neutrophil activation in the blood that peaked 8 hours after LPS administration.Soluble mediators of neutrophil responses and neutrophil activation begin to resolve 8-12 hours following LPS.However, neutrophil frequency remains elevated through 12 hours post-LPS, as neutrophils would not yet be anticipated to undergo homeostatic apoptosis by 12 hours following release into the periphery and apoptosis could be further delayed in response LPS 38 .
Brain Neutrophil Activation Following Low-Level Chronic LPS Brain neutrophils were assessed after 4 weeks of LPS injections at 4 hours, 8 hours, and 12 hours following the nal injection.Brain neutrophils were identi ed in sagittal sections via myeloperoxidase (MPO) staining using an antibody that has been validated to stain for neutrophils in mouse and human brain tissue (Fig. 3A) 18 .Neutrophils were counted across entire sagittal sections and were signi cantly elevated in the brain 4 hours following the nal LPS injection.Neutrophil frequencies were higher but not signi cantly higher than saline-injected mice at 4 hours and 8 hours following LPS administration, as neutrophils were higher in the 8-hour and 12-hour groups of salineinjected mice.Of note, increased peripheral neutrophil activation in the 8-hour and 12-hour groups of saline-injected mice were also observed at time of collection, suggesting a potential for neutrophil activation and brain in ltration with handling and injection-induced in ammation (Fig. 2A).Increased concentrations of neutrophils in the brain were observed mainly in the cortex (Fig. 3B), which remained signi cantly elevated in LPS-injected mice compared to saline-injected mice through 12 hours post LPS (Fig. 3B).Neutrophil CD11b expression measured on neutrophils isolated from brain tissue was signi cantly elevated 4 hours and 8 hours following the nal LPS injection, as was observed in blood neutrophils (Fig. 3C).Taken together these data indicate that neutrophils in ltrate the cortex and demonstrate increased activation in brain tissue by 4 hours following peripheral low-level LPS injection.

Discussion
Despite LPS as a common model for peripheral immune activation and subsequent neuroin ammation, no studies have investigated the role of neutrophils in mediating the periphery-brain axis of in ammation induced by chronic low levels of LPS.Neutrophils in the periphery and vasculature could contribute to neuroin ammation and neurodegeneration in multiple ways, including their role in blood ow stalling and their direct release of proin ammatory cytokines and factors that disrupt the blood brain barrier 12,39 .In addition, peripheral in ammation could result in increased neutrophil adhesion molecules that result in their extravasation into the brain and release of granule and NET components that may directly damage tissue.Neutrophils in the brain could impact the surrounding tissue in multiple ways.Their release of ROS, NETs, and cytokines can activate microglia, and their release of extracellular histones can induce neuronal apoptosis 17,40 .The matrix metalloproteinases (MMPs) and proteases released by neutrophils during degranulation or the generation of NETs results in breakdown of the extracellular matrix and neuronal damage 17,23 .
With this study, we sought to determine if low levels of peripheral LPS results in neutrophilic contributions to neuroin ammation.LPS is often used a general inducer of chronic in ammation in animal models, and LPS, derived from resident gram-negative bacteria in the gut, mouth, and skin, is also speci cally implicated in the pathogenesis of neurogenerative and neuroin ammatory diseases 41 .Models of various neurodegenerative diseases have demonstrated that chronic low-level LPS administered IP results in neuroin ammation, as evidenced by induced glial activation, cognitive dysfunction, cerebrovascular leakiness, and in ammatory cytokines in the brain 34 .Chronic peripheral LPS in these models also induced proteinopathy, with increased amyloid deposition in AD models and TDP-43 aggregation in ALS models 34 .The role of neutrophils in mediating CNS in ammation and neurodegeneration in response to low levels of chronic LPS in the periphery has never been examined.Here, we report that chronic, IP LPS injections for 4 weeks induces increased neutrophils in the periphery and brain, speci cally the cortex, in mice.In our preliminary assessments, we found that neutrophil activation in the periphery peaked at 4 weeks of injections, therefore limiting our brain investigations to 4 weeks, but future studies should investigate the role of fewer and additional injections on neutrophils in the brain.
There are multiple mechanisms for how peripheral LPS may result in increased neutrophils in the brain.It has been demonstrated that LPS signaling through the TLR-4 receptor increases CD11b expression on neutrophils, and the binding of CD11b/CD18 (Mac 1) to ICAM one mediates adhesion and transmigration, which are necessary for extravasation into tissues 42 .We observed increased CD11b expression on peripheral and brain neutrophils following chronic low level LPS.Therefore, it is possible that LPS increases neutrophil adhesion and extravasation into the brain through increased CD11b expression.A previous study demonstrated that neutrophils enter the brain in 5xFAD mice, a mouse model for AD, through binding of LFA-1 to integrins, and LFA-1 dependent neutrophil recruitment has also been observed in a model of LPS-induced lung in ammation 14 .We did not measure LFA-1 (CD11a/CD18) on the surface of neutrophils in this study.
However, this model can be used to further understand how peripheral in ammation may result in neutrophil migration into the brain and decipher which adhesion molecules are involved.In addition, peripheral in ammation can contribute to blood brain barrier (BBB) dysfunction, thus allowing for in ammatory cytokines and peripheral immune cells to more easily tra c into the brain and activate microglia that perpetuate neuroin ammation and continued BBB dysfunction 43 .Neutrophils have been found near areas of BBB dysfunction in AD mouse models but whether the BBB is a cause, or a consequence of neutrophil invasion remains to be determined 12 .
Increased CD11b mediates phagocytosis and oxidative burst in neutrophils and is increased on peripheral neutrophils in AD 37,44 , potentially due to increased TNFa [45][46][47] .In our study, TNFa increased 4 hours following the nal LPS injection but resolved by 8 hours post-LPS and CD11b expression was sustained through 8 hours post-LPS in both the periphery and brain but resolved by 12 hours post-LPS.This suggests that weekly LPS injections do not result in sustained elevations of TNFa and neutrophil activation at this very low dose.It will be interesting in future studies to determine how sustained exposure to subclinical in ammatory stimuli may alter these responses in comparison to intermittent stimuli, which we tested here.However, previous studies have demonstrated that exposure to this level of LPS early in life resulted in cognitive impairments 10 months later in mice 48,49 , suggesting that damage caused by leukocytes that in ltrate temporarily may result in sustained impairment or may synergize later with age to contribute to degeneration.Finally, CD11b expression on neutrophils increases with age, so CD11b expression and neutrophil activation in mice receiving low-level chronic LPS should be further examined at different ages to determine how age impacts this model 50 .
Overall, the data provided here are evidence that chronic, low-level LPS administered in the periphery increases neutrophil frequency and activation in the periphery and brain.While we did not study functionality of neutrophils in this study, the observed increase in NE is suggestive of increased NET release, which has been observed as a peripheral marker of neurodegeneration and neuroin ammation 40 .
NET release in the brain is known to contribute to damage, so future studies should investigate NETs in the brain following low levels of peripheral LPS exposure.Of note, neutrophils may also have suppressive functions and contribute to the resolution of in ammation 12 .
Studies have demonstrated that LPS may induce regulatory T cells that in turn result in production of IL-10 by neutrophils, and this interaction is mediated by CD11b 51 .However, two observations suggest that is not a main mechanism observed in our study: 1) IL-10 peaks at 4 hours post-LPS while neutrophils remain elevated, suggesting that, at least at later timepoints, neutrophils are not producing high levels of IL-10; and 2) Neutrophils that produce IL-10 were demonstrated to have decreased CD11b expression in a prior study 51 , and neutrophils in this model upregulated CD11b expression following LPS.Future studies should perform functional assays to assess NET release, phagocytic ability, and ability to suppress T cell function to fully elucidate how neutrophils contribute to in ammation in this model.
This study has several limitations, some of which have already been discussed.First, this is a small pilot study and although we included both male and female mice, we are underpowered to analyze them separately.Sex differences should be examined in the future to understand how this model may be applied to investigate sexual dimorphism in neuroin ammation.Second, we observed some activation of peripheral neutrophils and some higher neutrophil counts in the brain with some of the saline-injected groups.This makes the interpretations of sustained neutrophil in ltration into the brain beyond 4 hours challenging.However, it depicts the continued need for control groups when performing injections and studying in ammation, as the injection itself may be a mediator of in ammation.Finally, we are unable to speak to the longevity of neutrophil responses with this model based on this pilot.Future studies should investigate sustained neutrophil in ammation beyond 12 hours following LPS injections and the potential for impacts weeks to months after injection.However, this study provides the foundation for an experimental model to induce neutrophil in ltration and activation in the brain with a subclinical peripheral stimulus, which can be used to understand the role of neutrophils in mediating the peripheryneuroin ammation axis in different mouse models of disease.

Animals
All mice in the study were generations F3 and F4 from the MRI in-house maintained strain C57BL/6J.The in-house lines are refreshed from The Jackson Laboratory periodically to reduce genetic drift.The mice were housed in individually ventilated and air ltered cages in a super-barrier mouse room.All cages, bedding, water, and enrichment were autoclaved, or UV treated prior to contact with the mice.Mice always had free access to food and water.All mouse cages were only opened in the hood and all personnel were wearing autoclaved lab coats and used sterile gloves.LPS and saline mice were co-housed to reduce cage to cage bias.In the 8-week study (Fig. 1A), 8 females and 11 males received LPS, and 7 females and 8 males received saline.In the subsequent 4-week study (Fig. 1C), LPS injected mice had 6 females and 6 males received LPS 6 females and 5 males received saline.

LPS injections
Mice were weighed weekly for 0.5 mg/kg by weight calculations.Vaccine grade LPS from Escherichia coli 0111:B4 (InvivoGen) or United States Pharmacopeia (USP) sterile grade saline (Cytiva Z1376), were 0.22 uM sterile ltered prior to injections.Mice were injected IP with LPS or sterile saline once a week for 8 weeks for the preliminary study (Fig. 1A), and once a week for 4 weeks for the next iteration (Fig. 1C).

Blood and brain collection
Baseline blood was the day before the rst injection.In the initial experiment, submandibular blood was collected 12 hours after the 4-week and 8-week ( nal) LPS or saline injection.For the next iteration of the study, submandibular blood was collected at 12, 8, or 4 hours after the 4-week ( nal) LPS or saline injection just prior to euthanasia and brain collection.Following the submandibular bleeds, deep anesthetization was carried out with avertin via IP injection.A cardiac puncture was then performed to obtain additional blood prior to whole body perfusion with 20 mL of PBS to ush the vasculature (until uids ran clear).Following perfusion, the brain was then

Figures
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Figure 1 Study
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Figure 2 Increased
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