Neuroinflammatory and neurometabolic consequences from inhaled 2020 California wildfire smoke-derived particulate matter at a remote location


 Utilizing a mobile laboratory located >300 km away from wildfire smoke (WFS) sources, this study examined the systemic immune response profile, with a focus on neuroinflammatory and neurometabolic consequences, resulting from inhalation exposure to naturally occurring wildfires in California and Arizona in 2020. After a 20-day exposure period, WFS-derived particulate matter inhalation resulted in significant neuroinflammation while immune activity in the peripheral (lung, bone marrow) appeared to be resolved in C57BL/6 mice. Importantly, WFS exposure increased cerebrovascular endothelial cell activation and expression of adhesion molecules (VCAM-1 and ICAM-1) in addition to increased glial activation and peripheral immune cell infiltration into the brain. Flow cytometry analysis revealed proinflammatory phenotypes of microglia and peripheral immune subsets in the brain of WFS-exposed mice. Interestingly, endothelial cell neuroimmune activity was differentially associated with levels of PECAM-1 expression, suggesting that subsets of cerebrovascular endothelial cells were transitioning to resolution of inflammation following the 20-day exposure. Neurometabolites related to protection against aging, such as NAD+ and taurine, were decreased by WFS exposure. Additionally, increased pathological amyloid-beta protein accumulation, a hallmark of neurodegeneration, was observed. Neuroinflammation, together with decreased levels of key neurometabolites, have important implications in priming inflammaging and aging-related neurodegenerative phenotypes.


INTRODUCTION
Wildfire smoke (WFS) exposure poses an increasing threat to a growing and aging global population.
A demonstrable increase in the extent of wildfires and resultant damage has been documented in the United States over the past 15 years, an effect which correlates well with changing global temperatures. 1 In 2020, wildfires from the west coast generated smoke that engulfed much of the United States in high concentrations of particulate matter (PM), potentially impacting the health and well-being of 100's of millions of citizens for several months.
Wildfires release high concentrations of airborne particulates and gases derived from varying biomass and anthropogenic fuels (e.g., buildings, vehicles, etc) that create unfamiliar and toxic exposures to populations far away from the source. Both PM and carbon monoxide are stable components of WFS that can travel thousands of miles. Even at a distance, WFS exposures increase acute respiratory hospital admissions. 2,3,4,5 Additionally, underserved minority populations, including Native American communities, are disproportionately vulnerable to wildfire events. 6 Wildfires produce complex mixtures of inhaled toxicants that can not only damage the lungs but also promote systemic health effects. While most gaseous products remain localized, PM from WFS is carried far distances across the continent by prevailing winds. Short-and long-term pulmonary, cardiovascular, and neurological outcomes from inhaled ambient PM are well studied; however, relatively little is known about the impact of WFS-derived PM on neurological outcomes -including implications for priming agerelated disease sequelae. Ambient PM2.5 (PM less than 2.5 microns in aerodynamic diameter) has been associated with increased incidence of Alzheimer's disease and related disorders (ADRD), suicide, depression, psychosis, and other adverse neurological outcomes 7,8,9,10 . Impairment of blood brain barrier (BBB) and neuroinflammation has been implicated in pathophysiology of these neurological disorders.
More recently, neurometabolite alterations in relation to neuroinflammation are being recognized as key drivers underlying cellular senescence and neurodegenerative disorders 11,12,13 .
Prior studies suggest that inhaled PM induces proteolytic activity in the lung, leading to shedding of endogenous peptide fragments into the circulation that are bioactive and promote systemic endothelial inflammatory responses 14,15,16 . The BBB consists of tightly connected brain endothelial cells contouring the vasculature of the CNS, with astrocytic end feet surrounding this vasculature, and nearby microglia capable of monitoring local events. Trafficking of leukocytes is heavily regulated by the BBB 17  The present study exploited naturally-occurring wildfires in California and Arizona in October of 2020 to examine the neuroinflammatory consequences of WFS-derived PM at a site >1000 km away from the source. PM concentrations used in this study, due to the distant site and dilution factor, reflect a level lower than that experienced by tens of millions of people living in California, Oregon, and Washington during these wildfire events in 2020. Hallmarks of aging-and ADRD-related neurodegeneration, such as metabolite alterations, microglial activation, and pathological β-amyloid accumulation, were examined to gather supportive evidence as to the in vivo relevance of WFS exposure as an unrecognized contributor to neurological aging and degenerative sequelae.

Animals and Exposures.
Male C57BL/6 mice (Jackson Labs) at 2 months of age were housed in quarantine for one week prior to transport to the mobile laboratory ( Figure 1A) located in Paguate, NM (35°08'07.7"N 107°22'34.4"W; Figure 1A). Mice were housed in AAALAC-approved facilities, on a 12h light:dark cycle and provided a standard chow diet and water ad libitum. Mice were transported to the mobile laboratory 3-days prior to exposure initiation so that they could be acclimated to the chamber conditions. A total of 24 mice were used, evenly divided into Filtered Air (FA) control and concentrated PM2.5 exposed groups. All procedures were conducted with approval by the University of New Mexico Animal Care and Use Committee.
Exposures in the mobile laboratory were conducted in Hinners chambers with wire-bottom mouse cages; water was available to mice throughout the exposures, but food was withheld (4h/d). PM2.5 concentration was facilitated by a Harvard-type concentrator and mice were exposed whole-body for 4 h per day for 20 consecutive days 19 . Exposures began on October 6, 2020, and the final day was October 25, 2020. After the last round of exposures, mice were transported back to the UNM laboratories and euthanized the following day for tissue collection.
Particulate Matter Characterization. Exposure concentrations were measured in real-time with a DustTrak II (TSI, Inc; Shoreview, Minnesota), and 47 mm quartz filter weights collected for the duration of each daily exposure were used to gravimetrically confirm final daily averages. Filter samples were processed and analyzed in Class 100 clean rooms at the Michigan State University Exposure Science Laboratory. The samples were analyzed gravimetrically for particle mass using a microbalance (XPR6UD5, Mettler Toledo) in a temperature-and humidity-controlled clean laboratory as described in the Federal Reference Method 20 . PM samples collected on quartz filters were maintained at -40°C after sampling and were analyzed for carbonaceous aerosols (organic carbon and elemental carbon) by a thermal-optical analyzer (Sunset Laboratory) using the NIOSH 5040 method.
Particle samples collected on Teflon filters were wetted with ethanol and extracted in 1% nitric acid solution. The extraction solution was sonicated for 48 h in an ultrasonic bath, and then allowed to passively acid-digest for two weeks. Extracts were then analyzed for twenty trace elements using highresolution inductively coupled plasma-mass spectrometry (ELEMENT2, Thermo Finnigan). This analysis method incorporated daily quality assurance and quality control measures including field blanks, Type I water blanks, replicate analyses and external standards as described 21 .
Quantification of levoglucosan was performed in the MSU Mass Spectrometry and Metabolomics Core.
The details of sample extraction and analysis are described elsewhere 22 . In brief, for each sample, PM from approximately 100 mg of filter punches was extracted with 3 mL methanol containing 2 µg·mL -1 sedoheptulose anhydride as an internal standard (Millipore Sigma). One milliliter of the methanolic extract was dried down and derivatized with 100 µL of N-trimethylsilylimidazole (Millipore Sigma). One milliliter of the derivatized samples in splitless mode was analyzed using an Agilent 7890A GC/single quadrupole mass spectrometer with 5975C inert XL MSD. The mass spectrometer was operated using 70 eV electron ionization in selected ion monitoring mode for m/z 204 and 333. Quantification of levoglucosan, 3TMS derivative were based on a levoglucosan (Millipore Sigma) standard curve.
Lung Lavage, Cytokines, and Histopathology. Following euthanasia, lungs were lavaged with phosphate buffered saline as previously described. Total cell counts and differentials were conducted in a blinded manner. Cytokines were measured in BALF using an electrochemiluminescent assay Echocardiography. Before transport to the mobile exposure laboratory and after exposures just prior to euthanasia, mice were briefly anesthetized with isoflurane for doppler ultrasound assessment of cardiac function (Vevo LAZR, FUJIFILM VisualSonics). Anesthesia was maintained at a light level, targeting heart rate values above 400 beats per minute. Short-axis M-mode images were collected on all mice for assessment of cardiac output, ejection fraction, end-systolic dimensions and end-diastolic dimensions.
Brain tissue digestion for flow cytometry. Under isoflurane anesthesia, mice underwent transcardial ice-cold 0.1M PBS (pH=7.4) perfusion. One brain hemisphere was harvested in ice-cold HBSS buffer and processed immediately according to Miltenyi gentleMACS TM adult neural tissue digestion protocol, as described 23 . Briefly, brain tissues were minced with fine tip scissors and processed with enzymatic For intracellular staining, cells were fixed with Fixation buffer and then permeabilized using intracelullar fixation and permeabilization buffer set (eBioscience, USA). Cells were then stained with fluorochromeconjugated antibodies for the intracellular immune factors for another 1 h at room temperature in the dark. After another wash with 1x permeabilization buffer, cells were resuspended in 250-300µl FACs buffer and immediately proceeded for data acquisition. At least 50,000 live cell events were collected for each sample. Single-stained controls and isotype controls were used for laser compensation and data analysis. Data were acquired using the BD LSR Fortessa cell analyzer (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software v10.7.1.
Flow cytometry gating strategy. The gating strategy for determining different cell subsets in the brain tissues is similar described in our prior report 23 . Briefly, doublets (cell clumps) were excluded, and the live cells were identified based on their size, granularity (FSC v SSC) and negative viability dye staining. Cerebrovascular endothelial cells were identified based on negative expression of the common leukocyte marker, CD45 and positive staining for CD31 (PECAM-1, Platelet endothelial cell adhesion molecule-1). All CD45 + cells were first gated for PMN or neutrophil marker, Ly6G (1A8) staining, which were also verified by their positive CD11b staining. The population of CD45 + 1A8 -(leukocytes that are not neutrophils) were further analyzed to identify microglia with low or medium expression of CD45 (CD45 low/med , CD11b+) as distinguished from infiltrating macrophages/monocytes with CD45 high expression (CD45 high CD11b+). Additionally, infiltrating monocytes/macrophages were further analyzed for Ly6C expression to identify inflammatory monocytes (1A8 -CD11b + CD45 high Ly6C + ).
Median or geometric mean fluorescent intensities were plotted for activation markers or cytokine expression on these different immune and endothelial cell subsets.

Exposure Characterization:
The mobile laboratory-based concentration of PM2.5 led to 4-h exposure levels averaging 104 µg/m 3 across the 20-d period ( Figure 1B). During the peak of wildfire smoke transported to this region, high concentrations of 209 and 191 µg/m 3 were seen (first 2 days of exposure). We also measured the overall content of levoglucosan, a specific marker for woodsmoke, in pooled PM filter samples, which revealed that the first three days of exposure had a high contribution of wood burning in the PM2.5. Increased levoglucosan levels were also evident on exposure days 13-16.
Potassium levels in the WFS appeared elevated on days with greater wildfire contributions (Table S1) and correlations among K, Mg, and Mn were noted, consistent with previous studies of wildfire PM composition ( Figure S1) 30 . Modeling of wind trajectories for the initial period of the exposure identified a strong likelihood that woodburning-derived PM originated in southern California ( Figure 1C). Geospatial analysis of the western US revealed extensive smoke distribution during the exposure period.
Additional contributing wildfires were present in Colorado and Arizona. Importantly, mice were only exposed for four hours a day, so that the 24 h average PM2.5 exposure could be estimated as much lower, potentially <20 µg/m 3 (Table S1).

Pulmonary and Systemic Responses:
Histological assessment of lungs revealed a modest increase in the macrophage population in WFS-exposed mice compared to FA (Figure 2A,B). This was confirmed quantitatively with cells recovered in the BALF increased four-fold in WFS-versus FAexposed mice, principally due to macrophage elevations ( Figure 2C). The numbers of neutrophils were not elevated by WFS exposure (Table S2). However, neutrophil recruitment cytokines IL-17, MIP1, and MIP2 were statistically elevated ( Figure 2D), while TNFa and other conventional inflammatory mediators were unaltered (Table S2). The absence of neutrophils suggests that any acute pulmonary inflammatory phase had subsided, with effector responses trending downward towards resolution.
Additionally, the inflammatory cytokine IP10 was significantly elevated, indicating a persistent response in cellular subtypes of monocytes, T cells, NK cells, or eosinophils 31 . We questioned whether this inflammation was reflected systemically through bone marrow examinations. Interestingly, MIP2 and IP10 were elevated in the bone marrow of WFS-exposed mice ( Figure 2E). However, many other BALF, whole lung, regional brain tissue, and bone marrow inflammatory protein markers were unaltered by the 20-day WFS exposure (Tables S2-S5), including KC/Gro, TNFa, and IL-6, confirming that the overall pulmonary inflammatory response to this exposure was expectedly modest, or resolving from the earlier higher exposure concentrations. Additionally, echocardiography was performed to assess cardiac function; however, no significant changes were noted between FA and WFS mice after the 20-d exposure (Table S6) Neuroinflammaging: We assayed prefrontal cortices and hippocampal regions for inflammatory proteins and cerebelli for aqueous metabolites including specific assessments for NAD and related metabolites. Cytokines from the hippocampus showed no statistical differences (Table S4). However, prefrontal cortices showed a decrease in KC/Gro (Table S4). WFS-exposed mice displayed reduced NAD+, NADH, succinate, and taurine compared to FA-exposed mice ( Figure 3A), all of which have important implications for aging 12,13,32 . These aging-related metabolites prompted a direct examination of Aβ-42 levels, the more pathogenic peptide variant that aggregates faster, which was significantly increased in WFS-exposed mouse brain ( Figure 3A). Localization of increased Aβ-42 appeared proximal to cerebrovasculature together with greater staining for the early neurodegenerative disease marker sorcin 33 as also found in matched staining of APP/PS1 model mouse brain ( Figure 3C). Taken together, WFS exposure -from a source >1,000km away -gives rise to numerous markers of an accelerated inflammaging response with increased markers consistent with a neurodegenerative phenotype.
Given the proximity of these markers to the cerebrovasculature, we further wanted to assess indications of a neuroinflammatory response comparable to outcomes we have seen with other inhaled toxicant exposures 14,15,23 . Phenotypically, the neurovascular unit appeared similar to that previously seen after pulmonary particulate exposure, with extravascular albumin staining, increased GFAP process volume and greater IBA1 localization to indicate a reactive, neuroinflammatory state in WFSexposed mice ( Figure 4A). A neuroinflammatory state was affirmed with cytometric quantitation of microglia and CNS infiltrating peripheral immune cells (Supplemental Figure 2: gating strategy). An increase of microglial frequency along with their surface expression of CD45 were significantly elevated in WFS-exposed brains compared to FA controls ( Figure 4B). Moreover, intracellular levels of proinflammatory proteins CCL2 and iNOS were increased in microglia after WFS-exposure ( Figure 4B).
We validated microglial activation by measuring elevated surface expression of ICAM-1 and iNOS, though the increasing trend in TNFα expression did not reach significance ( Figure 4B).
Given peripheral immune reactivity ( Figure 2C), infiltration of peripheral immune cells into the CNS was also examined by cytometric analysis of the perfused brains. Contrary to the BALF, an increased influx of mature (1A8 high ) neutrophils was detected in WFS-exposed brains, displaying significantly higher levels of CD11b and MHC-II expression ( Figure 5A). The marker CD45 high includes various subsets of peripheral leukocytes, and no changes were observed on this macro level. However, further analysis of a CD45 high Cd11b + subset revealed significant increases in CNS-infiltrating Ly6C + inflammatory monocytes in the brain, suggesting an influx of these cells due to WFS exposure ( Figure 5B). Furthermore, there were significant increases in CD45 high LFA-1 + and CD45 high ICAM-1 + peripheral macrophages that exhibited a greater expression of MHC-II in WFS-exposed brains ( Figure 5C).
Flow cytometry also revealed a complex phenotype among brain endothelial cells (CD31 + /CD45 -) following 20-d WFS exposure, with subpopulations divided by their level of CD31 expression ( Figure   6A). WFS shifted CD31 + /CD45endothelial cells overall to a greater expression of CD31 ( Figure 6) consistent with vascular repair and restoration of the BBB, 34 with these pro-repair CD31 high /CD45cells expressing exhibiting reduced levels in most inflammatory markers ( Figure 6C). At the same time, CD31 med /CD45cells retained their inflammatory profile after WFS exposure with increases in CCL2, TNFα, iNOS, LFA-1 and VCAM-1 ( Figure 6B).

DISCUSSION
The recent spate of wildfires around the globe have provoked concern that smoke exposures may promote vascular and, by extension, neurological consequences 35,36 . While clear relationships between ambient air pollutants, such as PM2.5 and carbon monoxide, and increased incidence of ADRD have been reported 7,8,9 , there have been no specific assessments of wildfire smoke PM impacts on neuroinflammation or markers of ADRD. Findings of the present study confirm that the WFS-derived PM2.5 traveling from distant wildfires can promote numerous outcomes consistent with ADRD pathogenesis and neurological aging.
Neuroinflammation and key reduced metabolites occurred in otherwise healthy mice exposed for 80 total hours of WFS-derived PM2.5, including microglial activation and peripheral leukocyte invasion. The concentrations in our study averaged 104 µg/m 3 for only 4 h/d of exposure. Findings of a 13-day wildfire episode in Washington state found 24 h average PM2.5 to be 96 µg/m 3 (up from a baseline of 4.3 µg/m 3 ) with two counties experiencing peak concentrations near 400 µg/m 3 37, 38 . During this period, we estimate that over 1.6 million residents of Washington state experienced 24 h exposures well in excess of 100 µg/m 3 on average, and all 7 million people in the state were exposed to levels higher than our 24 h exposure average ( Figure S3). Similar exposures were seen for populations in Oregon and California. In addition to PM, carbon monoxide and potentially other gaseous pollutants can further contribute to neurological morbidity 39 . Notably, the long distance from the source and method of PM2.5 fractionation used in our study would lead to disproportionately low gaseous pollutants; thus, this study does not address potential additivity of the total pollutant atmosphere.
The pathways leading from inhaled pollutants to neurological outcomes are being discerned through mechanistic toxicological studies. We have suggested that circulating bioactive peptide fragments arise from pulmonary matrix metalloproteinase activity following inhalation of a diverse array of pollutants, including PM and ozone 14,23,40,41 . Endothelial VCAM-1 drives leukocyte invasion, which reduces BBB integrity and reactive consequences at the neurovascular unit; specific aging-related circulating factors may augment VCAM-1 signaling and promote BBB impairment 18 . Controlled exposures to woodsmoke caused a circulating bioactivity that induced endothelial VCAM-1 in ex vivo incubations, notably more potently than other emissions including diesel/gasoline emissions and ozone. 42 The present findings from flow cytometric assessment of VCAM-1 expression in CD31 med /CD45cerebrovascular endothelial cells confirms the in vivo relevance of this effect, but notably at a remarkably low concentration of PM2.5. Identification of evoked circulating factors after wildfire smoke exposure and the endothelial receptor interactions is essential to understanding pathways that confer susceptibility among individuals. Furthermore, the apparent initial steps of BBB repair and inflammation resolution, seen as an increase in CD31 high /CD45cells with reduced iNOS, CCL2, and TNFa expression after 20d of exposure, highlight another potential contributor to susceptibility, with genetic and lifestyle factors in the general population adding variability to the efficiency and quality of inflammatory resolution.
Endothelial cell responses are complex, with control and PM-exposed mice exhibiting relatively different levels of CD31, which drove the segregated analysis of CD31 med versus CD31 high . CD31 is not a passive, constitutive marker of endothelial cells in the brain, and it has been noted as a key player for maintaining BBB integrity 34 and neutrophil recruitment in inflamed brains 43 . Using a bifurcated analysis strategy for the endothelial cell analysis, we illustrate a potentially dynamic system that is crucial to acclimation to environmental stressors. Cells highly expressing CD31 were also downregulating other canonical inflammatory pathways, including CCL2 and iNOS. Thus, we postulate that subchronic exposure to inhaled PM from WFS likely leads to repair of the cerebrovascular endothelium, but little can be inferred from the present study design as to the timeframe of this resolution.
Despite the worst concentrations of the exposure occurring on the first two days of the 20-d exposure, there was little indication of resolution of microglial activation or the invading peripheral leukocytes.
Peripheral immune cells were not only in greater numbers in the brains of WFS-exposed mice, but they also expressed greater MHC-II protein on the surface, which is associated with the inflammatory response in neurodegenerative diseases 44,45 . Furthermore, peripheral immune cells expressed higher levels of inflammatory markers ICAM-1, iNOS, and CCL2 in WFS-exposed mice. These proinflammatory immune cells may interact with microglia and astrocytes and further propagate neuroinflammation. Eventually these immune cells may help resolve inflammation or injury in the brain, but the resolution of this infiltration may require months 46 . Repeated insults may therefore confound beneficial outcomes or escalated other pathologies such as Alzheimer's disease or multiple sclerosis 46 .
Understanding the molecular and cellular pathways regulating the resolution, as well as the range of environmental and pathological stressors that promote inflammation is, again, a priority for understanding how susceptibility to neurodegenerative disease arises.
Environmental stressors, including air pollution, have been associated with neurodegenerative diseases 9,39,47 , and the present study details pathways that may be points of vulnerability for individuals with genetic predisposition for ADRD. Genetic heritability is actually quite variable across different syndromes of dementia. Frontotemporal dementia only has clear heritability evidence in up to 30% of cases, most of which can be explained by single gene mutations (e.g., microtubule-associated protein tau and progranulin) 48 . Alzheimer's disease has a much higher heritable basis, between 60-80% 49 .
Thus, environmental factors and other pathologies must account for a substantial portion of the remaining incidence. Furthermore, even with the highly heritable Alzheimer's disease, the age of onset may be influenced by environmental factors interactions 50 . The elevation of Aβ-42 seen after WFS in mice is reminiscent of previous studies of acute ozone inhalation 23 . Aβ-42 is an early pathogenic indicator with aggregating potential in humans, but the existence of rodent Aβ-42 in response to air pollution is unclear as mice do not naturally form plaques 51 . Aβ-42 may be elevated due to increased production or reduced clearance, possibly a repercussion from microglial diversion to the neurovascular unit. Regardless of the mechanism, impairing BBB, driving neuroinflammation and reducing key metabolites may be central to environmental contributions to ADRD incidence; the sensitivity of individuals to these effects and the efficiency of resolution may influence susceptibility among individuals.
Compromised metabolism is a major hallmark of cellular aging. Neurons and neuro-supportive cells undergo numerous changes with aging including reductions of protein quality control and lysosomal dysfunction, along with DNA damage and epigenetic modifications. Genetic variants that influence neural longevity include FOXO3, IL-6, and TOMM40-APOE-APOC1. 52,53,54 However, it has been estimated that environmental contributions may account for as much as 70% of premature aging. 55 The aging neuron is vulnerable to reduced metabolic capacity, highlighted as a loss of nicotinamide adenine dinucleotide (NAD + ) concentrations. 56 This reduction in bioenergetics negatively impacts DNA repair, which appears central to a downward spiral of cellular functionality. NAD + levels can be augmented with precursor supplementation (nicotinamide mononucleotide) that reduces DNA damage and extends healthspan in mouse models. 57,58 Following WFS exposure, however, we see significant reductions in NAD + levels, along with NADH, succinate, and taurine. While this trend does not indicate aging, per se, it at least suggests that WFS exposure may add to the burden of aging-related metabolic impairments in the brain. More importantly, neurometabolite alterations are associated with oxidative damage and chronic neuroinflammation 11,32,59 . NAD + and taurine boost anti-inflammatory activity. Reduced levels of NAD + and taurine are associated with chronic neuroinflammation and drives neuroinflammaging, a lowgrade inflammation state that drives aging process 60,61 . NAD + is involved in stress resistance and synaptic plasticity and is downregulated in aged animals, while the conversion of NAD + to NADH is important for ATP generation 11 . Succinate is important for mitochondrial function and oxidative metabolism 62 . Taurine has been shown to be neuroprotective during aging 63 .
In summary, WFS exposure activated microglia, drove neural infiltration of inflammatory monocytes and MHC-II + cells, decrease neuroprotective metabolites, and increase Aβ-42 when examining the entire brain. Overall enhanced neuroinflammation from WFS exposure potentially contributes to neurological aging and pathology, and the effects demonstrated at environmentally-relevant concentrations warrant further exploration into long-term neurological sequelae and vulnerability for specific subpopulations, especially in the young and those of advanced age. Beyond simple metrics of plaque buildup, we identified key components of ADRD promotion from systemic inflammation to cerebrovascular endothelial activation and glial proinflammatory activation to downregulation of key energetic metabolites. Based on our novel findings neuroimmune activity and neurometabolism, these data point to fundamental effects of exposure to WFS leading to wide ranging pathology that may contribute to aging process.
The authors declare no competing interests with the results of this study and content of the manuscript.

Data Availability
Data are available upon request to the corresponding authors.  left lung lobe of mice exposed to A) filtered air or B) concentrated fine ambient particles. Slightly more alveolar macrophages (arrows) were present in WFS-exposed mice than filtered air control mice.  NADH, succinate, and taurine were all downregulated and amyloid beta (Aβ) levels were upregulated in WFS-exposed mice. B. Representative images of neurodegenerative pathogenic markers found at the neocortical neurovascular unit in WFS-exposed mice. Brain Aβ-42 (red) increases in WFS-exposed mice were observed only proximal to the neurovascular unit (endothelial ZO1 and astrocyte GFAP).
Additionally, the early pathogenic marker Sorcin increased (green), indicating ER stress / unfolded protein responses proximal to areas of Aβ-42 staining, which was also observed in unexposed APP/PS1 mice of the same age and background as a positive control, though they exhibited higher density Aβ-42 aggregation. N= 6 independent samples; two-tailed t-test; mean and SEM shown, log2 correction for normality.  surface expression of all CD45 + 1A8 + neutrophils in the brain due to WFS exposure. B. Inflammatory monocytes in the brain were increased in frequency with WFS exposure. C. CNS infiltrating CD45 high CD11b + macrophage/monocyte population shows increased MHC2 expression and frequency of LFA-1+ and ICAM-1+ peripheral leukocytes were also increased due to WFS exposure. N=4-5 independent samples; two-tailed t-test; mean and SEM are shown. Frequency of endothelial cells with high expression of CD31 was increased following WFS exposure, however, these endothelial cells displayed reduced levels of these proinflammatory factors. Increased frequency of ICAM-1 expressing endothelial cells were observed due to WFS exposure in this endothelial subset despite no changes in MHC2. C. Frequency of CD31 med endothelial were reduced following WFS exposure, however, these cells displayed increased proinflammatory phenotype such as increased levels of CCL2, TNFa and iNOS, although reduced levels of ICAM-1 and MHC2 were observed. N=4-5 independent samples; two-tailed t-test; mean and SEM are shown.  Ly6G (1A8) expression denoting neutrophils. CD45 + 1A8 + were further analyzed to identify neutrophil population with 1A8 high expression. CD45 + 1A8-population (leukocytes but not neutrophils) were further analyzed against CD11b expression to identify microglia CD45 med CD11b + population, as compared to CD45 high CD11b + macrophage/monocytes population. Inflammatory monocytes were identified based on their Ly6C high expression (CD45 + CD11b + Ly6C high ). Figure S3. Reanalysis of data reported by Liu and colleagues 35,36 from September 2020 wildfires revealed over 1.6 million people in Washington state were exposed to concentrations in excess of 100 µg/m 3 for the 13-day period.   Figure S3. Reanalysis of data reported by Liu and colleagues 35,36 from September 2020 wildfires revealed over 1.6 million people in Washington state were exposed to concentrations in excess of 100 µg/m 3 for the 13-day period.