World Trade Center Dust Induces Airway Inflammation while Promoting Aortic Endothelial Dysfunction

Abstract

Respiratory ailments have plagued occupational and public health communities exposed to World Trade Center (WTC) dust since the September 11, 2001 attack on the Twin Towers. The nature of these ailments is proposed to be induced by inhalation exposure to WTC particulate matter (WTC_PM), released during the collapse of the buildings and subsequent resuspension during cleanup. We investigated this hypothesis using both an in vitro and an in vivo mouse intranasal (IN) exposure model to identify the inflammatory potential of WTC_PM with specific emphasis on respiratory and endothelial tissue responses. In vitro studies identified WTC_PM exposure to be positively correlated with cytotoxicity and increased NO₂⁻ production in both BEAS-2B pulmonary epithelial cells and THP-1 macrophage cells. In vivo C57BL/6 mouse studies exhibited significant increases in inflammatory markers including increases in polymorphonuclear neutrophil (PMN) influx into nasal and bronchoalveolar lavage fluids (NLF and BALF), as well as increased total protein and cytokine/chemokines levels. Concurrently, NLF, BALF, and serum NO₂⁻ levels exhibited significant homeostatic temporal deviations with evidence of temporal aortic dysfunction in myography studies. Respiratory exposure to- and evidence -based retention of- WTC_PM may contribute to chronic systemic effects seen in mice, with resemblance to observed effects in WTC_PM-exposed human populations. Collectively, findings reported herein are reflective of WTC_PM exposure and its effect(s) on respiratory and aortic tissues, highlighting potential dysfunctional pathways that may precipitate inflammatory events, while simultaneously altering homeostatic balances. The tight interplay between these balances, when chronically altered, may contribute to- or result in- chronically diseased pathological states.

Background

Over a million tons of debris and airborne particulate matter (PM) were generated and/or removed from the World Trade Center (WTC) site within the first year after the collapse, exposing an estimated 400,000+ people, including first responders, residents, and workers engaged in the massive cleanup and building maintenance [1]. The debris, comprised of building materials, furniture and office equipment combustion residues, paper, and unburned jet fuel, were incorporated into WTC_PM during the collapse event [2]. No single element, compound, or factor has been implicated as being causal for the observed adverse health effects in WTC_PM-exposed human populations, but PM chemical composition and alkalinity can potentially provide links between exposure and subsequent symptoms that have been associated with WTC_PM exposure, or in relation to causality for the degradations of cellular and systemic response pathways. Some symptoms associated with more conventional ambient air PM exposure have been similar to those experienced by people exposed to WTC_PM and may be indicative of a common factor between PM exposure and WTC_PM exposure. Epidemiologic studies have demonstrated relationships between WTC_PM exposure and adverse health outcomes experienced by rescue workers and residents of the surrounding area and were found to be dominated by lower respiratory symptoms similar to those who suffer from PM exposure (coughing, wheezing, and aggravated asthma) [3].

Previous particle characterization studies have indicated WTC_PM to be highly alkaline (pH 9.2-12), derived from the compositional makeup of the dust with 50% being comprised of an alkaline mixture containing cement and gypsum dust and the other 40% being synthetic vitreous fibers [SVF]). Conventional ambient air PM respiratory effects studies have focused on neutral and/or acidic respirable particles <2.5 μm in aerodynamic diameter to identify the potential adverse respiratory effects. Uniquely, >99% of WTC_PM were >10 μm, with 59.1% of the PM mass ranging in size from 10-53 μm [4,5]. Due to compositional similarities between fractional size groups greater than 2.5 μm, physiological responses to such coarse particles would likely be due to respiratory tract deposition patterns that vary with particle size, whereby coarse thoracic PM (2.5 – 10 μm) and super-coarse (10-53 μm) fractions deposit most prevalently in the conductive airways of the upper respiratory tract and tracheobronchial tree, where sensory innervations are more dominant.

The conductive airways provide the first line of defense against inhaled PM, specifically maintained by their characteristic structural configurations, mucosal surfaces, prevalent innate immune cells, and antioxidant rich environment. However, WTC_PM exposures, particularly in the context of WTC First Responders, can overwhelm defensive capabilities, leaving the upper- and potentially lower-respiratory tract vulnerable. Understanding the interplay between particle retention and clearance mechanisms can determine appropriate courses of action and treatment for those exposed to WTC_PM. Considering WTC_PM is a highly heterogeneous mixture, conventional applications of treatment may or may not be successful considering the multitude and array of particles being exposed to the respiratory systems, and potentially other systems directly or indirectly.

The objective of this study was to investigate WTC_PM exposure and its role in inflammatory potentiation in both in vitro-cell and in vivo-mouse models, with specific emphasis on respiratory cardiovascular tissues. Investigating these interactions at the nasal-pulmonary interface can help illuminate long-term health issues associated with generic PM exposures, including central nervous system effects and/or co-exposure effects from the surrounding cleanup area.

To achieve an exposure method with relevance for human health outcomes, this study implemented intranasal (IN) instillation as a particle delivery mechanism based on the ability of these large alkaline particles to not only be deposited in the nasal cavity, but to also be aspirated into lower pulmonary areas. This exposure methodology provides a real-world exposure scenario based on the high potential for the overloading of particle clearance capacity, in both nasal and pulmonary tissues, that may have occurred in people caught in the dense WTC_PM plume. This research is critical to understanding causal mechanisms and/or events that may precipitate inflammatory cascades that, if occurring chronically, could shed light on processes involved in pulmonary and extra-pulmonary disease development.

Methods

In vitro Cell Lines:

BEAS-2B (ATCC®, Manassas, VA) cells were maintained in complete media containing Dulbecco’s Modified Eagle Medium (DMEM) according to manufactures protocol and seeded at 15-30x10⁴ cells/cm². THP-1 cells (ATCC®, Manassas, VA) were maintained in suspension cultures containing complete RPMI 1640 media and seeded at densities of 2-4x10⁴ cells/ml. Addition of PMA in DMSO (Sigma-Aldrich®, St. Louis, MO) was accomplished at a final concentration of 0.1% in media, with cells assayed 3 days post-PMA exposure. Cultures were maintained at 37°C in a humidified 5% CO₂ atmosphere.

CytoTox 96 Non-Radioactive Cytotoxicity Assay:

CytoTox 96® Non-Radioactive cytotoxicity assay kit (Promega, USA) was used in accordance with manufacturer protocol. Lysis 10X Solution (9% (v/v) Triton ®X-100 in water) was provided by the manufacturer for use as the positive control. Cytotoxicity percentage was calculated using the formula provided by the manufacturer (% cytotoxicity = [Abs of experimental sample/ Abs of maximum LDH release] x 100).

Colony Formation Unit Assay:

BEAS-2B cells were harvested and seeded at 300-400 cells per dish and incubated for 24 hours with addition of treatment media (31 μg-1 mg/ml of WTC_PM). Cells were stained with 0.5% crystal violet and visible colonies were counted after a 10-day incubation. Colonies were considered strong for scoring with 50 cells/colony.

Griess Reagent System:

Cell-free mouse NLF, BALF, and hemolysis-free serum were assayed using the Griess Reagent System (Promega, Madison, WI) and prepared according to manufacturer protocol. Concentrations of total nitrite were calculated from a standard curve established with serial dilutions of sodium nitrite starting at 100 μM and ending at 0.39 μM, with a limit of detection of 2.5 μM. Colorimetric optical density was read at 535 nm.

Animals:

Pathogen-free 8-10-week old and age-matched control male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed in an approved facility at NYUSOM and acclimated for 1-2 weeks under controlled temperature (22 ± 2°C) and relative humidity (30-50%) with a 12-hr light/ dark cycle prior to use in any experiments. Mice were provided ad libitum access to standard laboratory chow and filtered water. All protocols were approved by the NYU School of Medicine IACUCs.

Intranasal Instillation:

Mice were anesthetized in a closed container with 1-3% Isoflurane in oxygen (Butler Schein, Dublin, OH). Mice were affixed to a plexiglass board at a 45°angle. Top and bottom incisors were secured, and particle suspension delivered in a volume of 50 μl. Exposure frequency included a single IN instillation or 4 IN instillations over the course of one week (day 1, day 3, day 5 and day 7).

WTC_PM Particle Preparation:

Concentrations of 31 μg -1000 μg/ml were prepared from dry WTC_PM10-53μm or WTC_PM<53μm stocks and suspended in media 1 hour prior to in vitro exposures. For the purposes of the studies herein, all in vivo studies were performed with DPBS-suspended WTC_PM<53. Doses ranging from 31 μg – 4000 μg/50 μl were prepared from dry WTC_~53μm dust stocks and suspended in DPBS (or water for alkalinity studies) 1 hour prior to in vivo IN instillations. For alkalinity studies, sodium hydroxide pellets (Sigma-Aldrich®, St. Louis, MO) were dissolved in sterile water and diluted to 1.0 μM at a pH of 8.1, sterile filtered and intranasally instilled at 50 μl.

Animal Processing Post-Exposure:

Intranasally instilled animals were euthanized 24hours-post single or final exposure via intraperitoneal injection of pentobarbital (0.36mg/g). Serum, bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), whole lung and nasal cavities were collected and stored at −80°C. Of note, the nasal cavities is inclusive of the area from the cribriform plate to the nares, to include the ethmoturbinates, nasoturbinates, maxilloturbinates, and nasal vestibule. Whole blood collected from the vena cava was centrifuged at 3000 x g for 10 minutes. Serum was collected, double spun, isolated, and stored at −20°C to be evaluated for nitrite. Triple flush BALF and NLF samples using Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) were collected and placed at 4°C. Lavage fluids were centrifuged (15,000 x g for 5 minutes) for generation of cell-free supernatant and stored at −20°C for endpoint evaluation. For transpharyngeal nasal lavages, head and mandible were excised and cannula inserted into the posterior opening of the pharynx for nasal cavity flushing. Organs and intact nasal cavities were weighed, flash frozen in liquid nitrogen and stored at −80°C. For histopathologic valuations, lungs were fixed in situ with 10% formalin at a constant fluid pressure of 25cm. Whole lungs sections were processed and stained with H&E or PAS. All pulmonary tissues were semi-quantitatively evaluated by a certified histopathologist (Mass Histology Associates, Inc.; Worcester, MA), and graded accordingly to an endpoint: N/0= Normal; 1= Minimal; 2= Mild; 3= Moderate; 4=Severe.

Cellular Differentials and Cell Counts:

Differential slides were affixed with 100 μl of BALF or NLF, fixed in methanol and stained with Hemacolor (Harleco, Gibbs-town, NJ). Differential cell counts were performed under light microscopy with cells totals determined via hemocytometer. Cell viability was evaluated using Trypan blue staining (Sigma-Aldrich®, St. Louis, MO).

Total Protein Assay:

Epithelial permeability was assessed by the Bradford method, quantifying levels of total protein in BALF and NLF using a Coomasie Blue protein assay (Thermo Scientific, West Palm Beach, FL). Cell-free supernatant total protein was measured at an absorbance of 595 nm.

Enzyme-Linked Immunosorbent Assays (ELISA):

Protein levels of mouse TH1, TH2, and TH17 cytokines/chemokines from BALF and NLF (n=5; samples pooled) were determine using a MultiAnalyte ELISArray kit (Qiagen) according to manufacturer’s instructions. Colorimetric quantitation of 570 nm and 450 nm optical densities were used to adjust for wavelength correction. Reported ELISA values are relative optical density percentages relative to control mean values.

Inductively coupled plasma mass spectrometry (ICP-MS):

Whole lungs and nasal cavities were excised and trimmed for determination of wet/dry weight ratios as well as trace elemental analysis (Perkin Elmer NexION 350D) undergoing standardized drying and digestion protocols (Titan MPS Microwave) using tissue specific programs, and with Sc, In and TB serving as internal standards. Results are given in μg/g of dried tissue calculated by ICP-MS Syngistix V1.1 software.

Vascular Function and Graded Dose Responses:

Aortic pharmacological response was performed 1, 7, and 30 days following a single vehicle or single WTC_PM intranasal exposure. The thoracic aorta was excised, perivascular adipose tissue removed, and 2 mm cylindrical sections mounted onto myography chamber pins (DMT620M multi-channel myography system; DMT, Ann Arbor, MI) in a continuously oxygenated bath per standard assay procedures [6]. Standard incubation challenges were employed and drug stock concentrations of phenylephrine (PE) and acetylcholine (Ach) (Sigma-Aldrich®, St. Louis, MO) were administered in ascending concentrations. Vascular contractility was expressed as a percentage of the peak response to 100 mM KPSS. Half-maximal dilation and contraction values and maximum contraction and relaxation values were u sed to compare treatment groups [7].

Statistical Analyses:

Statistical Analyses were performed using GraphPad Prism® software (Version 5.0, GraphPad Software Inc.) or Microsoft Excel. All data are expressed as mean ± SEM. An unpaired t-test was used to determine differences within treatment groups with respect to the various intranasal treatments and control treatments. A one-way analysis of variance (ANOVA) with a Student-Newman-Keul’s post-hoc analysis was used to determine significant differences associated with multiple exposure groups as well as control groups. A repeated-measures two-way ANOVA with Bonferroni’s post-tests was used to evaluate vascular reactivity with respect to pharmacologic testing. Dixon and Grubbs analyses were used to screen for outliers. Differences were interpreted as statistically significant when p-values were below the threshold of ≤0.05.

Results

WTC_PM induces in vitro cytotoxicity in structural and immunologic cells

In vitro methods using BEAS-2B and THP-1 cell lines were employed to preliminarily investigate the cytotoxic potential of WTC_PM10-53 and WTC_PM<53 particle size groups, via LDH release, NO₂⁻ formation and colony formation. Figure 1A demonstrates increased LDH release in BEAS-2B bronchial epithelial cells with increasing WTC_PM concentrations in cell culture media. In comparison to control cells, LDH release increased from approximately 7% at 31 μg/ml to 35-50% at 1000 μg/ml across both particle size groups, with R² values of 0.82 and 0.73 for 10-53 μm and <53 μm groups, respectively. Figure 1B illustrates a positive correlation (R²=0.94) between increased BEAS-2B LDH production and increased NO₂⁻ production, as well as dose-dependent decreased colony formation units in Figure 1C, ranging from 93% clonogenic ability at 31 μg/ml down to 0% at both 500 and 1000 μg/ml. Similarly, THP-1 monocytes showed a dose-dependent increase in NO₂⁻ production (Figure 1D).

Figure 1. In vitro cytotoxicity markers 24 hours post-WTC_PM exposure.

A) BEAS-2B lactate dehydrogenase (LDH) release from WTC_{PM10-53 μm} and WTC_{PM<53 μm} exposed cells. Asterisks (*) indicate a statistically significant difference from 1 mg/ml exposures (p<0.05). B) Cytotoxicity and NO₂⁻ correlations with respect to WTC_PM10-53 exposure. C) Clonogenic survival assay assessment; photos unavailable. D) THP-1 activated monocyte production of NO₂⁻ in vitro in relation to WTC_PM10-53 exposure. Bars are mean ± SEM with n=3/group. Asterisks (*) indicate a statistically significant difference from control values (p<0.05). <LOD indicates below the limit of detection.

WTC_PM induces respiratory tract inflammation in vivo

Figures 2 A and B illustrate significant neutrophil influx in both upper and lower respiratory tissues of mice treated with WTC_PM in single or multiple intranasal instillations (IN). Nasal PMN infiltrates significantly increased from control baseline (~3%) to approximately 20-25% PMNs in single exposure dose categories of 125 – 1000μg. Similarly, pulmonary % PMN influx was significantly increased at doses >31μg. In comparing single and multiple dose groups, some groups receiving multiple IN instillations were found to have decreased % PMN influx relative to mic’ receiving the same dose in a single IN instillation (Supplemental Figure 1). In a 24-hour time course evaluation, mice exposed to a single dose of 125μg WTC_PM experienced peak NLF and BALF PMN influx 24 hours and 6 hours post-exposure, respectively (Figures 2C and D). In BALF, total cell count peak (5.9 x 10⁴ total cells) coincided with PMN influx (50%) and protein increases (17000μg/ml. ELISArray data in Figures 2E and F identify significantly increased cytokines in NLF (II α IL2, IL12, IL17A, TNFα and GM-CSF) and BALF (IL1α, IL2, IL4, IL6, IL12, IL17A, TNFα, G-vFSF and GM-CSF) in mice 24 hours after a single exposure to 250 or 1000 μg of WTC_PM. With regards to short- and long-term pulmonary injury indicators, wet/dry ratios (indicative of lung edema) were significantly higher in single exposure WTC_PM treated mice 24 hours post-exposure (Supplemental Figure 2). While data indicate dissipation of edema at 30 days post-exposure, a s nglt treatment of 1000 μg WTC_PM contributed to an 11% decrease in viable alveolar macrophages 30 days post-exposure (Supplemental Figure 3).

Figure 2. Biomarkers of inflammation in NLF and BALF of WTC_PM<53 exposed C57BL/6 mice.

A&B) Percent (%) polymorphonuclear neutrophil (PMN) influx comparisons in NLF and BALF 24 hours post-initial or final WTC_PM<53 exposure. Reported values are averages of individually measured lavage samples ±SEM with n=5-7. C&D) A 24-hour time course comparison of %PMNs, total protein, and total cells in single treatment (125 μg) mice. Total cell count unavailable for NLF. Time course values are averages of individually measured samples ±SEM with n=3-4. E&F) ELISArray cytokines and chemokines samples were pooled (n=5) and measured in duplicate 24 hours post-exposure in NLF and BALF, respectively (250 μg dose for NLF unavailable). Reported values are relative optical density percentages (relative to control mean values). Asterisks (*) indicate a statistically significant difference from control values (p<0.05).

Alkalinity, particles or metals as toxicity factors

With reference to pH, DPBS-suspended WTC particle pH remained neutral with a pH range of 6.8-7.3 (Figure 3A). Conversely, water-suspended particles exhibited a pH dependent increase in relation to increased WTC_PM concentrations, ranging from 6.5-10. Use of three different WTC_PM concentrations suspended in water (reflective of low, medium, and high pH) or DPBS (reflective of neutralized pH), identified BALF PMN influx differences between water and DPBS groups, with PMNs remaining similar across water-suspended groups (15-19% PMN) and varied in DPBS-suspended groups (9-39% PMN) Figure 3B). In NLF, nitrite levels of water suspended WTC_PM remained unchanged from control values. In medium (125μg) and high dose (1000μg) DPBS-suspended group, NLF total nitrite levels were significantly increased (~25μM) as compared to the DPBS control group (7 μM) (Figure 3C) . Conversely, BALF total nitrite levels significantly decreased in a dose dependent manner regardless of suspending vehicle (Figure 3D). Figure 3E further identifies WTC particles to be the main influencing toxicity factor with the most robust PMN response had by the DPBS-suspended (pH neutralized) WTC particle group. Evidence of particle penetration and retention into the upper and lower airways 24 hours and 90 days post-single exposure can be seen in Figures 4A and B. Graded pulmonary tissues collected 30 days post-WTC_PM exposure revealed minor to mild inflammation, no increased mucus production or fibrotic formations (Supplemental Figure 4 and Supplemental Table 1). Analysis of pulmonary insoluble and soluble particles revealed lung burden increases of Al (3964%), Cr (1172%), Ca (284%) and Mn (479%). Pulmonary levels of Al, Ti, Cr, Pb, Ba, Sr, Zn, Cu, Mo, Na, Mg, Ca, Mn, and Ni were all found to be significantly increased in higher exposure concentration groups of 1000μg (data not shown) and 4000μg (Figures 5A and B).

Figure 3. Evaluation of pH and/or particle effect in BALF.

A) pH of WTC_PM suspended in water or DPBS at varying concentations. B) %PMN comparison of water or DPBS suspended WTC_PM 24 hours post- single exposure. C&D) NLF and BALF NO₂⁻ evaluation of WTC_PM suspended in water or DPBS. Reported values are averages of individually measured samples ±SEM with n=5. E) pH effect vs. particle effect on PMN influx. All endpoints were evaluated 24 hours post single exposure. Symbols (*) and (#) indicate a statistically significant difference from vehicle control values or compared groups, respectively (p<0.05).

Figure 4. Gross microscopic examination of WTC particles in lavage fluids and pulmonary tissue.

A1-A4) BALF retrieved particles from WTC_PM<53 exposed mice 24 hours post-exposure. A5) BALF sample; phagosome encapsulated WTC particle within a macrophage. A6) NLF retrieved particles from WTC_PM<53 exposed mice 24 hours post-exposure. B1-3) Gross histopathological examination of whole lungs and particle retention. H&E staining. Red circles identify embedded WTC particles within pulmonary tissues of WTC_PM<53 exposed mice.

Figure 5. ICP-MS total lung burden of trace elements.

Whole lung analysis of WTC_PM exposed mice (4000 μg) 24 hours post-exposure for insoluble (A) and soluble (B) trace elements. All elements presented are statistically significant (p<0.05) in WTC_PM exposed mice compared to the control group. Below limit of detection is indicated by <LOD. Reported values are averages of individually measured samples ±SEM with n=4-5.

Potential indications of long-term effects on other organ systems

Preliminary BALF and serum total nitrite data in Figures 6A and B illustrate significantly lower levels of BALF and serum nitrite 24 hours post-WTC_PM exposure. A more than doubling of BALF and serum total nitrite occurred in the 1000μg exposure group 90 days post-exposure, as compared to controls. Figures 7A–C identifies maximum aortic contraction responses to phenylephrine (PE) as well as maximum relaxation responses to acetylcholine (Ach) in WTC_PM exposed mice at 24 hours, 7 days, and 30 days post single WTC_PM exposure. Figure 7A illustrates a lack of difference between control and WTC_PM exposed mice against increasing concentrations of PE and Ach, respectively, 24 hours post-WTC_PM exposure. Aortas tested 7 days post-WTC_PM exposure began exhibiting differences between WTC exposed and control groups with a more definitive difference between maximum PE and Ach responses (Figure 7B). These changes remained statistically insignificant, although differences in % change were larger between control and WTC_PM exposed groups. This relationship is more evident and sustained 30 days post-exposure, whereby vessel relaxation values are significantly different from control aortic vessel values at −7, −6, and −5M concentrations of PE and Ach, with approximately a 35% difference between control and WTC_PM exposed groups (Figure 7C). ICP-MS data from whole hearts revealed significant decreases in soluble Mg, K, Mn, Cr, and Zn and increased Cu, As, Se, P, and Ca and K in animals sacrificed 24 hours post-exposure as well as 30 days post-exposure (Supplemental Figures 5 and 6).

Figure 6. Time course comparisons of BALF and serum NO₂⁻.

Doses given as single or multiple dose exposures and evaluated 24 hours, 30 days or 90 days post-exposure. Samples were measured individually in triplicates using the Griess reagent assay. Reported values are averages of individually measured BALF samples ±SEM with n=4-6 and serum samples ±SEM with n=3. Asterisks (*) indicate a statistically significant difference from control values (p<0.05).

Figure 7. Temporal vascular response curves in response to PE and Ach.

Mice were exposed to single dose of 1000 μg WTC_PM. Vascular reactivity was measured at 24 hours, 7 days and 30 days post-exposure and analyzed by repeated-measures two-way ANOVA with Bonferroni’s post-tests. Reported values are averages of individually measured samples ±SEM with n=3/4. Asterisks (*) indicate a statistically significant difference from control value responses using 2way ANOVA (p<0.05).

Discussion

The WTC_PM exposure event was not a single exposure event, but a multiple exposure event, with continuous exposures through rescue/recovery operations, working on the central pile which burned well into early October 2001, and the year-long outdoor and indoor clean-up phases which are less well documented. Thus, formal human exposure estimates have not been identified, but have been categorized by amount of time spent occupationally on the WTC pile. Aside from a lack of understanding with regards to human dosimetry estimates, many challenges remain regarding routes of exposure, duration, ventilation rates, frequency, locality/temporality, temperature, and other forms of exposure.

Previous WTC_PM in vitro studies revealed exposure to WTC_PM10–53 increased inflammatory cytokine production in alveolar macrophages obtained from human subjects without WTC-exposure or pulmonary symptoms and suggested the large particle exposure may have contributed to the high incidence of lung injury in WTC exposed populations [8]. Cytotoxicity endpoints, including decreased cellular viability and increased apoptotic activity in pulmonary fibroblasts were also reported at doses similar to those tested in this study [9]. Initial rodent in vivo investigations examining WTC induced health effects were begun by Gavett et al., briefly 2 years after the collapse of the towers, and followed up by studies published by Cohen et al. and Vaughan et al., and culminating in a review of literature published by Lippmann et al. [10–12, 3]. The studies herein continue the investigation into respiratory and endothelial tissue impacts with evaluations focused at the nasal-pulmonary interface, identifying increased inflammatory parameters derived from WTC_PM exposure. The design of these assessments were based on previous WTC human health studies demonstrating a link between disease development, oxidative and inflammatory potentials of the dust itself. The data and information provided conclusively demonstrate nasal and pulmonary inflammation follow WTC_PM exposure. The mechanisms behind inflammatory outcomes may be related to respiratory oxidant stressors, brought about by a surplus of reactive electrophiles, mainly by both resident (epithelial and endothelial) cells and infiltrated leukocytes, all of which have been found to play substantial roles in tissue injury and abnormal tissue repair.

WTC_PM was found to be cytotoxic and induced nitritive stress parameters in pulmonary derived cells (BEAS-2B) and immunologic monocytic cells lines (THP-1). These in vitro parameters proved to be dose specific, supporting the idea that structural cells, as well as immunologic cells within pulmonary pathways may very well have been adversely affected in the WTC_PM exposure event. Typically, in normal and uninjured alveoli, a major part of the surface area is comprised of type I epithelial cells and cuboidal type II cells that are involved in surfactant production, fluid transport, and repopulation of the alveolar epithelium post-injury. In injured alveoli, the epithelia undergo apoptotic/necrotic events, basement membrane denudation, inflammatory cell influx, as well as macrophage and PMN activation [13,14]. During these events, proteases, oxidants, cytokines/ chemokines, and other inflammatory mediators are released, coupled with protein-rich fluid influx into the alveolar spaces. Many of these events have been documented within the scope of this investigation and establish a firm foundation linking WTC_PM exposure and respiratory tissue inflammation.

WTC_PM was found to induce inflammation in mouse nasal and pulmonary tissues, as evidenced by increased neutrophil influx in NLF and BALF. A typical dose-response is not as clearly visible in in vivo studies after single exposures to 125 μg as compared to in vitro dose-responses. Considerations for this include particle overloading after a certain dose as well as the nature of a non-homogenous mixture which may result in different response outcomes. Additionally, WTC_PM exposure was found to significantly increase alveolar macrophage cell death 30 days post-exposure. Changes in pulmonary macrophage cell populations have the potential to alter long-term immune cell responses to WTC_PM [15]. Both acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by a robust inflammatory response involving substantial infiltration of PMNs into the lung, which ultimately result in capillary-alveolar barrier dysfunction, followed by pulmonary edema, subsequently resulting in gas exchange dysfunction [13,16]. WTC_PM exposure also induced acute proinflammatory mediators and resulted in significantly increased whole lung wet/dry ratios, suggestive of excessive volume of fluid accumulation in the tissue, brought about by aberrant changes in pressures acting across microvascular walls. These changes can provoke epithelial integrity impairment in addition to molecular structural compromises involved in fluid and solute flux.

Inclusively, oxidative stress is known to increase production of inflammatory mediators within epithelial lung cells and immune cells, initiating and/or promoting mechanisms of disease, and has been described as a major contributing mechanism resulting in pathological outcomes associated with respiratory dysfunction [17,18]. What largely remains unknown is how oxidative stress mechanisms impact the nasal-pulmonary region, which remains especially true for WTC exposed cohorts. Under normal homeostatic conditions, reactive oxygen species are generated as byproducts of oxygen metabolism, with reactive nitrogen species generated as products of NO metabolism, and more specifically, nitrite production via oxidation of NO [19]. Cohen et al. identified the potential for a single high exposure to WTC_PM to alter pulmonary expression of genes associated with oxidative stress and immune function [20]. Within this investigation, WTC_PM was found to induce the nitric oxide (NO) metabolite NO₂⁻ in vitro in a dose dependent manner. In vivo, WTC_PM exposure produced decreased levels of mouse NLF and BALF NO₂⁻, an endpoint associated with pulmonary arterial hypertensive states [21–23]. Concurrently, pulmonary arteriopathy was found to be present in 58% of lung biopsies from non-FDNY WTC-PM exposed individuals [24]. Levels of NO₂⁻ were noted to be doubled in WTC_PM exposed mice 90 days post-exposure and may be a consequence of prolonged inflammatory responses including overproduction of nitric oxide (NO) and tissue injury brought about by the dust. This increase in NO₂⁻ has been correlated with asthmatic phenotypes [25,26]. Similarly, asthma and other respiratory-related conditions were found to be new onset cases brought about by exposure to WTC_PM in both adults and children [27–31]. While both pulmonary arteriopathy and asthmatic pathologies have markedly different clinical presentations, they share key pathological features (inflammation and smooth muscle cell constriction and proliferation), thought to be a consequence of either mechanical distal airway compression via remodeled pulmonary arteries or imbalances in vaso/broncho-constrictive mediators (increased endothelin-1 and decreased NO) [32,33].

Cardiovascular diseases are among the emerging health concerns from WTC_PM exposure [34–36]. NO, a product of endothelial NO synthase (eNOS) and a key signaling molecule involved in vascular homeostatic processes was found to be significantly decreased in serum NO₂⁻ 24 hours post-exposure. Conversely, mouse serum NO₂⁻ levels more than doubled 90 days post-WTC_PM exposure, mirroring NO₂⁻ level activities in NLF and BALF samples. Decreases in NO bioavailability have been found to be a hallmark feature in endothelial dysfunction preceding atherosclerotic events as well as an independent predictor of cardiovascular risk, attributed to NO synthesis reduction and reduced NO scavenging by ROS [37]. On the contrary, endothelial dysfunction has been associated with eNOS upregulation rather than downregulation, attributed to elevated levels of H₂O₂, a dismutation product of O₂.⁻[38,39]. The vascular myography studies herein suggest WTC_PM can induce endothelial dysfunction over time, given evidence of NO₂⁻ dysregulation and pharmacologic testing of vascular tone through vasoconstrictive and vasorelaxation mechanisms.

Due to the high alkaline nature of WTC_PM, it is important to discern whether exposure outcomes were driven by the presence of WTC particles or by the alkaline nature of the dust. Neutralized particles resulted in a doubling of % PMN influx into WTC_PM exposed lungs of mice as compared to animals exposed to WTC_PM suspended in water. Lending more evidential support for particle driven outcomes, neutralized particles independently induced increased NO₂⁻ production in mouse nasal cavities while simultaneously depleting levels of NO₂⁻ in lower airway tissues. What remains unanswered is why do neutralized WTC particles induce a larger inflammatory response? Metals identified in WTC_PM have been found to have long retention times in rat pulmonary tissues [40]. Multiple studies have identified metal solubility to increase with lower pH environments. Physiologically, these environments can be found within cellular lysosomal compartments. Hypothetically, solubilized metal ion release from lysosomal compartments into extracellular fluids may incite inflammatory pathways that would otherwise be kept in homeostatic balance. The addition of an acutely alkaline pH environment may hinder lysosomal capacities to degrade internal compartmental contents, resulting in a less robust inflammatory response.

It is important to note WTC_PM exposure was done using a novel technique with suspended WTC_PM delivered through IN instillation, providing the most optimal exposure scenario with relevance to both nasal and pulmonary tissues, as well as mimicking the significantly high incidence of particle overloading that occurred in those caught in the dense WTC_PM plume. Inhalation is a natural delivery mechanism for particles and has led to comparable/real world exposure scenarios, allowing for evaluations at all levels of the respiratory tract as well as deposition, clearance, kinetics, and calculated delivered dose studies [41, 42]. The largest limitation of inhalation studies, in comparison to studies presented here, is inhalation studies largely apply to fine and ultrafine fractions of particles. Due to the presence of much larger sized particles in WTC_PM, these larger particles may deposit on the outer nares of mice or clog their nasal passages, producing impacts mostly in the upper respiratory tract, limiting particle delivery to the lungs and other targets. Thus, particle suspensions were used, allowing for particles to be more equally distributed throughout the nasal cavity, as well as aspirated into the lower respiratory tract, reaching the lungs not as a sheet of liquid but rather aerosolized as large droplets and deposited in the lung as individual particles. However, it is important to recognize that the use of particle suspensions has its own limitations. During inhalation, particles are delivered as individual particles, which when deposited, have direct contact/ hits with epithelial surfaces. In the case of highly alkaline WTC_PM, this mode of deposition could produce intense, localized alkaline spots that could be more injurious than when in suspension. Using suspended particles, the impact of initial hits on airway epithelium may be reduced, potentially underestimating both acute and chronic outcomes from exposure to WTC_PM.

Conclusion

Most available data on WTC_PM exposed human cohorts have been from epidemiologic studies, with limited literature investigating causative mechanisms of disease. This investigation serves as the first study to systematically explore the particle-driven inflammatory effects of WTC_PM, as well as providing the first extensive data on acute and subchronic systemic responses related to WTC_PM exposure. These data further validate the toxic potential of a dust that was initially considered to be “harmless”, putting the health of the public at great risk, especially for rescue workers, cleanup crews, and local residents who were continuously exposed.

This study has demonstrated WTC_PM exposure alone to be an inducer of nitritive stress and inflammation in nasal, pulmonary, and to some extent cardiovascular tissues. In addition, in vitro studies using pulmonary and monocytic immune cell lines revealed increased susceptibility to cell injury and death in relation to WTC_PM exposure. Implications for other adverse health outcomes could include further cardiovascular homeostatic alterations as well as mental health outcomes, due to the location and proximity of CNS tissues (olfactory receptor neurons) in the nasal cavity. In addition, insoluble WTC_PM deposited on epithelial linings were not fully cleared, resulting in tissue particle retention. Subsequent particle translocation via anterograde transport by olfactory receptor neurons or organ-to-systemic distribution pathways should be investigated further.

Table 1. WTC_PM<53 mouse exposure matrices (A) and human equivalent dosing (HED; B).

HED (mg) calculations and ratios are derived from regulatory allometric body weight scaling factors of 0.67 (BW^0.67) and 0.75 (BW^0.75) and assuming an average mouse weight of 0.02 kg and 50 kg or 70 kg for humans.

A.
Exposure groups	Dosing Frequency	Sacrifice Timepoint	Mean sample size (n=)
Single exposure	Single exposure	24 hours., 7 days, or 30 days post-exposure	5
Multiple Exposures	Four exposures over the course of 7 days, every other day.	24 hours. post-final exposure	5
Multiple IN exposures + 90-day recovery period	Four exposures over the course of 7 days, every other day.	90 days post-final exposure	5

B.
Mouse IN dose (mg)	Mean HED (mg) (BW0.67)	Mean HED (mg) (BW0.75)	Mean total HED	Inhalable HED (mg/m3)
0.031	6.6	12.5	9.6	1.0
0.062	13.2	25.1	19.1	1.9
0.125	26.6	50.5	38.6	3.9
0.25	53.2	101.1	77.2	7.7
0.5	106.5	202.1	154.3	15.4
1	213.0	404.3	308.6	30.9
4	851.9	1617.2	1234.5	123.5

Table of Abbreviations

Abs

Absorbance

Ach

Acetylcholine

Ag

Silver

Al

Aluminum

ANOVA

Analysis of Variance

As

Arsenic

ATCC

American Type Culture Company

Ba

Barium

BALF

Bronchoalveolar Lavage Fluid

Be

Beryllium

BEAS-2B

Immortalized Human Bronchial Epithelial Cells

C57BL/6

C57BL/6 Inbred Mouse

Ca

Calcium

Cd

Cadmium

CD-X

Cluster of Differentiation

CNS

Central Nervous System

Co

Cobalt

COPD

Chronic Obstructive Pulmonary Disease

Cr

Chromium

Cu

Copper

DMEM

Dulbecco’s Modified Eagle Medium

DMSO

Dimethyl Sulfoxide

DPBS

Dulbecco’s Phosphate Buffered Saline

ELISA

Enzyme-Linked Immunosorbent Assays

FBS

Fetal Bovine Serum

Fe

Iron

G-CSF

Granulocyte-colony stimulating factor

GM-CSF

Granulocyte-Macrophage Colony Stimulating Factor

H&E

Hematoxylin and Eosin

HCl

Hydrochloric Acid

IACUC

Institutional Animal Care and Use Committee

ICP-MS

Inductively Coupled Plasma Mass Spectrometry

In

Indium ICP-MS Internal Standard

IN

Intranasal

iNOS

Inducible Nitric Oxide Synthase

IT

Intratracheal Instillation

K

Potassium

KO

Knock Out Mouse

KPSS

High Potassium Physiologic Salt Solution

LDH

Lactate Dehydrogenase

Mg

Magnesium

Mn

Manganese

Mo

Molybdenum

mRNA

Messenger Ribonucleic Acid

Na

Sodium

NaOH

Sodium Hydroxide

Ni

Nickel

NLF

Nasal Lavage Fluid

NO

Nitric Oxide

NO₂⁻

Nitrite

P/S

Penicillin/Streptomycin

PAS

Periodic Acid Schiff

Pb

Lead

PE

Phenylephrine

PM

Particulate Matter

PM_2.5

Particulate Matter <2.5μm

PM₁₀

Particulate Matter <10μm

PMA

Phorbol-12-Myristate-13-Acetate

PMN

Polymorphonuclear Neutrophil

PSS

Physiologic Salt Solution

ROS

Reactive Oxygen Species

Sb

Antimony

Sc

Scandium ICP-MS Internal Standard

Se

Selenium

SEM

Standard Error Mean

Sn

Tin

Sr

Strontium

Tb

Terbium ICP-MS Internal Standard

THP-1

Human Leukemic Monocyte Line

Ti

Titanium

Tl

Thallium

TNF

Tumor Necrosis Factor

V

Vanadium

WTC

World Trade Center

WTC_PM

World Trade Center Particulate Matter

WTC_PM<2.5

World Trade Center Particulate Matter <2.5 μm

WTC_PM10-53

World Trade Center Particulate Matter 10-53

WTC_PM<53

World Trade Center Particulate Matter <53 μm

Zn

Zinc