HSOA Journal of Toxicology: Current Research Prolonged Exposure to Ultrafine Black Carbon (UBC) in Combination with Microbial Proxies Trigger Oxidative, Proliferative, and Innate Immune Perturbations in Pulmonary Lung-Derived Cells

Prolonged Exposure to Ultrafine Black Carbon (UBC) in Combination with Microbial Proxies Trigger Oxidative, Proliferative, and Innate Immune Perturbations in Pulmonary Lung-Derived Cells. Abstract Particulate Matter (PM), one of the main criteria pollutants identified by the US-EPA, consists of particles produced from the incom - plete combustion of burning fuels. Ultrafine particles comprise a substantial proportion of outdoor PM, including ultrafine black car - bon or UBC (<100nm). Upon inhalation, UBC weaken lung health due to their minute size, their ability to enter the respiratory tract, and their capacity to translocate across distant, secondary targets. Data acknowledging cytotoxic carbonaceous nanoaerosol effects on pulmonary-immune cell behavior is indeed lacking. We hypoth esized that prolonged in vitro exposure to nanocarbon contributes to cellular stress, impairs cell-cycle activity, and significantly elicits a counter-response to short-term microbial proxies. Human (A549) and murine (LA4) epithelial lung cells, human THP1 monocytes, and murine RAW264.7 phagocytes were seeded on day zero then given UBC (at 0.3, 1.0, and 3.0μg/mL) on days 1, 4, and 7. For this set of assays, multiple markers of oxidative damage and proliferative activity were measured after continuous UBC insult. This showed an imbalance of cellular anti-oxidant/pro-oxidant levels, modification to inner mitochondrial membrane potential, and abnormal cell-cycle activity. In addition, UBC-treated cells were succumbed to three mi - crobe-like mimics on day 9. All extracellular measurements for the second section of our study were performed on day 10. Cumulative administrations to different microorganisms (LPS, Poly I:C, and Zy -mosan) following exposure to 3.0μg/mL-UBC generated dispropor tionate oxidant trends and increased cytokine production depending on cell type. To conclude, microbial challenges in the presence of UBC caused an observable shift for cellular behavior, suggesting that continuous ultrafine-PM exposure can potentially alter inflam matory function in vitro .


Introduction
The Texas Commission on Environmental Quality (TCEQ) requires that emissions from both "point" (a single, identifiable source) and "non-point" sources should be calculated and properly reported to the public. TCEQ's air quality division maintains that emissions originating from on-road mobile sources such as combustion-related emissions from tailpipes must be identified at the state level [1]. At the federal level, the National Ambient Air Quality Standards (NAAQS) were set as part of the Clean Air Act legislation several decades ago. The NAAQS statute established primary and secondary national air quality standards; particulate matter or PM is included as one of six criteria air pollutants. As stated by the United States Environmental Protection Agency (US-EPA), these outdoor pollutants are known to harm the environment and negatively affect public health [2,3]. Fine (<2.5µm in diameter) and coarse (<10µm diameter) PM is closely monitored and regulated across the US by this agency [2]; Inhaling PM 0.1 inflicts severe risks to a given population, where the lung is the primary target organ [5]. Pollution from PM at this aerodynamic diameter can easily pass through the throat and into the deeper cavities of the lung [6]. PM 0.1 or nanoaerosol fractions of pollutants can originate from natural sources such as forest fires and from human activities like agricultural practices and transport emissions [7,8]. A study focusing on the El Paso airshed, proximate to the Bridge of the Americas (BOTA), found that ultrafine particles represent 95% of the total particulates at the border crossing between Ciudad Juarez, Chihuahua, Mexico and Texas [9], pinning local residents at higher risks of exposure [10][11][12]. Particle surface area directly relates to the gravity of health-related conditions affecting residents in proximity to economically advancing and developing regions. Compromised functionality of the lung was reported by people living less than 100 meters away from heavy-traffic metropolitan areas [4].
Numerous research-based evidence using in vitro experiments stress the importance of exploring the harmful effects from nanoparticle exposure and linking these with declining human health. It has been reported previously that the majority of combustion by-products are comprised of UBC [6,13,14]. Ultrafine black carbon monomers (~14nm) and carbon black aggregates (<260nm) have been tested on macrophages and monocytes; only UFP are known to induce oxidative damage, cause nuclei deformation/shrinkage, and aggregation of chromatin structures seen in vitro [15,16]. Research concerning inhalable aerosol pollution provides insight whereby these may cause deleterious lung health [17][18][19][20][21][22][23][24][25]. Both metal oxide nanoparticles and urban UFP can easily access, transport across the blood barrier, and remain in the tissues/organs even after 24hr [26]; alarmingly, these materials may even reappear at different locations via macrophage-mediated transport [27].
The World Health Organization (WHO) stated that air pollution-related deaths are closely linked to exposures to Particulate Matter (PM), described as a mixture of carcinogens, carbon compounds, and other "health-harming substances" [28,29]. Carbon black or Ultrafine Black Carbon (UBC) is a soot-like PM that encompasses a significant percentage of PM, with primary particles sized less than 100nm [30]. Black carbon materials are in high demand for numerous industrial applications including the production of rubber, tires, and printing inks [31]. Black carbon interactions have shown that ambient particulate pollution, precisely from UBC exposure, can lead to serious health effects [32]. Moreover, in vitro and in vivo models have demonstrated that accumulation of UBC causes toxicity as well as mitochondrial dysfunction and DNA damage [31,33,34]. Since UBC is largely found suspended in the atmosphere, the most common health risks associated with UBC are often lung diseases [35,36]. Inhalation of PM leads to pulmonary inflammation can subsequently spread to systemic inflammation [37,38]. Pulmonary inflammation, essential to the immune response, mainly triggers biochemical reactions from negative conditions affecting the airways. Common respiratory illnesses such as acute lung irritation and asthma involve inflammation in the lungs leading to shortness of breath, cough, and wheezing [39].
According to the Canadian Centre for Occupational Health and Safety, there are four ways particulates deposit in the lungs when inhaled [40]. Particles sized 0.5µM or less are more commonly deposited in the bronchial region of the lungs through sedimentation. Smaller particles, on the other hand, can be transported by diffusion to the alveoli due to their extremely fine size [40]. Cells found in these regions can mediate several inflammatory responses in the presence of an irritant. Macrophages, or professional phagocytic cells, found in the lung include pulmonary alveolar macrophages play a role in maintaining lung immunity [41]. The presence of antigens or particulates in the lung leads to the activation of innate immunity, which is the first line of defense against a variety of dangers stemming from exteriororigins [42]. Both inflammation and oxidative stress contribute greatly to the injurious effects from air pollution on the respiratory system [27]. Innate immunity must be closely evaluated since chronic insult from external materials in the airways may impair pulmonary function.
Moreover, in vitro studies suggest that pulmonary fibrosis and carcinogenesis can be due to chronic inflammation brought upon by stress to internal cellular components [43][44][45]. This stress may contribute to a notable imbalance of antioxidants/pro-oxidants and measurable cell-cycle disturbances and are attributed to interactions with nano-or UFP [46,47]. The above in vitro models have detailed stress-induced generation of Reactive Oxygen Species (ROS) and subsequent oxidative damage from particle deposition while suggesting that they could impact downstream lung function. Nonetheless, the research mentioned above reported the detrimental effects at much shorter time frames (10-72 hours) and higher concentrations of nanocarbon exposures (10-500μg/mL) in comparison to our present work.
Long-term (>7 days), low-dose (<10μg/mL) in vitro studies investigating industrial activity, pulmonary pathology, and long-term Ultrafine Black Carbon (UBC) remain understudied. We predicted that sustained exposure from low-level concentrations of carbonaceous nanoparticles alter cellular function by affecting mitochondrial fitness and modifying cellular life-cycle. An in vitro regimen was used herein to obtain a closer estimation of cellular responses from contact to ultrafine aerosol by investigating cellular behavior upon additive exposure to nanocarbon particles at low doses. Human pulmonary epithelial cells (A549), THP1 human peripheral-blood monocytes (macrophage precursors), murine macrophages (RAW264.7), and murine pulmonary epithelial cell (LA4) lines were analyzed. The four different cell types were individually exposed to increasing amounts of UBC suspended in cell culture media (0.3, 1.0, and 3.0μg/mL) to the completion of 10 days. Oxidative stress indicators, such as ROS accumulation, free intracellular glutathione levels, mitochondrial membrane potential, as well as, proliferative activity (cell-cycle analyses) were conducted to address our conjecture. Results showed that the three low-dose concentrations of UBC administered over a 9-day period induced oxidative damage at a moderate rate and provoked a change to cell-cycle activity. GSH (reduced or free glutathione) levels severely increased compared to the untreated control yet decreased in a pollutant-dependent manner for all cell lines. Mitochondrial membrane health yielded depolarization across both human lung epithelial cells and monocytes; murine phagocytes remained in a hyperpolarized state (no significant damage to RAW264.7 mitochondrial fitness seen). These results indicated that sub toxic UBC availability disturbed intracellular health by causing moderate responses against

Nanocarbon material
Black carbon (Vulcan XC-72) purchased from Cabot Corporation (Billerica, MA) was used for all assays. Carbon nanoparticles, or UBC, have been characterized by Soto et al., by Transmission Electron Microscopy (TEM) using a Hitachi H-8000 analytical TEM operated at 200kV accelerating potential fitted with a goniometer-tilt stage and a Noran Energy Dispersive (X-Ray) Spectrometer (EDS) [48]. For this study, nanocarbon suspensions of 10mg/mL were sonicated in warm 1X PBS (Fisher Scientific, Pittsburgh, PA) and diluted to 100µg/mL. These were further diluted in complete cell culture media and subsequently re-homogenized prior to cell administration in order to lessen particle aggregation.
The incubation conditions were maintained at 37°C and a 5% CO 2 environment for all cells.

Exposure regimen
6-well plates (Fisher Scientific, Pittsburgh, PA) were used to seed at a density of 1,500 cells per well on day "0" and allowed to attach for 24hr prior to UBC-particle treatments. Depending on the assay type, the following groups consist of: [A] (1) untreated cells; (2) H 2 O 2 -treated cells; (3) 0.3µg/mL UBC-treated cells; (4) 1.0µg/mL UBC-treated cells; and (5) 3.0µg/mL UBC-treated cellsor [B] (1) untreated cells; (2) 3.0µg/mL UBC-treated cells; (3) 3.0µg/mL UBC+M 1 cells; (4) 3.0µg/mL UBC+M 2 cells; and (5) 3.0µg/mL UBC+M 3 treated cells. On day 1, the cells were either treated with all three UBC concentrations where two groups per cell type remained untreated to serve as controls for set [A]or treated with the highest concentration of UBC where one group remained untreated to serve as a "media only" control for set [B]. It follows for set [A]: experimental wells were replenished with UBC (0.3, 1.0, and 3.0µg/mL) on days "4" and "7" and untreated groups received only fresh media. On the 9 th day, one set of untreated wells was exposed to hydrogen peroxide (10mM H 2 O 2 ) as a positive control for toxicity unless otherwise indicated. On the 10 th day, all 5 groups were harvested, centrifuged, and re-suspended in 1-2mL of ice-cold 1X PBS for subsequent assays. For set [B]: experimental wells received sustained UBC (at 3.0µg/mL) on days "4" and "7" where untreated control groups received fresh media. On the 9 th day, one set of wells was exposed to 10mM H 2 O 2 as a positive control for toxicity unless otherwise noted. The microbial stimuli section below describes the additive administrations of 3.0µg/mL UBC in detail with three different microbe-like (M 1 , M 2 , or M 3 ) infections. On the 10 th day, all groups were harvested, centrifuged, and re-suspended in 1mL of ice-cold 1X PBS to conduct experiments. Aliquots involving only supernatant studies were stored at -20ºC immediately after collection; these were assayed within 4-5 weeks.

Microbial stimuli
Following nine days of sustained UBC treatments, the cells were treated with three Toll-like receptor (or TLR) ligands. The different TLRs were administered to the UBC-treated cells for 24hr (from day 9 to day 10). The following were used as proxies to mimic microbial contaminants suspended in phenol-red free compete DMEM: lipopolysaccharide to simulate a bacterial-like infection (or LPS); polyinosinic-polycytidylic acid to mimic a viral-like infection (or Poly I:C); Zymosan (Zymo) of yeast origin to simulate a fungal-like infection (Fisher Scientific, PA). The treatments for these microbes were handled at the following final concentrations: 0.5X LPS, 500µg/mL Poly I: C, and 250µg/mL Zymo. The TLR ligands that were targeted include TLR-2, 3, and 4, where LPS binds TLR-4, Poly I:C interacts with TLR-3, and Zymo targets TLR-2. All TLRs in this study are found to be membrane-bound, while only Poly I: C can be internalized into cellular phagosomes as well. Every microbial stimulus is labeled and written throughout the manuscript, as indicated: M 1 (LPS), M 2 (Poly I:C), and M 3 (Zymo).

ROS detection using confocal imaging
Reactive Oxygen Species (ROS) generation was qualitatively presented using a fluorescent dye-based assay. Digital high-resolution fluorescence images were captured by using a confocal microscope equipped with an EC Plan-Neofluar 40x magnification/1. 30

Intracellular oxidative damage
The content of intracellular stress by reduced glutathione (free GSH) depletion after exposure to nanocarbon particles was conducted via GSH-Glo Glutathione Assay (Promega Corporation, Madison, WI). The luminescent-based method determined the level of free GSH from cells in suspension and directly measured anti-oxidants produced in response to oxidative stress. After 9 days of UBC treatment (see Exposure regimen), the cells were harvested and transferred to white wall 96-well plates (Fisher Scientific, Pittsburgh, PA). According to the manufacturer's instructions, the first component (GSH-Glo) was added and the plates incubated at Room Temperature (RT) for 30min. The second component (luciferin detection reagent) was added to the wells and incubated at RT for an additional 10-15min. The samples were read via Luminoskan Ascent Software v2.6 (Thermo Fisher Scientific Ascent Luminometer v2.5). Free GSH concentrations (in µM) were determined by interpolation from a generated GSH standard curve. Experimental samples along with associated controls were assessed in parallel.

Mitochondrial membrane health
Analysis of mitochondrial health after exposure to nanocarbon was conducted via MitoProbe JC-1 Assay Kit (Life Technologies Corporation, Carlsbad, CA) and flow cytometry. This quick-stain method determined the percentage of cells in suspension with damaged mitochondrial membranes or depolarized profiles. Following 9 days of UBC treatment (from Exposure regimen above), the cells were harvested and transferred to flow cytometry tubes (Fisher Scientific, Pittsburgh, PA) in triplicates. 2.0µL of JC-1 (5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcar-bocyanine iodide) cationic dye was added to each sample for a final concentration of 2.5µM and then incubated at 37ºC with 5% CO 2 for 30min. The cells were rinsed in 1X PBS and read immediately via flow cytometry (Beckman Coulter FC500 Flow Cytometer Cytomics Analyzer System). Approximately 10,000 to 100,000 events (cells) were acquired per sample and analyzed by utilizing CXP software (Beckman Coulter). Each experimental sample along with the associated controls were performed in parallel.

Cytometric proliferative analysis
Perturbations to normal cellular cycle progression were assessed using a Nuclear Isolation Medium (NIM) containing the DNA intercalating agent, DAPI, and flow cytometry. The NIM-DAPI reagent stains and cross-links cell nuclei allowing the quantification of DNA content in a cell population. Lung-derived cells (1,500 cells/well) were treated with different concentrations of UBC for 9 days (see Exposure regimen). The cells were harvested on day 10 and rinsed in 1mL of 1X PBS. 100µL of re-suspended cells in 1X DMEM (phenol-red free) and 200µL of nuclear isolation medium and 4',6-diamidino-2-phenylindole solution (NIM-DAPI; Beckman Coulter) were gently mixed in flow-cytometry tubes (Fisher Scientific, Pittsburgh, PA) just before measurement using a Gallios Flow Cytometer (Beckman Coulter). NIM-DAPI-stained cells were read after excitation. About 1x10 4 to 1x10 5 events were collected and analyzed per sample using the Kaluza Flow Cytometer Software (Beckman Coulter). Experimental samples along with associated controls were similarly examined. RAW264.7 murine macrophage cycle distributions are shown: Hypodiploid, diploid, hyperdiploid, and tetraploid.

Extracellular oxidative stress
The production of a stable nitrite metabolite (NO 2 -) using the Griess Reagent System (Promega Corporation, Madison, WI) was measured spectrophotometrically (Molecular Devices). The sulfanilamide and N-1-Napthylethylenediamine Dihydrochloride (NED) colorimetric assay is used to detect the extracellular production of the NO 2 -stress marker. Following 9 days of UBC treatment (see Exposure regimen), the cells were exposed to three microbes for 24hr. After, the supernatants were collected and re-plated on 96-well plates (Fisher Scientific, PA). As per the manufacturer's instructions, sulfanilamide and NED solutions were added to all samples. A color change was seen after the addition of NED. A microplate reader was used to analyze the samples at 540nm (SPECTRA Max190 Microplate Spectrophotometer). NO 2 -levels were calculated (in µM) from a sodium NO 2 -ladder provided by the system. Each experimental sample along with associated controls were simultaneously examined.

ROS monitored via carboxy-H 2 DCF-DA
ROS accumulation was measured using the fluorescence-based 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate or carboxy-H 2 DCF-DA (Fisher Scientific, PA) fluorescein analog reagent. The carboxy-H 2 DCF-DA reagent is transformed intracellularly by esterases into a green fluorescent form (carboxy dichlorofluorescein) upon oxidation. Negative charges impede any leakage of this fluorescent molecule. Following 9 days of UBC treatment (see Exposure regimen section), the cells were further treated with three distinct microbes for 24hr. After incubation, the samples were collected, washed, and transferred to black wall 96-well plates (Fisher Scientific, PA). Each experimental sample and corresponding control (untreated cells) were simultaneously evaluated. The suspensions were gently mixed and read via Fluoroskan Ascent Software v2.6 (Thermo-Fisher Scientific Ascent Fluorimeter, v2.5) at 490nm excitation and 520nm emission wavelengths. ROS generation) was determined by continuously scanning the plates over a 12hrperiod (collected at time points 240, 360, 480, 600, and 720min) while the incubation temperature inside the Fluorimeter remained constant at37°C. The left panel shows linear curves for Fluorimeter Units (FU)over 720min, and the right panel further expresses this data as the sum of area under the curve or ΣAuC (FU*min).

Cytokine analyses
Pro-inflammatory and anti-inflammatory cytokines were both accessed via commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kits (BD Biosciences Pharmingen, CA and R&D Systems, MN). Interleukin-1 beta and tumor necrosis factor alpha (IL-1β and TNF-α) as well as interleukin-4 and transforming growth factor-1 beta (IL-4 and TGF-1β) were measured. ELISA analyses were performed according to manufacturer protocols. In brief, clear ELISA plates (Fisher Scientific, PA) were coated overnight with the corresponding capture antibody at 4°C. The wells were blocked at room temperature; the cultured supernatants from all the 3.0µg/ mL UBC-treated cells containing individual microbes (M 1 , M 2 , and M 3 ) were added. In addition, the plates were incubated alongside a biotin-conjugated anti-cytokine antibody followed by a streptavidin-horseradish peroxidase conjugate (HRP-label). The TMB chromogenic substrate solution-containing 3.3', 5.5'-tetramethylbenzidine-was used to develop a colorimetric output after 15-30min (Fisher Scientific, PA). A microplate reader was then used to measure absorbance at a wavelength of 450nm (SPECTRA Max190 Microplate Spectrophotometer). Individual cytokine concentrations were concurrently calculated (in µM) from a cytokine standard provided by each of the ELISA kits. Untreated controls along with all experimental groups were analyzed in parallel.

Phagocytic/endocytic activity
Cellular phagocytic activity (ability to engulf other particles) was determined using the Vybrant Phagocytosis Assay (Life Technologies Inc., NY). This method measures bioparticles' engulfment using a fluorescent label. The fluorescently labeled particles are directly proportional to the amount of phagocytosis or internalization activity detected across the cell samples. Cells were seeded at a density of 1x10 4 per well (or 100µL from a 1x10 5 cells/mL solution) in 96well black-walled plates (Fisher Scientific, PA) and incubated for 2hr with fluorescein-labeled Escherichia coli Bio Particles (K-12 strain) at 37°C. The cells containing bioparticles were subsequently treated with trypan blue in order to quench any E. coli bioparticles that were not internalized for 1min (fluorescent particles engulfed by the cells are exclusively measured). The solutions were vacuum aspirated from each well; the plates were analyzed immediately using the Fluoroskan Ascent Software v2.6 (Thermo Fisher Scientific Ascent Fluorimeter, v2.5) at 490nm excitation and 520nm emission wavelengths. A media control, a 10mM H 2 O 2 control, and three respective experimental (0.3-3.0µg/mL UBC) treatments were studied concurrently.

Statistical approach
Significant differences between control groups (untreated control or toxicity control) and experimental groups were evaluated using R Project for Statistical Computing by applying a one-way ANOVA with a post-hoc Tukey Kramer test at a significance level of *p<0.05. For non-transformed data (non-normally distribution), a non-parametric Kruskal-Wallis test and non-parametric post-hoc multiple comparisons test (significances at p<0.05) was used to determine statistical significance. The GraphPad Prism (Project Software v5, San Diego, CA) application was used to represent averaged values, including bars showing standard error of the mean (±SEM). Lastly, statistical comparisons against the media control group alone are indicated by a star (*) and against the 3.0µg/mL-UBC alone are specified by double daggers ( ‡) where deemed applicable.

Results
Excess oxidative damage can promote the generation of Reactive Oxygen Species (ROS), cause stress, and elicit responses that may impact cellular health. Consequently, ROS was determined qualitatively using microscopy and the CellROX Deep Red dye, which generates red ROS fragments for visualization. Following 9 days of UBC treatment, all cells were prepared and confocal images were acquired on the tenth day. Untreated cells, 10mM H 2 O 2 -treated cells, and the highest UBC-treated groups for each cell type are shown/as seen in figure 1 (A-L). Red species appear to be heavy in some regions and absent in others. Human epithelial line (A549) and monocytic (THP1) media control groups show background ROS fragments indicative of long-term incubation. The murine epithelial line (LA4) showed a higher amount of ROS staining for the UBC experimental group relative to the untreated control. On the other hand, murine RAW264.7 macrophages had relatively equal ROS species across both control groups (media control and H 2 O 2 control) along with the UBC experimental sets. Reduced glutathione or free GSH levels were quantified to measure intracellular damage as a result of 9 days of continuous UBC exposures. All samples were prepared for free GSH bioluminescent analysis on the tenth day. Media only (untreated) cells, 10mM H 2 O 2 -treated cells, and all three UBC-treated groups across all cell lines are depicted in figure 2 (A-D). Free GSH levels were between 2 and 12.5µM for human A549 cells and between 2.5 and 15µM for the murine LA4 cells; between 1.0 and nearly 3.0µM for the THP1 monocytes; and finally between 0.4 and almost 0.8µM for the RAW264.7 murine macrophages. Significant values showing differences between the untreated and the UBC-treated groups were reported for the following cell lines: A549 (low-UBC level), LA4 (low-UBC level), THP1 (low-and mid-UBC levels), and for murine RAW264.7 macrophages (low-UBC level). Both A549 and LA4 samples showed about the same range of free GSH content (<15µM); the THP1 and RAW264.7 yielded less than 3.0µM GSH. Refer to table 1 for additional GSH quantitation.
To assess further cellular fitness after nine days of incubation, mitochondrial health was evaluated. Mitochondrial health can be assessed using several approaches; JC-1 dye was used to examine UBC effects on mitochondrial membrane potential or ΔΨm. ΔΨm is commonly measured using cationic dyes, which accumulate at the negatively charged mitochondrial matrix. As shown in figure 3 (A-D), the extent of mitochondrial depolarization (damage) profiles fluctuated across the cells. In comparison to the matched untreated controls, a statistically significant increase in mitochondrial damage was seen for A549 epithelial cells across all concentrations of UBC ( Figure 3A); no change was observed for LA4 epithelial cells ( Figure 3B). For the human THP1 monocytes, a significant increase in mitochondrial damage was seen at the highest UBC level ( Figure 3C). The extent of health was inversely related to toxicant concentration ( Figure 3D) for murine macrophages. Refer to table 2 for additional mitochondrial potential information.
Total cell cycle distribution was investigated to evaluate any changes to cellular conditions potentially induced by low-dose, additive UBC. The nuclear isolation medium containing DAPI, a DNA intercalator, was used after 9 days of contaminant exposure in cells treated with three different concentrations of UBC (0.3, 1.0, and 3.0µg/mL). This examined the progression of each cell-cycle phase by quantifying nuclear DNA content via NIM-DAPI fluorescent signal. Cells emitting low levels of DAPI-DNA fluorescent signal converge with cells undergoing apoptosis potentially carrying fragmented DNA or a sub-G0/G1 cell subpopulation. Cells with high DAPI-fluorescence represented cells experiencing mitosis. In a single-parameter flow cytometry histogram, using an FL-9 detector and 405nm excitation laser for DAPI, the sub-G0/G1 (hypodiploid) subpopulation is located to the left side of the G0/G1 phase. Each dataset (per cell line) was bar plotted to depict cell counts (y-axis) and distributed using four cell-cycle subpopulations across five groups (x-axis), see figure 4(A-D).
The following DNA proliferation analyses compared the experimental UBC-treated cells against non-treated cells of normal cycling profile consistently across all tested samples. After RAW264.7 cells were exposed long-term to UBC (0.3, 1.0 and 3.0µg/mL), changes in the sub-G0/G1 and G0/G1 phases were imperceptible at all the UBC concentrations tested figure 4 (A,B,D). In contrast, the percentage of RAW264.7 cells in the S phase presented significant Note: On day 10, the cells were harvested and analyzed via bioluminescent-based glutathione assay (A-D). Free GSH concentrations were calculated. Tukey graphs display the mean of 6-8 replicates. Data represent two independent experiments. Statistical significance displayed as *p≤0.05 compared to media control. A 1-way Anova was conducted across all cell lines. increments for all UBC groups whereas a significant drop was depicted across all UBC groups in the G2/M phase ( Figure 4C). Additionally, the UBC-exposed A549 cells displayed a dose-dependent increase of the sub-G0/G1 population significantly different at the highest UBC treatment; a significant decrease in the G0/G1 population at the highest dose was also observed. No modification was detected in the S phase. However, the G2/M subpopulation was significantly lower at the 1.0µg/mL and 3.0µg/mL UBC concentrations for the A549 cells in Supplemental Information 1 or SI 1. Moreover, the UBC-treated LA4 cells exhibited a similar tendency, where the sub-G0/G1 subpopulation was increased in a dose-dependent manner, significant at the 3.0µg/mL UBC. No significant changes to the S and G2/M phases were observed for LA4 (SI 2). The UBC-treated THP1 macrophage precursors displayed a significant increment in the sub-G0/G1 phase at the highest UBC concentration (SI 3). Monocytes elicited a significant decrease in the G0/G1 subpopulation at the highest UBC concentration; no significant changes to the S and G2/M phases were observed. The highest UBC dose at 3.0µg/mL caused a significant increased shift in the sub-G0/G1 and G0/G1 phases for the A549, LA4, and THP1 cells (SI 1-3) but not the murine RAW264.7 (Figures 4).
Across these, UBC induced significant anti-proliferative activity as evidenced by an arrest in the S phase ( Figure 4).
Since immune mediators initiate biochemical signaling, the extracellular presence of nitrite or NO 2 was quantitated to evaluate innate immunity. After 9 days of cumulative UBC treatments, the samples were treated with three different microbial products (3.0µg/mL UBC-treated+M 1-3 groups); supernatants were collected for nitrite content analysis on the tenth day. Untreated, 3.0µg/mL UBC-treated, and the highest UBC treated +microbe groups are shown for each of the cell lines in figure 5(A-D). Extracellular NO 2 -levels were between 2.0 and 3.5µM for A549 cells and between 2.0 and 15µM for LA4 cells; between 0.2 and 0.6µM for human THP1 monocytes; and between 4.0 and 10µM for the murine RAW264.7 macrophages. Statistically significant values between the media control and experimental UBC +microbe treatments were found for the murine LA4 (M 1 -and M 2 -UBC groups) and the murine RAW264.7 (M 1 , M 2 , M 3 -UBC groups). Both LA4 and RAW264.7 showed a 5 to 10-fold increase in the production of NO 2 - (Figure 5B and 5D). A549 and THP1 cell lines did not show statistical differences compared to the corresponding untreated controls ( Figure 5A and 5C).
Reactive Oxygen Species (ROS) were quantitatively assessed to show the potential impact on innate immunity from UBC using the carboxy-H 2 DCF-DAassay where Fluorimeter Units (FU) indicated ROS. After 9 days of continuous exposure to UBC, the cells were prepared for ROS analysis on the tenth day. Untreated, 3.0µg/mL UBC-treated, and 3.0µg/mL UBC +microbe groups for each cell line are shown in figure 6 (A-D). ROS indicator units designated the level of cellular stress steadily increasing over a period of 12hr (720min). ROS reached almost 800 FU after the 720min readings for all cells.
The data was further analyzed to depict the summation of all areas under the curve (Σ of AuC) to assess statistical differences between every experimental group against the untreated control. As seen on the right panel of figure 6, the AuC analysis found: Significant decrease   in A549 for one of the UBC +microbes group; an increase in LA4 for one of the UBC +microbes group; no significant effects were found for THP1; finally, a significant increase in the RAW264.7 lines for two UBC +microbe groups. All Σ AuC calculations indicated elevated ROS at specific experimental samples compared to the untreated control as time progressed.
Inflammatory cytokine levels were evaluated as a third innate immune biomarker. After 9 days of cumulative UBC treatments, the cells were treated with three different microbial proxies (3.0µg/mL UBC-treated +M 1-3 groups); accordingly, the corresponding supernatants were collected for cytokine content assessments following the tenth incubation day. Untreated (media only), 3.0µg/mL UBC-treated, and the highest UBC treated +M 1-3 groups are shown for each of the cell lines in figure 7(A-D) and figure 8(A-D). Figure 7 depicts cytokine concentrations (in µM) that were induced by all cell lines in response to the highest UBC concentration. Neither human A549 nor murine LA4 epithelial lung cells showed significant effects in cytokine production (neither IL-1β nor TNF-α) compared to the media control. THP1 monocytes expressed a significant increase in the Th1 cytokine TNF-α (pro-inflammatory) with no change to TGF-β; RAW264.7 expressed a significant increase in the Th2 cytokine IL-4 (anti-inflammatory) only. Next, cytokine presence was further assessed testing media control groups against the 3.0µg/mL UBC +microbial proxies (M 1-3 ). Figure 8 depicts cytokine induction across all lines in response to 3.0µg/mL UBC-treated +microbe stimulus. The A549 epithelial cells exposed to the 3.0µg/mL UBC treated +M 1-3 groups showed a significant increase compared to the media control for IL-1β; no change was observed for TNF-α. The LA4 epithelial cells exposed to the 3.0µg/mL UBC treated +M 1-3 groups showed a significant increase compared to the control for both IL-1β(M 1 ) and TNF-α cytokines (M 1 ). THP1 monocytes exposed to the 3.0µg/mL UBC treated+M 1-3 groups indicated significant increments compared to the control for IL-1β (M 2,3 ) and TNF-α cytokines (M 1,2 ); no change was evident for TGF-β. RAW264.7 phagocytes exposed to the 3.0µg/ mL UBC treated +M 1-3 groups indicated significant increments compared to the control for TNF-α (M 1,3 ) and IL-4 (all) cytokines; no change was evident for IL-1β.
Phagocytosis or endocytosis is a process whereby a cell internalizes extracellular material by drawing the material inward while engulfing around it. Therefore, as a final measure of UBC impact on innate immunity, cellular capability to phagocytose material was determined following UBC administrations. Following 9 days of cumulative UBC exposure, the cells were prepared for phagocytic activity (endocytosisin epithelial lines) on the tenth day. Untreated cells, 10mM H 2 O 2 -treated cells, and UBC-treated cells are shown in Supplemental Information 4 (SI 4). No significant modifications to phagocytic/ endocytic behavior were observed across A549, LA4, or the THP1 monocytes. All UBC groups yielded a statistical decrease in phagocytosis for RAW264.7.

Discussion
The preferred carbon nanomaterial suspensions were chosen to depict dose-responses and were based on previously published data, where the low concentrations approximated potential pollutant presence in the lung [49]. The cumulative doses were at least one log lower in reference to other in vitro respiratory immune models; hence, these findings contribute to understanding how nano-pollutant impacts lung-derived immortalized cells [49]. Nine days of exposure maximized potential UBC impact while minimizing effects from extended incubation. This experimental design consisted of controlled administrations of UBC at low concentrations (0.3-3.0µg/mL) compared to high doses reported prior to this project (10-500µg/mL). Additionally, the following work was conducted over a longer duration unlike existing literature showing acute treatments (10-72 hours). The cell lines tested were chosen because epithelial cells and leukocytes are the first in contact with inhaled particulate matter; both contribute to stress and inflammation in pulmonary tissues [50]. Of importance, airway epithelial cells are essential health defense against infections, participating in innate immunity [51]; macrophages are the most abundant immune cells of the lung, essential for systemic immune responses [52].The lungs are continuously exposed to a diverse array of microbes and organic/inorganic particulate materials [53]. To find if continuous exposure of lung-derived immortalized cells to UBC alters innate immune function, several immune parameters were measured following a 9-day in vitro regimen of either UBC alone or UBC in combination with microbial challenges. In the second half of this study, cellular innate immunity as a function of oxidative impact, ROS accumulation, cytokine production, and phagocytosis in response to accumulating UBC in the presence or absence of an additional pathogen-like proxy were evaluated. This part of the study consisted of controlled dosages of UBC at 3.0µg/mL "plus" shortterm pathogen-mimics to assess cell performance from cumulative exposure to UBC on the tenth day of this experimental design.
Under homeostasis, ROS is continuously produced mainly in the mitochondria but its balance is controlled by several scavenging enzymatic systems [54]. For decades, ROS was identified as harmful to cells; however, they also function as second messengers regulating a number of signaling physiological pathways [55,56]. Elevated levels of ROS results in oxidative stress leading to senescence, lipid peroxidation, and DNA damage, concurrently contributing to several human degenerative illnesses, like chronic inflammation, aging, and cancer [15,57]. It was reported that the mechanism of action used Note: Upon day 10, the cell supernatants were collected and cytokine levels were measured via spectrometric ELISA assays (A-D). Cytokine content in µM was calculated and the data is displayed as the mean of 4-6 replicates. Data represent at least 2 experiments completed independently. Statistical significance displayed as *p≤0.05 in comparison to media control. See Appendix A for additional information. by urban ambient air (PM) eliciting DNA damage was mainly oxidative stress [58,59,60]. In addition, a previous study indicated that the UFP (57 nm soot fraction) from vehicle emissions provoked systemic oxidative stress and DNA damage [61]. We sought to analyze intracellular ROS levels in cells exposed to UBC by using the Cell-Rox Deep Red fluorescent probe and confocal microscopy ( Figure 1). Upon oxidation, a red fluorogenic signal indicating oxidative stress was emitted and a visual ROS staining pattern was recorded. The red species reflect where ROS are more heavily concentrated inside the cell, as opposed to cytosolic dispersal. UBC resulted in significant stress in the murine LA4 epithelial line as evidenced by the visible fluorescent species. Neither human A549 epithelial cells nor THP1 monocytes were greatly affected by ROS upon exposure to long-term UBC. Their corresponding UBC-treated groups showed less red species; the cells appear to contain a lower concentration of ROS fragments.This might suggest that the two cell lines could be protected by the compounded UBC exposure following ourextended protocol. The present study exhibited that UBC has the ability to induce pro-oxidants mainly for the LA4 line. These epithelial cells originate from the thin layer of the protective barrier inairway cells, cover the surface of the lungs, and provide structural integrity. In contrast, after the UBC insult on the immune system line, RAW264.7, ROS levels were not dramatically altered. Macrophages and neutrophils increase the production of ROS after detection of pathogens [62], and use ROS as critical antimicrobial mediators (pro-oxidants) to eliminate bacteria [63].
Our speculation remained that the macrophages used in this study seemed unable to recognize UBC as potential pathogens and, therefore, do not accentuate ROS production under pollutant presence. Again, this was a qualitative analysis whereby ROS fragments were recorded visually; additional experimentation portrayed support of these images further in this study, where it appears that oxidant levels were promulgated to offset UBC damage in cells.
Despite the minimal toxicity of UBC across most cell lines, we surmised that UBC could deleteriously affect cellular production of anti-oxidants due to ultrafine black carbon's extremely small size. Intracellular damage was assessed by measuring the level of reduced GSH. Glutathione balance is essential to the function of normal biological mechanisms, although an imbalance in overall glutathione ratios contributes to DNA damage and apoptosis [64]. A change in free glutathione concentration is of importance when assessing toxicological responses; this antioxidant biomarker induces ROS accumulation and potentially nanomaterial-mediated cell death [65,66]. Oxidative injury as a function of antioxidant production from cells exposed to UBC was measured by the bioluminescent activity of intracellular glutathione. Reduced glutathione is an antioxidant metabolite capable of preventing or neutralizing damage caused by ROS. More than 90% of the total glutathione pool exists in healthy tissues in its reduced form (GSH) and less than 10% in its disulfide form (GSSG) [67,68]. Oxidative stress prompts intracellular glutathione production and is essential to keep an overall balance to the redox status and protect intra-and extracellular environment. Certain levels of oxidative stress and subsequent glutathione depletion serve as triggers for increased glutathione production (to replace antioxidant activity) while addressing redox imbalance. Thus, increased levels of free GSH suggests ongoing cellular oxidative stress. Figure 2 indicated that human (A) and murine epithelials (B) and phagocytes (D) were all significantly higher due to UBC interactions after nine days, notably at the lowest level of UBC, 0.3µg/mL. Only the human THP1 monocytic line (C) showed a significant increase in free GSH concentration compared to the untreated control for both 0.3 µg/mL and 1.0µg/mL UBC treatments. While GSH content was higher compared to the media control groups across all cells (except at the highest 3.0µg/mL-toxicant dose), GSH depletion is observed prominently and is directly related to increasing UBC levels. The highest UBC level showed an equal background GSH amount as the untreated cell control for each line. These patterns demonstrate that UBC inflicts a special phenotype across the immortalized cell lines that might suggest less susceptibility or resistance against long-term toxicant insult across all lung-derived cells forthe highest 3.0µg/mL-UBC concentration. This modification is not entirely favorable considering the alterations imposed on cell-cycle progression (below) and induction of cellular senescence [69], in response to UBC accumulation. UBC-induced oxidative damage did not seem to supersede the cells' innate ability to neutralize ROS downstreamand could suggest a unique aspect concerning maintenance of a steady-state redox balance.
Although UBC was not found to be particularly cytotoxic for the immortalized cell lines, it remained possible that intrinsic cell health was negatively impacted. Cumulative exposure of epithelial cells and (pre)-phagocytes following UBC exposure were conducted to evaluate mitochondrial membrane potential (or ΔΨm). Mitochondria are important organelles in all eukaryotic cells and play a crucial role as regulators in the control of cellular homeostasis [70]. ΔΨm should be relatively stable; often, alterations reflecting normal physiological activity may be present. Sustained ΔΨm fluctuations in a cell, however, may be injurious [71][72][73][74]. An enduring rise or fall of ΔΨm vs. normal background levels may lead to loss of viability causing an increment in various detrimental pathologies downstream [75]. As an indicator for ΔΨm, the membrane-permeant JC-1 polychromatic fluorescent probe is widely used to assess mitochondrial health and to monitor cells with depolarized (damaged) mitochondria via flow cytometry [76]. The JC-1 approach highlighted that LA4 cells are not significantly affected by the nanocarbon particles ( Figure 3A). Dissimilarly, the human epithelial cells, murine macrophages, and human monocytes were all compromised figure 3 (A, C and D). The A549 and THP1 lines showed a nominal increase in mitochondrial depolarization with high UBC; the RAW264.7 macrophages showed an inverse dose-dependent alteration to mitochondrial health upon UBC exposure. The data indicated that only three cell lines presented a significant change in ΔΨm, particularly at the highest concentration of UBC. These findings supported that UBC induced significantly adverse dose-dependent increase to mitochondrial mechanisms (more damage to membrane potential) for both A549 human epithelial line and non-adherent THP1 monocytes compared to the control. Anunexpected increase in mitochondrial health was seen for the adherent murine RAW264.7 macrophages compared to the control for all concentrations of black carbon. Interestingly, an inverse dose-dependent increase compared to each experimental UBC-treatment was observed for RAW264.7. Literature suggests that some cells are capable of partially recovering from previously depolarized states [77]; the study showed that mitochondrial damage (or depolarization) in neurons could return to a healthy state and appeared to be highly dependent on ion (Na + /Ca 2+ ) channel flux. This particular phenotype substantially minimizes toxicity in culture and intracellular receptors [77]. Mitochondrial health trends showed significant modifications in all cell types except LA4 compared to the control ( Figure 3B). Based on this investigation, it was imperative to report that the highest UBC dose significantly distressed subcellular compartments across most of the cells used in this study. The murine macrophages seemed to recuperate or supersede mitochondrial damage after 9 days of nanocarbon insult.
A meticulous analyses of the cell-cycle profiles affected by longterm exposure to UBC help identify additional effects of nanoparticles on cultured cells. Controlled cell proliferation is fundamental for the development and homeostasis of multicellular organisms [78,79]. Therefore, proliferative analyses serve as key indicators of cellular fitness and can assist in the identification of certain proteins and pathways involved in the potential death or survival of cells after exposure to cytotoxic agents [80]. Formerly, it has been shown that UFP such as TiO2, elemental carbon, commercial black carbon, diesel exhaust-PM, and urban dust cause cytoskeletal toxicity in vitro in macrophages, which led to an impaired cell proliferation, and compromised phagocytic activity [81][82][83]. Additionally, carbon nano materials induce cellular cycle arrest in G1 phase, conveying a loss of cell viability through DNA damage/apoptosis in fibroblasts [84]. In the present study, UBC treatments were administered and cell-cycle progression were evaluated through the quantification and concurrent analysis of cellular DNA content via flow cytometry. Overall, these assays demonstrated that UBC inflicted unexpected alterations to cell-cycle profiles after 9 days of treatment across the diverse cells in this study. The RAW264.7 murine macrophage cell line displayed an anti-proliferative effect indicated by an arrest (or increase) in the hyperdiploid S phase shown in figure 4(A-D), and consequently a decrease in the tetraploid G2/M subpopulation. This confirms that UBC interferes with cellular synthesis. Murine macrophages were the only cell types to display this trend in our studies and potentially supported that the G2/M checkpoint involving Cdk1/Cyclin B within the mammalian cell cycle's regulatory machinery is affected [85]. In contrast to the phagocytes, the murine LA4 lung cells along with the human A549 lung cells and THP1 monocytes displayed a dose-dependent increment in the sub-G0/G1 apoptotic subpopulation as well as a significant decrease in the diploid G0/G1 phase in both A549 and THP1 cells (SI). An increment of sub-G0/G1 and G0/G1 are indicative of DNA fragmentation and a synthesis inhibitory effect, respectively. The A549, LA4, and THP1 lines showed no change to the S phase at all the UBC concentrations tested. A549 cells showed a significant dose-response decline in the G2/M subpopulation. As expected, when comparing the untreated control cells with Camptothecin-(CPT) treated cells, a positive control for cytotoxicity, significant changes to normal cell-cycle trends were induced across almost all the cell-cycle facets of the cell lines reviewed. Altogether, these results indicated that UBC shifted normal cycle distributions and provoked distinct alterations across each cell type. The most remarkable changes were for the anti-proliferative tendency (an arrest in the S phase) observed for the RAW264.7 macrophages and a dosage-response increment to DNA fragmentation/apoptotic profiles detected across the A549, LA4, and THP1 monocytes (SI, [1][2][3][4]. This behavior could potentially indicate that the G1/S checkpoint involving Cdk2/Cyclin E of the cell-cycle machinery might be disturbed or the Anaphase-Promoting Complex (APC) at the spindle checkpoint could be targeted [85], a component for future research.
A comprehensive examination assessed the impact on lung-derived immortalized cells after nano-pollutant +microbial challenges based on a 10-day post-exposure model. The innate immune system consists of soluble proteins that bind leukocytes to microbial products migrating through the blood stream to inflammation sites, where they may ingest and kill particulates [86]. Innate immune responses were measured after UBC "plus" TLR-ligand treatments as a function of ROS, NO 2 -(a form of RNS), and inflammatory cytokines. The TLR ligands included in this project were: M 1 (LPS), M 2 (Poly I:C),and M 3 (Zymosan or Zymo). The highest UBC concentration (3.0µg/ mL) plus three types of microbial proxies (microbe-like groups M 1-3 ) were given to the cells; experiments were conducted on the tenth day post-incubation. A 3.0µg/mL UBC concentration was selected to identify conjoined effects from microbial stimuli upon respiratory cells, as follows: [1] ROS levels (depicted as collective Σ AuC) were higher for continuous UBC +M 1-3 treatments; [2] NO 2 -content was statistically induced for UBC plus the microbe-mimics across most cell types; [3] cytokine production was also significantly increased compared to the untreated control; [4] phagocytic activity showed a significant dose-dependent decline for RAW264.7. UBC +M 1-3 presence altered cellular conditions by significantly increasing ROS (carboxy-H 2 DCF-DA), elevating oxidative stress (NO 2 -), and causing notable alterations to cytokine production (Th1 vs. Th2). These findings indicated that UBC impaired mammalian cell function, with each species responding differently to individual microbial challenges, subsequently provoking phagocytic decline across RAW264.7.Stress to external cellular environment was detected using the extracellular NO 2 -marker. A change in nitrite content is relevant when assessing toxicological responses, since it is a stress marker induced by ROS to stimulate nanomaterial-mediated cell death [65,66]. As shown in responded to all three microbial stimuli in the presence of UBC, minimally and insignificantly, UBC +microbes did not impede reactivity of the cells to produce nitrite. LA4 responded to LPS (M 1 ) and Poly I:C (M 2 ) in the presence of UBC, giving a significant increase in nitrite levels. This could predict a close interaction between the nanocarbon pollutant and PAMP presence. All three UBC +microbe challenges were significantly lower compared to the UBC-treated group, as portrayed by double daggers for LA4. In the leukocyte profiles, THP1 monocytes responded to all microbial stimulants but to a lesser degree in contrast to the 3.0µg/mL UBC alone; RAW264.7 phagocytes generated a statistical increase in NO 2 -concentration following all three infections even in the presence of UBC compared to the untreated groups. The LPS (M 1 ) and Zymo (M 3 ) groups for these murine macrophages yielded a statistical induction of the nitrite metabolite compared to the 3.0µg/mL UBC alone, as indicated by double daggers. Thus, the UBC + PAMP co-exposure promoted innate immunity as a function of nitrite production, mainly for the murine-specific immortalized cells and appears to modulate NO 2 -responses to TLR-2, 3, and 4 across individual cell types. NO 2 -is known to control either the replication or elimination of intracellular microbial pathogens [87,88]. Although primarily generated by macrophages to kill bacteria and parasites, nearly every type of cell produces NO 2 -in the body. Nitric Oxide Synthase (NOS) is a calcium-dependent enzyme that may catalyze the release of nitric oxide in the pulmonary airway and induce pro-inflammatory cytokines [89]; pro-oxidants amplify inflammatory responses by provoking cellular dysfunction under this state termed "nitrosative stress" agent [90]. An exposure to a nanosized pollutant and subsequent infection with a pathogen-like proxy could endanger and impair antimicrobial defenses as observed in vivo [91], due to a decreased expression of nitric oxide and phagocytic activity in alveolar macrophages. Cumulative and additive insults of UBC +microbes prompt extracellular stress together, yet human-derived cells could be capable of adjusting to increasing levels of UBC. The lack of significant NO 2 -metabolite in the A549 and THP1 cells might hint that an anti-oxidative effect occurred likely due to continuous aggregation of the pollutant [91]. A study suggesting arginine bioavailability (a marker of NO synthetic capacity) also associated with markers of inflammation [92]. The production of NO may activate/amplify inflammatory circuits by modifying protein structure and gene expression; this could in turn deplete arginine and arginase causing parasite death, for example [93]. Recently, research showed that the diffusion of NO induced an effective parasite killing ability from bystander cells and NO-producing cells alike [93]. NO and consequently NOS may not be targeted by the human cells, yet the murine cells (LA4 and THP1) seem to be under greater stress in the presence of UBC +microbial stimuli. Every cell type was able to produce nitricoxide following cumulative nano carbon and microbial exposures, suggesting the cells are not inhibited by UBC in the ircapacity to signal an innate immune response.
ROS can elicit extreme biological damage by modifying DNA, proteins, and lipids. Nonetheless, ROS also play beneficial roles including involvement in innate immunity. The classic example of this antimicrobial activity is the destruction of microorganisms occurring in phagolyso some structures of innate immune cells when superoxide converts to a potent cytotoxic molecule, hypochlorous acid [94]. To assess whether continuous exposure to UBC could exacerbate or inhibit ROS in response to microbial presence, 3.0µg/ mL UBC-treated cells were challenged using TLR ligands. Particularly, intracellular formation of pro-oxidants was evaluated, where a non-fluorescent indicator converted into a green-fluorescent form when intracellular esterases remove an acetate group (ROS activity). Fluorescent products remain intracellular due to negative charges that impede leakage. Figure 6 (A-D) represents ROS formation by the cells 12hr following immediate harvesting of the cells protocol (left panel). The data was analyzed to determine the sum of the areas under the curve or Σ AuC in FU * min (right panel) to evaluate ROS in cells overa 720min period. The results varied by cell line and gave significant values depending on distinct treatment types. The Σ AuC reached almost 300,000 FU * min across all cell types. The human pulmonary cells (A549) produced significantly less ROS in response to 3.0µg/mL UBC alone and UBC in combination with LPS (M 1 group) compared to the untreated group ( Figure 6A); the murine pulmonary cells (LA4) produced significantly more ROS when treated with UBC +Zymo (M 3 group) relative to the untreated control ( Figure 6B); no significant differences to ROS production by human monocytes (THP1) compared to media-only group were found ( Figure 6C). The most noticeable ROS response stemmed from the RAW264.7 macrophages, where the 3.0µg/mL UBC-treated group significantly reduced ROS production relative to the media control. The microbial M 1 group and M 3 (LPS, Zymo) stimulants yielded significant increase in ROS in comparison to the untreated control ( Figure 6D). Each cell type was able to generate oxidative species following both UBC and microbial challenges; this assessment found that the exposure did not inhibit cellular activity to signal innate immune function. The data demonstrate that cumulative administrations of the highest UBC concentration over a 9-day period had little effect on oxidant production (THP1) and may help neutralize (A549 and RAW264.7) cell damage relative to the media control. However, when UBC exposure is combined with one type of TLR ligand (LPS, Poly I:C, or Zymo), the generation of oxidative species might be impeded (no effect compared to control) or otherwise increase normally. This behavior might signal against a deleterious defense mechanism from particle-media interactions between the UBC aggregates, microbial stimulants, and the cells [95,96]. Due to black carbon's adsorptive properties, UBC might act as an antioxidant supplement, whereby the negative impact from oxidative species could be bypassed or even neutralized [97,98]. Black carbon's physiochemical properties yield energetically favorable, hydrophobic, and non-soluble particle formation (nonpolar interactions); free radicals are interestingly scavenged by nano-aggregate formations [95,96]. This behavior could explainthe observed protection against radical species for the UBC +microbe groups in the A549 cells and the THP1 monocytes.
A balance between Th1 and Th2 is crucial for immune control; abnormal responses may lead to a variety of inflammatory conditions, like chronic bronchitis and asthma [99]. Cytokines fulfill biological effects including cell proliferation and death; they have been implicated with inflammatory response, immunological conditions, and in diseases such as cancer [85]. To evaluate the potential pro-inflammatory activity of UBC, the production of IL-1β and TNF-α were assessed by ELISA. As shown in figure 7, the 9-day exposure to continuous UBC did not provoke an innate cytokine response from the A549 or LA4 cells. A statistically significant increase in TNF-α production was induced in THP1 cells by UBC alone, see figure 7.
A possible anti-inflammatory response was tested by measuring the production of both IL-4 and TGF-β. As shown in figure 7D, only the RAW264.7 were provoked to secrete IL-4, an anti-inflammatory cytokine, in response to cumulative UBC exposure. Moreover, cytokine production by lung-derived cells was evaluated to discover the effects of UBC in combination with TLR-2, 3, and 4 ligands, see figure  8. A549 produced IL-1β in response to all three PAMPs but did not produce TNF-α ( Figure 8A). LA4 produced both IL-1β and TNF-α but only in response to LPS ( Figure 8B). THP1 generated IL-1β in response to Poly I:C and Zymo and TNF-α in response to LPS and Poly I:C. No significant effect observed for the TGF-β cytokine (Figure 8C). Lastly, RAW264.7 produced only a TNF-α in response to the LPS and Zymo PAMPs. All microbes induced the generation of IL-4 after M 1 , M 2 , and M 3 challenges; no effect was observed for the IL-1β cytokine for RAW264.7 ( Figure 8D). Cumulative 3.0µg/mL UBC induced a cytokine response by both leukocytes. In addition, all cell types showed high levels of cytokines following UBC treatment +microbial challenges, hinting that they are not modified or inhibited by UBC in the irability to function as innate immune cells. Together, these results portrayed increased pro-inflammatory cytokine production and increased anti-inflammatory cytokine production in RAW264.7 macrophages after the combined exposures to both UBC (long-term) and PAMPs (short-term).
Internalization of foreign materials upon long-term, additive UBC doses was measured in this final study. Although a primary function of monocytes and macrophages, phagocytosis can be performed by non-professional phagocytes, like epithelial cells [100,101]. Therefore, as a final measure of UBC impact on innate immunity, the ability of cells to phagocytize additional material was investigated (Supplemental Information, SI 4). No significant values were calculated for either epithelial lines, although there was a slight increase on phagocytic activity upon increasing UBC treatments for both. THP1 monocytes yielded a minimal downward shift in phagocytic activity. In contrast, the measurements showed theRAW264.7 professional phagocytes significantly diminish in a dose-dependent trend. (Almost a 50% reduction in phagocytic activity occurred relative to the media control.) Thus, the most phagocytic cell line in this analysis exceptionally inhibited ingesting additional foreign material after UBC internalization. Even though our culture techniques were conducted using an adherent macrophage line, specific mechanisms describing UBC engulfment remain unknown. Other work indicates that most NPs may be internalized by macropinocytosis if the particle is about 50nm in size [91]. This uptake occurred concomitant to generation of pro-inflammatory mediators: If an infection compromises this activity, cellular impairment follows and antimicrobial responses prevented. Since phagocytic activity decreased, so could production of extracellular NO. Finally, this dataset proposes that low nanoparticle uptake (phagocytic activity in macrophages) coincides with a senescent phenotype observed previously [69].
In the present investigation, low-dose, prolonged 9-day UBC exposure caused moderate ROS protection by the cells (determined by confocal imaging) and induced oxidative damage according to each metabolite-oxidative stress measurement. Murine LA4 and human (A549 and THP1) cells appeared to be prone to damage from toxicant exposure determined by JC-1, whereas mitochondrial membrane health was not severely compromised in murine RAW264.7 macrophages. Interestingly, the cells used in this study may be adapting to a new hyperpolarized state. In comparison to the untreated control, RAW264.7 also indicated an S phase arrest and significant depletion to G2/M subpopulation as validated by cell-cycle cytometry. Assays observing insult from other ultrafine nanomaterials done in vitro showed that cells could potentially benefit upon elevating ROS levels from baseline ROS (9-15%) to higher ROS content (29-33%); a counter-response was produced within the cells' machinery that prolongs proliferation and increases cell differentiation [15,46]. Moreover, ROS could impact intracellular signal transduction processes by promoting moderate oxidant species, increasing proliferating cell nuclear antigen, and elevating cellular subpopulation within the S phase [46]. Thus, this study is in agreement with a currently atypical phenotype observed only for RAW264.7 phagocytes following a nine-day contamination period [69]. This characteristic discovery is not entirely favorable considering the negative alterations imposed from diminished enzyme activity [69], in response to long-term UBC. The complete toxicant effects provide insight into in vitro impacts after nano-pollutant aggregate formation, where: Damage was moderately high across epithelial cells; minimal low-level oxidative stress was present; damage to mitochondrial membrane (ΔΨm) with increasing UBC was observed in human cells; and non-uniform permutations to individual cell-cycle trends were also reported. UBC-induced damage did not seem to supersede their innate ability to neutralize ROS suggesting a unique aspect concerning maintenance of a steady-state redox across human cell lines. No overt prevention to the innate cytokine responsewas exhibited in the presence of cumulative 3.0µg/mL UBC and subsequent 24hr compounded TLR ligations (M 1 , M 2 , or M 3 infections). Further, our studies found that the combined effects originating from UBC and microbe-like exposures did not hinder ROS generation. This work highlighted that native, terminally differentiated tissue cells may not ultimately elicit the same responses due to a modified upwards arginase activation and a lowered pathogen-killing capacity of the cells in question (mainly macrophages). If both ROS and NO x levels increase yet lessen phagocytosis, then the balance between both Th1 and Th2 markers is greatly compromised [91,102,103]. Arginine and by consequence the bio-available level of arginase are both of special interest and further experimentations remain [104][105][106][107][108][109][110].
The authors originally postulated that sustained UBC +microbial treatments might incapacitate the cells from distinguishing pathogenic infections as potential dangers and consequently disable stress responsesfrom the combined effects of the aforementioned irritants. However, the compilation of these measurements helped reveal unique perspectives concerning how chronic and extensive exposure to airborne irritants may elicit impairment across various cell lines in a non-monotonic manner.Overall, UBC in combination with microbe-like proxies may not only trigger local inflammatory reactions in vitro but further initiate detrimental complications in lung tissues and consequently systemic extrapulmonary maladies.