Autophagy is an intracellular process that helps maintain cellular homeostasis. In this study, we measured autophagic flux and cellular injury in alveolar epithelial cells (AEC) and lung adenocarcinoma (A549) cells exposed to polystyrene nanoparticles (PNP: 20 nm, carboxylated, near-infrared dye-labeled) and a secondary insult to explore the effects of limited autophagic capacity. Rat AEC monolayers (RAECM) and A549 cells were exposed for 24 hrs to i) PNP, ii) rotenone (RT: inhibits mitochondrial respiratory chain complex I) or tunicamycin (TN: disrupts protein synthesis and induces the unfolded protein response), or iii) RT or TN for an additional 24 hrs after 12 hrs of PNP preincubation. Release of lactate dehydrogenase (LDH) served as an index of cellular damage. Autophagic flux was assessed using confocal imaging to quantify the rate of autophagosome production. PNP taken up into AEC and A549 cells induced autophagy, eliciting increased autophagic flux and reaching steady-state at ~10-24 hrs. When RAECM or A549 cells were exposed to RT or TN alone for up to 24 hrs, autophagy was also activated, reaching steady-state autophagic flux similar to PNP exposure alone. In the presence of both PNP and RT (or TN), steady-state autophagic flux was similar to that with PNP, RT or TN alone. Cellular damage due to RT or TN, in both AEC and A549 cells, became more severe after PNP preexposure, indicating that induction of autophagy by PNP makes AEC and A549 cells more susceptible to injury by each of two secondary insults due to finite autophagic capacity.

Plastic pollution presents a looming danger to the environment and virtually all life on planet Earth. Especially pernicious are nanoplastics (NPs), which are plastic fragments with dimensions ≤1 μm. Conventional detection methods are ineffective for NPs, while their high specific surface area renders them efficient carriers of toxic substances; additionally, they may even be inherently toxic. Although NP waste chiefly arises from environmental weathering of larger plastic fragments, most published studies employed manufactured pristine NPs of uniform size and shape. Furthermore, almost all NP effects were studied using polystyrene (PS) as a convenient model material, despite PS accounting for <6% of all plastic pollution. There is thus an urgent need to expand investigations of environmental NP pollution and effects on biota. The present work provides a comprehensive roadmap for studying the effects of "real-world" NP pollution on living systems, using, for example, lung alveolar epithelial cells on which such NPs deposit by breathing ambient air. Herein, we describe detailed in-house methods to fabricate various NPs that are weathered with UV light and O3 gas exposure to more closely mimic real environmental NPs. We also illustrate a simple and straightforward bioelectrical method for assessing passive and active ion transport properties of primary rat lung alveolar epithelial cell monolayers as a model for the distal mammalian lung exposed to one of the generated NPs. This protocol allows researchers to rapidly and more accurately assess the biological impact of various simulated environmental NPs on a vulnerable air-blood barrier in the lung. Key features • Many simulated weathered environmental NPs can be produced at high concentrations (up to 120 mg/mL) and yields (up to 12 mg/g bulk plastic). • Any plastic waste can be "nano-sized" with this protocol and then studied for impacts on active and passive ion transport properties of cell monolayer models. • Methods described herein are very relevant for studying environmental pollution effects, since NPs are found in many different shapes, sizes, and compositions. • NP weathering and generation methods do not require any expensive or specialty lab instruments.

Introduction: About 0.25 megatons of plastic waste from consumer and industrial sources found in waterways, including sub-millimetric plastic particles generated from environmental degradation, may pose significant health risks. The biological and ecological impacts of materials <1 μm (nanoplastics) are of particular concern because they are generally too small for most conventional filtration processes and challenging to collect, detect and identify. Furthermore, their high surface area-to-volume ratio makes them efficient carriers for toxic substances. Despite this, most of the studies in recent literature reviews employed manufactured nanoplastics of uniform size and shape, which do not reflect actual environmental plastic waste. A systematic analysis of environmental nanoplastic impacts on biota would be vital in understanding the full life cycle of plastic pollution. In this study, we investigated how different nanoplastics affect the ion transport properties of lung alveolar epithelium. Methods: We crushed and pulverized cuvettes (polystyrene (PS)), droppers (polyethylene (PE)), centrifuge tubes (polypropylene (PP)), and containers (polyethylene terephthalate (PET)) prior to irradiation and ozone treatment. These nanoplastics in water were twice-filtered with 800 nm polyethersulfone filters and concentrated by evaporation. The resulting nanoplastic solids were weighed and re-suspended in 1 mL mQ water. They were then ultrasonicated for 15 minutes to break up any aggregates. We measured size distribution, surface charge and shape of the nanoplastics using a zeta sizer/DLS and electron and/or confocal microscopy. For ease of detection, these nanoplastics were also stained with Rhodamine B by heat swelling at 70°C for several hours, followed by dialysis or size-exclusion chromatography to remove unbound dye. Commercial 20 nm spherical polystyrene nanoparticles (PNP) with -10 mV zeta potential were used for comparison purposes. Effects of nanoplastics and PNP on primary cultured rat alveolar epithelial cell monolayers were studied by measuring transepithelial resistance (Rt) and spontaneous potential difference (PD) for up to 48 hours after adding nanomaterials to the apical fluid of the monolayers, while control monolayers received only culture fluid apically. Active ion transport rate (Ieq) was estimated as the ratio PD/Rt. Results: Typical size, polydispersity (%PD), morphology and absolute value of zeta potential for our nanoplastics are 120-350 nm, 11-25%, dendritic and 10-13 (±8) mV. PNP exposure led to a rapid decrease in Rt by ~70% at 30 minutes and a slow return to control level thereafter, whereas Ieq decreased by ~50% at 30 minutes and returned to control level very slowly. PP exposure also led to rapid fall in Rt by ~60% with very slow return to control level, but without an effect on Ieq. PS resulted in a slower decrease in Rt by ~40% at 8 hours, followed by return to control level by 48 hours, without affecting Ieq. The other nanoplastics (PE and PET) tested did not cause significant changes in either Rt or Ieq following apical exposure. Conclusions: These observations are consistent with the hypothesis that the material, size, shape and surface charge of nanomaterials influence their effects on the bioelectric properties of the alveolar epithelium. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Autophagy, a housekeeping mechanism, is crucial in the maintenance of normal cellular function. Although the involvement of autophagic processes is recognized in numerous diseases, it is unknown how cellular homeostasis might be affected by alterations in available autophagic activity and/or autophagic capacity. In this study, we measured autophagic flux and cellular damage in primary cultured monolayers of rat alveolar epithelial cells (AEC) exposed separately and sequentially to two different autophagy inducers. Rat AEC monolayers were exposed apically for 24 hrs to (1) polystyrene nanoparticles (PNP, 20 nm, carboxylated, near-infrared dye-labeled), (2) tunicamycin (TN, a disruptor of protein synthesis and inducer of the unfolded protein response, at 1-15 μg/mL), or (3) TN (1-15 μg/mL) after 12 hrs of PNP pre-incubation. Release of the cytoplasmic enzyme lactate dehydrogenase (LDH) was used as an indicator of cellular damage and quantified by an LDH assay from Dojindo (Rockville, MD). Autophagic flux was assessed by live cell imaging using confocal microscopy and 0.1 μM DAPRed (Dojindo; Rockville, MD) in the presence (for 1 hr) and absence of 40 μM chloroquine. Serial z sections were collected over the entire cell volume of live single cells to detect DAPRed (autophagosome) fluorescence. PNP were taken up into AEC, where their cytosolic presence induced a gradually increased autophagic flux, reaching a steady state at ~10-24 hrs. When AEC were exposed to TN alone for 24 hrs, autophagy was also activated. Cellular damage in the presence of TN, determined by increased LDH release, revealed dose-dependent injury with LD50 of 8.12 μg/mL over 24 hr TN exposure. When AEC were pre-exposed to PNP for 12 hrs and subsequently exposed to TN, increased LDH release was seen with LD50 of 3.95 μg/mL. In summary, the intracellular presence of PNP taken up from apical fluid of rat AEC monolayers activated autophagy. When cellular protein synthesis was disrupted with TN alone, autophagy was also activated. Furthermore, TN induced dose-dependent cellular damage in AEC. However, after pre-exposure to PNP, TN induced greater cellular damage observed as increased LDH release. These results suggest that autophagic capacity is limited and that pre-induction of autophagy by inhaled PNP makes AEC more susceptible to secondary injury by TN. These findings are consistent with the hypothesis that environmental stressors (e.g., ambient air nanoparticles) exert their harmful effects, at least in part, by reducing available autophagic activity/capacity, thereby causing nanoparticle-exposed AEC to be more susceptible to secondary injury. Funding: NIH; WRMPPF. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer's disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models. Key features • Allows real-time qualitative imaging of autophagic flux at single-cell level. • Primary cells and cell lines can also be utilized with this technique. • Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.

We have shown previously that lung adenocarcinoma (A549) cells internalize polystyrene nanoparticles (PNP). Once intracellular, PNP stimulate autophagy in A549 cells and over time are delivered to lysosomes, with subsequent egress from the cells by lysosomal exocytosis. We also found evidence that A549 cell exposure to either PNP or ambient air pollution ultrafine particles (UFP) leads to lysosomal dysfunction. In the current study, we assessed if there is a potential limit to autophagic capacity in A549 cells. In the presence of chloroquine (40 μM, 1 hr), A549 cells were exposed to a fluorescent marker, DAPRed (Dojindo; Rockville, MD), that labels autophagosomes and autolysosomes. Autophagic activity was assessed based on the fluorescence intensity of DAPRed, which was measured in serial z sections over the entire cell volume in live, single cells using confocal laser scanning microscopy. Single exposure (50 nM Rapamycin as positive control, 80 μg/mL of 20 nm carboxylated PNP, or 1 μg/mL of UFP (<180 nm, collected in Los Angeles)) and double exposure (Rapamycin + PNP or Rapamycin + UFP) models were utilized to determine the kinetics of autophagic activity over 24 hr. Data on autophagic activity were also obtained by measurement of fluorescence intensities using LC3-GFP-RFP in response to PNP/UFP exposures. LC3-GFP-RFP was transduced (700:1 virus particle per cell ratio; Thermo Fisher Scientific; Waltham, MA) into A549 cells and expression of LC3-GFP-RFP was assessed using confocal microscopy. We found that autophagic activity remained relatively constant in unexposed A549 cells (negative control) as opposed to a rapid increase (>3 times negative control) with a peak at ~3-5 hr post exposure in Rapamycin-exposed cells. Both PNP and UFP exposure resulted in gradual elevations in autophagic activity, reaching a steady state (at ~2.5 times negative control) at ~8-10 hr post exposure with no appreciable change from ~10 to 24 hr. Combining Rapamycin with PNP or UFP caused a rapid rise in autophagic activity (peaking <5 hr post exposure); however, no difference in steady state autophagic capacity (~2.5 times negative control) was observed in comparison to either PNP or UFP exposure over 24 hr. Similar time courses of elevated autophagic activity were observed by increased fluorescence of LC3-GFP-RFP in response to 24 hr exposure to Rapamycin, PNP or UFP. In addition, higher expression of LC3-RFP indicated higher levels of autophagosome fusion with autolysosomes (increased autophagic flux). These data suggest that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic capacity, thereby making nanoparticle-exposed cells more susceptible to secondary injury.

Autophagy, a homeostatic mechanism, is crucial in maintaining normal cellular function. Although dysregulation of autophagic processes is recognized in certain diseases, it is unknown how maintenance of cellular homeostasis might be affected by the kinetics of autophagic activity in response to various stimuli. In this study, we assessed those kinetics in lung adenocarcinoma (A549) cells in response to exposure to nanoparticles (NP) and/or Rapamycin. Since NP are known to induce autophagy, we wished to determine if this phenomenon could be a driver of the harmful effects seen in lung tissues exposed to air pollution. A549 cells were loaded with a fluorescent marker (DAPRed) that labels autophagosomes and autolysosomes. Autophagic activity was assessed based on the fluorescence intensity of DAPRed measured over the entire cell volume of live single cells using confocal laser scanning microscopy (CLSM). Autophagic activity over time was determined during exposure of A549 cells to single agents (50 nM Rapamycin; 80 μg/mL, 20 nm carboxylated polystyrene NP (PNP); or, 1 μg/mL ambient ultrafine particles (UFP) (<180 nm)), or double agents (Rapamycin + PNP or Rapamycin + UFP; concomitant and sequential), known to stimulate autophagy. Autophagic activity increased in all experimental modalities, including both single agent and double agent exposures, and reached a steady state in all cases ~2 times control from ~8 to 24 hrs, suggesting the presence of an upper limit to autophagic capacity. These results are consistent with the hypothesis that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic response to additional stimulation, thereby making nanoparticle-exposed cells more susceptible to secondary injury due to autophagic overload.

Rationale: We have previously reported that polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell (AEC) monolayers (RAECM) in a time-, dose-and size-dependent manner without the involvement of endocytic process(es).Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and are exocytosed in a cytosolic [Ca 2+ ]-dependent manner.In this study, we explored PNP interactions with A549 cells with a focus on the time course of activation of autophagy.Methods: A549 cells were exposed apically to 20 nm carboxylated PNP, followed by live cell imaging.Serial z sections over the entire cell volume were performed in live single cells by confocal laser scanning microscopy.PNP uptake was investigated using pharmacologic inhibitors of endocytic processes and by colocalization of PNP with early endosome marker Rab5a-GFP.Activation of autophagy was determined by colocalization of autophagy marker LC3-GFP with PNP, while the level of autophagic activity was assessed using DAPRed (Dojindo).PNP egress was measured in the presence and absence of ATP-induced elevations in intracellular [Ca 2+ ].Results: PNP uptake was decreased by cytochalasin D, an inhibitor of macropinocytosis, but not by inhibitors of clathrin-or caveolin-mediated endocytosis.Partial colocalization of PNP with Rab5-GFP was observed after five hr PNP exposure.Three hours after PNP exposure, PNP-induced activation of autophagy was indicated by colocalization of LC3-GFP and PNP.At 24 hr of PNP exposure, PNP-filled autophagosomes almost entirely filled the cytosol.Autophagic activity did not change in control (unexposed) cells over 24 hr.In PNP-exposed cells, autophagic activity gradually increased after ∼6 hr, reaching steady state at 2.5 times control after ∼8 hr and remaining elevated through 24 hr of PNP exposure.Egress of PNP resulted in a 90% decrease in intracellular PNP content at 24 hr and was not affected by elevations in cytosolic [Ca 2+ ].Conclusions: Similar to primary rat AEC, A549 cells internalize extracellular PNP, although there are differences in the pathway/kinetics of uptake, intracellular handling and egress.The presence of PNP in A549 cells increased autophagic flux in a time-dependent manner.These data showing time-dependent activation of autophagy by PNP suggest that autophagy-dependent cellular defense mechanisms against other simultaneous insults may be compromised, limiting the ability of cells to cope with cellular stress in the presence of nanoparticles.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.

Introduction. Strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity is an important target in the next future. Objective: To prove that autophagic processing of nanoparticular is essencial for maintenance of alveolar epithelial cells. Material and Methods: Utilizing confocal microscopy, we quantitatively assessed uptake, processing and egress of near infrared (NIR) fluorescence-labeled polystyrene nanoparticles (PNP) in live primary rat alveolar epithelial cell (AEC) monolayers (RAECM) after apical exposure. Intracellular PNP content was assessed by measuring the time courses of NIR fluorescence intensity for PNP of 20, 100 and 200 nm and for apical [PNP] (20 nm) of 40, 80 and 160 ng/mL. PNP content and colocalization with intracellular vesicles (including autophagosomes, lysosomes, Golgi and endoplasmic reticulum) in PNP-exposed AEC were determined over the entire cell volume via z-stacking. Involvement of endocytosis in PNP uptake was tested by pharmacologic inhibition of classical endocytotic pathways. Colocalization of early endosome marker Rab5-GFP with PNP in apically exposed AEC was investigated for up to 3 hr. The role of autophagy in intracellular processing of PNP or ambient pollution particles (APP, diameter ≤200 nm) was assessed using LC3 (microtubule-associated protein 1A/1B light chain 3B)-II immunolabeling and inhibitors of autophagosome formation (3-methyladenine (3-MA)) or autolysosome formation (bafilomycin or chloroquine). Mechanisms of PNP uptake into and egress from RAECM were further studied by inhibition of microtubule polymerization required for movement of PNP-filled intracellular vesicles (e.g., autophagosomes) and mobilization of intracellular [Ca2+] known to speed up exocytosis. Isotropic cuvette-based microfluorimetry was used to determine intracellular [PNP] from PNP content assessed by anisotropic confocal microscopy. Results: Uptake rates and steady state intracellular content decreased as PNP size increased from 20 to 200 nm. Uptake rates and steady state intracellular content increased with increased apical [PNP] (20 nm) and were unaffected by inhibition of endocytic pathways. <3% of total intracellular PNP colocalized with Rab5 positive intracellular vesicles post-PNP exposure for up to 3 hr. Both PNP and APP exposure led to marked increases in LC3-II expression, and PNP increasingly co-localized with autophagosomes and/or lysosomes over time. PNP egress exhibited both fast [Ca2+]-dependent release and a slower diffusion-like process. Inhibition of microtubule polymerization curtailed initial rapid PNP egress, causing elevated vesicular and intracellular PNP content. Interference with autophagosome formation led to slower PNP uptake and markedly decreased steady state intracellular PNP content. At steady state, cytosolic [PNP] was higher than apical [PNP], and vesicular [PNP] (~80% of intracellular PNP content) exceeded both cytosolic [PNP] and intracellular [PNP]. Conclusions: These findings are consistent with the hypotheses that (1) autophagic processing of nanoparticles is essential for maintenance of AEC integrity, (2) altered autophagy and/or lysosomal exocytosis may lead to AEC injury and (3) intracellular [PNP] in AEC is regulable, suggesting strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity.

Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Using confocal microscopy, we quantitatively assessed uptake, processing, and egress of near-infrared (NIR)-labeled carboxylated polystyrene nanoparticles (PNP) in live alveolar epithelial cells (AEC) during interactions with primary rat AEC monolayers (RAECM). PNP fluorescence intensity (content) and colocalization with intracellular vesicles in a cell were determined over the entire cell volume via z stacking. Isotropic cuvette-based microfluorimetry was used to determine PNP concentration ([PNP]) from anisotropic measurements of PNP content assessed by confocal microscopy. Results showed that PNP uptake kinetics and steady-state intracellular content decreased as diameter increased from 20 to 200 nm. For 20-nm PNP, uptake rate and steady-state intracellular content increased with increased apical [PNP] but were unaffected by inhibition of endocytic pathways. Intracellular PNP increasingly colocalized with autophagosomes and/or lysosomes over time. PNP egress exhibited fast Ca 2+ concentration-dependent release and a slower diffusion-like process. Inhibition of microtubule polymerization curtailed rapid PNP egress, resulting in elevated vesicular and intracellular PNP content. Interference with autophagosome formation led to slower PNP uptake and markedly decreased steady-state intracellular content. At steady state, cytosolic [PNP] was higher than apical [PNP], and vesicular [PNP] (~80% of intracellular PNP content) exceeded both cytosolic and intracellular [PNP]. These data are consistent with the following hypotheses: 1) autophagic processing of nanoparticles is essential for maintenance of AEC integrity; 2) altered autophagy and/or lysosomal exocytosis may lead to AEC injury; and 3) intracellular [PNP] in AEC can be regulated, suggesting strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity.

RationaleTo understand the contribution to lung pathogenesis of ambient ultrafine air pollution particles, knowledge of basic mechanisms underlying how inhaled nanoparticles interact with alveolar epithelial cells (AEC) is needed. We have reported that polystyrene nanoparticles (PNP) at the apical surface of primary rat alveolar AEC monolayers (RAECM) are taken up in a manner dependent on exposure time, dose and particle size via a non‐endocytic ‘diffusional’ process, induce autophagy, and exit via both fast intracellular calcium‐dependent lysosomal exocytosis and a slower (‘diffusional’) process. In this study, we further investigated intracellular fate of PNP.MethodsRAECM were apically exposed to near‐infrared labeled PNP (80 μg/mL; carboxylated; 20 nm). AEC were subjected to confocal imaging throughout the volume of the cells. In each z plane, total intracellular fluorescence intensity as well as vesicular and cytosolic PNP were determined and normalized to total intracellular PNP (PNPic). Volumes of endosomal, autophagosomal and lysosomal compartments, and colocalization of PNP in cytosolic vs vesicular compartments, were assessed using an early endosome marker (Rab5) tagged with green fluorescent protein (GFP), quinacrine, microtubule‐associated protein 1 light chain 3 (LC3B)‐GFP, and Magic Red (a lysosomal marker). [PNPic] was then determined by an isotropic method (cuvette‐based microfluorometry) utilizing AEC lysates as a function of time. [PNP] in vesicles ([PNPv]) and cytosol ([PNPc]) were estimated from the respective fractions of compartmental volumes and co‐localized PNP in each compartment.ResultsAfter 12 hr, [PNPic] reached a plateau of 1.84 mg/mL. Distribution of intracellular PNP was ~80% in intracellular vesicles (primarily lysosomes) and ~20% in cytosol at steady state, equivalent to 8.86 mg/ml and 0.58 mg/mL for [PNPv] and [PNPc], respectively. About 3% of total intracellular PNP was observed in Rab5‐GFP positive early endosomes by 3 hr after PNP exposure, but inhibition of classical endocytosis pathways did not alter PNP uptake kinetics. In contrast, blockade of autophagosome formation by 3‐methyladenine (3MA) markedly reduced PNP uptake and decreased steady state [PNPic] by ~75% with no detectable vesicular PNP. Inhibition of autophagosome‐lysosome fusion by bafilomycin lowered steady state PNPic by ~20% compared to control, with ~81% in cytosol and ~19% in vesicles.SummaryIn AEC exposed to PNP, [PNPv] exceeds both [PNPc] and [PNPic], with [PNPc]>>apical [PNP]. Cytosolic presence of PNP triggers autophagosome formation, leading to PNP accumulation in lysosomes. Inhibition of autophagy leads to decreased PNP entry and [PNPic]. These data suggest that intracellular processing of PNP is regulable, offering possible approaches to amelioration of inhaled nanoparticle‐related cytotoxicity in AEC.Support or Funding InformationFunding: NIH; Will Rogers Institute; Hastings and Whittier FoundationsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.