Pulmonary Exposure of Mice to Ammonium Perfluoro(2-methyl-3-oxahexanoate) (GenX) Suppresses the Innate Immune Response to Carbon Black Nanoparticles and Stimulates Lung Cell Proliferation

Abstract

Background:

Per- and polyfluoroalkyl substances (PFAS) have been associated with respiratory diseases in humans, yet the mechanisms through which PFAS cause susceptibility to inhaled agents is unknown. Herein we investigated the effects of ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), an emerging PFAS, on the pulmonary immune response of mice to carbon black nanoparticles (CBNP). We hypothesized that pulmonary exposure to GenX would increase susceptibility to CBNP through suppression of innate immunity.

Methods:

Male C57BL/6 mice were exposed to vehicle, 4 mg/kg CBNP, 10 mg/kg GenX, or CBNP and GenX by oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) was collected at 1 and 14 days post-exposure for cytokines and total protein. Lung tissue was harvested for histopathology, immunohistochemistry (Ki67 and phosphorylated (p)-STAT3), western blotting (p-STAT3 and p-NF-κB), and qRT-PCR for cytokine mRNAs.

Results:

CBNP increased CXCL-1 and neutrophils in BALF at both time points evaluated. However, GenX/CBNP co-exposure reduced CBNP-induced CXCL-1 and neutrophils in BALF. Moreover, CXCL-1, CXCL-2 and IL-1β mRNAs were increased by CBNP in lung tissue but reduced by GenX. Western blotting showed that CBNP induced p-NF-κB in lung tissue, while the GenX/CBNP co-exposed group displayed decreased p-NF-κB. Furthermore, mice exposed to GenX or GenX/CBNP displayed increased numbers of BALF macrophages undergoing mitosis and increased Ki67 immunostaining. This was correlated with increased p-STAT3 by western blotting and immunohistochemistry in lung tissue from mice co-exposed to GenX/CBNP.

Conclusions:

Pulmonary exposure to GenX suppressed CBNP-induced innate immune response in the lungs of mice yet promoted the proliferation of macrophages and lung epithelial cells.

Introduction

Air pollution continues to be one of the most pressing public health topics on a global scale as the World Health Organization estimates that more than 7 million people worldwide die annually from exposure to air pollution [1]. Decades of scientific evidence have converged in defining and solidifying the link between exposure to air pollution and the onset of adverse health outcomes, such as exacerbation of asthma and cardiovascular disease. Upon encountering professional phagocytes and the lung epithelium, inhaled air pollution particles have been shown to activate distinct cellular signaling pathways that lead to the production of cytokines and chemokines that mediate the infiltration of inflammatory cells into the lung. For example, the influx of neutrophils into the lung from the vasculature is a key innate immune response to air pollution ultrafine particles or engineered nanoparticles [2]. Acute neutrophilic inflammation is a classic host defense mechanism against inhaled microbes and, under the appropriate circumstances, is followed by resolution of the inflammatory response. However, prolonged exposure to air pollution particles can modulate the innate immune response of the lungs, leading to chronic pulmonary and systemic inflammation [3–6]. Co-exposure to other inhaled toxicants can also trigger disruptions in the immune system that can directly or indirectly modulate the initial immune response to inhaled ultrafine air pollution particles. The resulting dysregulated immune response may potentially increase susceptibility to lung diseases.

Per- and polyfluoroalkyl substances (PFAS) are persistent chemicals in ‘non-stick’ products such as Teflon^™ and firefighting foams that cause immunosuppression in mice [7–9] and have been associated with susceptibility to asthma and lung infections in humans [10–13]. Representative “legacy” PFAS compounds, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are presumed immune hazards to humans by the US National Toxicology Program (NTP) after findings from both animal and human studies provided evidence of suppressed T cell-dependent antibody response (TDAR) and suppressed vaccine response [8, 9, 14], including a decrease in vaccine-associated antibody concentrations in exposed children [15, 16].

Although many of the “legacy” PFAS compounds have been or are being phased out of production, emerging PFAS compounds such as hexafluoropropylene oxide dimer acid (HFPO-DA), commonly referred to by the trade name ‘GenX’, have recently gained attention due to industrial release, environmental contamination of air and water, and presence in blood samples from humans [17, 18]. The ammonium salt of HFPO-DA (GenX) is termed ammonium perfluoro(2-methyl-3-oxahexanoate). While the majority of release has been into water, GenX is also emitted from industrial sources into the atmosphere, bringing into question the potential consequences of pulmonary exposure. Several studies in the United States and Europe have documented the atmospheric release of GenX. For example, an investigation by the North Carolina Department of Environmental Quality (NC-DEQ) demonstrated up to 1046 kg of GenX air emissions were released from an industrial production facility in the Cape Fear River basin in 2017 [19]. Since then, NC-DEQ has required GenX emissions to be reduced to 10.46 kg per year at the industrial production facility via a 2019 Consent Order [20]. Another recent study provided data suggesting the occurrence of air dispersion of GenX in West Virginia that was detected as far as 29 km from a production facility [21]. In addition, atmospheric emission and detection of GenX was documented in the Netherlands near a fluoropolymer production plant [22]. A separate investigation suggested atmospheric emission as a potential source of GenX detected in/on all grass and leaf samples near a production plant [23]. Currently, due to lack of data concerning the immunomodulatory effects of GenX, it is difficult to fully and accurately assess the potential for inhaled GenX to impact aspects of the immune system. Evidence of immunotoxicity of GenX has been observed in in vivo studies when daily administration of GenX by oral gavage resulted in suppressed TDAR and decreased spleen weight in female C57BL/6 mice. However, GenX-exposed C57BL/6 male mice of the same study did not exhibit the suppression of TDAR but showed a significant increase in T lymphocyte numbers [24].

The idea that oral or inhalation exposure to PFAS is potentially hazardous to our immune system is widely acknowledged [25]. But this common agreement on PFAS-induced immune dysfunction relies disproportionately on data from studies involving mostly oral exposure. Additionally, while new studies continue to further characterize the immunotoxic potential of various PFAS chemicals, there is still a lack of studies that focus on elucidating the effects of PFAS exposure on the innate immune response, especially when PFAS exposure coincides with inhalation of air pollution particles. In this study, we aimed to investigate the potential immunomodulatory effect of GenX on the innate immune response induced by carbon black nanoparticles (CBNP), a surrogate for ultrafine air pollution particles.

Materials and Methods:

GenX:

GenX (Ammonium perfluoro(2-methyl-3-oxahexanoate), molecular formula C₆H₄F₁₁NO₃ (CAS number 62037-80-3) was purchased from Synquest Laboratories (Alachua, FL). The acid form of GenX is also referred to as hexafluoropropylene oxide dimer acid (HFPO-DA) with the molecular formula C₆HF₁₁O₃. GenX was dissolved in deionized water to achieve the stock concentration of 100 mg/mL. Further dilutions were achieved in phosphate-buffered saline (PBS). A GenX dose of 10 mg/kg body weight delivered to the lungs of mice by oropharyngeal aspiration (OPA) was used in the present study. This dose was within the dose range routinely used for oral gavage (1-100 mg/kg) and 10 times lower than that used in a previous study that investigated GenX exposure using 100 mg/kg by oral gavage [24].

Carbon Black Nanoparticles:

Carbon black nanoparticles (CBNP), sold under the product name Raven 5000 Ultra II Powder, were purchased from Columbian Chemicals (Marietta, GA). Characterization data was previously reported by our laboratory [26]. CBNPs possessed a mean diameter of ~8 nm, had an external surface area of 350 m²/g, and were insoluble in water. Carbon black nanoparticles were sonicated in a cup horn sonicator (Q500, Qsonica, Newtown, CT) for 10 minutes at 60 amps and vortexed immediately before delivery to mice. The CBNP concentration delivered to the lungs of mice by oropharyngeal aspiration (4 mg/kg body weight) was selected based on several studies that reported an observable CBNP-induced immune response [27, 28].

Animal care:

Male C57BL/6J mice (10 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited animal facility, which was pathogen-free, humidity/temperature controlled and supplied food and water ad libitum. All animal procedures were approved by the NC State University Institutional Animal Care and Committee (IACUC). Mice were housed 5 per cage according to their respective treatment groups – vehicle control (1d), vehicle control (14d), GenX (1d), GenX (14d), CBNP (1d), CBNP (14d), GenX + CBNP (1d), and GenX + CBNP (14d).

Exposure of mice to GenX and CBNP:

GenX or CBNP were suspended in Dulbecco’s phosphate buffered saline (Genesee Scientific, Morrisville, NC) to achieve final concentrations of 10 mg/kg and 4 mg/kg, respectively. Carbon black nanoparticles were sonicated in a cup horn sonicator (Q500, Qsonica, Newtown, CT) for 10min at 60 amps and vortexed immediately before delivery to mice. Mice were exposed to 50 μl vehicle control, CBNP, GenX, or both by oropharyngeal aspiration (OPA) under isoflurane anesthesia. OPA is a well-established surrogate method for inhalation for pulmonary delivery of particles and other agents to the lungs of mice [29]. Necropsy was performed at 1 and 14 days after exposure to observe acute and sub-chronic responses.

Necropsy and sample collection:

Necropsy was performed at 1 and 14 days after exposure to observe pro-inflammatory and pro-fibrotic responses in the lungs of mice, respectively. Mice were euthanized with an intraperitoneal injection of pentobarbital. Bronchoalveolar lavage fluid (BALF) was collected from each mouse by cannulating the trachea and lavaging the lungs with 0.5ml of chilled DPBS two times. The BALF was centrifuged at 1200 RPM for 5 minutes, and the resulting supernatant was transferred to a separate set of tubes and utilized to analyze protein, LDH, and cytokines/chemokines. The cell pellet was resuspended in 500 μL of PBS and used to analyze inflammatory cells and assess mitotic figures in macrophages. For histopathology, the left lung lobe was fixed in neutral buffered formalin (VWR, Radnor, PA) for 24hrs, then transferred to 70% ethanol for three days before being embedded in paraffin. For mRNA analysis, a right superior lung lobe was stored in RNAlater (Fisher Scientific, Waltham, MA). For protein analysis, the right medial and inferior lung lobes were snap-frozen in liquid nitrogen and stored at −80°C. Serum from blood was collected using the BD Microtainer Capillary Blood Collector (BD, Franklin Lakes, NJ).

BALF inflammatory cell differentials and mitotic figures:

For evaluation of lung inflammatory cells, 100ul of BALF was centrifuged onto glass slides using a Cytospin 4 centrifuge (ThermoFisher, Waltham, MA). The slides were then fixed and stained with the Diff-Quik stain set (Siemens, Newark, DE). Average total cell counts and differential cell counts were quantified by using an Olympus light microscope BX41 (Center Valley, PA) with three representative photomicrographs taken at 100x magnification and every cell type (macrophages, neutrophils, eosinophils, and lymphocytes) counted using ImageJ software with the Fiji expansion [Eliceiri/LOCI group, University of Wisconsin-Madison, Madison, WI). Mitotic macrophages were quantified by counting every macrophage in any stage of mitosis on each slide.

Cytokine analysis in BALF:

DuoSet enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) were used according to the manufacturer’s protocol to quantify protein levels of cytokines C-X-C motif chemokine ligand 1 (CXCL1), C-C motif chemokine ligand 2 (CCL2), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), osteopontin (OPN), and tumor necrosis factor alpha (TNF-α) from BALF. Cytokine concentrations were derived from the absorbance values measured at 450nm with a correction at 540nm using the Multiskan EX microplate spectrophotometer (ThermoFisher, Waltham, MA). Concentration values were derived from standard curves according to the manufacturer’s protocol.

qRT-PCR:

Applied Biosystems high-capacity cDNA reverse transcription kit (ThermoFisher Scientific, Waltham, MA) was used to create cDNA from the mRNA isolated from the right lung lobes using Quick-RNA^™ MiniPrep (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. The FastStart Universal Probe Master (Rox) (Roche, Basel, Switzerland) was then used to run Taqman qPCR on the Applied Biosystems QuantStudio3 Real-Time PCR System Thermal Cycling Block (ABI, Foster City, CA) to determine the comparative C_T (ΔΔC_T) fold change expression of CXCL1, CXCL2 and IL-1β normalized to GAPDH as the endogenous control.

Immunoblotting:

Whole lung lysate was prepared from snap-frozen right lung lobe. Frozen lung samples were transferred to 1.5 mL centrifugation tubes containing lysis buffer (20mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 1mM Na₃VO₄, 1x Halt^™ Protease Inhibitor Cocktail, in DPBS). One 35mm stainless steel bead was inserted into each 1.5 mL centrifugation tube containing lung sample and lysis buffer to facilitate the tissue homogenization process. Mini Bead Mill Homogenizer (VWR International) was used with a speed setting of 4 for 1 minute. The resulting mixture was spun down at 12,000 RPM for 4 minutes. The protein concentration of the supernatant was determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA). Absorbance was read at 450nm with a correction at 540nm using the Multiskan EX microplate spectrophotometer (ThermoFisher, Waltham, MA). Samples were loaded onto a Novex^™ 4–12% SDS-PAGE gel (Invitrogen, Carlsbad, CA), separated by electrophoresis, and transferred onto PVDF membranes. Membranes were blocked for one hour and incubated overnight in 1:1000 dilution of rabbit or mouse primary antibodies purchased from Cell Signaling Technology (phosphorylated (p)-STAT3 at Tyr705, STAT3, phosphorylated (p)-NF-κB at Ser536, NF-κB, and β-actin). Following primary antibody incubation, the membranes were washed and incubated in 1:2500 dilution of anti-rabbit (Cell Signaling Technology, Danvers, MA) or anti-mouse (Santa Cruz Biotechnology, Dallas, TX) with horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence (ECL) Prime Western Blotting Detection Reagent (Cytiva, Marlborough, MA) was used to facilitate HRP-induced chemiluminescence and resulting signals were captured using Amersham Imager 680 (GE Life Sciences, Marlborough, MA). Semi-quantitative densitometry was performed using ImageJ and Adobe Photoshop.

Histopathology and immunohistochemistry:

The left lung was cut into three cross sections, which were embedded in paraffin, and 5-micron histologic sections were mounted on charged glass slides. Sections were stained with hematoxylin and eosin (H&E) to assess pro-inflammatory tissue reactions. Chromogenic Immunohistochemistry (IHC) was performed on paraffin-embedded lung tissues using specific antibodies against the cell proliferation marker Ki-67 and phospho-STAT3 (pSTAT3). For Ki-67 immunostaining, sections were deparaffinized, retrieved with citrate, and stained on a Biocare IntelliPATH ^™ Autostainer Stainer (Pacheco, CA). Staining with the primary rabbit anti-Ki-67 polyclonal antibody (Biocare, Catalogue # CRM325C; 1:100 dilution) for 30 minutes at room temperature was followed by the use of the Vector ImmPRESS^® HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Burlingame, CA) using DAB chromogen per the manufacturer’s instructions. Three rinses were between steps with tris buffered saline (Thermo Fisher Scientific, Waltham, MA). Sections were counterstained with Mayer’s hematoxylin (Thermo Fisher Scientific) for 5 minutes, dehydrated, cleared, and mounted with Permount (Thermo Fisher Scientific). On Ki-67 immunostained slides, all positively stained cells for the protein Ki-67 were counted and categorized as either: airway epithelial, alveolar epithelial, or macrophages. Photomicrographs were taken at 100x magnification using the Olympus BX41 light microscope (Center Valley, PA). IHC for p-STAT3 was carried out using the Leica Bond III Autostainer system. Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Heat induced antigen retrieval was performed for 20 minutes at 100°C using Bond-Epitope Retrieval solution 2 pH-9.0 (AR9640) followed by a 5 min Bond peroxide blocking step (DS9800). After pretreatment, slides were incubated for 1 hour with the p-STAT3 antibody (Cell Signaling Technology, 9145S). Antibody detection with 3,3’-diaminobenzidine (DAB) was accomplished using the Bond Intense R detection system (DS9263) supplemented with Novolink Polymer (Leica, #RE7260-K) secondary. Stained slides were dehydrated and coverslipped with Cytoseal 60 (8310-4, Thermo Fisher Scientific). A negative control contained no primary antibody. p-STAT3 positive cells were counted, and cell types (bronchiolar epithelium, alveolar epithelial cells, or macrophages) were noted per ten, 40X objective fields using an Olympus BX53 microscope.

Statistical Analysis:

One-way ANOVA with a Tukey’s post hoc test or Student’s t-test was utilized to evaluate differences between treatment groups (GraphPad Prism, version 5.0, La Jolla, CA).

Results

Pulmonary exposure to GenX selectively decreases CBNP-induced neutrophil presence in BALF from the lungs of mice.

Male C57BL/6 mice were exposed by oropharyngeal aspiration to vehicle control, GenX (10 mg/kg), CBNP (4 mg/kg), or a combination of GenX and CBNP prior to necropsy at 1 and 14 days after exposure. GenX or the combination of GenX and CBNP caused a significant increase in total protein levels in the BALF of mice at 1 day but not at 14 days post exposure (Fig. 1A). Histopathology of lung tissue sections stained with H&E indicated focal inflammation in the bronchoalveolar regions of lungs from mice treated with CBNP or GenX/CBNP (Fig. 1B). Differential counting of BALF cells from the lungs of mice did not reveal any significant differences in macrophage counts across treatment groups at 1 day post exposure time point. However, there was a significant decrease in macrophage counts between CBNP-exposed and GenX/CBNP-exposed groups 14 days after exposure (Fig. 2A). While there were significant increases in the number of neutrophils induced by CBNP at both 1 and 14 days after exposure, co-exposure to GenX and CBNP significantly decreased the number of neutrophils in the BALF compared to CBNP alone (Fig. 2B). However, exposure to GenX alone did not result in any significant differences in the number of neutrophils observed in the BALF of mice compared to that of the vehicle control group at both 1 and 14 days after exposure (Fig. 2B).

Fig. 1.

Lung injury and inflammation in mice at 1 day or 14 days post exposure to GenX, CBNP, or GenX and CBNP. (A) Total protein in BALF (ug/ml). *p<0.05 or **p<0.01 compared to vehicle and #p<0.05 comparing CBNP group to GenX + CBNP co-treatment group as determined by one-way ANOVA with Tukey’s test for post hoc analysis. (B) Hematoxylin and eosin-stained lung sections showing focal inflammation associated with agglomerates of CBNP in the bronchoalveolar region of the lungs (arrows). Black bar indicates 20 μm.

Fig. 2.

Differential cell counts in BALF from mice at 1 day or 14 days post exposure to GenX, CBNP, or GenX and CBNP. (A) Macrophage counts: #p<0.05 comparing between treatment groups as determined by one-way ANOVA with Tukey’s test for post hoc analysis. (B) Neutrophil counts. *p<0.05, ****p<0.0001 compared to vehicle, ####p<0.0001 comparing CBNP group to GenX + CBNP co-treatment group as determined by t-test

GenX reduces CBNP-induced CXCL-1 protein in the BALF and CXC chemokine mRNAs in lung tissue.

CXCL-1, a chemokine that plays an important role in recruitment and activation of neutrophils [30], was increased in the BALF of mice exposed to CBNP (Fig. 3). The GenX/CBNP co-exposure significantly reduced CBNP-induced CXCL-1 protein levels in BALF at both 1 day and 14 days post exposure periods (Fig. 3). The changes in CXCL-1 protein in BALF (Fig. 3) and CXCL-1, CXCL-2, and IL-1β mRNA in lung tissue (Fig. 4) corroborated the CBNP-induced neutrophilic infiltration in BALF that was suppressed by GenX (Fig. 2). BALF protein levels of CCL2, a monocyte chemoattractant, were also induced by both CBNP and GenX at 1 day after exposure. At the same time, the combination of CBNP and GenX additively increased CCL2 compared to exposures to either agent alone (Fig. 5A). In addition, GenX, either alone or in combination with CBNP, significantly increased osteopontin (OPN), a matricellular cytokine, in the BALF of mice at 1 day post exposure (Fig. 5B). Pro-inflammatory cytokines IL-6 and TNF-α in BALF were also analyzed by ELISA but were not significantly induced or suppressed by CBNP or GenX (data not shown). Collectively, these data demonstrated that the suppression of CBNP-induced CXCL-1 by GenX was selective to this chemokine and not observed with other cytokines and chemokines in BALF analyzed.

Fig. 3.

CXCL-1 secreted protein levels (pg/ml) in BALF collected from mice at 1 day post exposure and 14 days post exposure to vehicle, GenX, CB, or GenX/CBNP co-exposure. ***p<0.001, ****p<0.0001 compared to vehicle, ##p<0.01, ###p<0.001 comparing between treatment groups as determined by one-way ANOVA with Tukey’s test for post hoc analysis.

Fig. 4.

CXCL-1, CXCL-2, and IL-1β mRNA levels in lung tissue at 1 day post exposure and 14 days post exposure to vehicle, GenX, CB, or GenX/CBNP co-exposure. *p<0.05, **p<0.01, ****p<0.0001 compared to vehicle; #p<0.05, ##p<0.01, or ####p<0.0001 comparing between treatment groups as determined by one-way ANOVA with Tukey’s test for post hoc analysis.

Fig. 5.

CCL-2 and OPN secreted protein levels (pg/ml) in BALF collected from mice 1-day post exposure to GenX, CB, or GenX/CBNP co-exposure. (A) CCL-2 in BALF. ***p<0.001 compared to vehicle, ###p<0.001 comparing between treatment groups as determined by one-way ANOVA with Tukey’s test for post hoc analysis. (B) OPN in BALF. *p<0.05 compared to vehicle determined by one-way ANOVA with Tukey’s test for post hoc analysis.

GenX stimulates the proliferation of lung macrophages and alveolar epithelial cells.

When analyzing H&E-stained Cytospin slides of BALF cells, we observed macrophages in various phases of mitosis, particularly in the GenX treatment groups (Fig. 6A). Upon further quantitative evaluation, it was found that the BALF from GenX-exposed mice and GenX/CBNP co-exposed mice of the 1-day post exposure group contained significantly greater numbers of macrophages with mitotic figures (Fig. 6B). CBNP exposure did not result in a significant change in the number of observed mitotic figures compared to the vehicle control group. Furthermore, there were no significant differences in the number of mitotically active macrophages among the treatment groups 14 days after exposure (Fig. 6B). To further confirm our observation, immunohistochemistry was performed on lung sections for Ki-67 protein, a nuclear protein commonly targeted as a marker of cell proliferation [31]. Ki-67 immunostaining revealed proliferating macrophages and alveolar epithelial cells in the bronchoalveolar region of mice treated with GenX (Fig. 7A). Quantitative assessment showed that GenX exposure caused a significant increase in the numbers of Ki-67 positive macrophages in the lungs of mice 1 day after exposure (Fig. 7B). While GenX caused an increase in Ki-67 positive macrophages, neither CBNP nor GenX/CBNP co-exposure caused a significant increase in the number of Ki-67 positive macrophages at 1-day post-exposure compared to vehicle-treated mice (Fig. 7B). At 14 days post exposure, GenX caused a significant increase in the number of Ki-67 positive macrophages; however, the magnitude of the increase was not as evident as that observed at 1-day post exposure (Fig. 7B). In contrast, GenX and GenX/CBNP co-exposure, but not CBNP alone, caused a significant increase in Ki-67 positive alveolar epithelial cells at 1-day post exposure (Fig. 7C). There were no significant differences in the number of Ki-67 positive alveolar epithelial cells among treatment groups at 14 days post-exposure (Fig. 7C). GenX, CBNP, or GenX/CBNP co-exposure all caused significant increases in Ki67 positive airway epithelial cells at 1 day post-exposure, but none of these treatments caused a significant increase in Ki67 immunostaining in the airway epithelium at 14 days post-exposure (Fig. 7D).

Fig. 6.

Mitotic macrophages in BALF from mice after exposure to GenX, CB, or GenX/CBNP co-exposure. (A) Representative microscopic images of Diff-Quick-stained alveolar macrophages from the BALF of mice treated with 10 mg/kg GenX at 1 day post exposure (1000X magnification). Arrows indicate macrophages undergoing various stages of mitotic division. (B) Quantification of mitotic macrophages from Cytospins of BALF collected from mice 1 day or 14 days post exposure to GenX, CB, or GenX/CBNP co-exposure. *p<0.05, **p<0.01 compared to vehicle, #p<0.05 comparing CBNP group with GenX and CBNP co-exposure group as determined by t-test. Black bar indicates 10 μm.

Fig. 7.

Ki-67 immunostaining to determine cell proliferation in the lungs of mice after exposure to GenX, CB, or GenX and CBNP. A) Microscopic images of lung sections 1 day post exposure to treatments stained with Ki-67. Red dashed arrows indicate representative Ki-67 positive macrophages, black dashed arrows represent macrophages with CBNP, and green solid arrow indicates representative Ki-67 positive epithelial cells. B) Quantification of Ki-67 positive lung macrophages, C) alveolar epithelial cells, and D) airway epithelial cells from microscopic images. N = 4-5 animals per group, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared to each cell type’s respective vehicle group, ####p<0.0001 comparing each cell type’s respective treatment groups as determined by one-way ANOVA with Tukey’s test for post hoc analysis. Black bar indicates 20 μm.

GenX stimulates activation of STAT3 protein and reduces phosphorylation of NF-κB.

Whole lung lysates were analyzed via western blot for activation levels of STAT3 protein, a transcription factor implicated in cellular proliferation [32, 33] to investigate possible molecular mechanisms involved in the observed proliferative effects exhibited by GenX on lung macrophages and epithelial cells. Semi-quantitative densitometry of phosphorylated and total STAT3 protein detected by western blotting showed significantly increased phosphorylation of STAT3 protein in the lung lysate samples from mice exposed to either GenX-alone or co-exposed to GenX and CBNP (Fig. 8A and B). CBNP significantly increased the phosphorylation status of NF-κB when compared to vehicle control (Fig. 8A and C). The GenX/CBNP co-exposure displayed a ~55% decrease in phosphorylation status of NF-κB when compared to CBNP exposure and was not statistically significant compared to vehicle control or CBNP alone (p=0.1166). Immunohistochemistry showed intense p-STAT3 staining in alveolar macrophages, alveolar epithelial cells, and airway epithelial cells in the GenX, CBNP, and GenX/CBNP treatment groups (Fig. 9). In contrast, lung tissue from vehicle control had only weak p-STAT3 immunostaining. A semi-quantitative pathology assessment showed that most of these cells were alveolar epithelial cells, followed by macrophages and airway bronchiolar epithelial cells (Supplemental Table 1).

Fig. 8.

Protein analysis by western blot of whole lung lysate collected from mice 1-day post exposure to GenX, CB, or GenX/CBNP co-exposure. (A) Western blot compilation of proteins p-STAT3, STAT3, p-NF-κB, NF-κB and β-actin analyzed from lung lysate collected from mice 1 day following exposure. (B) Densitometry comparison of the average expression of p-STAT3 versus total STAT3. (C) Densitometry comparison of the average expression of p-NF-κB versus total NF-κB. N=4-5, *p<0.05, **p<0.01 compared to vehicle control determined by t-test.

Fig. 9.

Immunohistochemistry of p-STAT3 in lung tissue sections from mice 1-day post exposure to GenX, CB, or GenX and CBNP. Red dashed arrows indicate representative pSTAT3 positive macrophages, black dashed arrows represent macrophages with CBNP, and green solid arrow indicates representative p-STAT3 positive epithelial cells. Black bar indicates 20 μm.

Discussion

The pulmonary immune system plays a significant role in determining one’s susceptibility to the impact of inhaled environmental agents. Through continuous monitoring and dynamic responses by professional phagocytes and lung epithelium, the pulmonary immune homeostasis is delicately maintained to ensure optimized physiological functions of the lungs. Any imbalance, such as suppression of the immune system, can increase susceptibility to disease. The current study aimed to elucidate the consequences of pulmonary exposure to GenX, an emerging PFAS, and the impact on the innate immune response to inhaled carbon black nanoparticles (CBNP), which we used as a surrogate for the carbonaceous core of ultrafine air pollution particles. Our findings suggest that pulmonary exposure to GenX increases susceptibility air pollution particles by suppressing neutrophilic infiltration into the lung (a key innate response orchestrated by macrophage- and epithelial-derived chemokines), and further by increasing the proliferation of lung macrophages and epithelial cells. This theoretical concept is illustrated in Fig. 10 with an emphasis on lung macrophages. To our knowledge, this is the first study that has investigated the consequences of pulmonary exposure to GenX (or any PFAS) with emphasis on innate immune response to inhaled nanoparticles.

Fig. 10.

Hypothetical illustration of macrophage polarization induced by GenX exposure that involves switching of the pro-inflammatory phenotype to a proliferative phenotype. GenX suppresses the production of neutrophil chemokines by macrophages (e.g., CXCL-1), while activating STAT3 and cell proliferation. It is proposed that a similar mechanism could also apply to lung epithelial cells (not shown).

PFAS consist of over 4,700 compounds that are known to be key components of widely used commercial stain-proof (Stainmaster^™) and non-stick (Teflon^™) applications as well as fire-fighting foams [34]. Due to these applications over decades, PFAS are found ubiquitously in the environment and contaminate necessities such as food and drinking water [17]. Moreover, various emerging PFAS have been detected in the blood of adults and children [18]. Epidemiological studies in human populations and toxicological studies with experimental animals provide strong evidence that humans exposed to PFAS are at risk for immunosuppression [35]. The epidemiologic evidence shows that serum PFAS are associated with cancer in adults [10, 36], decreased lung function among children with asthma [10, 11] and increased respiratory tract infections in children [13]. A study of Norwegian adolescents showed a positive correlation between PFAS and asthma, as well as between PFOS and nickel allergy [12]. All of these adverse health outcomes are linked to immunosuppression. Studies with mice show that oral exposure to PFAS causes tumors and suppresses adaptive immune responses at serum concentrations within the reported range of exposed humans [7]. For example, PFOA has been reported to suppress IgM antibody synthesis in female mice [8]. Additionally, it has been suggested that suppression of T-cell dependent antibody production in female mice by PFOA is mediated by disruption of B-cell/plasma cell function [37]. There is some evidence that oral or dermal PFAS exposure can cause adverse respiratory effects in mice. For example, oral exposure of mice to PFOA in chow has been shown to cause lung inflammation [38]. Dermal exposure to PFOA has been reported to augment the lung hypersensitivity response to ovalbumin in mice, suggesting that PFOA enhances IgE response to environmental allergens [39]. While PFAS have been shown to suppress the adaptive immune system, little is known regarding the impact of PFAS on the innate immune system.

The adverse health effects of legacy PFAS such as PFOA and PFOS have been reported by the scientific community and have gained the attention of both the general public and policymakers. However, such is not the case with emerging PFAS compounds such as GenX. Additionally, although previous epidemiological and experimental studies indicated immunomodulatory effects of PFAS, including GenX [14–16, 24, 40, 41], most of these PFAS exposure studies only considered or utilized oral ingestion as the route of exposure. This is logical, given that PFAS are released into waterways adjacent to industrial manufacturing facilities. However, most recently, the industrial release of GenX into the atmosphere from production facilities has generated concern for inhalation as a significant route of exposure [19, 21–23].

We observed a significant reduction of CBNP-induced neutrophil presence coinciding with significant reduction of CBNP-induced neutrophil chemoattractant CXCL-1 in BALF collected from mice exposed to either GenX alone or GenX/CBNP combination via oropharyngeal aspiration. Increases in CXCL-1 and neutrophils induced by CBNP were most prominent at 1 day post-exposure and declined by 14 days post-exposure. Yet, suppression of CBNP-induced CXCL-1 and neutrophil numbers by GenX were significant at both time points. This indicates that the suppressive effect of GenX on CBNP-induced neutrophilia was sustained for two weeks post-exposure, although to a lesser degree. Additionally, mRNA levels of CXCL-1, as well as CXCL-2 and IL-1β were induced by CBNP and suppressed by GenX. CXCL-1 and CXCL-2, as well as IL-1β, play important roles in neutrophil recruitment [42,43]. In contrast to neutrophil numbers that were increased by CBNP and suppressed by GenX, no significant difference in the number of macrophages or monocytes in BALF were observed. BALF protein levels of CCL-2 (a monocyte chemokine) were significantly increased by CBNP in the absence or presence of GenX. Moreover, BALF levels of OPN (a pro-fibrotic cytokine) were induced by GenX in the absence or presence of CBNP. These observations suggest that the suppressive effects of GenX may be specific to chemokines that play a role in neutrophil recruitment (CXCL-1, CXCL-2, IL-1β). While our data suggest that suppression of CBNP-induced CXC chemokines (CXCL-1 and CXCL-2) or IL-1β by GenX might play a role in the inhibition of neutrophil infiltration into the lungs of mice, further work with knockout mice or pharmacological inhibitors would be needed to confirm the roles of each of these chemokines.

In rodent models, CXCL-1 plays a major role in the host immune response in recruiting and activating circulating neutrophils as part of the innate immune response against invading pathogens [42]. Specifically, by binding and activating CXCR2 receptors on neutrophils as well as binding to heparin sulfates on surfaces of endothelium and epithelium, CXCL-1 plays a critical role in neutrophil diapedesis from the systemic circulation into the lung by facilitating the adhesion of circulating neutrophils to the surface of the endothelium and epithelium [44–46]. Inhibition of CXCL-1 using a blocking antibody resulted in reduced neutrophilia in rats exposed to LPS while CXCR2-null mice displayed no alveolar neutrophilia upon LPS exposure [47, 48]. CXCR2-deficient mice were also more vulnerable to Candida albicans infection that coincided with decreased infiltration of neutrophils into the infected sites [49]. In humans, modulation in levels of IL-8/CXCL-8, an ortholog of murine CXCL-1, is implicated in various lung pathologies such as pulmonary fibrosis, acute respiratory distress syndrome (ARDS), and sepsis [50–53]. Elevated levels of CXCL-8 correlated with increased neutrophil numbers in BALF of ARDS patients [54]. Considering their well-documented roles as important components of the innate immune response, the suppression of both CBNP-induced CXCL-1 levels and CBNP-induced neutrophil presence in the lung by GenX observed in this study clearly demonstrates an immunomodulatory effect in the context of pulmonary exposure that could compromise host defense against inhaled air pollution particles and/or infectious microbes. We observed a reduction in CBNP-induced phosphorylation of NF-κB by western blotting. NF-κB is a key transcription factor that mediates the production of CXCL-1 and other cytokines/chemokines [55–57]. The current investigation did not clarify if the reduction in phosphorylation status of NF-κB by GenX was the mechanism that mediated reduction in CXCL-1 levels. Further investigation is needed to confirm the role of NF-κB in GenX induced suppression of CXCL-1 and determine the mechanism through which GenX reduces NF-κB activation. Regarding the latter, PFOS has been reported to up-regulate gene expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which has been shown to suppress NF-κB activation [58,59]. Therefore, it would be valuable to determine whether GenX upregulates Nrf2 and whether this serves as a potential mechanism for suppressing CBNP-induced NF-κB activation in the lungs of mice.

Another important role of neutrophils that has been documented is highlighted in recent studies suggesting that neutrophils may be capable of interacting with cells of both the innate and adaptive immune systems. For example, in an in vivo study involving the adaptive immune response, neutrophils were found to be capable of phagocytosing and presenting antigens into major histocompatibility complex I (MHC I) and cross-prime CB8+ T cells [60]. In addition, a study involving mice infected with vaccinia virus produced a similar finding showing that neutrophils can generate antigen-specific CB8+ T-cell responses [61]. Neutrophils have also been observed to present antigens to CD4+ T cells both directly [62] or indirectly by forming a hybrid phenotype with dendritic cells [63]. Furthermore, neutrophils are involved in phagocytosis and subsequent processing of antigens while also influencing dendritic cell- and macrophage-derived antigen presentation, resulting in modified CD4+ T-cell and B-cell responses [64]. Such evidence suggests that neutrophils have important roles in innate and adaptive immune responses.

Previous research regarding the immunomodulatory effects of GenX and other PFAS involved endpoints related to the adaptive immune system, specifically showing suppression in the T-cell dependent antibody response (TDAR) [8, 24, 65]. Our study showed that pulmonary exposure to GenX in mice resulted in suppression of neutrophil recruitment into the lung, coinciding with decreased protein levels of CXCL-1. Considering the documented roles that neutrophils have in modulating the adaptive immune response, our findings from the current study investigating the effect of pulmonary exposure to GenX on the innate immune response are potentially important for understanding the immunosuppressive effect of PFAS and GenX on the adaptive immune response. It may be possible that exposure to GenX may result in immunomodulatory effects on both innate and adaptive immune responses by specifically targeting neutrophils, particularly since these immune cells can modulate both types of immune responses. A more comprehensive study investigating how GenX-induced suppression of neutrophil recruitment affects innate and adaptive immune responses may provide insight.

Interestingly, we also found increased numbers of mitotically active macrophages and alveolar epithelial cells in the lungs of GenX-exposed mice. This discovery was initially made through observations of increased mitotic figures in macrophages from BALF Cytospins of GenX-exposed mice and subsequently confirmed by Ki-67 immunohistochemistry, which revealed significantly increased proliferation of both macrophages and alveolar epithelial cells in the lung tissue. Increases in mitotic figures and Ki67 immunostaining were most prominent at 1 day post-exposure and declined by 14 days post-exposure. It is still unclear why macrophages and epithelial cells of GenX-exposed mice show increased proliferation. Previous studies have demonstrated that macrophages can undergo a self-renewing process via local proliferation [66–70]. Considering that our histological and cytotoxic analyses do not show any signs of overt toxicity or insult that may require such a self-renewing process, our findings suggest that exposure to GenX-induced proliferation of macrophages is aberrant and could be linked to fibroproliferative diseases such as fibrosis. Increases in proliferating lung macrophages have been observed in patients with idiopathic pulmonary fibrosis [71]. Likewise, lung epithelial cell proliferation induced by GenX could increase lung disease susceptibility, including cancer. For example, A recent study involving A549 lung cancer cells showed that exposure to PFAS, including GenX, can result in epigenetic modifications and dysregulate cell proliferation [72]. Additionally, human breast epithelial cells exposed to PFOA displayed PPAR-α-dependent proliferation coinciding with increased cyclin D1 and CDK4/6 levels [73]. Currently, PFAS is recognized by the Agency for Toxic Substances and Disease Registry (ATSDR) to potentially increase the risk of developing kidney or testicular cancer in humans [74]. Indeed, a landmark epidemiology study showed that individuals in the Parkersburg, WV cohort had increased incidence of testicular cancer that correlated to high levels of legacy PFAS [75]. Further investigation should determine the mechanisms through which GenX or other PFAS cause lung macrophage and epithelial cell proliferation and identify biomarkers associated with proliferation that might give clues to lung disease outcomes and susceptibility.

While the mechanism of GenX-induced lung cell proliferation remains to be determined, GenX likely activates one or more intracellular signaling pathways that stimulate cell proliferation. We demonstrated by western blotting that oropharyngeal aspiration of GenX, CBNP or GenX/CBNP combination resulted in significantly increased phosphorylation of STAT3 in lung tissue from mice. While GenX or CBNP individually stimulated an increase in p-STAT3 in lung tissue, the combination of GenX and CBNP resulted in the highest levels of p-STAT3. Furthermore, immunohistochemistry showed that increased p-STAT3 in mice treated with GenX or GenX and CBNP was localized in lung macrophages, alveolar epithelial cells, and airway epithelial cells. Therefore, the activation of STAT3 by GenX, with or without CBNP, could be a mechanism of GenX-induced cell proliferation in the lungs of mice. Considering that activation of STAT3 is often implicated with cellular proliferation and cancer [76–78], such observation may provide a possible mechanistic detail on previously documented dysregulation of cell proliferation following exposures to PFAS, including GenX. However, the precise role of STAT3 in GenX-induced lung cell proliferation remains to be elucidated.

The single OPA dose of GenX delivered to the lungs of mice in the present study (10 mg/kg body weight) was likely high compared to doses that would be encountered from environmental or occupational exposures. Oral exposure to GenX represents a higher exposure scenario compared to inhaled GenX. However, exposure could occur from inhaled indoor dust containing GenX released from household products or inhaled aerosolized GenX from contaminated water (e.g., shower water). Studies using oral gavage of PFAS in mice have routinely used between 1 and 100 mg/kg body weight. For example, a GenX dose of 100 mg/kg/day by oral gavage described in the investigation by Rushing et al. [24] was reported to decrease TDAR in mice. Due to the lack of an available reference dose for pulmonary exposure of mice via oropharyngeal aspiration, we initially performed a preliminary experiment with mice using 50 mg/kg GenX delivered by OPA (data not shown). There was no mortality with 50 mg/kg, yet this dose caused transient lethargy post-anesthesia after the OPA procedure prior to full recovery. Future investigations using pulmonary exposures (inhalation, oropharyngeal aspiration, or intranasal aspiration) should consider the potential effects of GenX or other PFAS over a lower dose-response range. The exposure dose of CBNP used in the present study represents a high single dose that has been routinely used for other carbon-based nanoparticles [27–29]. CBNP have been used as a surrogate for the carbonaceous core of air pollution particles and are therefore relevant to human exposures. The time points evaluated in our study were chosen to capture acute pro-inflammatory (1 day) or pro-fibrotic (14 days) effects following a single pulmonary exposure. Our findings of significant effects of GenX were primarily limited to acute (1 day) effects on CBNP-induced inflammation. Future work should focus on sub-chronic effects of GenX in the lung (weeks to months) over repeated low dose exposures and determine whether pre-exposure to GenX (either through inhalation or oral exposure) would affect the immune response to inhaled particles.

In conclusion, our results reveal that pulmonary exposure to GenX exhibits immunosuppressive effects on the host’s innate immune response to inhaled CBNP by reducing CXCL-1 and subsequent neutrophil recruitment to the lungs of mice. Since neutrophils are a key early responding immune cell that mediate the innate immune response to inhaled particles, bacteria and viruses, GenX-induced immunosuppression may increase susceptibility to lung injury due to potentially dysregulated response towards inhaled air pollution particles or engineered nanoparticles. Additionally, we found that pulmonary exposure to GenX stimulated the proliferation of alveolar macrophages and lung epithelial cells. Regarding macrophages, the hypothetical mechanism illustrated in Fig. 10 proposes that GenX, and perhaps other PFAS, causes a phenotypic shift from a pro-inflammatory phenotype to a proliferative phenotype that could have important implications for susceptibility to lung diseases such as asthma, fibrosis and cancer.