Carbon Nanotube Immunotoxicity in Alveolar Epithelial Type II Cells Is Mediated by Physical Contact-Independent Cell-Cell Interaction with Macrophages as Demonstrated in an Optimized Air-Liquid Interface (ALI) Coculture Model

Abstract

There is a need for the assessment of respiratory hazard potential and mode of action of carbon nanotubes (CNTs) before their approval for technological or medical applications. In CNT-exposed lungs, both alveolar macrophages (MФs), which phagocytose CNTs, and alveolar epithelial type II cells (AECII cells), which show tissue injury, are impacted but cell-cell interactions between them and the impacted mechanisms are unclear. To investigate this, we first optimized an air-liquid interface (ALI) transwell coculture of human AECII cell line A549 (upper chamber) and human monocyte cell line THP-1 derived macrophages (lower chamber) in a 12-well culture by exposing macrophages to CNTs at varying doses (5-60 ng/well) for 12-48 h and measuring the epithelial response markers for cell differentiation/maturation (proSP-C), proliferation (Ki-67), and inflammation (IL-1β). In optimal ALI epithelial-macrophage coculture (3:1 ratio), expression of Ki-67 in AECII cells showed dose dependence, peaking at 15 ng/well CNT dose; the Ki-67 and IL-1β responses were detectable within 12 h, peaking at 24-36 h in a time-course. Using the optimized ALI transwell coculture set up with and without macrophages, we demonstrated that direct interaction between CNTs and MФs, but not a physical cell-cell contact between MФ and AECII cells, was essential for inducing immunotoxicity (proliferative and inflammatory responses) in the AECII cells.

Keywords: MWCNT; air–liquid interface; alveolar epithelial cells; alveolar macrophages; carbon nanotube; coculture; immunotoxicity; inhalation toxicity.

Figure 1

Diagrammatic representation of the air–liquid Interface (ALI) coculture model. The ALI culture was setup by placing transwell (0.4 µm pore size) inserts in individual wells of a 12-well culture cluster. Human THP-1 differentiated macrophages (15 × 10³ cells) were seeded in a given culture cluster well (lower compartment) and treated with CNT suspension at a defined test dose in a defined volume (315 µL) of complete RPMI medium. The transwell chamber (upper compartment) seeded with A549 alveolar epithelial type II cells (45 × 10³ cells) in 50 µL of complete RPMI medium was then inserted. The defined total volume of the RPMI medium (315 + 50 = 365 µL) allowed creation of an air–liquid interface for the epithelial cells in the transwell in continuity with the macrophages in the lower chamber.

Figure 2

(A–H) Dose–response analysis for optimization of CNT dose in the ALI coculture model set up as described in Figure 1. The THP-1 derived macrophages seeded in individual wells in the culture cluster were treated (in triplicate) with an increasing dose of CNTs (5–60 ng/well). The cellular response in A549 cells (AECII cells) was tracked for expression of the marker for cell proliferation (Ki-67) using immunofluorescence microscopy as shown in the following figure panels corresponding to varying CNT doses: untreated control (A), 5 ng (B), 10 ng (C), 15 ng (D), 20 ng (E), 40 ng (F), 60 ng (G). Dose–response was assessed by plotting Ki-67 expression levels at different doses of CNT exposure (H). Each response marker was measured in triplicate A549 cultures, with 5 spots imaged per culture. Red and green fluorescence represented expression of proSP-C (cytoplasmic) and Ki-67 (nuclear), respectively, whereas blue fluorescence (DAPI stain) represented the nucleus. ProSP-C expression was tracked qualitatively as an index of desired cell differentiation/maturation of the AECII cells. Statistical significance was based on p ≤ 0.05. p-values indicate extent of statistical significance as compared to the control.

Figure 3

(A–H) Temporal analysis for optimization of the time required to elicit cell proliferation response to CNT treatment in alveolar epithelial cells in the ALI coculture model set up as described in Figure 1. The THP-1-derived macrophages seeded in individual wells in the culture cluster were treated with a defined dose of CNTs (15 ng/well) and including both untreated control and Pluronic F-127 (vehicle)-treated control. The response in terms of expression of the marker for cell proliferation (Ki-67) was tracked in the A549 cells (AECII cells) in the transwell; the temporal response was quantified at the following time points: 12 h (A,E), 24 h (B,F), 36 h (C,G), and 48 h (D,H). The response markers were measured in triplicate A549 cultures, with 5 spots imaged per culture. Ki-67 expression detectable within 12 h reached its maximum at 24 h. Red and green fluorescence represented expression of proSP-C (cytoplasmic) and Ki-67 (nuclear), respectively, and the blue fluorescence represented the nucleus. ProSP-C expression was tracked qualitatively as an index of desired cell differentiation/maturation of the AECII cells. Statistical significance was based on p ≤ 0.05.

Figure 4

(A–H) Temporal analysis for optimization of the time required to elicit inflammatory response to CNT treatment in alveolar epithelial cells in the ALI coculture model set up as described in Figure 1. The THP-1 derived macrophages seeded in individual wells in the culture cluster were treated with a defined dose of CNTs (15 ng/well) and including both untreated control and Pluronic F-127 (vehicle)-treated control. The response in terms of expression of the marker for inflammatory response (IL-1β) was tracked in the A549 cells (AECII cells) in the transwell; the temporal response was measured at the following time points: 12 h (A,E), 24 h (B,F), 36 h (C,G), and 48 h (D,H). IL-1β expression in A549 cells detectable within 12 h reached its maximum at 24 h. The response markers were measured in triplicate A549 cultures, with 5 spots imaged per culture. Red, green, and blue fluorescence represented expression of proSP-C, IL-1β, and nucleus, respectively. ProSP-C expression was tracked qualitatively as an index of desired cell differentiation/maturation of the AECII cells. Statistical significance was based on p ≤ 0.05.

Figure 5

(A–H) Role of macrophages in eliciting cell proliferation response to CNT treatment in alveolar epithelial cells in the ALI coculture model set up as described in Figure 1 with modification in terms of macrophages. The ALI system was set up with or without macrophages in the lower chamber and with A549 cells (AECII cells) in the upper chamber (transwell) and run for 36 h using a defined dose of CNTs (15 ng/well) and including both untreated control and Pluronic F-127 (vehicle)-treated control. The response was tracked in terms of expression of the marker for cell proliferation (Ki-67) in the A549 cells (AECII cells) in the transwell. The response markers were measured in triplicate A549 cultures, with 5 spots imaged per culture. Red, green, and blue fluorescence represented expression of proSP-C, Ki-67, and nucleus, respectively. ProSP-C expression was tracked qualitatively as an index of desired cell differentiation/maturation of the AECII cells. The role of macrophages was assessed based on the Ki-67 expression levels in presence or absence of macrophages in the lower chamber. Ki-67 expression in epithelial cells was detected only when macrophages were included (lower figure (B,D,F,H)) and was not detected in absence of macrophages (upper figure (A,C,E,G)) in the ALI set up. This suggested an effector role of macrophages in eliciting epithelial response to CNTs in a physical contact-independent manner via diffusible soluble mediator(s). Statistical significance was based on p ≤ 0.05.

Figure 6

(A–H) Role of macrophages in eliciting inflammatory response to CNT treatment in alveolar epithelial cells in the ALI coculture model set up as described in Figure 1 with modification in terms of macrophages. The ALI system was set up with and without macrophages in the lower chamber and with A549 cells (AECII cells) in the upper chamber (transwell) for 36 h using a defined dose of CNTs (15 ng/well) and including both untreated control and Pluronic F-127 (vehicle)-treated control. The response was tracked in terms of expression of the marker for inflammatory response (IL-1β) in the A549 cells (AECII cells) in the transwell. The response markers were measured in triplicate A549 cultures, with 5 spots imaged per culture. Red, green, and blue fluorescence represented expression of proSP-C, IL-1β, and nucleus, respectively. ProSP-C expression was tracked qualitatively as an index of desired cell differentiation/maturation of the AECII cells. The role of macrophages was assessed based on the IL-1β expression levels in the presence or absence of macrophages in the lower chamber. IL-1β expression in epithelial cells was detected only when macrophages were included (lower figure (B,D,F,H)) and was not detected in absence of macrophages (upper figure (A,C,E,G)) in the ALI set up. This suggested an effector role of macrophages in eliciting epithelial inflammation response to CNTs in a physical contact-independent manner via diffusible soluble mediator(s). Statistical significance was based on p ≤ 0.05.