Protein corona of airborne nanoscale PM2.5 induces aberrant proliferation of human lung fibroblasts based on a 3D organotypic culture.

soma:li2018-3dhlf-001

Primary human lung fibroblasts in a 3D fibrin-hydrogel organotypic culture

OX:li2018-3dhlf-ros-dose

ROS fluorescence in 3D-cultured HLFs across an NPM-protein corona dose series

Intracellular ROS labelled with DCFH-DA in 3D-cultured HLFs exposed for three days to the NPM-protein corona at 5, 10 and 20 ug/ml NPM. ROS increased positively with NPM concentration. Fluorescence intensity is reported only in Fig. 5A with images in Fig. 5F-H, so no numeric value is recorded. Significance versus control was marked *P < 0.05, **P < 0.01 in the figure legend without per-group attribution in the text.

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Evidence

OX:li2018-3dhlf-ros-modulators

ROS fluorescence in 3D-cultured HLFs with vitamin C or TGF-beta1

ROS labelled with DCFH-DA in 3D-cultured HLFs exposed for three days to the NPM-protein corona (20 ug/ml NPM), to the same corona co-treated with vitamin C 0.1 mg/ml, or to TGF-beta1 2 ng/ml. Vitamin C repressed ROS generation and TGF-beta1 promoted it, reproducing the corona response. Values reported only in Fig. 5B with images in Fig. 5C-E; none recorded.

Exposure conditions

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Evidence

OX:li2018-3dhlf-gsh

GSH fluorescence in 3D-cultured HLFs across the NPM-protein corona dose series and with vitamin C or TGF-beta1

Intracellular glutathione labelled with ThioGlo-1 in 3D-cultured HLFs after three days. GSH depletion was detected in every NPM-protein corona group (5, 10 and 20 ug/ml NPM) and in the TGF-beta1 group, accompanying the ROS rise; vitamin C co-treatment suppressed the depletion. Values reported only in Fig. 6A-B with images in Fig. 6C-H; none recorded.

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Evidence

OX:li2018-3dhlf-mda

Intracellular MDA in 3D-cultured HLFs across the NPM-protein corona dose series and with vitamin C or TGF-beta1

Malondialdehyde, a lipid peroxidation end product, measured by ELISA after three days. Despite the ROS rise and GSH depletion, the NPM-protein corona induced no MDA increase at any dose, and neither vitamin C nor TGF-beta1 changed MDA. This negative result is the basis for the authors' conclusion that the corona produces a limited ROS increase without oxidative damage. Values reported only in Fig. 7A-B; none recorded.

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Evidence

OX:li2018-3dhlf-8ohdg

Intracellular 8-OH-dG in 3D-cultured HLFs across the NPM-protein corona dose series and with vitamin C or TGF-beta1

8-hydroxydeoxyguanosine, the most commonly measured marker of oxidative DNA damage, measured by ELISA after three days. The NPM-protein corona induced no variation in 8-OH-dG at any dose, and neither vitamin C nor TGF-beta1 changed it. Values reported only in Fig. 7C-D; none recorded.

Exposure conditions

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Evidence

GE:li2018-3dhlf-asma-dose

Alpha-SMA expression in 3D-cultured HLFs across an NPM-protein corona dose series

Alpha-smooth muscle actin immunofluorescence in 3D-cultured HLFs after three days of exposure to the NPM-protein corona at 5, 10 and 20 ug/ml NPM. The corona stimulated HLFs to express alpha-SMA and expression was positively correlated with the NPM exposure concentration. Alpha-SMA is a key protein of activated, not quiescent, fibroblasts and a fibrosis marker, so this is the proliferation pathway the authors propose. Fluorescence intensity reported only in Fig. 8A with images in Fig. 8F-H; no numeric value recorded.

Exposure conditions

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Evidence

GE:li2018-3dhlf-asma-modulators

Alpha-SMA expression in 3D-cultured HLFs with vitamin C or TGF-beta1

Alpha-SMA immunofluorescence in 3D-cultured HLFs after three days of exposure to the NPM-protein corona (20 ug/ml NPM), the same corona co-treated with vitamin C 0.1 mg/ml, or TGF-beta1 2 ng/ml. Vitamin C repressed alpha-SMA secretion, tying the induction to the ROS it also suppressed; TGF-beta1 promoted alpha-SMA, reproducing the corona effect. Values reported only in Fig. 8B with images in Fig. 8C-E; none recorded.

Exposure conditions

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Evidence

KE:mie-oxidative-stress

Increased oxidative stress

KE:ke-increased-proliferation

Increased cell proliferation

KER:li2018-oxidative-stress-to-proliferation

Increased oxidative stress leads to increased cell proliferation in 3D-cultured human lung fibroblasts

Dose-response by MTT after three days of exposure in the 3D fibrin construct: cell viabilities of 103%, 109% and 113% of control at NPM-protein corona concentrations of 5, 10 and 20 ug/ml NPM, with the 20 ug/ml group significant versus control at p < 0.01. The specificity controls were flat: naked NPM at 20 ug/ml gave 104% and pure serum 105%, so neither component alone reproduced the effect. Vitamin C co-treatment lowered viability relative to the 20 ug/ml corona group, and TGF-beta1 2 ng/ml reproduced the whole response. No numeric values are available for the intervening ROS or alpha-SMA steps, which are reported as fluorescence bar charts only, so the quantitative chain stops at the proliferation endpoint.

PROTOCOL:li2018-3dculture-001

3D organotypic fibrin-hydrogel culture of human lung fibroblasts

Human lung fibroblasts were suspended at 2 x 10^6 cells/ml, mixed with the exposure solution, fibrinogen (10 mg/ml) and thrombin (100 U/ml) in a ratio of 60:20:19:1 on ice, and seeded as 15 ul aliquots in a microfluidic chip (AIM Biotech, Singapore) for imaging or 50 ul aliquots in a 96-well plate for quantitative assays. Gelation ran 1 h at 37 C, after which the construct was filled with HLF culture medium, replaced daily, and cultured for three days. Fibrinogen was dissolved in FBS-free HLF medium; thrombin in DPBS containing 0.1% BSA.

Equipment: Microfluidic chip (AIM Biotech, Singapore); Fibrinogen (Sigma) and thrombin (Sigma); Confocal microscope (Olympus IX81)

PROTOCOL:li2018-mtt-001

MTT assay for cell proliferation of 3D-cultured HLFs

After three days of exposure the cell medium was removed by pipetting and 100 ul of gel dissociation saline solution (Biovision) was added. Mixtures were incubated at room temperature for 5-10 min and pipetted with a 1 ml tip until the fibrin matrix dissolved, then centrifuged at 1,000 x g for 5 min at 4 C and the supernatant removed. Cell proliferation was measured with an MTT kit (Nanjing Jiancheng, China) and reported as cell viability relative to the untreated control.

Equipment: Microplate reader; MTT assay kit (Nanjing Jiancheng); Gel dissociation saline solution (Biovision)

PROTOCOL:li2018-dcfhda-001

DCFH-DA fluorescence detection of intracellular ROS in 3D-cultured HLFs

Intracellular reactive oxygen species were labelled with DCFH-DA (Merck KGaA, Germany). Images of fluorochrome-labelled ROS in 3D-cultured HLFs were captured on a confocal microscope (Olympus IX81) at x10 magnification and processed with Imaris software. For quantification, 3D-cultured HLFs on cell plates were released from the fibrin matrix with gel dissociation saline solution and ROS fluorescence intensity was read on a fluorescence reader (FLx800; BioTek Instruments).

Equipment: Confocal microscope (Olympus IX81); Fluorescence reader (FLx800, BioTek Instruments); DCFH-DA probe (Merck KGaA)

PROTOCOL:li2018-thioglo-001

ThioGlo-1 fluorescence detection of intracellular GSH in 3D-cultured HLFs

Intracellular glutathione was labelled with ThioGlo(TM)-1 (Merck KGaA, Germany). Images of fluorochrome-labelled GSH in 3D-cultured HLFs were captured on a confocal microscope (Olympus IX81) at x10 magnification. GSH fluorescence intensity was quantified on a fluorescence reader (FLx800; BioTek Instruments) after the cells were released from the fibrin matrix.

Equipment: Confocal microscope (Olympus IX81); Fluorescence reader (FLx800, BioTek Instruments); ThioGlo-1 probe (Merck KGaA)

PROTOCOL:li2018-elisa-001

ELISA quantification of MDA and 8-OHdG in 3D-cultured HLFs

Concentrations of malondialdehyde (MDA, a lipid peroxidation end product) and 8-hydroxydeoxyguanosine (8-OHdG, a marker of oxidative DNA damage) were measured with ELISA kits according to the manufacturer's instructions (Nanjing Jiancheng, China), on 3D-cultured HLFs recovered from the fibrin matrix by gel dissociation and centrifugation.

Equipment: Microplate reader; MDA and 8-OHdG ELISA kits (Nanjing Jiancheng)

PROTOCOL:li2018-asma-if-001

Alpha-SMA immunofluorescence in 3D-cultured HLFs

Alpha-smooth muscle actin was detected with a fluorescent anti-alpha-SMA agent (Abcam, USA) in HLFs cultured in the microfluidic chip. Nuclei were counterstained with DAPI (Invitrogen) and live cells labelled with an Invitrogen live-cell dye. 3D fluorescence images were captured on a confocal microscope (Olympus IX81) at x10 magnification and processed with Imaris software; the fluorescence intensity of alpha-SMA expression was analysed with ImageJ software.

Equipment: Confocal microscope (Olympus IX81); Imaris software (Bitplane Scientific Software); ImageJ software