titanium dioxide nanoparticles enhance macrophage ... bianchi.pdf · titanium dioxide nanoparticles...

Titanium dioxide nanoparticles enhance macrophage activation by LPS through a TLR4-dependent intracellular pathway

Massimiliano G. Bianchia, Manfredi Allegrib, Anna L. Costac, Magda Blosic, Davide Gardinic, Camilla Del Pivoc, Adriele Prina-Mellod, Luisana Di Cristoa, Ovidio Bussolatib, Enrico Bergamaschia

aUnit of Occupational Medicine, Department of Clinical and Experimental Medicine and bUnit of General Pathology, Department of Biomedical, Biotechnological and Translational Sciences, University of Parma; cInstitute of Science and Technology for Ceramics (CNR-ISTEC), National Research Council of Italy, Faenza(RA); dCentre for Research on Adaptive Nanostructures and Nanodevices (CRANN) and School of Medicine, Trinity College Dublin, Dublin

Grant NMP4-SL-2012-280716

UNIVERSITA’ DEGLI STUDI DI PARMA

Sanowork

http://cordis.europa.eu/fp7/home_en.html


Bacterial Lipopolysaccharide (LPS or endotoxin)

BACKGROUND

Po

lysa

cch

arid

e t

ail

• A common environmental PAMP (macrophage activator)

• Component of the outer membrane of Gram– bacteria

• Elicits strong inflammatory response in competent cells

Pyrogenic moiety

Activation of Toll-like Receptor 4 pathway in Macrophages LPS

TLR4

CD

14

ADAPTOR

MKKs

Pro-inflammatory gene transcription

Gram-

Lysosomes

Endosomes

NFkB Nos2Il6, TNF

IN

OUT

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Lipid A



TiO2 nanoparticles (NPs) at a glance…..

BACKGROUND

• One of the most common manufactured metal-based NPs worldwide

50,400 tons in 2010; expected to increase to 201,500 in 2015

• Used in several industrial applications

Electronics, solar cells, paints, textiles… Food, cosmetics, toothpaste…… Antibacterial and anti-polluting coatings

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Intrinsic Physico-chemical properties

Bio-activities- Inflammation- Oxidative stress - Cancerogenicity

Direct particle-cell interaction

Delivery of bio-active molecules



TiO2 nanoparticles and the paradigm of “protein corona”

BACKGROUND

A role for environmental contaminants in TiO2 NP effects?Sanowork

Biological behavior

size

shap

e

surface chemistry

surf

ace

co

atin

gs

Adapted from Nel A.E. et al., Nat. Materials 2009

Environmental/Biological Matrix

Nanostructuredmaterial



To asses the effects of TiO2 NP and LPS on murine macrophage Raw 264.7

AIM

Biological parameters evaluated

Cell end points

Cytotoxicity Inflammatory markers

- Cell viability - NO production - Pro-inflammatory genes - Cytokine secretion

Raw 264.7Immuno-competent cells

Express TLR4 receptors

TiO2 NPsLPS TiO2 NPs + LPS

Experimental design

Cell monolayer

SanoworkBianchi et al., Toxicol. Res. 4, 385-398, 2015



Physico-chemical properties of NAMA41® and Aeroxide® P25

BACKGROUND

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Deionized waternatural pH Deionized watermedium pH Complete culture medium

TiO2 NP pHSize

(d. nm)PdI

ζ pot.(mV)

pHSize

(d. nm)PdI

ζ pot.(mV)

pHSize

(d. nm)PdI

ζ pot.(mV)

NAMA41® 3,9 45 0,48 41,2 7,3 9864 0,76 -15,9 7,3 1962 0,98 -10,9

DEV. ST 1 0,09 0,0 2390 0,30 0,4 147 0,03 0,5

Aeroxide®P25 6,5 286 0,30 37,4 7,7 3425 0,36 -11,0 7,7 532 0,53 -10,8

DEV. ST 4 0,04 0,9 226 0,10 0,1 16 0,11 0,4

TiO2 NPXRD phase distribution Density

(g/cm3)SSABET (m2/g) dBET (nm)

Anatase (%) B=Brookite R=Rutile (%)

NAMA41® 84 16, B 3.98 154 10

Aeroxide® P25 83 17, R 4.10 60 24

Mean size distribution by intensity and ζ potential for 0.125 mg ml−1 of NAMA41® and Aeroxide® P25 dispersed in deionized water and complete culture medium

Bianchi et al., Toxicol. Res. 4, 385-398, 2015



RESULTS Cytotoxicity

Not fluorescent Em λ=590 nm

ResorufinResazurin

Resazurin assay

Ex λ=536 nm

10 20 40 80 10 20 40 80 10 20 40 80

0

20

40

60

80

100

120

Control LPS 1 ng/ml LPS 10 ng/ml

*

Ce

ll vi

abili

ty(%

of

con

tro

l)

P25® do not markedly affect cell viability up to 48h

Effects of P25® and LPS on cell viability

P25® µg/cm2

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*p < 0.05 vs. Untreated cultures




+ + + + + + LPS® 1 ng/ml

P25 µg/cm2 5 10 20 40 80 5 10 20 40 80

40kDa-

140kDa- - Nos2

- Actin

48h

0.0

0.5

1.0

1.5

2.0

2.5

No

s2/A

ctin

pro

tein

(a.u

.)

0.0

0.2

0.40.4

0.8

1.2

No

s2/G

ap

dh

mR

NA

*

###

$24h

0

5

10

15

20

25

Nit

rite

s,

M

**§§

###

$$48h

1 1 10 10

+ + +P25® 80 µg/cm2

LPS ng/ml

RESULTS Inflammatory markers

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Effects of P25® and LPS on NO production

*p < 0.05 vs. Untreated cultures###p < 0.001 vs. cultures treated with LPS 1 ng/ml alone $p < 0.05 vs. LPS 10 ng /ml alone

*

**p < 0.01, ***p < 0.001 vs. untreated cultures ##p < 0.001 vs. cultures treated with LPS 1 ng/ml alone $$p < 0.01 vs. LPS 10 ng /ml alone.

P25® synergize the LPS-mediated stimulation of Nos2 gene/protein expression and of NO production




P25® synergize also the secretion of inflammatory cytokines induced by LPS

Effects of P25® and LPS on cytokine secretion

TNF-alpha

0

1000

2000

3000

4000

5000

***

***

1 1 10 10LPS ng/ml

TiO2 NPs 80 g/cm2 + + +

***

$$$

$$$

TNF-

(p

g/m

l)

IL-6

0

50

100

150

200500

1000

1500

2000

1 1 10 10LPS ng/ml

TiO2 NPs 80 g/cm2 + + +

***

***

$$

$$$

ns

IL-6

(p

g/m

l)

1 1 10 10

+ + +P25® 80 µg/cm2

1 1 10 10

+ + +P25® 80 µg/cm2

LPS ng/ml


48h 48h

LPS ng/ml

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*p < 0.05, ***p < 0.001 vs. untreated cultures; ##p < 0.01, ###p < 0.001 vs. cultures treated with LPS 1 ng/ml alone; $p < 0.05, $$$p < 0.001 vs. LPS 10 ng/ml alone




The synergistic effect of P25® on LPS-dependent macrophage activation is shared by NAMA41® (another industrial preparation of TiO2 NPs)

A comparison between the effects mediated by P25® and NAMA41®Data 1

0

1

2

3

4

5

Nit

rite

s,

M

0

1000

2000

3000

4000

5000

TNF-

(p

g/m

l)

Data 2

0.000

0.005

0.010

0.015

No

s2/G

ap

dh

mR

NA

LPS 1 ng/ml

NAMA41® µg/cm2

+ + + + + +

5 10 20 40 80 5 10 20 40 80

LPS 1 ng/ml

NAMA41® 80 µg/cm2

P25® 80 µg/cm2

+ + +

+ +

+ +

48h

48h

24h


*** *****

##

###

ns

ns

***

###

###

*****

***

### ###

Sanowork**p < 0.01, ***p < 0.001 vs. untreated cultures;##p < 0.01, ###p < 0.001 vs. cultures treated with LPS 1 ng/ml alone. Bianchi et al., Toxicol. Res. 4, 385-398, 2015



Role of TLR4 on the P25®-mediated synergistic induction of Nos2: effect of polymyxin B

RESULTS Mechanism characterization

Polymyxin 50 µg/ml + + + +

LPS 1 ng/ml + + + +

P25® 80 µg/cm2 + + + +

Nos2 -

Actin -

48hPolymyxin B

Pro-inflammatory gene transcription

LPS

CD

14

ADAPTOR

P25® enhance macrophage activation by LPS via a TLR4-dependent mechanism

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TLR4

IN

OUT

Suppression of Nos2 induction by Polymyxin




Effect of cytoskeletal disorganization on NO production and P25® internalization


w/o

inh

ibit

or

Cyt

och

alas

in5

µg

/ml

P25® 10 µg/cm2 + LPS 1 ng/ml

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Cytochalasin blokes the endocytosis of P25®




Effect of cytoskeletal disorganization on NO production and P25® internalization


1

+ P25® µg 80/cm2

LPS ng/ml 1 10 10

+ +

Endocytosis blockade inhibits the synergistic effect of P25® on LPS-

dependent NO production

Involvement of an intracellular site

48h

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*p < 0.05, **p < 0.01,***p < 0.001 vs. untreated cultures; ###p < 0.001 vs. cultures treated under the same conditions without the inhibitor

0

5

10

15

20

W/O Ihibitor

Cytochalasin 5g/ml

1 1 10 10LPS ng/ml

TiO2 NPs 80 g/cm2 + + +

Nit

rite

s,

MN

itri

tes,

µM

* ***

***

***

***

ns ns ns ###**

######***

w/o Inhibitor




Role of MKKs in the LPS and P25® effects


p-p38-

p38-

Tub-

p-ERK 1/2-

ERK 1/2-

Tub-

6h6h

0

5

10

15

20p

-p38/(

p38/T

ub

)

Fo

ld c

ham

ge

0

5

10

15

20

p-p

38/(

p38/T

ub

)

Fo

ld c

han

ge

LPS 1 ng/ml + +P25® 80 µg/cm2 + +

LPS 1 ng/ml + +P25® 80 µg/cm2 + +

LPS 1 ng/ml

NO in

hibito

r M

U01

26 1

M

SB20

3580

2

0

50

100

150

200

***

ns

Nit

rite

s

(% o

f u

nin

hib

ite

d v

alu

es)

LPS 1 ng/ml + P25 80 g/cm2

NO in

hibito

r M

U01

26 1

M

SB20

3580

2

0

50

100

150

200

**

***Nit

rite

s(%

of

un

inh

ibit

ed

valu

es)

LPS-P25® synergy involves p38 but not ERK 1/2 MAPK

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**p < 0.01 and ***p < 0.001 vs. cultures incubated with the same doses of LPS and TiO2 NPs in the absence of inhibitors

LPS 1 ng/ml

NO in

hibito

r M

U01

26 1

M

SB20

3580

2

0

50

100

150

200

***

ns

Nit

rite

s

(% o

f u

nin

hib

ite

d v

alu

es)




• TiO2 NP synergize LPS inflammogenic activity

SUMMING UP

• The effect requires TLR4 signalling, phagocytosis and the phosphorylation of p38 MAPK

- Enhanced NO production, pro-inflammatory gene expression, cytokine secretion

- TLR4 inhibitors, blockade of TiO2 NP internalization and Inhibition of p38 phosphorylation prevent macrophage activation

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Do TiO2 NP bind LPS ?

Hypothesis




PRELIMINARY RESULTS P25®-LPS binding

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Assessment of LPS corona on P25® by SDS-PAGE and Silver Staining

P25 (100 µg/ml)LPS (1ng/ml)

1h (37°C) Centrifugation-10000 rpm-4°C-7 min

P25 + LPS corona

Repeatedwashes and

centrifigations

Free LPS

LPS hard corona

TiO2 NP bind LPS and are likely responsible for LPS intracellular

delivery



TiO2 NP as “TROJAN HORSES”

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• TiO2 NP are able to deliver high amounts of LPS in to the cell

- LPS corona formation on TiO2 has been demonstrated

SUMMING UP




A working model….

Free LPS“Out-door” activation of TLR4 receptors on plasma membrane

Free LPS + TiO2@LPS“Out-door”+ “In-door” activation of TLR4 receptors in endosomal compartments

Nanomaterials change the biopersistence

and/or bioavailability of PAMPs

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Biological effects depend (also) from

the bioactive molecules present in

the tissue(contaminants,

PAMP, etc.)

Exploitable for modulating

inflammatory responses ?

CONCLUSIONS




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Thank you all…..!!



titanium dioxide nanoparticles enhance macrophage ... bianchi.pdf · titanium dioxide nanoparticles...

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