NK cells from homozygous NLG4-/- (KO) mice inhibit liver ... · killings and consequently decreased...

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J. Amer, A. Salhab, R. Safadi 17/06/2016 Eilat The Liver Unit, Hadassah Medical Center, Jerusalem NK cells from homozygous NLG4-/- (KO) mice inhibit liver fibrosis through PI3K/AKT/mTOR pathway and decreased interleukin-4

Transcript of NK cells from homozygous NLG4-/- (KO) mice inhibit liver ... · killings and consequently decreased...

J. Amer, A. Salhab, R. Safadi

17/06/2016Eilat

The Liver Unit, Hadassah Medical Center, Jerusalem

NK cells from homozygous NLG4-/- (KO) mice inhibit liver fibrosis through PI3K/AKT/mTOR pathway and decreased

interleukin-4

Abstract MP-196: Recombinant β-neuroxin as a therapeutic target to inhibit fibrosis throughalleviation in NK cells cytotoxicity following inhibition of neuroligin-4 (nlg4) receptorPresenter:Johnny Amer (Israel)Abstract MP-197: Tgf-beta2 inhibition in endothelial cells ameliorates liver fibrogenesis and inflammationPresenter:Anne Dropmann (Germany)

Abstract MP-199: Association of controlled attenuation parameter and glycosylated hemoglobin in patients with fatty liverPresenter:Anita Arslanow (Germany)

Abstract MP-201: Early changes in non invasive assessment of liver fibrosis in hepatitis c virus-infected patients treated with daas:preliminary reportsPresenter:Claudia Iegri (Italy)

Abstract MP-213: Survival of apoptosis-primed activated hepatic fibroblasts is bcl-xl dependentPresenter:Anja Moncsek (Switzerland)

Abstract MP-202: The change of liver and spleen stiffness measured by shear-wave elastography after transjugular intrahepaticportosystemic shunt predicts outcome.Presenter:Jonel Trebicka (Germany)

Disclosures

None

Proposal: NLG4 is mediating HSC/NK synapse

Autism

Schizophrenia

NLGN4

NK

75% of LX2 (HSCs)

HSC

aSMA NLG4 bNeuroxinNLG4 & bNeuroxin

aSMANLG4 bNeuroxin

Confocal NK/HSC interplay: NLG4:bNeuroxin are involving immune Synapse

Gene expressions of impaired NK cells in cirrhosisNeuroligin 4: is a postsynaptic membrane protein that

mediates synapse formation between neurons

TRENDS in Neurosciences 2006

To assess the role of PI3K/AKT/mTOR pathway &

NLG4 in NK cell killing

AIM

WT NLG4-/-

CCl4 i.p injections 2X/wk for 6 wks

Livers Serum

ALT levels

Lymphocytes immune alterations aSMA activations and expressions PI3K/AKT/mTOR expressions

DESIGN

Methods

Serum ALT levels .

RT-PCR is used to quantify and to analyze the gene

expression of liver aSMA and PI3K/AKT/mTOR.

Fluorescence –activated cell sorting (FACS) is used to

evaluate the changes on the NK cells. Count, activations

(CD107a), CD8, CD4, F4/08 (monocytes) and primary

HSCs activations, apoptosis (Annexin V) and

proliferations (CFSE)

RESULTS

0

0.4

0.8

1.2

1.6

wt naïve wt fib(1w) wt fib(5w) Nlg4-/- naïve Nlg4-/- fib(1w) Nlg4-/- fib(5w)

aSM

A/b

Act

inra

tio

0

200

400

600

800

WT NAÏVE WT FIB(1W) WT FIB(5W) NLG4-/- NAÏVE NLG4-/- FIB(1W) NLG4-/- FIB(5W)

Seru

m A

LT

(IU

/ml)

WT Naïve CCl4 (6w)

NLG4-/-

Naïve CCl4 (6w)

P<0.05

P<0.05

In vivo CCl4 model: WT vs. NLG4-/-

0

5

10

15

20

Naïve WT WT 6wks Naïve -/- NLG-/-6wks

%

NK+ CD3-

0

10

20

30

40

CD8 CD4 F480

%

Naïve WT WT 6wks

Naïve -/- NLG-/- 6wks

P=0.05

FACS analysis of liver lymphocytes

0

2000

4000

6000

8000

10000

WT NLG4-/-

aSMA MFI

0

5

10

15

20

25

30

35

40

WT NLG4-/-

% aSMA/ANN-V+

0

2

4

6

8

10

12

WT NLG-/-

CFSE (FOLD decrease)

Isolated pHSCs from livers of NLG4-/- mice showed:

0

5

10

15

20

WT NLG4-/-

mTOR

mR

NA

exp

ress

ion

0

20

40

60

80

WT NLG4-/-

PI3K

mR

NA

exp

ress

ion

0

2

4

6

WT NLG4-/-

Akt 1

mR

NA

exp

ress

ion

0

20

40

WT NLG4--/-

% IL-4N

K1

.1 e

xpre

ssio

ns

(

(FA

CS

Liver NK cells PI3K/AKT/mTOR pathway

0

20

40

60

B1 C1 D1 E1

HSC apoptosis:aSMA+/Ann V+ (%)

0

2

4

6

8

B1 C1 D1 E1

NK activation:NK+/aKIR+ (%)

0

20

40

B1 C1 D1 E1

NK adhesion on HSC (%)

WT HSC

WT vs. NLG4-/- NK

NK cell WT WT-/- -/-CCl4 - +- +

Homozygous NLG4-/- mice exert anti-fibrotic profile throughincrease in NK cells and decreased CD8 cells.

Alterations in NK cells phenotypes were observedthrough increased activity associated with increased pHSCskillings and consequently decreased in aSMA quantitations.

NK activations and potentials to kill are suggested to beassociated with elevated expressions of PI3K/AKT/mTORaccompanied in decreased in the pro-fibrogenic marker; IL4.

NLG4 modulations could be a potential target for fibrosistreatments.

Conclusions

Acknowledgments

[email protected]

Holy Family Hospital, Nazareth

Nils Brose; Abteilung Moleklare Neurobiolgie, Germany

Thank you