Imperial College Academic Trainees Annual Event

The critical roles of TNF receptors in experimental acute lung injury

Dr Brijesh PatelClinical Research Fellow

Wellcome Trust Clinical PhD Programme

Anaesthetics, Pain Medicine, and Intensive Care

ALI/ARDS

ALI/ARDS is characterised by:• acute onset• hypoxaemia• bilateral chest radiographic changes• normal left atrial pressures

Has a mortality of over 35% despite lung protective ventilation strategies.

Mulitiple aetiologies can lead to ALI/ARDS.• Direct – pneumonia; aspiration pneumonitis• Indirect – non-pulmonary sepsis; trauma; transfusion related; pancreatitis

Alveolar epithelial damage is a prerequisite to pulmonary oedema formation in all aetiologies of ALI/ARDS.

p55

TNFTNF

FADD

TRADD

Caspase 8/10

Apoptosis

Caspase 3

TRAF2

RIPRIP

NEMO/IKKNEMO/IKK MKK3MKK3

p38p38

MAPKMAPKNFκBNFκB

cFLIPcFLIP

MEKK1/7MEKK1/7

JNKJNK

AP1AP1

Cell Survival

TNF receptor signalling

p75

Aims & Hypotheses

1. Do the two TNF receptors have distinct roles in experimental ALI/ARDS induced by acid aspiration?

2. What are the cellular mechanisms through which TNF receptor mediate their effects during experimental ALI/ARDS?

Experimental model of acid aspiration

Carotid LineTracheostomy

Intraperitoneal line for anaesthesia

Airway Pressure Transducer

Flow Transducer

Pneumotache

Flow Control

Inspiratory valveExpiratory valve

Setup for murine ventilation

Experimental Protocol

Baseline Respiratory Mechanics & Arterial Blood Gas

Acid Instillation

0 30 60 90 120 150 180

ABG

Mins

Respiratory Mechanics Measurements

GA + Setup for murine ventilation

Vt – 8mls/kgPEEP – 3cmH2O

p55KO shows improved respiratory physiology and p75KO show worse respiratory mechanics

Significant interaction P-value (P<0.0001) by 2-way ANOVA; *** P<0.001 vs WT Acid by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

0

50

100

150

200

250

30 60 90 120 150 180

Ela

sta

nc

e C

ha

ng

e (

%)

Time post instillation (minutes)

Baseline

WT Saline

WT Acid

p55KO Acid

p75KO Acid

DKO Acid

0

100

200

300

400

500

Pa

O2:

FiO

2 R

ati

o

***

Acid +/- + + + +-

Mouse strain

WT

p55

KO

p75

KO

DK

O

WT

0

2

4

6

8

We

t:D

ry

Ra

tio

**

Acid +/- + + + +-

Mouse strain

WT

p55

KO

p75

KO

DK

O

WT

p55KO shows improved alveolar capillary barrier permeability

BA

LF

Pro

tein

(m

g/m

l)

0

1

2

3

*

Acid +/- + + + +-

Mouse strain

WT

p55

KO

p75

KO

DK

O

WT

• P<0.05; ** P<0.01 vs WT Acid by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

Alveolar fluid clearance – an epithelial functional marker

Tight Junctions ENaC Na/K ATPase Aquaporins

Alveolus

Type 2 Alveolar Epithelial Cell

Type 1 Alveolar Epithelial Cell

Pulmonary capillary

Interstitium

Na+ H2O

Ex vivo measurement of alveolar fluid clearance

02468

101214161820

No

Dru

g C

on

tro

l

Am

ilo

rid

e

Iso

pro

tere

no

l

AF

C (

%/3

0m

in)

**

**

ΔVinstillate = AFC = ΔCAlb = ΔF = (F30-F0)/F30

** P<0.01 vs No Drug Control by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

* P<0.05 vs WT Acid by 1-way ANOVA with Bonferroni test; Mean±SD; N=6-7

The p55 receptor mediates alveolar epithelial dysfunction in ALI

0

2

4

6

8

10

12

Alv

eola

r F

luid

Cle

aran

ce

(%/3

0min

ute

s)

*

Acid +/-

Mouse strain

+

WT

+

p55

KO

-

WT

p75

KO

+

0

25

50

75

100

125

BA

LF

RA

GE

(n

g/m

l)

Acid +/-

Mouse strain

+

WT

+

p55

KO

-

WT

p75

KO

+

*

Summary – Roles of TNF receptors in early ALI/ARDS

1. TNF receptors show opposing roles in pulmonary oedema formation in a model of direct ALI/ARDS induced by acid aspiration.

2. p75 TNF receptor deletion leads to worsening respiratory mechanics.

3. p55 TNF receptor deletion leads to reduced levels of alveolar epithelial injury as assessed by a functional marker i.e. AFC and a soluble marker i.e. RAGE.

4. This study provides a rationale for specific p55 TNF receptor blockade on the alveolar epithelium in ALI/ARDS states.

Acknowledgements

Professor Masao Takata – Magill Chair in Anaesthesia

Dr Michael Wilson – Lecturer in Physiology

and the rest of the critical care group

0

500

1000

1500

2000

2500

3000

3500

4000

BA

LF

MIP

-2 (

pg

/ml)

Acid +/- + + + +-

Mouse strain WT

p55

KO

p75

KO

DK

O

WT

0500

10001500200025003000350040004500

BA

LF

KC

(p

g/m

l)

Acid +/- + + + +-

Mouse strain WT

p55

KO

p75

KO

DK

O

WT

p75 receptor attenuates alveolar inflammation

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

BA

LF

IL

-6 (

pg

/ml)

Acid +/- + + + +-

Mouse strain WT

p55

KO

p75

KO

DK

O

WT

**

Future work

1. Early phase of ALI/ARDS:

• Investigate the influence of TNF p55 receptor death domain signalling in type 1 and type 2 AECs.

2. Extend the investigation into the longer term roles of TNF receptors in experimental ALI/ARDS:

• A resolving model of experimental acid aspiration.• The role of the p75 TNF receptor in promoting resolution and repair.