Amlan Chakraborty1,2, Jennifer C. Boer2, Simon G. Royce3, Cordelia Selomulya1, Magdalena Plebanski4*

1Department of Chemical Engineering, 2Department of Immunology and Pathology, 3Department of Pharmacology. Monash University, Clayton, Victoria 3800, Australia; 4School of Health and Biomedical Sciences and Enabling Capability platforms, Biomedical and Health Innovation, RMIT University, Melbourne, Victoria 3083, Australia. *Corresponding author: magdalena.plebanski@rmit.edu.au

Effect of a Small Natural Dietary Compound on Lung Pathology in Airway Inflammation

AbstractBackground: Bronchodilators and corticosteroids reduce respiratory symptoms and

exacerbations in diseases associated with airway inflammation but are ineffective in a

subset of patients. There is a need to develop new and safe anti-inflammatories.

Aim: To investigate whether a small natural dietary compound (SNDC) can attenuate

inflammation in a model of acute lung injury, when delivered directly into the lung.

Methods: SNDC was delivered in a model of acute lung injury (ALI) involving an i.n.

sensitization with LPS (n=4 mice/group). Each group was subjected to a pulmonary

function test (PFT) and immunohistochemistry for pro-inflammatory cytokines. It was

hypothesised that the anti-inflammatory role of SNDC was mediated by dendritic cells

(DC). To explore this, GMCSF induced Bone Marrow derived Dendritic Cells

(BMDCs) were treated with LPS &/or SNDC and the cells analysed by flow cytometry.

Results: LPS treatment of mice resulted in increased lung resistance, and this was

significantly decreased (p<0.0001) by co-administration with SNDC. In BMDCs,

inflammatory activation markers were upregulated by LPS, and co-administering

SNDC prevented this activation.

Discussion: One of the causes of airway inflammation is exposure to

lipopolysaccharide (LPS-principal component of Gram negative bacteria) which

activates the DCs into an inflammatory state. Our result show that airway constriction

and distensibility of the lung is normalised with SNDC treatment and suggest this

effect may be mediated by DC.

Conclusion: SNDCs warrant exploration as potential future anti-inflammatory

compounds to treat steroid resistant inflammatory lung diseases.

AIMS

• To investigate whether a small natural dietary compound

(SNDC) treatment is able to abrogate airway inflammation

induced by lipopolysachharide (LPS).

• To study the effect of SNDC in down regulating inflammation in

a model of Antigen Presenting Cells in-vitro.

METHODS

• For Aim 1, 50 ml of 100mM SNDCs were delivered in a 24 hour

model of Acute Lung Injury induced by i.n. administration of 5 mg

LPS. The different groups are represented in Figure 1. Post

sensitization with LPS and/or treatment, the mice were subject to a

Pulmonary Function Test. The BAL fluid was collected and

differential cell count was done on the cells. The mice were culled

followed by isolating the lung. Two lobes from both the right and

left lung were paraffin embedded. Whole lung sections (4 mm) was

stained with H&E and PAS/Alcian blue followed by analysis using

Olympus BX50 microscope.

AMREPflow

To determine production of pro-inflammatory cytokines such as IL-

1b, IL-6 and TNF-a in lung tissue, paraffin-embedded tissue was

processed and immuno-stained followed by detection using DAKO

Rapid EnVision immunohistochemistry (IHC) procedure. The

sections were counterstained with Mayer’s hematoxylin. The slides

were then scanned using Leica Biosystems at 40X magnification and

analysed using the Positive pixel count algorithm in Aperio

Imagescope. The value of the total number of strong positive signal

across the smaller airways were measured and analysed using a one-

way ANOVA.

• For Aim 2, Day 6 Bone Marrow Derived Dendritic Cells were

cultured using 10ng/ml GMCSF (Figure 2). The cells were then

stimulated with or without LPS and SNDCs or together. After 24

hours of incubation, the cells were harvested, stained and

analysed using Flow cytometry using BD Fortessa X20. Cells

were gated as DCs (CD11c+MHCII+ and GR1-) and the activation

markers studied were CD40, CD80 and CD86.

LPS (i.n) 5 mg

In 50 ml

LPS 5 mg + SNDC

100 mM (i.n)

In 50 ml

Day 0

Saline (i.n)

In 50 ml

SNDC 100 mM (i.n)

In 50 ml

Day 2Day 1

• Lung function test with

Methacholine.

• BALF differential cell

count.

• Immunohistochemistry

(IL1b, IL6, TNFa, TGFb).

• H&E and Pas stain

Day 3

• Lung function test with

Methacholine.

• BALF differential cell

count.

• Immunohistochemistry

(IL1b, IL6, TNFa,

TGFb, CD68, TSLP).

• H&E and Pas stain

N=4/group. All conditions delivered with vehicle (saline)

LPS group LPS + SNDC group Saline group (vehicle control) SNDC group

CD11c

MH

CII

GR-1

SS

C-A

Day 1- Cull mice

Culture Bone marrow cells

with GM-CSF

Day 3- Replenish

MediaDay 6- Addition of

conditions Day 7- Cell harvesting

and flow cytometry

Figure 2:Culturing Bone marrow derived dendritic cells

n=4/group.

Mean±SEM.****p<0.

0005, ***p<0.005

Control

SNDC

LPS

LPS +

SNDC

PAS/Alcian Blue:

Goblet cellsH&E: Alveolar

area

H&E: Interstitial

breakH&E: Airway

lumenIL-1b IL-6 TNF-a

IL1b

(T

ota

l in

te

ns

ity

of s

tro

ng

po

sit

ve

)

Co

ntr

ol

LP

S

SN

DC

LP

S+S

ND

C

0

5 0 0 0 0 0

1 0 0 0 0 0 0

1 5 0 0 0 0 0

n = 4 m ic e /g ro u p .

M e a n S E M .

1 0 s e c t io n s / lu n g o f m ic e

* * * p < 0 .0 0 1 , * * * * p < 0 .0 0 0 1

*** ********

IL6

(T

ota

l in

te

ns

ity

of

str

on

g p

os

itv

e)

Co

ntr

ol

LP

S

SN

DC

LP

S+S

ND

C

0

1 .01 0 7

2 .01 0 7

3 .01 0 7

4 .01 0 7

n = 4 m ic e /g ro u p .

M e a n S E M .

1 0 s e c t io n s / lu n g o f m ic e

* * p < 0 .0 1 , * * * * p < 0 .0 0 0 1

******

**

**

TN

Fa

(T

ota

l in

te

ns

ity

of s

tro

ng

po

sit

ve

)

Co

ntr

ol

LP

S

SN

DC

LP

S+S

ND

C

0

5 0 0 0 0 0

1 0 0 0 0 0 0

1 5 0 0 0 0 0

2 0 0 0 0 0 0 **

**n = 4 m ic e /g ro u p .

M e a n S E M .

1 0 s e c t io n s / lu n g o f m ic e

* * p < 0 .0 1

Nu

mb

er

of

Go

ble

t c

ell

s/1

00m

m

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0 .0

0 .5

1 .0

1 .5**** ****

****

Alv

eo

lar a

re

a (m

m2

)

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

** **

*

Air

wa

y a

re

a (m

m2

)

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

** **

**

A lv e o la r s e p ta tio n c o u n t

Alv

eo

lar s

ep

tati

on

(pe

r m

m)

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

5

1 0

1 5

2 0

2 5**** ***

***

A lv e o la r c o u n t

Alv

eo

lar c

ou

nt(

pe

r m

m2

)

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

2 0

4 0

6 0

8 0*** ***

**

n=4 mice/group.

Mean±SEM.

10 sections/lung of mice.

****p<0.0001,

**p<0.01, *p<0.5

B M D C

CD

40

MF

I

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

* ** *

B M D C

CD

80

MF

I

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

* ** *

B M D C

CD

86

MF

I

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0 ** *

Mean±SD, n=3/group, One way ANOVA with post-hoc

un-paired t-test to test within group. *p<0.5, **P<0.05,

***p<0.005, ****p<0.0001.

RESULTS

Control

LPS LPS +SNDC

SNDCControl SNDC

LPS LPS+SNDC

C o lla g e n F ib r e D e n s ity

Co

lla

ge

n I

MF

I

Salin

e (

veh

icle

)

LP

S+veh

icle

SN

DC

+veh

icle

LP

S+S

ND

C+veh

icle

1 5

2 0

2 5

3 0

3 5 *

** *

Collagen

Lung Tissue

ROI’s selected around

small airways and

interstitial space excluding

blood vessels and larger

airways.

Mean±SD, n=4/group, One

way ANOVA to test in

between strains. *p<0.5,

**P<0.05.

INTRODUCTION

Acute Lung Injury (ALI) is characterised by airway inflammation and is

very common with over four million deaths per year1. Airway

inflammation is mainly contributed by lipopolysaccharide (LPS) which is

the cell wall of gram (-)ve bacteria2,3. LPS which is a TLR4 agonist, is

known to increase airway hyper-responsiveness (AHR)4. Current

treatments include the use of bronchodilators and anti-inflammatory

drugs such as corticosteroids (e.g. prednisone, beclomethasone,

fluticasone) and cromolyn in paediatric therapy. In adults, beta-2-agonists

(SABA) and leukotriene receptor agonists (LTRA) is used. However,

prolonged use of these drugs has shown to cause adverse effects5 and has

led to the development of resistance6.

Here we demonstrate the use of a small natural dietary compound in

attenuating airway inflammation in a model of Acute Lung Injury (ALI)

using LPS to induce inflammation.

C D 1 1 b+M a c r o p h a g e s

CD

40

MF

I

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

5 0 0

1 0 0 0

1 5 0 0 ******

****

C D 1 1 b+M a c r o p h a g e s

CD

80

MF

I

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

2 0 0 0

4 0 0 0

6 0 0 0 ******

****

C D 1 1 b+M a c r o p h a g e s

CD

86

MF

I

Un

treate

dL

PS

SN

DC

LP

S+S

ND

C

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0 ********

1. Ferkol T, Schraufnagel D. The Global Burden of Respiratory Disease. Annals of the American Thoracic Society, 11(3), 404-406 (2014).

2. Allen IC. Bacteria-Mediated Acute Lung Inflammation. In: Mouse Models of Innate Immunity: Methods and Protocols. Allen, IC Ed.

(Humana Press, Totowa, NJ, 2013) 163-175.

3. Knapp S. LPS and bacterial lung inflammation models. Drug Discovery Today: Disease Models, 6(4), 113-118 (2009).

4. Starkhammar M, Larsson O, Kumlien Georén S et al. Toll-Like Receptor Ligands LPS and Poly (I:C) Exacerbate

Airway Hyperresponsiveness in a Model of Airway Allergy in Mice, Independently of Inflammation. PLOS ONE,

9(8), e104114 (2014).

5. Dahl R. Systemic side effects of inhaled corticosteroids in patients with asthma. Respiratory

Medicine, 100(8), 1307-1317).

6. Barnes PJ. Corticosteroid resistance in patients with asthma and chronic obstructive

pulmonary disease. Journal of Allergy and Clinical Immunology, 131(3), 636-645).

REFERENCE

In comparison to LPS induced airway inflammation,

• The number of goblet cells, alveolar area, airway area and airway resistance were brought down to normal with SNDCs.

• Reduced alveolar septation and number of alveoli/mm2 was also brought to normal.

• Levels of pro-inflammatory cytokines IL-1b, IL-6, TNF-a and BAL inflammatory cells such as neutrophils and alveolar macrophages were reduced with SNDCs.

• More collagen I deposition in LPS group than control which is persistent with SNDCs.

M e th a c h o lin e (m g /m l)

Rs (

cm

H2

O.s

/ml)

P B S 3 .1 2 5 6 .2 5 1 2 .5 2 5 5 0

0

5

1 0

1 5

2 0

s a lin e (v e h ic le )

S N D C + v e h ic le

L P S + v e h ic le

L P S + S N D C + v e h ic le

****

***

R e s is ta n c e

SNDCs reduced expression of activation markers CD40, CD80 and CD86 on Bone Marrow Derived Dendritic Cells (characterised as CD11c+MHCII+GR1-) and

Bone Marrow Derived Macrophages (characterised as CD11c-MHCII+GR1-CD11b+).

Figure 1: Study design to evaluate the anti-inflammatory effect of SNDCs

BA

L c

ell

s x

10

6

Co

ntr

ol

LP

S o

nly

SN

DC

LP

S+S

ND

C

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

N e u tro p h il

L y m p h o c y te s

M a c ro p h a g e s

E o s in o p h il********

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