Conclusion

Report
Asthma and Anti-TNF-α Therapeutics
Jessie Scott
MCB 5255
Overview

Asthma

TNF-α

Anti-TNF-α Therapies

Paper 1

Paper 2

Future Directions

Specific Aim
Asthma

Allergic respiratory disease with complex
cytokine interactions, involving airway
inflammation, mucus hypersecretion,
reduced breathing ability, and airway
remodeling in response to allergens

2 types:

Mild-Moderate asthma: involves Th2
lymphocytes, eosinophils, and high levels of
IgE.



Treatable by corticosteroids
Severe Refractory Asthma: involves Th1
lymphocytes, Th17 lymphocytes, neutrophils
and some eosinophils, and TNF-α

Not treatable by corticosteroids

Heterogeneous phenotypes
Need to distinguish between these two
types for appropriate treatment
Hansbro et al. 2011
TNF-α and asthma

Effects of TNF-α on the respiratory system

It causes the recruitment of pro-inflammatory
cells and affects airway remodeling – typical
asthma symptoms

Inhaled recombinant human TNF-α has been
demonstrated to cause a decrease in forced
expiratory volume (FEV) and increased
neutrophil and eosinophil recruitment
Brightling et al. 2008
Effect of control or tumour necrosis factor alpha (TNFα) inhalation upon log PC20 expressed as
percentage of the baseline value after 24 and 48 hours.
Thomas P S , and Heywood G Thorax 2002;57:774-778
Copyright © BMJ Publishing Group Ltd & British Thoracic Society. All rights reserved.
(A) Effect of control (open bars) or tumour necrosis factor alpha (TNFα, shaded bars) inhalation
on change in percentage neutrophils from baseline for the 3 days of the study (ANOVA, p<0.01;
post hoc Bonferroni corrected t test, *p<0.05,**p<0.02).
Thomas P S , and Heywood G Thorax 2002;57:774-778
Copyright © BMJ Publishing Group Ltd & British Thoracic Society. All rights reserved.
TNF-α

Starts as transmembrane TNF
(tmTNF)

Cleaved by TNF-α converting
enzyme (TACE), then it becomes
soluble TNF (solTNF)

Binds to TNFR1 or TNFR2,
stimulates transcription of
proinflammatory genes

Extracellular domains of TNFR can
be cleaved to regulate TNF-α
levels
http://www.ajpcr.com/Vol4Suppl1/551.pdf
TNF-α Signaling

TNF- α binds to TNFR1 or TNFR2

TNFR1 is expressed on all cells and primarily
binds solTNF

TNFR2 is expressed primarily on immune cells
and binds both solTNF and tmTNF

TNFR1 signaling can lead to apoptosis
activation through the caspase 8 pathway

TNFR1 and TNFR2 both lead to the expression
of inflammatory genes and cell survival
tmTNF

tmTNF has been implicated in tumor immune evasion in some
cases

tmTNF binds to TNFR2, these receptors are involved in Treg
functions and myeloid-derived suppressor cell (MDSC)
accumulation – these cells allow for immune evasion of tumor
cells (Hu et al. 2013)

tmTNF was found to increase the suppressive effects of MDSC
while solTNF did not

This increase was stopped by TNFR2 inhibition

A mutant non-cleavable tmTNF also led to increased
tumorigenesis in cell culture

Another study found that breast cancer cells displayed high
levels of tmTNF (Yu et al. 2013)

A monoclonal antibody to tmTNF demonstrated tumor
suppression and even regression in a mouse model bearing breast
cancer tumors
http://www.jleukbio.org/content/88/5/827/F1.large.jpg
Anti-TNF-α Treatments



Etanercept – human fusion protein made of TNF-αR2 and the Fc region of IgG1

Subcutaneous injection

Non-selective for tmTNF or solTNF

Doesn’t activate complement system
(Brightling et al. 2008)
Infliximab – chimeric monoclonal antibody made up of human Ig constant
region, 2 mouse variable regions to TNF-α

Can trigger complement system

Non-selective for tmTNF or solTNF
DN-TNF – dominant negative TNF, mutated TNF that forms heterotrimers with
native TNF to decrease its binding efficiency to TNFRs
http://media.pharmacologycorner.com/wp-content/uploads/2009/05/tnfmoa7.gif
Paper 1

Goal: To determine the differences (if any) between anti-TNF therapy and
corticosteroid therapy on airway remodeling in asthma.

Conducted comparisons and looked at statistical significance between etanercept
and each other experimental situation (untreated asthma, healthy control, and
dexamethasone) for different tissue remodeling factors.
Figure 1

This displays how the experimental
model for asthma was generated

Mice were treated once a week
with IP OVA for 2 weeks

They were then treated with
aerosolized OVA for 30min/day for
3 days each week for 10 weeks

During the last two weeks, mice
also received etanercept,
dexamethasone, or saline IP

Mice were then sacrificed and
airway epithelia was examined
Figure 2

Data displaying the basement membrane thickness, smooth
muscle thickness, and epithelial thickness of airways from
asthmatic mice that were untreated, or treated with steroids or
etanercept.

Etanercept demonstrates reduced basement membrane thickness
and epithelial thickness compared to the untreated asthma.

The steroid demonstrates decreased basement membrane,
smooth muscle, and epithelial thickness compared to the
untreated asthma

Conclusion: All data is statistically significant
Etanercept vs Healthy Control

Conclusion:
Etanercept
treatment could
not reduce
subepithelial
muscle and
basement
membrane
thickness compared
to healthy control.

Etanercept could
revert remodeling
so that epithelial
layer thickness and
goblet cell number
were not
significantly
different
compared to
healthy control.
Etanercept vs Untreated Asthma

Conclusion:
Etanercept
treatment
significantly
reduced:

epithelial and
basement
membrane
thickness

goblet cell number

Mast cell number

Etanercept could
not reduce
subepithelial
muscle layer
thickness
Etanercept vs Dexamethasone

Conclusion: No
statistical significant
difference between
treatments on:

epithelial thickness

basement membrane
thickness

mast cell number

However, steroid
treatment was better
for decreasing
subepithelial muscle
layer thickness
Summary of Paper 1


Anti-TNF-α treatment with etanercept led to a decrease in:
epithelial and basement membrane thickening

Mast cell number

Goblet cell number (better than steroid treatment)

It could not decrease subepithelial muscle layer thickness significantly like
dexamethasone could; it is comparable to dexamethasone in all other
measures

Similarities may be due to steroid-caused reduction in TNF-α levels
Paper 2

Goal: To determine whether inflammation is mediated by solTNF or tmTNF and
investigate if dominant-negative TNF (DN-TNF) therapy can reduce TNF inflammation
Figure 1

Used TNF knockout mice (no tm or
solTNF) and tmTNF knock in mice
to study effects of solTNF and
tmTNF on eosinophil and
macrophage recruitment, and lung
activity of eosinophil peroxidase

Conclusion: solTNF mediates
allergic lung inflammation
symptoms: eosinophil and
macrophage recruitment and EPO
activity
Figure 2

Stimulated asthma with ovalbumin
immunization and challenge in Balb/c
mice

Treated mice with etanercept, a nonselective anti-TNF treatment

Used XENP1595 and XENP346 as
dominant negative control of soluble
TNF (DN-TNF)

DN-TNF worked just as well as
etanercept for reducing eosinophil
recruitment and activity

DN-TNF worked better than etanercept
in reducing lymphocyte recruitment

Conclusion: soluble TNF mediates
inflammatory lung response, these side
effects are controlled well by DN-TNF
therapy
Figure 3
http://www.frontiersin.org/files/Articles/30094/fphar-03-00156-r4/image_m/fphar-03-00156-g005.jpg

Lung resistance and compliance was measured by
plethysmography

Mice treated with etanercept or DN-TNF had
better compliance and less resistance than mice
that were untreated

Conclusion: etanercept and DN-TNF both increase
lung compliance, therefore solTNF is what
mediates airway resistance
Figure 4

Treated with etanercept or DN-TNF
at time of challenge didn’t yield as
good results as treatment at the
same time as immunization and
challenge (see previous figures)

Conclusion: treatment at time of
immunization and challenge
worked better, but etanercept and
DN-TNF were both able to reduce
eosinophil recruitment
Figure 5: H&E Staining

H&E staining demonstrating areas of eosinophil infiltration

Conclusion: Anti-TNF treatments reduced eosinophil infiltration.
Figure 5: PAS Staining

PAS staining demonstrating areas of goblet cell hyperplasia and hypersecretion of
mucus

Conclusion: Anti-TNF decreased goblet cell hyperplasia and mucus secretion the most.
Figure 6

Inflammatory cytokines and IgE
from BAL samples were
measured by ELISA.

Conclusion: Anti-TNF treatments
reduced inflammatory cytokines
and IgE levels.
Paper 2 Summary

Soluble TNF mediates the inflammatory effects of TNF

Regulation of solTNF through DN-TNF therapy can reduce asthma symptoms
such as eosinophil recruitment, EPO activity, macrophage recruitment, goblet
cell hyperplasia, mucus secretion, and lung compliance.

Etanercept and other non-selective anti-TNF therapies are not recommended
for asthma treatment because of the negative side effects from
immunosuppression

However, DN-TNF may be an effective asthma therapy because it preserves
the innate immune activity of tmTNF while reducing asthma symptoms
Future Directions

A suitable treatment for Severe Refractory Asthma still does not exist, etanercept and
infliximab are useful in some cases, but not all

The risk-benefit ratio for using non-selective anti-TNF therapies is not ideal

Mediating soluble TNF decreases the inflammation, but does not affect susceptibility
to infection – DN-TNF may be the solution to severe refractory asthma treatment

Specific Aim: Stimulate tumor formation in mice using ultraviolet irradiation on TNFknockout mice, tmTNF knock in mice, and while treating wild-type mice with DN-TNF
or etanercept to observe any differences in the rate of tumor formation. If the
tmTNF knock in mice and the DN-TNF treated mice form less tumors, then tumor
suppression is mediated by tmTNF.

This will help clarify whether DN-TNF could be used as a safe and effective asthma
treatment
Thank you!
Works Cited

Brightling C, Berry M, and Amrani Y. (2008). Targeting TNF-alpha: a novel therapeutic approach
for asthma. The Journal of allergy and clinical immunology 121:5-10.

Hansbro PM, Kaiko GE, Foster PS. (2011). Cytokine/anti-cytokine therapy – novel treatments
for asthma? British Journal of Pharmacology 163:81-95.

Hu X, Li B, Li X, Zhao X, Wan L, Lin G, Yu M, Wang J, Jiang X, Feng W, Qin Z, Yin B, Li Z. (2014).
Transmembrane TNF-a promotes suppressive activities of myeloid-derived suppressor cells via
TNFR2. Journal of Immunology 192:1320-1331.

Maillet I, Schnyder-Candrian S, Couillin I, Quesniaux VFJ, Erard F, Moser R, Fleury S, Kanda A,
Dombrowicz D, Szymkowski DE, Ryffel B. (2011). Allergic lung inflammation is mediated by
soluble tumor necrosis factor (TNF) and attenuated by dominant-negative TNF biologics.
American Journal of Respiratory Cell and Molecular Biology 45: 731-739.

Thomas PS and Heywood G. (2002). Effects of inhaled tumour necrosis factor alpha in subjects
with mild asthma. Thorax 57:774-778.

Yilmaz O, Karaman M, Bagriyanik HA, Firinci F, Kiray M, Turkeli A, Karaman O, Yuksel H. (2013).
Comparison of TNF antagonism by etanercept and dexamethasone on airway epithelium and
remodeling in an experimental model of asthma. International Immunopharmacology 17: 768773.

Yu M, Zhou X, Niu L, Lin G, Huang J, Zhou W, Gan H, Wang J, Yin B, Li Z. (2013). Targeting
transmembrane TNF-a suppresses breast cancer growth. Cancer Research 73:4061-4074.

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