Cytokines and
Inflammatory Bowel
Disease
Alexander Lind
University of Connecticut MCB5255 2014-04-09
Overview
Background information about cytokines
Cytokines and IBD
Article 1
Article 2
Future studies, specific aims
Cytokines
Cell
communication
and regulation
Involved in
nearly all
biological
processes:
Embryonic
development
Stem cell
differentiation
Inflammatory
responses
Used as
prognostic and
therapeutic
agents in human
disease
http://www.genecopoeia.com/product/search/pathway/h_inflamPathway.php
Cytokine storm
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180813/
http://www.cytokinestorm.com/
Pro-inflammatory/ Anti-inflammatory cytokines
Overview
Background information about cytokines
Cytokines and IBD
Article 1
Article 2
Future studies, specific aims
Cytokine imbalance
contributes to IBD
through:
Macrophage,
Dendritic cell
recruitment and
activation
T-cells differentiation
 Crohn's disease Th1 cell mediated
response
Ulcerative ColitisTh2 cell mediated
response
http://www.nature.com/nrgastro/journal/v7/n2/fig_tab/nrgastro.2009.218_F1.html
CROHN'S DISEASE
ULCERATIVE COLITIS
•Th1 cell induced differentiation
mediated by IL-12 and IL-23
•Th2 atypical immune response
•Lack of increased IFN-γ expression
(abundant in CD) rather than the
elevation of IL-4 the Th2-defining
cytokine
•Classical pro-inflammatory cytokines,
such as IL-1, IL-6, and TNF-α
•Th2 cytokines IL-10 and IL-13 play a
key role, increased IL-13 production
by NKT cells
•TH17 cells producing IL-17
•Characterized by enhanced
production of IFN-γ and TNF-α
http://www.nature.com/nrd/journal/v5/n3/fig_tab/nrd1986_ft.html
http://www.clinsci.org/cs/118/0707/cs1180707f03.htm
TNF-α
•TNF-α is upregulated in both
CD and UC
•Mostly produced
by activated
macrophages
• Transmembrane
protein, activated
through proteolytic
cleavage
• Play an important
role in the
pathogenesis of
IBD
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731177/
Anticytokine therapies
•Monoclonal anti-TNF-alpha antibodies has showed good
results in both inducing and maintaining remission
• Do not act only by neutralizing TNF-alpha:
- Induce monocyte and T-cell apoptosis
- Antibody-dependent cell-mediated cytotoxicity
•Unclear why certain anti-TNF therapies are effective in
IBD where as other are not
Problems with current Anti TNF-α
therapies
• Liver Injury Secondary to Anti-TNF-Alpha Therapy in Inflammatory Bowel
Disease: A Case Series and Review of the Literature. Parekh et. al. 2014
•Incidences of serious infections and tuberculosis among patients receiving
anti-tumor necrosis factor-α therapy. Yoo et. al 2014
•Recurrence of active tuberculosis following resumption of anti-TNFα therapy in a patient with Crohn's disease. Masia et. al. 2014
•Tuberculosis infection following anti-TNF therapy in inflammatory bowel
disease, despite negative screening. Debeuckelaere et. al. 2013
•Immune-mediated inflammatory reactions and tumors as skin side effects of
inflammatory bowel disease therapy Marzano et. al. 2014
Overview
Background information about cytokines
Cytokines and IBD
Article 1
Article 2
Future studies, specific aims
Article 1
“Orally delivered siRNA targeting
macrophage Map4k4 suppresses systemic
inflammation” by Aoudi et.al. 2009
Investigates the potential RNA interference to
decrease inflammatory response
http://silence-therapeutics.com/platform-technologies/rna-interference/
Figure 1. Experimental design
Figure 2. Orally delivered Map4k4 siRNA containing vectors can be phagocytized by
macrophages and silence messenger RNA expression
Figure 2. (Continued)
Conclusion: In vitro treatment of Map4k4 siRNA can silence
messenger RNA expression and inhibit LPS induced TNFα
production.
Figure 3: What effect does Map4k4 silencing have on LPS activation of
previously known pathways for regulation of Tnf-α production?
Conclusion: Map4k4 is a new target for suppression of Tnf-α expression,
activation occurs independently of previously known pathways
Figure 4. Oral
administration of Map4k4
siRNA decreases mRNA
expression in lung, liver
and spleen.
Figure 4. ( Continued)
Conclusion: Demonstrated that macrophages in the gut internalize orally
delivered siRNA, undergo RNA interference mediated gene silencing and
migrate into tissues throughout the body.
Figure 5: What is the effect on TNFα expression after Map4k4
silencing?
Conclusion:Map4k4-siRNA administration protects mice from LPS-induced
lethality through inhibition of TNF-α in macrophages
Conclusions from article 1
The results in this articles describes a new strategy for oral delivery of siRNA
to weaken inflammatory responses in human disease
 Identification of Map4k4, a previously unknown mediator of cytokine
expression.
 Silencing of Map4k4 in macrophages in vivo protected mice from
lipopolysaccharide-induced lethality by inhibiting of TNF-α
This article demonstrated promising results for anti-TNF-α protein therapeutics.
Development of GeRP-mediated delivery of siRNA show promising results as
inflammatory decreasing agents for several disease including IBD.
Overview
Background information about cytokines
Cytokines and IBD
Article 1
Article 2
Future studies, specific aims
Article 2
“Gene silencing of TNF-alpha in a murine model of acute colitis
using a modified cyclodextrin delivery system” by MacCarthy et.al.
2013
Investigates possibility of silencing TNF-α trough RNA
interference in IBD
Figure 1. Stability and delivery of siRNA
siRNA has short half-life time in plasma due to nuclease
degradation
Cyclodextrin, natural occurring oligosaccharides
http://pubs.rsc.org/en/content/articlepdf/2010/MB/C001050M
Figure 2.Investigation of vector-siRNA complex
stability in simulated intestinal fluids
Figure 3. Quantification
of TNF-α and IL-6
expression in LPS
stimulated cells
Figure 4. Examination of inhibitory
effect of TNF-α siRNA on LPSinduced cytokine secretion and
expression
Conclusion: TNF-α siRNA reduced LPS
induced cytokine expression of TNF-α and
IL-6
Figure 5. Clinical response after TNF-α siRNA delivery in
DSS treated mice
A trends towards clinincal improvement could be observed
after TNF-α siRNA delivery: increased body weight,
reduced colon weight
Figure 6. Decreased TNF-α and
IL-6 expression in proximal colon
after TNF-α -siRNA delivery to
DSS-treated mice
Conclusions from Article 2
 Showed in vitro studies that delivery of TNF-α siRNA could interfere with
LPS induced activation of pro-inflammatory cytokines
Intrarectal administration of TNF-siRNA in DSS-treated mice gave indications
of:
1) clinical improvements of IBD associated features
2) Lower expression of TNF-α and IL-6 in proximal colon
This article demonstrated promising results of a CD-based siRNA delivery system for
silencing of pro-inflammatory cytokine TNF-α. Potentially used in future treatment of
IBD.
Overview
Background information about cytokines
Cytokines and IBD
Article 1
Article 2
Future studies, specific aims
Future studies
 There is a problem with side effects using current therapies
 RNA interference silencing Mp4k4 and TNF-α show promising results
Combination of silence several genes? Other ways affecting TNF-α expression?
Specific aim: To use RNA interference
technique to try and silence gene
expression of transcription factor LITAF
known to regulate TNF-α expression
References
•
Genecopoeia. Cytokines and Inflammatory Response http://www.genecopoeia.com/product/search/pathway/h_inflamPathway.php
• Kindt TJ et.al.. Kuby Immunologi 6th edition, Figure 12.
• Morens DM et.al. The 1918 influenza pandemic: Lessons for 2009 and the future. Critical Care Medicine.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180813/
• Osterholm et. al.. Proposed Mechanism of the Cytokine Storm Evoked by Influenza virus. New England Journal of Medicine
http://www.cytokinestorm.com/cytokine_storm.html
• Audet CM et. al. Interplay between pro-inflammatory cytokines and growth factors in depressive illnesses. Cellular neuroscience.
http://journal.frontiersin.org/Journal/10.3389/fncel.2013.00068/full
• Melmed GY et.al. Future biologic targets for IBD: potentials and pitfalls. Nature Reviews Gastroenterology and Hepatology.
http://www.nature.com/nrgastro/journal/v7/n2/fig_tab/nrgastro.2009.218_F1.html
• Monteleone G et.al. T-cell-directed therapies in inflamatory bowel diseases. Clinical science. http://www.clinsci.org/cs/118/0707/cs1180707f03.htm
• Joshua R et.al. Evolving knowledge and therapy of inflammatory bowel disase. Nature reviews.
http://www.nature.com/nrd/journal/v5/n3/fig_tab/nrd1986_ft.html
• Sanchez-Munoz et.al. Role of cytokines in inflammatory bowel disease. World J Gastroenterology. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731177/
• RNA interference. Silence therapeutics. http://silence-therapeutics.com/platform-technologies/rna-interference/