Transcript Alteration of Chemotaxis in the Gut of IBD Patients

Alteration of Chemotaxis in the
Gut of IBD Patients
KRISTEN DOSTIE
Overview
 Introduction
 What do we know about neutrophil chemotaxis in




IBD?
Paper 1
Paper 2
What is still unknown about neutrophil chemotaxis
in IBD?
Specific Aim
The Inflammatory Response
http://www.uic.edu/classes/bios/bios100/lecturesf04am/inflammation01a.jpg
How are neutrophils recruited from the
bloodstream to the affected area?
 Chemotaxis!

Movement of a cell in response to a chemical stimulus
 CXC chemokines are ligands for CXCR receptors on several different cell types
 Promote neutrophil migration
Neutrophil infiltration is largely mediated
through binding of CXCR receptors
Nature Reviews Immunology 13, 649–665 (2013)
Overview
 Introduction
 What do we know about neutrophil chemotaxis in




IBD?
What is still unknown about neutrophil chemotaxis
in IBD?
Paper 1
Paper 2
Specific Aims
What is generally known about neutrophil
chemotaxis in IBD?
 Invasion of neutrophils into the
mucosal epithelium and intestinal
lumen correlates with disease activity
 Neutrophil infiltration disrupts
epithelial barrier integrity

Allows lumenal proteins and
microorganisms to breach the submucosa
 Neutrophils within the mucosa and
submucosa produce proinflammatory
signals that perpetuate neutrophil
recruitment to the affected area
What is generally known about neutrophil
chemotaxis in IBD?
 Neutrophils release a plethora of inflammatory mediators that can exacerbate
inflammation
Inflammatory Bowel Disease. Volume 19, Number 7, June 2013
Overview
 Introduction
 What do we know about neutrophil chemotaxis in




IBD?
Paper 1
Paper 2
What is still unknown about neutrophil chemotaxis
in IBD?
Specific Aims
Paper 1
Paper 1 Background
 Neutrophils produce
proteases that cleave
collagen into prolineglycine-proline (PGP)


Matrix metalloproteinases
(MMPs)
Prolyl endopeptidase (PE)
 PGP has been shown to
be a chemoattractant for
neutrophils via CXCR1/2
in several lung diseases
 Goal: to investigate the role of PGP in neutrophil
recruitment associated with IBD
Figure 1: Is protease expression upregulated in
the colon of IBD patients?
Conclusions:
 MMP8 is
upregulated in both
inflamed and noninflamed IBD
intestinal samples
 MMP8 present in
IBD patients exists
dominantly in its
active form
Figure 1 (cont’d): Where do these proteases
localize within the intestine?
MMP8: weak
expression in
neutrophils and
inflammatory
cells
MMP9:
neutrophils
PE: neutrophils
and epithelial
cells
Figure 2: Are products of MMP and PE activity
increased in IBD?
 N-Ac-PGP (an acetylated form of PGP) expression is
increased in IBD patient intestinal samples.
Figure 3: Do IBD neutrophils produce more proteases and
their subsequent products compared to healthy controls?
Proteases
Chemoattractants
Conclusion: IBD neutrophils produce significantly higher levels of
MMP8, MMP9, and N-Ac-PGP in conditioned medium
Figure 4: Protease expression in a DSSinduced colitis model
• PE: no significant difference in
expression
• MMP9: significant increase in
expression
• MMP8: no significant difference in
expression
• Conclusion: protease expression
in DSS-induced colitis colon
samples is comparable to human
IBD tissue levels.
Figure 5: Localization of protease expression in
DSS-induced colitis colon tissue
• MMP8: leukocytes and
epithelial cells
• MMP9: leukocytes
• PE: leukocytes and
epithelial cells
• Conclusion:
localization of proteases
is comparable to
patterns in human
disease.
Figure 5 (cont’d): Chemoattractant expression in
DSS-colitis colon tissue
Conclusion: N-Ac-PGP and PGP are generated in DSS colon samples, but not
at significant levels.
Figure 6: PGP neutralization as a
therapeutic for IBD
Conclusion: PGP neutralization reduced disease activity index (DAI),
shortening of the colon, histopathological scores, and neutrophil infiltration in
the colon.
Figure 7: The PGP generation cascade leads to
neutrophil chemotaxis
Conclusions from Paper 1
 Components of the PGP generation pathway (matrix
metalloproteinases and PE) are present in intestines of
human IBD patients and DSS-induced colitis mice.
 N-Ac-PGP-mediated neutrophil recruitment likely plays
a role in the perpetuation of intestinal inflammation of
IBD
 PGP neutralization reduced neutrophil infiltration and
disease activity indices in a DSS-induced colitis model
Overview
 Introduction
 What do we know about neutrophil chemotaxis in




IBD?
Paper 1
Paper 2
What is still unknown about neutrophil chemotaxis
in IBD?
Specific Aim
Paper 2
Lumican
 Found in connective tissue rich in fibrillar
collagens
 Deficiency impairs connective tissue function
 Promotes neutrophil migration by interacting
with neutrophil migration receptor MAC1 or
CD11b or CD18
 Expression is upregulated in epithelial cells
after injury
 Goal: characterize the role of lumican in
regulating immune response during
inflammation
http://onlinelibrary.wiley.com/doi/10.1111/febs.12210/full
Figure 1: Summary of TNBS colitis model
Figure 2: Response to intrarectal TNBS treatment
using saline or ethanol as controls
Conclusion: Lum -/- TNBS-treated mice displayed more pronounced
adverse effects to TNBS treatment than the WT strain.
Figure 3: Macroscopic appearance of Lum +/+ and
Lum -/- colons from saline vs. TNBS-treated mice
Figure 4: Increased inflammation of colons in
Lum+/+ and Lum-/- mice
Conclusion: colonic edema and swelling is increased in Lum-/- mice.
Figure 5: Is early acute inflammatory response T
cell-mediated or innate immune driven?
Rag1-/- mice do not
have mature B or T
cells.
Conclusion: Early stages of TNBS induced colitis involves an innate
immune response
Figure 6: Histology of colon cross sections from
Lum+/+ and Lum-/- mice
Figure 7: Inflammation scores and MPO
expression
Conclusion: Lum-/- colons have more tissue damage but less
neutrophil infiltration after TNBS colitis induction.
Figure 8: Induction of proinflammatory
cytokines in inflamed colonic tissue
Conclusion: TNBS colitis induced CXCL1/KC and TNFα production in
Lum+/+ but not Lum-/- colonic tissue.
Figure 9: NF-κB activation in Lum +/+ vs.
Lum-/- mice
1º peritoneal cells
Conclusion: delayed nuclear localization of NF-κB in Lum-/- colonic
samples suggests a weak innate immune response in the absence of
lumican.
Conclusions from Paper 2
 Lumican deficiency leads to decreased neutrophil
infiltration but increased severity of colitis
 Lumican plays a role in regulation of inflammation
in colitis
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

CXCL1
TNF-α
Neutrophil recruitment
What is still unknown about neutrophil
chemotaxis in IBD?
 Well, a lot of things.
 ECM proteins and their degradation products


Several are ECM proteins are upregulated in inflamed colonic tissue
of IBD patients
Neutrophils themselves produce proteases whose products further
perpetuate their infiltration
 Role of upregulated ECM proteins in the perpetuation of
neutrophil infiltration in IBD is poorly studied.
 Mechanism of action of ECM proteins/protease products
on neutrophil recruitment is poorly understood
Specific Aim and Future Directions
 Specific Aim
 Determine a set of ECM proteins and their degradation
products upregulated in inflamed colonic tissue in IBD that
can be chemoattractants for neutrophils
 Potential experiments
 Electric cell-substrate impedance sensing (ECIS)/Taxis to
determine potency of chemoattraction induced by various
ECM proteins and degradation products
 Determine mechanism of action of these proteins by blocking
CXCR receptors
Thanks for listening!