KRISTEN DOSTIE Alteration of Chemotaxis in the Gut of IBD Patients.
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Transcript of KRISTEN DOSTIE Alteration of Chemotaxis in the Gut of IBD Patients.
KRISTEN DOSTIE
Alteration of Chemotaxis in the Gut of IBD Patients
Overview
IntroductionWhat do we know about neutrophil
chemotaxis in IBD?Paper 1Paper 2What 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?
CXC chemokines are ligands for CXCR receptors on several different cell types Promote neutrophil migration
Chemotaxis! Movement of a cell in response to a chemical stimulus
Neutrophil infiltration is largely mediated through binding of CXCR receptors
Nature Reviews Immunology 13, 649–665 (2013)
Overview
IntroductionWhat do we know about neutrophil
chemotaxis in IBD?What is still unknown about neutrophil
chemotaxis in IBD?Paper 1Paper 2Specific 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
IntroductionWhat do we know about neutrophil
chemotaxis in IBD?Paper 1Paper 2What is still unknown about neutrophil
chemotaxis in IBD?Specific Aims
Paper 1
Paper 1 Background
Neutrophils produce proteases that cleave collagen into proline-glycine-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 non-inflamed 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?
MMP9: neutrophils
PE: neutrophils and epithelial cells
MMP8: weak expression in neutrophils and inflammatory 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 DSS-induced 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
IntroductionWhat do we know about neutrophil
chemotaxis in IBD?Paper 1Paper 2What 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?
Conclusion: Early stages of TNBS induced colitis involves an innate immune response
Rag1-/- mice do not have mature B or T cells.
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 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!