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   

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

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