Journal Club Fall 2013

7/29/2019 Journal Club Fall 2013

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Participation of the Receptor for Advanced Glyca

Products in Efferocytosis

Arnaud Friggeri, Sami Banerjee, Subrata Biswas, Andressa de Freitas, Gang Liu, Angelika Bierhaus

Journal of Immunology (2011) 186: 6191-6198

Mohit Koladia

Advisor: Dr. Stefan Vetter


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Efferocytosis

Clearance of apoptotic cells

Resolution of inflammation

Defects/Alteration-

Acute & chronic inflammatory diseaseslike rheumatoid arthritis and acute lung

injury

Ravichandran K S J Exp Med 2010;207:1807


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RAGE

Expression Low levels: Macrophages

High levels: Pulmonary epithelial cells

Upregulation leads to potentiation of inflammation associate

diseases

HMGB1: Ligand for RAGE Activates Macrophages, Neutrophils, etc.

Binds to phosphatidylserine on apoptotic cells and decreases clearance by macrophages

C-terminal domain of HMGB1 is required for inhibiting efferocytosis (same motif is required f


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Hypothesis

Phagocy

te

Apoptotic

Cell

Engulfment of Apoptotic cell


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Questions addressed in the Pap

1. Whether RAGE is involved in efferocytosis or not?

2. Determine if RAGE influences efferocytosis.

3. How does RAGE recognize and bind to apoptotic

4. Determine whether RAGE binds to PS on apoptot5. If RAGE enhances efferocytosis in vivo?


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Materials & Methods

Animal study: male C57BL/6 wild type and RAGE knInduction of apoptosis Neutrophils were isolated from BM cell suspension and incubated at 43 C for 60 min in se

Thymocytes were incubated with 1 uM dexamethasone

InVitro experiments Full length human RAGE was expressed in HEK 293 cells using FG12 vector

Cell adhesion assay: to determine adhesion of apoptotic cells to RAGE

Solid phase ELISA: to check binding of RAGE to phospholipids

InVivo experiments Apoptotic neutrophils were injected intratracheally to WT or RAGE knockout mice for assa

Apototic thymocytes were injected i.p. and peritoneal lavage was used for assay


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Materials & Methods

Efferocytosis assay Apoptotic neutrophils/thymocytes were added to 24 well plate containing macrophage

grown on coverslip

Incubate at 37 C for 2 hrs

Non-ingested cells removed by washing with PBS

Cells on coverslip is fixed and stained with Wright-Giemsa stain

Blind observer counts the cells and phagocytic index is calculated


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RAGE deficient macrophages have decreased ability to engulf a


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Efferocytosis is influenced by RAGE


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Conclusion

RAGE deficient macrophages showed decreased ability to engulf apoptotic

compared to RAGE expressing macrophages

The ability of RAGE to enhance uptake of apoptotic cells was not only spec

macrophages as shown by incubation with bone marrow derived macropha

Inhibitory effects of anti-RAGE Abs suggest ligand binding to extracellular d

responsible for efferocytosis


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RAGE expression enhances efferocytosis in HE

Target: Thymocytes


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Conclusion

RAGE expressing HEK293 cells had significantly greater ability to engu

thymocytes than cells transfected with control vector

Overexpression of RAGE enhances efferocytosis even in non-professio


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AGEs inhibits efferocytosis by macroph

Conclusion

BSA AGE dose dependen

of macrophages to phag

Target: Neutrophils


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sRAGE enhances efferocytosis by macrop

(nM)

Target: Thymocytes


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Conclusion

sRAGE does not block RAGE mediated efferocytosis

sRAGE increased the phagocytosis of apoptotic cells by RAGE+/+ and

These data suggest that promoting effect of sRAGE in efferocytosis is

mechanisms independent of presence of RAGE on macrophage


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RAGE binds to Phosphatidyl Serine


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Apoptotic cells bind to RAGE by interactions


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Conclusion

sRAGE bind to PS in a dose dependent manner but minimally bind to PC anRAGE specifically binds to PS only

Annexin V competitively decreased binding of sRAGE to PS

This suggests RAGE to be novel receptor for PS which facilitates recognitionfor clearance by efferocytosis


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RAGE participates in Efferocytosis under in viv


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Conclusion

Phagocytosis of apoptotic neutrophils by alveolar macrophages was signi

RAGE-/- mice compared to control RAGE+/+ mice

Phagocytosis of apoptotic thymocytes by peritoneal macrophages was sig

in RAGE-/- mice compared to control RAGE+/+ mice


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Summary

Demonstrate a novel role of RAGE in enhancing efferocytosis through facilit

recognition of apoptotic cells by macrophages

Novel study that suggests that RAGE may be directly involved in resolution

by facilitating the clearance of apoptotic cells

The authors also concludes that therapies like administration of sRAGE may

removing dying cells from tissue injury sites


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Critique 1

It is well established that R

If we believe authors claim th

efferocytosis, we might expect to see

index in lung tissue as compared


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Critique 2

The authors have not discussed the relative percentage of apoptotic cells prese

and in vivo conditions

The amount of cells undergoing apoptosis may be relatively high under in vivo

conditions when compared to in vitro condition

I think the experiments conducted in the in vivo inflammatory models in rats/mic

substantiated the authors claim more effectively


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Acknowledgement

Dr Stefan Vetter

Dr Estelle Leclerc

Dr Jagdish Singh

All gra


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Questions & Discussion


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Isolation of Neutrophils


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Efferocytosis enhances the production o


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Conclusion

Efferocytosis enhanced IL-10 production in both RAGE+/+

RAGE/ macrophages

Documents

Journal Club Fall 2013