journal club fall 2013
TRANSCRIPT
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YouClas
Month day, year
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