TY - JOUR
T1 - Issues with the Specificity of Immunological Reagents for NLRP3
T2 - Implications for Age-related Macular Degeneration
AU - Kosmidou, Cassandra
AU - Efstathiou, Nikolaos E.
AU - Hoang, Mien V.
AU - Notomi, Shoji
AU - Konstantinou, Eleni K.
AU - Hirano, Masayuki
AU - Takahashi, Kosuke
AU - Maidana, Daniel E.
AU - Tsoka, Pavlina
AU - Young, Lucy
AU - Gragoudas, Evangelos S.
AU - Olsen, Timothy W.
AU - Morizane, Yuki
AU - Miller, Joan W.
AU - Vavvas, Demetrios G.
N1 - Funding Information:
This work was supported by: NEI R21EY023079-01A1, R01-EY025362-01 (DGV); the Yeatts Family Foundation (DGV, JWM); the Loefflers Family Fund (DGV, JWM); the 2013 Macula Society Research Grant award (DGV); a Physician Scientist Award from RPB and the Alcon Research Institute Young Investigator Award (DGV), an unrestricted grant from the Research to Prevent Blindness foundation (JWM); NEI grant EY014104 (MEEI Core Grant), the Bayer Healthcare Global Ophthalmology Awards Programme (DEM) and the STAMATIOU foundation scholarship (NEE). The authors would like to thank and acknowledge Dr. S.A. Trauger from the Small Molecule Mass Spectrometry facility at Harvard University for the protein sequencing analysis.
Publisher Copyright:
© 2017 The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection. Furthermore, NLRP3 was below detection limits in ex vivo macular RPE from AMD patients, as well as in human induced pluripotent stem cell (hiPSC)-derived RPE from patients with overactive NLRP3 syndrome (Chronic infantile neurologic cutaneous and articulate, CINCA syndrome). Evidence presented in this study provides new data regarding the interpretation of published results reporting NLRP3 expression and upregulation in RPE and addresses the role that this inflammasome plays in AMD pathogenesis.
AB - Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection. Furthermore, NLRP3 was below detection limits in ex vivo macular RPE from AMD patients, as well as in human induced pluripotent stem cell (hiPSC)-derived RPE from patients with overactive NLRP3 syndrome (Chronic infantile neurologic cutaneous and articulate, CINCA syndrome). Evidence presented in this study provides new data regarding the interpretation of published results reporting NLRP3 expression and upregulation in RPE and addresses the role that this inflammasome plays in AMD pathogenesis.
UR - http://www.scopus.com/inward/record.url?scp=85040513596&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85040513596&partnerID=8YFLogxK
U2 - 10.1038/s41598-017-17634-1
DO - 10.1038/s41598-017-17634-1
M3 - Article
C2 - 29323137
AN - SCOPUS:85040513596
SN - 2045-2322
VL - 8
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 461
ER -
