PMID- 30735689
OWN - NLM
STAT- MEDLINE
DCOM- 20200313
LR  - 20200313
IS  - 1872-7905 (Electronic)
IS  - 0022-1759 (Linking)
VI  - 467
DP  - 2019 Apr
TI  - Optimized protocols for studying the NLRP3 inflammasome and assessment of 
      potential targets of CP-453,773 in undifferentiated THP1 cells.
PG  - 19-28
LID - S0022-1759(18)30403-4 [pii]
LID - 10.1016/j.jim.2019.02.002 [doi]
AB  - The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates 
      production of the pro-inflammatory cytokine IL-1beta. To overcome both the 
      variability among primary immune cells and the limitations of genetic 
      manipulation of differentiated human or murine macrophages, we developed a 
      simplified, reliable and relevant cell-based model for studying the NLRP3 
      inflammasome using the undifferentiated human myelomonocytic cell line THP1. 
      Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome 
      activation occurred in response to canonical NLRP3 activation stimuli including 
      nigericin, ATP, and urea crystals, culminating in pro-IL-1beta cleavage, 
      extracellular release of mature IL-1beta, and pyroptosis. We used this THP1 cell 
      system to investigate potential targets of the potent, NLRP3 inflammasome 
      selective inhibitor CP-456,773. We optimized a viral shRNA transduction method 
      for gene expression knockdown (KD), and the KD of NLRP3 itself eliminated 
      inflammasome activation and IL-1beta production. NLRP3 inflammasome activation and 
      CP-453,773 pharmacology were not altered in ABCb7- or ABCb10-deficient THP1 
      cells, eliminating these gene products as candidate pharmacological targets of 
      CP-453,773. For ABCb10, we confirmed our results using CRISPR/CAS9-mediated 
      ABCb10 knockout (KO) THP1 sub-lines. In summary, undifferentiated THP1 cells are 
      fully competent for activation of the NLRP3 inflammasome and production of IL-1beta, 
      without differentiation into macrophages, and we describe optimized KD and KO 
      methodologies to manipulate gene expression in these cells. As an example of the 
      utility of undifferentiated THP1 cells for investigations into the biology of the 
      NLRP3 inflammasome, we have used this cell system to rule out ABCb7 and ABCb10 as 
      potential targets of the NLRP3 inflammasome inhibitor CP-453,773.
CI  - Copyright (c) 2019 Elsevier B.V. All rights reserved.
FAU - Guzova, Julia A
AU  - Guzova JA
AD  - Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United 
      States; Department of Pharmacology & Experimental Therapeutics, Boston University 
      School of Medicine, Boston, MA 02118, United States.
FAU - Primiano, Michael J
AU  - Primiano MJ
AD  - Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United 
      States.
FAU - Jiao, Aiping
AU  - Jiao A
AD  - Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United 
      States.
FAU - Stock, Jeffrey
AU  - Stock J
AD  - Department of Discovery Sciences, Pfizer, Groton, CT 06340, United States.
FAU - Lee, Chiachin
AU  - Lee C
AD  - Department of Discovery Sciences, Pfizer, Groton, CT 06340, United States.
FAU - Winkler, Aaron R
AU  - Winkler AR
AD  - Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United 
      States.
FAU - Hall, J Perry
AU  - Hall JP
AD  - Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United 
      States. Electronic address: james.p.hall@pfizer.com.
LA  - eng
PT  - Journal Article
DEP - 20190205
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Inflammasomes)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (NLR Family, Pyrin Domain-Containing 3 Protein)
RN  - 0 (NLRP3 protein, human)
RN  - 0 (Sulfonylurea Compounds)
RN  - 268B43MJ25 (Uric Acid)
RN  - RRU6GY95IS (Nigericin)
SB  - IM
EIN - J Immunol Methods. 2019 May;468:67. PMID: 30947983
MH  - Cell Differentiation/drug effects
MH  - Cell Survival/drug effects
MH  - Cells, Cultured
MH  - HEK293 Cells
MH  - Humans
MH  - Inflammasomes/*drug effects/metabolism
MH  - Lipopolysaccharides/antagonists & inhibitors/pharmacology
MH  - NLR Family, Pyrin Domain-Containing 3 Protein/*antagonists & 
      inhibitors/metabolism
MH  - Nigericin/antagonists & inhibitors/pharmacology
MH  - Sulfonylurea Compounds/*pharmacology
MH  - Uric Acid/antagonists & inhibitors/pharmacology
OTO - NOTNLM
OT  - ABCb10
OT  - ABCb7
OT  - CP-456,773
OT  - Interleukin-1beta (IL-1beta), NLRP3 inflammasome
OT  - THP1
EDAT- 2019/02/09 06:00
MHDA- 2020/03/14 06:00
CRDT- 2019/02/09 06:00
PHST- 2018/11/14 00:00 [received]
PHST- 2019/02/04 00:00 [revised]
PHST- 2019/02/04 00:00 [accepted]
PHST- 2019/02/09 06:00 [pubmed]
PHST- 2020/03/14 06:00 [medline]
PHST- 2019/02/09 06:00 [entrez]
AID - S0022-1759(18)30403-4 [pii]
AID - 10.1016/j.jim.2019.02.002 [doi]
PST - ppublish
SO  - J Immunol Methods. 2019 Apr;467:19-28. doi: 10.1016/j.jim.2019.02.002. Epub 2019 
      Feb 5.