DOI: 10.5061/dryad.hmgqnk9rz
title: Blockade of IKK signaling induces RIPK1-independent apoptosis in human macrophages

description: Regulated cell death in response to microbial infection plays an important
 role in immune defense and is triggered by pathogen disruption of
 essential cellular pathways. Gram-negative bacterial pathogens in the
 Yersinia genus disrupt NF-κB signaling via translocated effectors injected
 by a type III secretion system, thereby preventing induction of cytokine
 production and antimicrobial defense. In murine models of infection,
 Yersinia blockade of NF-κB signaling triggers cell-extrinsic apoptosis
 through Receptor Interacting Serine-Threonine Protein Kinase 1 (RIPK1) and
 caspase-8, which is required for bacterial clearance and host survival.
 Unexpectedly, we find that human macrophages undergo apoptosis
 independently of RIPK1 in response to Yersinia or chemical blockade of
 IKKβ. Instead, IKK blockade led to decreased cFLIP expression, and
 overexpression of cFLIP contributed to protection from IKK
 blockade-induced apoptosis in human macrophages. We found that IKK
 blockade also induces RIPK1 kinase activity-independent apoptosis in human
 T cells and human pancreatic cells. Altogether, our data indicate that, in
 contrast to murine cells, blockade of IKK activity in human cells triggers
 a distinct apoptosis pathway that is independently of RIPK1 kinase
 activity. These findings have implications for the contribution of RIPK1
 to cell death in human cells and the efficacy of RIPK1 inhibition in human
 diseases.
description: # Data from: Blockade of IKK signaling induces RIPK1-independent apoptosis
 in human cells. We have submitted our raw data for the luminescent
 Caspase-Glo 3/7 Assay Kit by Promega (`Caspase-Glo.xlsx`), luminescent
 cell viability CellTiter-Glo 2.0 Assay Kit by Promega
 (`CellTiter-Glo.xlsx`), colorimetric ELISA Assay Kit by R&D Systems
 for the human cytokine TNFa (`ELISA_TNF_hMDM_Infection.xlsx` ,
 `ELISA_TNF_THP-1.xlsx`) and for the human cytokine IL-18
 (`ELISA_IL-18_THP-1.xlsx`), colorimetric LDH Cytotoxicity Detection Kit by
 Sigma (`LDH_BMDM_Infection.xlsx`, `LDH_hMDM_Infection.xlsx`,
 `LDH_hMDM_Stim.xlsx`, `LDH_THP-1_Stim.xlsx`), and fluorescent Propidium
 iodide (PI) uptake cytotoxicity assay (`PI_Uptake_hMDM`). Apart from the
 PI Uptake data file (`PI_Uptake_hMDM`), each file is made up of an excel
 sheet with multiple tabs, starting with summarized compiled data
 (**Summary**), followed by data from individual experiments, labeled by
 date (YYYY/MM/DD). The PI Uptake data file (**PI_Uptake_hMDM**) does not
 contain a summary tab, but it does contain a guide tab with a key for
 abbreviations, followed by data from individual experiments, labeled by
 date (YYYY/MM/DD). ## Description of the data and file structure We have
 enclosed datasets for multiple assays, as described above. **Cell types
 are as follows**: human monocyte-derived macrophages (hMDMs: each unique
 healthy donor is indicated as ND###, and each donor is highlighted in the
 corresponding color of the colored dot displayed in the manuscript figure
 graphs), THP-1 cells (various genotypes), PANC-1 cells, and Jurkat T
 cells. Please disregard any other highlighting or colors, as they were
 used for personal reference. **The various genotypes for THP-1 cells are
 as follows**: WT THP-1 (wild-type cells), RIPK1-/- Clone 1
 (CRISPR-mediated knockout, single-cell clone, labeled as clone 2B8),
 RIPK1-/- Clone 2 (labeled as clone 2F11), CFLAR-/- Clone 1 (labeled as
 1B4) , CFLAR-/- Clone 2 (labeled as 2C3), plenti-CFLAR
 (CFLAR-overexpressing cells, bulk population, also abbreviated as pCFLAR),
 plenti-mCherry (mCherry-overexpressing WT control cells), FADD-/- Clone 1
 (labeled as 1B7), FADD-/- Clone 3 (labeled as 1C10), and TRADD-/- (bulk
 population). We also utilized siRNA-mediated silencing of the gene BID
 (siBid) in WT THP-1 cells. **Inhibitor, stimulation, and infection labels
 and abbreviations are as follows**: * UT = Untreated, UI or Mock =
 Uninfected, TX = Triton, L = LPS, LI = LPS+IKKi, LIN = LPS+IKKi+Nec-1, LZ
 = LPS+ZVAD, LZN = LPS+ZVAD+Nec-1 * T = TNF, TI = TNF+IKKi, TIN =
 TNF+IKKi+Nec-1, TSm = TNF+Smac Mimetic (LCL161), TSmN = TNF+Smac Mimetic +
 Nec-1 * Chx = Cycloheximide, Apt-1 = Apostatin-1 TRADD inhibitor **For
 ELISA assays**: cytokine concentration values were calculated from the
 detected values through nonlinear transformation and interpolated using
 GraphPad Prism. ND indicates that the values were not detected, due to
 falling outside of the standard curve. ## Sharing/Access information Refer
 to manuscript for further details and methods, which is available on
 bioRxiv:
 [https://www.biorxiv.org/content/10.1101/2023.06.20.545781v1](https://www.biorxiv.org/content/10.1101/2023.06.20.545781v1)