DOI: 10.5061/dryad.p2ngf1vv4
title: IAP in Crohn's disease

description: The study examines the effect of cellular inhibitor of apoptosis protein
 (cIAP) inhibition on monocyte and PBMC cytokine production and cell death
 in healthy and Crohn's disease subjects. PBMCs from healthy controls
 were exposed to 4h of doses ranging from 0.1 to 1.0 micromolar of SMAC
 mimetics (SM) LCL161 and BV6 and co-incubated with LPS. SM consistently
 and dose-dependently reduced mRNA production of TNF-a, IL6 and IL-1b as
 determined by standard qPCR. A similar effect was seen on protein
 expression of TNF-a as determined by ELISA. To determine if this reduction
 in cytokine production was secondary to cell death, standard flow
 cytometry using ANX5 antibodies and nuclear dye 7AAD was performed. PBMCs
 were exposed from 4-16 hours to SM and/or LPS. Overall, neither of the
 stimulations LPS, SM or combined LPS/SM induced cell death. To determine
 if the effect was specific to LPS-induced signaling, PBMCs from healthy
 controls and from patients with inactive Crohn’s disease were exposed to
 LPS, MDP and entero-adhesive E. coli. Only LPS-induced TNF expression was
 inhibited by BV6. This effect was seen in both healthy controls and
 Crohn's patients. Since monocytes are a major source of TNF-a in the
 intestine and important for intestinal inflammation and treatment
 responses in Crohn’s disease we went on to examine how TNF-a production
 was affected in primary human monocytes. SMs reduced LPS-induced TNF-a
 mRNA expression in monocytes as determined by routine qPCR. We screened
 for different known modulators of IAP signaling (infliximab, zVAD, GSK
 872, Nec1s, and NSA) by exposing PBMCs to these inhibitors and/or LPS and
 SMs. Only the RIPK1 inhibitor Nec1s was able to reverse SM-inhibition of
 LPS-induced TNF mRNA expression. Further, by standard Western blotting, we
 found that SM decreased the p-p65/total p65 ratio suggesting downstream
 inhibition of NFkB. To test if RIPK1 is involved in LPS-mediated TNF-a
 production we tested TNF-a mRNA expression by routine qPCR in the monocyte
 cell line THP1. Experiments were done on THP1 wild-type cells and THP1
 RIPK1 K/O cells. RIPK1 K/O increased LPS-mediated TNF-a expression. In
 conclusion, SM can inhibit TNF-a, IL-1b and IL-6 expression without
 induction of cell death. The effect might in part be mediated RIPK1. The
 data set provides the qPCR, ELISA and flow cytometry data in the order
 mentioned above. Each experiment is labelled according to cells used,
 exposure to inhibitors, length of stimulation where appropriate. qPCR data
 are normalized to LPS-only exposed cells, protein levels as pg/mL, flow
 cytometry data are shown as % cells, and Western blot quantification as
 pixels (ImageJ). 
description: PBMCs and monocytes were havested from healthy subjects and
 patients with Crohn's disease. Cells were then cultured and exposed
 to SMAC mimetics LCL161 or BV6 as indicated and/or inhibitors of upstream
 or downstream pathways (infliximab,
 zVAD, GSK 872, Nec1s, and NSA) as indicated. Cytokine
 production (TNF-a, IL-6, IL-1b) was measured by qPCR and ELISA (only
 TNF-a) as indicated. Cytotoxiciity was assessed by flow cytomertry
 using ANX5 antibodies and nuclear dye
 7AAD as indicated. NFkB p65 and p-p65 expression was measured
 by standard Western blotting. Readouts were as follows: qPCR data normlized to LPS-only exposed cells,
 protein levels as pg/mL, flow cytometry data as % cells, and Western blot
 quantification as pixels (ImageJ). 
description: XML
description: # IAP in Crohn's disease ### Brief summary of dataset contents,
 contextualized in experimental procedures and results. The study examines
 the effect of cellular inhibitor of apoptosis protein (cIAP) inhibition on
 monocyte and PBMC cytokine production and cell death in heathy and
 Crohn's disease subjects. PBMCs from healthy controls were exposed to
 4h of doses ranging from 0.1 to 1.0 micromolar of SMAC mimetics (SM)
 LCL161 and BV6 and co-incubated with LPS. SM consistently and
 dose-dependently reduced mRNA production of TNF-a, IL6 and IL-1b as
 determined by standard qPCR. A similar effect was seen on protein
 expression of TNF-a as determined by ELISA. To determine if this reduction
 in cytokine production was secondary to cell death, standard flow
 cytometry using ANX5 antibodies and nuclear dye 7AAD was performed. PBMCs
 were exposed from 4-16 hours to SM and/or LPS. Overall, neither of the
 stimulations LPS, SM or combined LPS/SM induced cell death. To determine
 if the effect was specific to LPS induced signaling, PBMCs from healthy
 controls and from patients with inactive Crohn’s disease were exposed to
 LPS, MDP and entero-adhesive *E coli*. Only LPS-induced TNF expression was
 inhibited by BV6. This effect was seen in both healthy controls and
 Crohn's patients. Since monocytes are a major source of TNF-a in the
 intestine and important for intestinal inflammation and treatment
 responses in Crohn’s disease we went on to examine how TNF-a production
 was affected in primary human monocytes. SMs reduced LPS-induced TNF-a
 mRNA expression in monocytes as determined by routine qPCR. We screened
 for different known modulators of IAP signaling (infliximab, zVAD, GSK
 872, Nec1s, and NSA) by exposing PBMCs to these inhibitors and/or LPS and
 SMs. Only the RIPK1 inhibitor Nec1s was able to reverse SM-inhibition of
 LPS induced TNF mRNA expression. Further, by standard Western blotting, we
 found that SM decreased the p-p65/total p65 ratio suggesting downstream
 inhibition of NFkB. To test if RIPK1 is involved in LPS-mediated TNF-a
 production we tested TNF-a mRNA expression by routine qPCR in the monocyte
 cell line THP1. Experiments were done on THP1 wild-type cells and THP1
 RIPK1 K/O cells. RIPK1 K/O increased LPS-mediated TNF-a expression. In
 conclusion, SM can inhibit TNF-a, IL-1b and IL-6 expression without
 induction of cell death. The effect might in part be mediated RIPK1. The
 data set provides the qPCR, ELISA and flow cytometry data in the order
 mentioned above. Each experiment is labelled according to cells used
 (PBMCs, monocytes, THP1), then comes the readout (e.g., TNF), the
 inhibitor used (e.g., LCL161) and finally the method (e.g. PCR). These
 parameters are always mentioned in this order and separated by
 "/". qPCR data are normalized to LPS-only exposed cells, protein
 levels as pg/mL, flow cytometry data are shown as % cells, and Western
 blot quantification as pixels (ImageJ). Abbreviations used follow standard
 abbreviations as used in the text above. ## Description of the Data and
 file structure Results from each experiment is listed in the order
 explained in the abstract and in the above sections. The data set provides
 the qPCR, ELISA and flow cytometry data. Each experiment is labelled
 according to cells used (PBMCs, monocytes, THP1), then comes the readout
 (TNF, IL6, IL1b, cell death (ANX5+/AAD-), p65, p-p65), the inhibitor used
 (e.g., LCL161, BV6, Nec1s) and finally the method (PCR, ELISA, flow
 cytometry). These parameters are always mentioned in this order and
 separated by "/". qPCR data are normalized to LPS-only exposed
 cells, protein levels as pg/mL, flow cytometry data are shown as % cells,
 and Western blot quantification as pixels (ImageJ). Describe relationship
 between data files, missing data codes, other abbreviations used. Be as
 descriptive as possible. N/A ## Sharing/access Information Links to other
 publicly accessible locations of the data: N/A Was data derived from
 another source? No. If yes, list source(s): N/A