IAP in Crohn's disease
Data files
Sep 12, 2023 version files 27.59 KB
Abstract
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).
README: 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).
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Methods
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).
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