IAP in Crohn's disease

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).