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P327. Unravelling the mechanism of action of infliximab in Crohn's disease and healthy controls following in vitro culture with blood-enriched dendritic cells

S. Peake1, D. Bernardo2, E. Mann2, J. Landy1, H. Omar2, S. Knight2, A. Hart1, 1St Mark's Hospital, IBD Unit, Harrow, United Kingdom, 2Imperial College, APRG, London, United Kingdom

Background

Dendritic cells (DC) play a key role in discriminating between commensal microorganisms and harmful pathogens. DC phenotype and cytokine production determine the type of immune response (immunity/tolerance) elicited by T-cells following antigen presentation. DC also direct effector T-cells to target tissues to perform their function via imprinting tissue-specific homing markers. In CD, dysregulation of the immune response to gut microbiota & aberrant immune cell trafficking play a central role in disease pathogenesis. Infliximab (IFX) is a widely used and effective treatment for CD. Its mechanism of action is unclear.

We investigated the effect of IFX on phenotype & ongoing cytokine production of human blood-enriched DC from patients with active CD & HC.

Methods

Low density cells (LDC), which are enriched for DC, were obtained following Ficoll & Nycoprep separation of blood from patients with active CD (CDAI >220) & HC.

LDC were cultured (0.5×106 cells/ml) with IFX (1, 10, 100 µg/ml and basal) for 24hr. Activation marker (CD40, CD80, HLADR), TLR receptor (TLR2, 4) & homing marker (CCR4, 5, 7, 8, 9, 10, β7) expression was quantified. Natural ongoing intracellular cytokine production (TNFα, TGFβ and IL-6, -10, -12, -15) was assessed via intracellular staining and flow cytometry. Cytokine secretion was measured on cell-free culture supernatants via Multiplex.

Unpaired t-test and one-way ANOVA statistical analyses were applied.

Results

TNFα and IL-6 were increased in culture supernatants from CD although their intracellular ongoing cytokine production was decreased. LDC from CD had decreased β7 (gut-homing integrin) expression.

Following IFX culture, LDC decreased β7 expression (HC only) and reduced CCR9 intensity ratio. There was a trend towards reduction in TLR2 and 4 expression in CD (not statistically significant). IL-12 production by LDC from HC was increased following IFX culture.

There was a marked reduction in TNFα in cell supernatant following culture with IFX & a reduction in IL-2.

Conclusion

Increased TNFα and IL-6 in culture supernatants from CD patients coupled with a decrease in ongoing production by DC suggests a negative cytokine feedback system.

Reduced expression of CCR9 and elevated production of IL-12 in LDC cultured with IFX, shows reduced affinity for gut-homing and increased immunogenicity and may suggest a possible mechanism for IFX-induced paradoxical inflammation.

The reduction in TNFα in the supernatant following IFX culture is likely to represent cytokine neutralization.