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PTH-106 Unravelling the Mechanism of Action of Infliximab in Crohn’S Disease and Healthy Controls Following in Vitro Culture with Blood-Enriched Dendritic Cells
  1. S T C Peake1,
  2. D Bernardo2,
  3. E Mann2,
  4. J Landy1,
  5. H Omar2,
  6. S C Knight2,
  7. A L Hart1
  1. 1IBD Unit, St Mark’s Hospital
  2. 2APRG, Imperial College, London, UK

Abstract

Introduction 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 elicited by T-cells following antigen presentation. DC also direct the 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 role in disease pathogenesis. Infliximab (IFX) is an effective treatment for CD but its mechanism of action is unclear. In this study we investigated the in-vitro effect of IFX on phenotype & ongoing cytokine production of human blood-enriched DC from patients with active CD and healthy controls (HC)

Methods Low density cells (LDC), enriched for DC, were obtained following Ficoll & Nycoprep separation of blood from patients with active CD (CDAI > 220) and HC. LDC were cultured (0.5x106cells/ml) with IFX (1.10&100μg/ml&basal) for 24hr. Activation marker (CD40, CD80, HLADR), TLR receptor (TLR2.4) and homing marker (CCR4.5.7.8.9.10, β7) expression was quantified by flow cytometry. Natural ongoing intracellular cytokine production (TNFα, TGFβ&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 & CCR9 intensity ratio (dose-dependent). There was a trend towards reduction in TLR2 and 4 expression (not statistically significant). IL-12 production by LDC from HC was increased following IFX culture. There was a marked reduction in TNFα and IL-12 in cell supernatant following culture with IFX

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 β7 expression on LDC from CD patients may suggest that DC have already been recruited to the site of mucosal inflammation. Further experiments are needed to confirm this. 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 most dramatic effect of IFX was a reduction in TNFα in the supernatant, which is likely to represent neutralisation of the cytokine

Disclosure of Interest None Declared.

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