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DOP014. The role of optineurin in macrophage cytokine secretion and Crohn's disease

T.S. Chew1, A.M. Smith1, G.W. Sewell1, N.R. O'Shea1, S.L. Bloom2, A.W. Segal1, 1University College London, Centre for Molecular Medicine, London, United Kingdom, 2University College London Hospital, Department of Gastroenterology, London, United Kingdom

Background

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. We previously showed that macrophages from CD patients secrete lower levels of proinflammatory cytokines due to vesicle trafficking defects [1], which may be responsible for the deficient neutrophil recruitment in bowel [2] and decreased bacterial clearance observed in CD patients compared to healthy controls (HC) [1] [2]. To investigate the low cytokine secretion, we performed transcriptomic analysis in CD and HC macrophages and identified a subgroup of CD patients with low expression of optineurin (OPTN).

Methods

OPTN was knocked down in THP-1, acute monocytic leukaemic cells by electroporation with siRNA then stimulated with heat killed E. coli (HkEc) and Pam3. Optn−/− and Optn+/+ littermate mice were gavaged with Citrobacter rodentium, which induces a large bowel colitis. Optn−/− and Optn+/+ bone marrow derived macrophages were stimulated with E. coli to investigate cytokine secretion and vesicle trafficking. Confocal microscopy of TNF and early endosome antigen 1 (EEA1) was performed in Optn−/− and Optn+/+ macrophages.

Results

siRNA knockdown resulted in reduction of OPTN protein levels by 60% and after stimulation resulted in significantly reduced TNF release with HkEc (p = 0.02) and Pam3 (p < 0.01) compared to mock electroporation. C. rodentium colitis resulted in greater weight loss (p < 0.0001), mortality (p = 0.007) and colitis scores (p = 0.04) in the Optn−/− mice. This was associated with decreased serum TNF (p = 0.03) and IL6 (p = 0.02) levels in the Optn−/− mice despite similar levels of bowel TNF and IL6 mRNA expression. Optn−/− macrophages secrete less TNF (p = 0.006) and IL6 (p = 0.02) despite similar levels of mRNA expression. Confocal microscopy of TNF and EEA1 showed decreased colocalisation of TNF and the endosomal compartment in Optn−/− macrophages (p = 0.04) after bacterial stimulation.

Conclusion

OPTN has a known role in vesicle trafficking and bacterial handling [3] [4]. The C. rodentium colitis model shows that OPTN plays a role in enteric bacteria handling, most likely via defective cytokine secretion of tissue resident macrophages. This defect is likely due to impaired trafficking of TNF from the Golgi complex to the endosomal compartment. Diminished OPTN expression in humans may increase the risk of developing CD via impaired cytokine secretion and defective bacterial clearance.

1. Smith, A.M. et al., (2009), Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease, Journal of Experimental Medicine, 1883–97.

2. Marks, D.J. et al., (2006), Defective acute inflammation in Crohn's disease: a clinical investigation, Lancet, 668–78.

3. Sahlender, D. A. et al., (2005), Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis, Journal of Cell Biology, 285–95.

4. Wild, P. et al., (2011), Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth, Science, 228–33.