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P043. Induction of mucosal inflammation by Damage-Associated Molecular Patterns (DAMPs): Intestinal Epithelial Cells (IEC)-derived IL‑1α as a new player in the pathogenesis of IBD

M. Scarpa1, S. Kessler2, T. Sadler2, C. Fiocchi2, E. Stylianou2

1Istituto Oncologico Veneto IOV I.R.C.C.S., Padova, Italy; 2Cleveland Clinic Foundation, Pathobiology/NC22, Cleveland, United States

Background: Much of the work on IBD pathogenesis is focused on the role of microbial factors, such as Pathogen-Associated Molecular Patterns (PAMPs) from the gut microbiota. However, an inflammatory response can be induced not only by PAMPs, but also intracellular alarm signals arising from non-physiological cell death, tissue damage, or stress, the so called DAMPs. Necrotic cell death releases several endogenous pro-inflammatory DAMPs, including proteins, DNA, RNA, extracellular matrix components and lipid mediators, all of which contribute to the pathogenesis of injury and exacerbate an initial insult. Moreover, sterile inflammation secondary to ongoing tissue damage is thought to contribute to many chronic diseases. We hypothesize that DAMPs may also play a role in the intestinal inflammatory response.

Methods: Necrotic cell (NC) supernatants were prepared from HT-29 cells and freshly isolated human IEC, suspended at 108 cells/ml and subjected to 5 freeze/thaw cycles. Cells were then centrifuged and supernatants collected. Human Intestinal Fibroblasts (HIF) were used as targets of DAMP-derived signals by exposing them to NC supernatants for 24 h. IL‑6 and IL‑8 levels in the cultures were measured by ELISA. Blockade with specific antibodies to TLR2, TLR4, RAGE, IL‑1RI, IL‑1α and IL‑1β was performed by incubating these with HIF or NC supernatants.

Results: HIF secreted large amounts of IL‑6 and IL‑8 (up to 120,000 pg/ml) in response to NC supernatants from HT29 cells or freshly isolated IEC. Neutralization studies revealed that a functional IL‑1RI, but not TLR2, TLR4 or RAGE, was critical for IL‑6 and IL‑8 production by HIF. Both IL‑1RI agonists, IL‑1α and IL‑1β, could trigger IL‑6 and IL‑8 production but only IL‑1α, and not IL‑1β, was obligatory for the induction of this effect with NC. Control experiments excluded a role of histones, chromatin, mitochondria, uric acid, HMGB1, IL‑33, caspase‑1 and ER stress in the response to necrotic IEC.

Conclusions: These results show the unexpected, highly selective and potent pro-inflammatory activity of IEC-derived IL‑1α, an intracellular non-secreted cytokine that belongs to the “alarmin” group of DAMPs. Thus, IL‑1α emerges as a potential major inducer of mucosal inflammation in all gastrointestinal diseases where IEC death occurs, such as IBD.