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Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury

ABSTRACT: Intestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Small Intestine Epithelium

DISEASE(S): Ischemia

SUBMITTER: Anna M Kip

LAB HEAD: Kaatje Lenaerts

PROVIDER: PXD022999 | Pride | 2021-01-11

REPOSITORIES: Pride

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			Action	DRS
	191011_MK_SIO14_P1_DM1.raw	Raw
	191011_MK_SIO14_P1_DM2.raw	Raw
	191011_MK_SIO14_P1_GM1.raw	Raw
	191011_MK_SIO14_P1_GM2.raw	Raw
	191011_MK_SIO14_P2_DM1.raw	Raw

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Project description:Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM antibodies recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild type C57BL/6 mice. We identified a novel IgM monoclonal antibody (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1-/- mice. Monoclonal Ab B4 was found to specifically recognize mouse annexin IV. Pre-injection of recombinant annexin IV blocked IR injury in wild type C57BL/6 mice, demonstrating the requirement for recognition of this protein in order to develop IR injury in the context of a complex natural antibody repertoire. Humans were also found to exhibit IgM natural antibodies that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target antigen that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury. Keywords: Natural antibodies, Annexin, Ischemia Reperfusion, Inflammation, Complement In the study presented here, a total of 4 custom spotted antigen slides were hybridized with known antibodies.

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