Ichinose, M.Suzuki, N.Wang, T.Wright, J.A.Lannagan, T.R.M.Vrbanac, L.Kobayashi, H.Gieniec, K.Ng, J.Q.Ihara, S.Mavrangelos, C.Hayakawa, Y.Hughes, P.Worthley, D.L.Woods, S.L.2021-12-172021-12-172021Scientific Reports, 2021; 11(1):7200-1-7200-112045-23222045-2322https://hdl.handle.net/2440/133771The intestinal stroma provides an important microenvironment for immune cell activation. The perturbation of this tightly regulated process can lead to excessive inflammation. We know that upregulated Toll-like receptor 4 (TLR4) in the intestinal epithelium plays a key role in the inflammatory condition of preterm infants, such as necrotizing enterocolitis (NEC). However, the surrounding stromal contribution to excessive inflammation in the pre-term setting awaits careful dissection. Ex vivo co-culture of embryonic day 14.5 (E14.5) or adult murine intestinal stromal cells with exogenous monocytes was undertaken. We also performed mRNAseq analysis of embryonic and adult stromal cells treated with vehicle control or lipopolysaccharide (LPS), followed by pathway and network analyses of differentially regulated transcripts. Cell characteristics were compared using flow cytometry and pHrodo red phagocytic stain, candidate gene analysis was performed via siRNA knockdown and gene expression measured by qPCR and ELISA. Embryonic stromal cells promote the differentiation of co-cultured monocytes to CD11b(high)CD11c(high) mononuclear phagocytes, that in turn express decreased levels of CD103. Global mRNAseq analysis of stromal cells following LPS stimulation identified TLR signaling components as the most differentially expressed transcripts in the immature compared to adult setting. We show that CD14 expressed by CD11b⁺CD45⁺ embryonic stromal cells is a key inducer of TLR mediated inflammatory cytokine production and phagocytic activity of monocyte derived cells. We utilise transcriptomic analyses and functional ex vivo modelling to improve our understanding of unique molecular cues provided by the immature intestinal stroma.en© The Author(s) 2021. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Transcriptome; inflammationIntestinesMonocytesCells, CulturedStromal CellsAnimalsMice, Inbred C57BLHumansMiceEnterocolitis, NecrotizingInflammationCoculture TechniquesInfant, NewbornInfant, PrematureGene Regulatory NetworksTranscriptomeJournal article10.1038/s41598-021-86675-42021-12-17572216Suzuki, N. [0000-0001-9566-8828] [0000-0002-8402-4530]Lannagan, T.R.M. [0000-0002-8206-8898]Kobayashi, H. [0000-0002-1717-4870]Gieniec, K. [0000-0002-6518-5443]Ng, J.Q. [0000-0003-2302-2643]Hughes, P. [0000-0001-7324-3626]Worthley, D.L. [0000-0003-0374-9124]Woods, S.L. [0000-0002-8955-2017]