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PReterm Enteroids to determine the Mechanism of Necrotising EnteroColitis

Periodic Reporting for period 1 - PREMNEC (PReterm Enteroids to determine the Mechanism of Necrotising EnteroColitis)

Reporting period: 2018-07-01 to 2020-06-30

What is the problem/issue being addressed?
NEC, a serious inflammatory mediated bowel disease, is a leading cause of death in preterm infants. NEC affects ~10% of preterm infants (<32 weeks gestation), with ~30% requiring surgery, and a mortality rate of ~20%, with significant morbidity in survivors (e.g. adverse cognitive outcome and cerebral palsy). The resulting societal cost of NEC per infant is >€124,600 in Europe[1]. Previous work[2], including my research[3–7], have shown associations between bacterial colonisation and NEC onset. I recently showed altered microbial development in NEC[3] and sepsis[7], with Bifidobacterium colonisation protective of disease onset. However, because such studies are observational and dependent on non-invasive sampling (e.g. stool), the analysis is limited to associations and the specific mechanisms of potential protection remain unknown.

A major obstacle preventing mechanistic and translational research is the lack of robust models for studying the preterm gut, especially in recapitulating necrosis and gut immaturity (e.g. poor barrier integrity), which are main factors involved in NEC onset. To overcome this long-standing hurdle, I will employ an ex vivo model derived from primary preterm human intestinal enteroids (PHIEs) that allows co-culture with specific enteric organisms associated with the cause or prevention of NEC. The project will directly analyse NEC and non-NEC resected tissue (otherwise discarded) using a powerful systems biology approach, overcoming the limitations of any single technique. The PHIE model will facilitate an understanding of the underlying mechanisms of the disease and allow microbial mediated strategies for disease prevention to be interrogated (e.g. a Bifidobacterium probiotic to promote gut maturation).

1. Ganapathy V et al. 2012. Breastfeed Med. 7:29–37. 2. Warner BB et al 2016. Lancet 387:1928–36 3. Stewart CJ et al. 2016. Microbiome. 4:67. 4. Stewart CJ et al. 2013. Arch Dis Child Fetal Neo. 98:F298-303. 5. Stewart CJ et al. 2012. Acta Paediatr. 101:1121–7. 6. Stewart CJ et al. 2013. PLoS One 8:e73465. 7. Stewart CJ et al. 2017 Microbiome. 5:75.


Why is it important for society?
NEC is the leading cause of death in preterm infants and survivors have long-term morbidity, including short bowel syndrome and neurological developmental delays. This places significant challenges on the patient and the family, as well as the resulting short- and long-term societal costs.


What are the overall objectives?
1. Use multi-omic analysis to directly characterise tissue and determine differences at the site of necrosis compared to adjacent healthy tissue and gut resections from non-NEC preterm infants
2. Isolate crypt cells from resected tissue to establish PHIE lines from preterm infants
3. Use PHIE co-culture model to interrogate the potential mechanisms of NEC and prevention strategies


Conclusions of the action
Objective 1: I have sequenced the microbiome from NEC and non-NEC preterm infants and showed NEC cases have lower diversity, significantly altered microbiome profiles, and increased Proteobacteria. This has been published in a respected peer-reviewed journal - https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-019-1426-6. I have also generated data using RNA-seq of fresh tissue showing that host gene expression at the site of NEC is different to location of insult in non-NEC surgeries within preterm infants (unpublished data).

Objective 2: I have established 10 preterm human intestinal enteroid lines and frozen additional tissue for future generation of lines. The lines generated range from gestational ages of 23 – 33 weeks.

Objective 3: I have employed the PHIE co-culture model and found stool from NEC infants results in barrier breakdown and translocation of bacteria, whereas control stool does not. Work investigating the role of specific bacteria of interested was halted due to covid.
Objective 1: 16S rRNA gene sequencing was performed on formalin fixed parrafin embedded tissue from NEC and non-infants in order to profile the microbiome at the site of disease (rather then e.g. stool samples). This analysis found NEC cases have lower diversity, significantly altered microbiome profiles, and increased Proteobacteria. This supports that the microbiome is associated with disease progression and validates previous work using non-invasive stool samples. This has been published in a respected peer-reviewed journal (see dissemination below)

Objective 2: This aimed to, for the first time, culture human intestinal enteroids from preterm infants. I am delighted to report that I have established 10 preterm human intestinal enteroid lines and frozen additional tissue for the future generation of lines. The lines generated range from gestational ages of 23 – 33 weeks and will provide a hugely valuable resource for my future work and for the work of others in the field. Indeed, in light of covid, we are using the lines as a model to study covid infection.

Objective 3: While this final objective was cut short due to covid, I have employed the PHIE co-culture model and found stool from NEC infants results in barrier breakdown and translocation of bacteria, whereas control stool does not. I also performed RNA-seq on the preterm HIEs and found lines derived from preterm infants with NEC is different to non-NEC infants, suggesting features of NEC pathogenesis may be recapitulated in the model. I also found preterm lines differ from adult lines, adding further evidence for using these lines in subsequent work.


Dissemination
During the course of this 2 year Fellowship I have published >20 peer reviewed publications and spoke at several leading national and international meetings. The results related specifically to this Fellowship were published as research articles, reviews, a BioRxiv pre-print, and book chapters, as detailed below:

Stewart CJ, Fatemizadeh R, Parsons P, Lamb CA, Shady DA, Petrosino JF, Hair AB. Using formalin fixed paraffin embedded tissue to characterize the preterm gut microbiota in necrotising enterocolitis and spontaneous isolated perforation using marginal and diseased tissue. BMC Microbiology. 2019; 4;19(1):52.

Masi AC, Stewart CJ. The role of the preterm intestinal microbiome in sepsis and necrotising enterocolitis. Early Human Development. 2019; 138:104854

Fofanova TY, Stewart CJ, Auchtung JM, Wilson RL, Britton RA, Grande-Allen KJ, Estes MK, Petrosino JF (2019) A novel human enteroid-anaerobe co-culture system to study microbial-host interaction under physiological hypoxia. bioRxiv. doi: https://doi.org/10.1101/555755

Stewart CJ , Ramani S, Estes MK (2019) Establishing human intestinal enteroid/organoid lines from preterm infant and adult tissue. In: Innate Lymphoid Cells: Methods and Protocols. Springer Nature. United Kingdom. In Press.

Nelson A, Stewart CJ (2019) Microbiota analysis using sequencing by synthesis: from library preparation to sequencing. In: Innate Lymphoid Cells: Methods and Protocols. Springer Nature. United Kingdom. In Press.
Schematic workflow to generate preterm human intestinal enteroids (HIEs) and perform experiments