Nutritional regulation of intestinal development begins in utero with exposure to protein-rich amniotic fluid and continues after birth with human milk and/or infant formula1,10. These developmental processes are essential for continued cellular differentiation of the gut and development of mucosal immunity18. In the healthy term infant, the continuum of enteral stimulation is continued postnatally, whereas the preterm infant is typically supported on parenteral nutrition with limited enteral stimulation in the first few weeks of life3. In addition, postnatal exposure to environmental organisms in the neonatal intensive care unit and the routine use of antibiotics can lead to aberrant intestinal development, microbial colonization and risk of intestinal disease in the preterm infant3,6. Hence, it is imperative to understand the transcriptional responses of the preterm gut so that specific nutritional practices can be employed in order to optimize intestinal development.

Sensitive noninvasive tests will become critical tools in tailoring nutritional interventions, including pre- and probiotics, in order to promote intestinal development and maturation in the growing infant. As part of this effort, our laboratory has developed a molecular methodology that utilizes stool samples containing intact sloughed epithelial cells in order to noninvasively quantify intestinal gene expression profiles in both the human infant11,12 and adult19. Systems biology approaches, such as computational linear discriminant analysis (LDA), were used to identify the best single genes and two- to three-gene combinations for distinguishing term breast-fed versus formula-fed groups11. In addition, putative “Master” regulatory genes were identified using coefficient of determination (CoD) analysis11. Collectively, these approaches can be used to identify mechanistic pathways of intestinal development in the first few months of life and to assess the impact of nutrition and other environmental exposures on the microbiome in the developing gut12. In this study, we have extended upon our initial observations by unraveling previously inaccessible complexities in the term vs preterm infant intestinal transcriptome by non-invasively interrogating the infant intestine using RNA-Seq, rather than gene microarray11.

In order to develop a more comprehensive understanding of the complexity of transcriptome profiles in the intestine, we utilized neonatal stool samples containing intact sloughed epithelial cells and generated large-scale RNA-Seq genome profiles. For this purpose, poly A+ mRNAs were first copied into DNA sequences, randomly sheared, attached to linkers and directly sequenced. Sequences were compared with the reference human genome and the density of corresponding reads determined. Furthermore, using this form of global digital transcriptome profiling, we documented the host transcript abundance and alternative splicing in healthy term infants at 12-weeks postnatal age and extremely preterm infants (24–30 weeks gestational age) at 2–5 weeks postpartum. Although the precise origin of exfoliated cells is not known, results from our previous study11 and reported herein, indicate that genes associated with discrete epithelial cell types (absorptive enterocytes, goblet cells, enteroendocrine cells and Paneth cells) are detectable. Thus, it is likely that transcriptome signatures of both the small and large intestine can be monitored over time.

The examination of global alterations in gene expression offers insight into the effects of premature birth and the resultant influence of environmental exposures uniquely experienced by the preterm infant (e.g. antibiotics, other medications, prolonged period of parenteral nutrition) on intestinal mRNA profiles. RNA-Seq (validated by qPCR) revealed that following an enrichment process, reads from stool derived RNA are of human origin. Unlike RNA extracted from human cell cultures or surgical specimens, where the quality and quantity of RNA is usually high and RNA degradation can be controlled with tissue handling20, infant stool represents a unique biological sample. Typically, expressed host transcripts consist of a narrow stretch of RNA that is rarely longer than several hundred bp. While exon-exon junctions in principle can be detected, we noted that less than 5% of transcripts exhibited their splice variants. In this respect, non-invasive gene expression analysis using infant stool appears to be more challenging than analysis of formalin fixed, paraffin embedded (FFPE) tissue21,22,23. Unlike FFPE tissue blocks that can yield large quantities of fragmented DNA and RNA sufficient to explore complete topologies of expressed genes and local properties of DNA, infant stool requires careful enrichment of human RNA. Our experience with gene expression in the infant gut indicates that next generation sequencing provides a robust non-invasive glimpse into the host transcriptome. We expect that the development of novel methodologies for library preparation will allow us to further elucidate the physiology of the developing infant intestine.

For obvious reasons, directly examining the host epithelium in the human preterm infant is unlikely, as intestinal biopsies are not routinely performed unless medically indicated. Therefore, noninvasive methodologies currently provide us the best snap shot of infant gene expression11 and host-microbe dynamic interactions12 in an in vivo setting. Although future well-controlled studies are needed to evaluate environmental/dietary exposures, this study highlights the potential of using the described noninvasive technology. We evaluated genes that were over-expressed in preterm vs. term (Table 1) or term vs. preterm intestine (Table 2). Although none of the infants were clinically ill at the time the stool samples were collected, one of the major categories of genes overexpressed in preterm vs. term samples was immune function. Because of the vast number of exfoliated epithelial cells shed from the lining of the intestine on a daily basis, it is unlikely that changes in cell composition, e.g., contribution of inflammatory cells from the submucosa, directly contributed to alterations in gene expression. Several cytokines, including IL1α and IL-33 were up-regulated in preterm vs. term. In addition, several genes that regulate the expression of cytokines and other immune genes were expressed at 3- (NFKB1α) to 6-fold (CASP1) higher levels in preterm vs. term infant exfoliated cells. Previous studies have shown that immortalized cells isolated from fetuses (H4 cells) or tissue explants from fetuses mount a more robust proinflammatory cytokine response (IL-8) after inflammatory stimulation with lipopolysaccharide or IL1β than cells from adult tissue (Caco-2) or explants from older children24. The excessive inflammatory response of the immature intestine is in part due to a developmental under-expression of IkB25 as well as overexpression of the NFkB/MyD88 innate inflammatory genes (TLR2, TLR4, MyD88, TRAF-6, NFkB1 and IL-8) and under-expression of negative regulator genes (SIGIRR, IRAK-M, A-20 and TOLLIP) in fetal intestine relative to older children8. Thus, it appears that activation status of the intestinal innate immune response may contribute to excessive inflammation in the immature intestine in response to colonizing bacteria, which is a hallmark of NEC8.

In exfoliated cells of term infants, up-regulated immune genes were associated with balancing the immune system, e.g. promoting T-cell development (LCP2; 3.6-fold greater in term than preterm), while inhibiting macrophage activation (LENG9; 16-fold greater in term than preterm). The majority of genes were involved in cell turnover, by regulating proliferation and apoptosis. One of the most highly differentially-expressed genes was an anti-apoptotic factor (MTRNR2L6), which was 5-fold higher in term than preterm. Another notable gene is SP3 (~2-fold higher in term than preterm), which is a transcription factor that can be regulated through short-chain fatty acid - acetylation, potentially supporting the role of these products of microbial metabolism in regulating normal gut growth in term infants26.

In summary, we provide incontrovertible evidence that whole-genome sequencing of stool-derived RNA can be used to generate a global transcriptome gene expression signature in neonates. We have also compared for the first time, the intestinal global transcriptome in individual term and preterm and pooled term infants. Our findings provide insight into the global patterns of gene expression that vary in exfoliated epithelial cells of term and preterm infants. We anticipate that the described noninvasive RNA sequencing-based approach will enable elucidation of how the bedside clinical management of an extremely preterm infant population influences intestinal gene expression. With this understanding, dietary and medical practices can be evaluated that optimally promote intestinal development and, ideally, identify those clinical practices that approximate as closely as possible the development of the healthy, term breast fed infant. The possible uses of non-invasive high-throughput RNA sequencing data are vast and include early detection (screening), monitoring disease progression, risk assessment and diet-dependent interaction between gut microbiota and host epithelium. We propose that stool samples containing exfoliated cells have the potential for generating comprehensive, diagnostic gene sets for the noninvasive identification/prediction of different intestinal phenotypes in infants.