Abstract

Objective

   Necrotizing enterocolitis (NEC) is the most frequent life-threatening
   gastrointestinal disease experienced by premature infants in neonatal
   intensive care units all over the world. The objective of the present
   study was to take advantage of RNA-Seq data from the analysis of
   intestinal specimens of preterm infants diagnosed with NEC. Function
   enrichments with Gene Ontology and the Kyoto Encyclopedia of Genes and
   Genomes were used to analyse previous data in order to identify
   biological and functional processes, which could provide more insight
   into the pathogenesis of NEC in infants.

Results

   Gene set enrichment analysis indicated that the most significant
   biological pathways over-represented in NEC neonates were closely
   associated with innate immune functions. One of the striking
   observations was the highly modulated expression of inflammatory genes
   related to the IL-17 pathway including such as pro-inflammatory
   cytokines (CXCL8), chemokines (CXCL5 and CXCL10) and antimicrobials
   (DEF5A, DEF6A, LCN2, NOS2) in the intestine of neonates diagnosed with
   NEC. Interestingly, the increase in IL-17 expression appeared to be
   under the IL-17F form, as reported in Crohn's disease, another
   inflammatory bowel disease. Further investigation is thus still needed
   to determine the precise role of IL-17F and its downstream targets in
   NEC.

   Keywords: Human intestine, Preterm newborn, Transcriptomics, Gene
   expression, Interleukin-17

Introduction

   Necrotizing enterocolitis (NEC) is the most common life-threatening
   condition of premature infants occurring in neonatal intensive care
   units [[35]1–[36]3]. This gastrointestinal disease is characterized by
   severe intestinal inflammation, intestinal necrosis and high morbidity
   [[37]4]. The infants who survive NEC are at a higher risk for
   developing short bowel syndrome, impaired growth and neurodevelopmental
   outcomes [[38]5, [39]6]. Several epidemiological investigations have
   suggested that preterm birth, enteral feeding and abnormal bacterial
   colonization are likely to play major roles in the pathogenesis of NEC
   [[40]7, [41]8]. However, the precise mechanisms leading to the
   development of NEC remain to be elucidated.

   One of the greatest challenges for neonatologists is to identify early
   reliable clinical signs and symptoms of NEC [[42]1, [43]2]. In this
   context, the ahead of time clinical manifestations of NEC are
   relatively nonspecific and at diagnosis NEC is often already at an
   advanced stage due first to the initially insidious condition and then
   to the fierce progression of the disease [[44]9, [45]10]. One strategy
   to prevent or treat NEC would be to develop an early diagnostic tool
   allowing the identification of preterm infants either at risk of
   developing NEC or at the onset of symptoms to aid in the diagnostic
   dilemma and treatment.

   Recently, we used the high-throughput sequencing of RNA transcripts
   (RNA-Seq) approach to determine the complete gene expression profiles
   of ileal specimens, which were resected from preterm infants diagnosed
   with NEC vs non-NEC conditions [[46]11]. This recently important work
   has led to the identification of potential biomarkers for the
   prediction of the disease. The objective of the present study was to
   provide new insights into the molecular mechanisms of NEC
   pathophysiology by conducting function enrichment analyses with Gene
   Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG).
   Our analysis revealed that immune and inflammatory responses were
   strongly modulated in the small intestine of neonates with NEC,
   supporting the finding that NEC development is related to the
   immaturity of the intestinal mucosa challenged by microbial dysbiosis
   [[47]12, [48]13]. Interestingly, we also observed that several
   interleukin-17 (IL-17) target genes, including chemokines and
   antimicrobial proteins, were strongly modulated in the small intestine
   of neonates with NEC. Pro-inflammatory IL-17 has been linked to the
   pathogenesis of diverse autoimmune and chronic inflammatory diseases
   while being essential for host defense against microbial colonization
   [[49]14, [50]15]. Our results suggest a role for IL-17 signaling,
   specifically IL-17F, in the development of NEC.

Main text

Materials and methods

Study population and informed consent

   As detailed in the previous report [[51]11], the present collaborative
   study recruited premature infants from neonatal intensive care units at
   the Centre Hospitalier Universitaire de Sherbrooke (Sherbrooke, QC,
   Canada), Erasmus MC-Sophia Children’s Hospital (Rotterdam, The
   Netherlands), Children’s Hospital of Eastern Ontario (Ottawa, ON,
   Canada) and Hôpital Pierre Zobda-Quitman (Fort-de-France, Martinique),
   between October 2008 and May 2013. After the approval of the
   Institutional Review Committee for the use of human material in each
   center, written informed consent from parents or guardians was obtained
   for each patient.

   Premature infants who had undergone bowel resection were eligible for
   the study. The diagnosis and staging of NEC were based according to the
   Bell's criteria [[52]9]. Freshly resected intestinal specimens taken
   from the ileum were conserved in RNAlater (Ambion) before RNA
   extraction. Preterm patients who had undergone bowel resection for
   stage III acute NEC were referred to as NEC, and preterm patients who
   had undergone resection for diseases other than NEC were included as
   control patients (CTRL) as detailed in [[53]11].

Sample preparation and RNA sequencing

   The procedures for sample preparation, library preparation and
   sequencing, and data analysis were performed as previously described by
   Tremblay et al. [[54]11].

Functional pathway enrichment analysis

   The bioinformatics software DAVID 6.8 ([55]https://david.ncifcrf.gov/)
   [[56]16] was used to perform Gene Ontology (GO) analysis and KEGG
   pathways enrichment analysis of differentially expressed genes (DEGs)
   identified in the pathogenesis of NEC.

Data validation by quantitative polymerase chain reaction (qPCR)

   All qPCR reactions were performed as previously described [[57]11]. The
   genes investigated were chemokine (C-X-C motif) ligand 8 (CXCL8) and 10
   (CXCL10), alpha-defensin 5 (DEFA5) and 6 (DEFA6), interleukin-17A
   (IL-17A) and IL1-7F (IL-17F), interleukin 6 (IL6), lipocalin 2 (LCN2),
   nuclear factor kappa B subunit 1 (NFKB1), regenerating islet-derived 3
   alpha (REG3A), ribosomal protein lateral stalk subunit P0 (RPLP0),
   ribosomal protein 23 (RPS23), and tumor necrosis factor (TNF). Primers
   (listed in Additional file [58]1: Table S1) were generated using the
   primer formation software Primer3 ([59]http://bioinfo.ut.ee/primer3).
   Differences in gene expression were evaluated by comparing the
   expression of control and NEC samples using the equation
   R = (E[target])^∆Ct target/(E[reference])^∆Ct reference) [[60]17].
   Samples were normalized to a set of 2 reference genes, RPLP0 and RPS23
   [[61]18, [62]19].

Results

RNA-Seq analysis and identification of DEGs

   The data for RNA-Seq analysis on ileum of preterm infants with NEC vs
   without NEC (CTRL) have been deposited at the National Center for
   Biotechnology Information's Gene Expression Omnibus and are accessible
   through GEO Series accession number [63]GSE64801
   ([64]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse64801). In
   total, 804 DEGs were identified (p < 0.05), 383 upregulated and 421
   downregulated [[65]11].

GO and KEGG pathway analyses in NEC samples

   In the previous study, IPA analysis revealed an impaired immune
   response in the intestine of NEC neonates where several related
   pathways and biological functions were found to be altered in the
   samples of NEC infants [[66]11]. To gain more insight in the function
   pattern of the DEGs obtained previously, GO and KEGG pathway analyses
   were conducted by DAVID. Among the 151 GO "Biological Process" terms
   enriched in DEGs, the top 8 terms, which were selected based on their
   highest p values, were highly associated with innate and adaptive
   immune as well as inflammatory responses (Fig. [67]1a). The same
   relationship with the immune responses was also noted with KEGG
   analyses (Fig. [68]1b). Intriguingly, a further detailed analysis of
   the DEGs revealed that many of those genes were highly associated with
   the signature target genes of IL-17 (Fig. [69]2), suggesting a role for
   this cytokine in the pathogenesis of NEC. Among these IL-17 target
   genes, changes in expression of pro-inflammatory cytokines, chemokines,
   antimicrobials and matrix metalloproteinase were identified.

Fig. 1.

   [70]Fig. 1
   [71]Open in a new tab

   Functional pathways in NEC samples. The negative logarithm of p-values
   (Fisher's test) calculated for the 8 most significant pathways
   overrepresented in small intestinal NEC samples compared to control
   non-NEC samples for GO Biological process (a) and KEGG (b) pathways.
   Dotted line: -Log 0.05 corresponds to 1.3

Fig. 2.

   [72]Fig. 2
   [73]Open in a new tab

   Association of DEG identified in NEC samples and IL-17 target genes.
   IL-17 receptor (IL-17R) can bind to IL-17A, IL-17F or IL-17A/F to
   activate various signaling pathways leading to the regulation of IL-17
   targets genes involved in tissue remodeling and anti-microbial activity
   as well as mediators of inflammation as adapted from [[74]15, [75]20].
   Genes identified as being significantly modulated in NEC samples
   compared with control non-NEC samples are labeled in green and red
   while those for which no significant differences were notes are labeled
   in grey

Validation of the gene expression profile in NEC samples

   To further investigate the gene expression profile identified in
   RNA-Seq analyses, we used qPCR to test representative DEGs known to be
   involved in IL-17 signaling in NEC samples as depicted in Fig. [76]2.
   As shown in Fig. [77]3a, qPCR analyses confirmed the upregulation of
   many of the IL-17 downstream genes in NEC samples. Incidentally,
   transcript expression of the two main IL-17 cytokines, IL-17A and
   IL-17F was also investigated and revealed a significant induction of
   IL-17F in NEC samples (Fig. [78]3b) confirming alterations in the IL-17
   signature gene profile in the intestinal samples of patients with NEC.

Fig. 3.

   Fig. 3
   [79]Open in a new tab

   Differential expression of IL-17-related genes assessed in intestinal
   NEC and non-NEC control samples by real-time qPCR. Transcript levels of
   IL-17 target genes (a) and IL17A and IL17F transcripts (b) in NEC
   relative to control. Results are expressed as a scatter dot plot
   showing individual data and as median with 95% confidence interval. *
   and ** are p < 0.05 and p < 0.01, respectively, using the one sample
   Wilcoxon test

Discussion

   NEC is a complex and a potentially fatal disease that remains a major
   cause of morbidity in neonates. Although significant effort has been
   made on the further understanding of this disease, no effective
   treatment or further diagnostic tools have been discovered [[80]3]. In
   the present study, we performed GO and KEGG pathway analyses on RNA-seq
   data that was previously done with the aim to further deepen our
   knowledge of the molecular mechanisms underlying NEC pathogenesis. GO
   and KEGG pathway analyses showed strong enrichment in genes involved in
   innate and adaptive immune responses, consistent with a
   disproportionate inflammatory response following an exposure to luminal
   microorganisms [[81]11]. Indeed, excessive inflammatory response and
   overexpression of proinflammatory cytokines are the main
   pathophysiological characteristics of NEC that contribute to damage the
   intestinal epithelium [[82]13]. Moreover, by reviewing in detail all
   enriched genes in these pathways, we highlighted several target genes
   of the IL-17 pathway [[83]20], which were modulated in NEC, suggesting
   a role for IL-17 signaling in the pathophysiology of this severe
   inflammatory disease.

   The IL-17 family of cytokines consists of six structurally related
   proteins, IL-17A through IL-17F [[84]15]. IL-17A and IL-17F are the
   most related members and they are usually secreted as homodimers or
   heterodimers by various innate immune cells [[85]21] including those
   residing in the gut [[86]22]. One of the major functions of IL-17 is to
   provide a protective inflammatory response to the host against
   pathogens [[87]23–[88]25]. However, despite the protective effects of
   IL-17 during infection, there is emerging evidence that dysregulated
   IL-17 expression could worsen the gravity of some diseases [[89]26,
   [90]27], namely NEC [[91]28, [92]29]. It is noteworthy that IL-17
   exerts only modest activity by its own, its immunomodulatory role
   arising from synergistic action with other cytokines [[93]24]. In
   preterm infants, IL-17 level was found to be increased after red blood
   cell transfusion that could lead to a higher risk of developing NEC
   [[94]30]. Moreover, an increased IL-17 level has been related to
   impairment of enterocyte tight junctions and increased epithelial cell
   apoptosis through Toll-like receptor 4-dependent Th17 polarization in
   mouse and human NEC models [[95]31] consistent with its key role in
   host defence [[96]24]. Indeed, as reviewed recently, although evidence
   for the involvement of adaptative immunity in NEC initiation and
   perpetuation is still insufficient, the potential for a pathogenic role
   of Th17 responses is pointed out [[97]25]. In our study, we showed that
   the transcript expression of IL-17F, but not IL-17A, is upregulated in
   the intestine of premature infants diagnosed with NEC. Distinct roles
   between IL-17A and IL-17F have been previously demonstrated in
   IL-17F-deficient mice [[98]32, [99]33]. Upregulated IL-17F expression
   was also reported in intestinal samples obtained from patients with
   active Crohn's disease [[100]34]. In the experimental colitis model
   induced by DSS, IL-17F^−/− mice were protected, whereas IL-17-knockout
   mice showed a more severe condition [[101]32]. In addition, a recent
   study showed that a potential genetic variant of IL-17F, but not
   IL-17A, was associated with an increased severity of NEC [[102]35].
   Taken together, these observations support a potential involvement for
   the upregulated expression of IL-17F in the development of NEC. Based
   on the similarity between NEC and Crohn's disease [[103]11], one may
   speculate that as proposed for Crohn's disease [[104]36], the
   development of specific inhibitors for IL-17F could become a potential
   option for treating NEC although it is important to consider that other
   organs such as the lung or the brain could also be affected by IL-17
   blockade [[105]37, [106]38].

   Upregulation of IL-17 controls the expression of inflammatory genes in
   the majority of non-hematopoietic cells [[107]39]. The trademark of
   IL-17 signaling is induction of pro-inflammatory cytokines, chemokines,
   antimicrobial proteins and inflammatory effectors, to maintain mucosal
   barrier integrity against infections [[108]20, [109]40]. Among the
   IL-17 signature genes, we have found the transcription factor NFKB1,
   chemokines such as CXCL8, CXCL10 as well as a plethora of antimicrobial
   molecules or regulators (DEFA5, LCN2, NOS2, REG3A) to be upregulated in
   NEC intestinal samples as compared to control. These observations
   suggest that IL-17 could be involved in a protective mucosal reaction
   towards pathogenic microorganisms in the intestine of NEC infants.
   Unexpectedly, we observed that expression levels of IL-17-related
   pro-inflammatory cytokines (CSF2, CSF3, and TNF) were either
   downregulated or unchanged in NEC tissues. In the early phase of
   infection, these cytokines have been reported to be involved in
   neutrophil recruitment for an effective innate immunity and rapid
   control against pathogens [[110]41, [111]42]. One possible explanation
   of this discrepancy could be the time lapse between early induction of
   these cytokines and the resection of tissue that occurred at the late
   phase of the disease.

   In conclusion, this study has led to the identification of a
   characteristic IL-17 core gene signature in the pro-inflammatory
   intestinal microenvironment of the premature infant with NEC.
   Furthermore, it appears that it is IL-17F, rather than IL-17A, which is
   consistently involved in the pro-inflammatory cascade in neonates
   affected with NEC. However, further investigations need to be
   undertaken to validate the role of IL-17F in the development of NEC at
   the functional level.

Limitations

   Intestinal samples from premature infants diagnosed with NEC are
   difficult to obtain [[112]43]. It is noteworthy that the small cohort
   of control and NEC patients represents a limitation in interpreting the
   results. A larger cohort which could also include NEC patients at
   earlier stages would help to better document the role of IL-17 in NEC
   initiation as well as increase the statistical power to better detect
   differentially expressed genes.

Supplementary Information

   [113]13104_2021_5489_MOESM1_ESM.docx^ (13.9KB, docx)

   Additional file1: Table S1. Primers used for qPCR.

Acknowledgements