Epigenome-Microbiome crosstalk: A potential new paradigm influencing neonatal susceptibility to disease

<p>Preterm birth is the leading cause of infant morbidity and mortality. Necrotizing enterocolitis (NEC) is an inflammatory bowel disease affecting primarily premature infants, which can be lethal. Microbial intestinal colonization may alter epigenetic signatures of the immature gut establishing inflammatory and barrier properties predisposing to the development of NEC. We hypothesize that a crosstalk exists between the epigenome of the host and the initial intestinal colonizing microbiota at critical neonatal stages. By exposing immature enterocytes to probiotic and pathogenic bacteria, we showed over 200 regions of differential DNA modification, which were specific for each exposure. Reciprocally, using a mouse model of prenatal exposure to dexamethasone we demonstrated that antenatal treatment with glucocorticoids alters the epigenome of the host. We investigated the effects on the expression profiles of genes associated with inflammatory responses and intestinal barrier by qPCR-based gene expression array and verified the DNA modification changes in 5 candidate genes by quantitative methylation specific PCR (qMSP). Importantly, by 16S RNA sequencing-based phylogenetic analysis of intestinal bacteria in mice at 2 weeks of life, we showed that epigenome changes conditioned early microbiota colonization leading to differential bacterial colonization at different taxonomic levels. Our findings support a novel conceptual framework in which epigenetic changes induced by intrauterine influences affect early microbial colonization and intestinal development, which may alter disease susceptibility.</p

M_PI effects on systemic immune and inflammatory cytokine expressions by multiplex immunoassay.

<p>Multiplex immunoassay of immune and inflammatory cytokine expressions in serum collected from preweaned GF and M_PI-L, H and SPF. The levels of these cytokines are presented as mean ± SEM. One-way ANOVA with post-hoc Tukey’s honest significance (HSD) test was used to compare the groups.</p

Comparison of bacterial communities following transfaunation of germ free mice with M_PI fecal lysates.

<p>A: The relative abundance of different bacterial phyla in input M_<b>PI</b>-L,-H fecal samples and from M_PI-L, H and SPF preweaned mouse pups is shown. B: PCoA analyses of the M_PI-L and M_PI-H in original human infants, transfaunated dams and respective pups based on Bray Curtis dissimilarities among samples given presence/absence of major taxa present in at least one sample. Axis 1 explained 27.5% of variation and axis 2, 18.1%.</p

NF-κB activation in M_PI-L, -H and SPF GF ileum mucosa by immunohistochemistry.

<p>A: NF-κB activation was analyzed by immunohistochemical staining of 5 micron paraffin-embedded distal ileum tissue from GF, M_PI-L, H and SPF mouse. Representative areas (n = 3/per group) are shown (magnification 400x), black arrows indicate nuclear translocation of the phosphorylated NF-κB p65 subunit. B: Nuclear translocation of pp65 was quantified by ImmunoRatio tool from Image J software and presented as % of pp65 positive nuclei/total nuclei. C: Growth curve of M_PI-L (n = 20) and M_PI-H (n = 15) from birth to weaning. One-way ANOVA with post-hoc Tukey’s HSD test was used to compare the groups.</p

MetaCore gene enrichment analysis of ileum microarray data representing microbiota effects in preweaned M_PI-L and M_PI-H-gnotobiotic mice.

<p>M_<b>PI</b>-L and M_<b>PI</b>-H induced differentially expressed genes were analyzed by GeneGO MetaCore software. Panel A: Top 10 overrepresented GO biological processes in preweaned M_PI-L, H vs GF mice. Panel B: Top 10 scored overrepresented networks in preweaned M_PI-L, H vs GF mice. Panel C: Comparison of the differentially expressed genes in M_PIL_GF and M_PI-H_GF presented in inflammatory response-related networks using the IPA program. Orange line: activation; Blue line: inhibition; Grey: not present.</p

The composition of microbiota in M_PI-L and M_PI-H.

<p>The unique bacterial species are in Italic.</p><p>The composition of microbiota in M_PI-L and M_PI-H.</p

Epo treatment reduced apoptosis and up regulated Bcl-2 expression in experimental neonatal rat ileum.

<p>(A) Cleaved caspase-3 staining of ileum section with NEC score 0 as described in Materials and Methods, with or without Epo administration (+Epo vs. –Epo). Original magnification, ×40. Scale bar = 50 µm. (B) The percentage of positive cleaved caspase-3 enterocytes per HPF. Data are shown as the mean ± SD obtained from 3 separate experiments with minimum 10 HPFs. * indicate <i>p</i><0.01 by Student’s <i>t</i>-test. (C) Corresponding intestinal lysates from the same set of pups were collected and subjected to immunoblotting for Bcl-2 and β-actin. Representative immunoblots from three experiments with similar results are shown (n = 10 animals for each treatment group). (D) Densitometric values were normalized to β-actin. * depicts <i>p</i><0.05 by Student’s <i>t</i>-test.</p

Epo treatment reduces autophagy in serum deprived IEC-6 cells.

<p>IEC-6 cells were incubated in serum free media (SF), complete media+rapamycin (CM+Rap, Rap: 100 nmol/l), complete media (CM) and serum free media+Epo (0.6 U/ml, 6 U/ml, 60 U/ml) (SF+0.6U, SF+6U, SF+60U) for 6 hours. After treatment, IEC-6 cells were fixed and stained for LC3 (green dots), nuclei were stained with DAPI (blue). Autophagosome formation in IEC-6 cells as reflected by LC3II puncta was observed using confocal microscopy. Images are representative of 3 separate experiments. The number of LC3II puncta was quantified and counted using ImageJ software in minimum 300 cells for each condition. Data are presented as means ± SD. * and ** indicate <i>p</i><0.05 and <i>p</i><0.01 respectively, compared with serum starved cells by Student’s <i>t</i>-test. Original magnification, ×600. Scale bar = 20 µm.</p

Increased autophagy and apoptosis in experimental neonatal rats with NEC.

<p>Representative slides for MF pups and NEC pups processed by H&E, Beclin 1 (Bec1), LC3, TUNEL and cleaved caspase-3 (Cas3) staining. Immunofluorescence of TUNEL positive enterocytes is shown in red and DAPI nuclear counterstaining is pseudo-colored green (TUNEL). In MF pups, the villi structure was intact (H&E, left panel); intestinal epithelial cells exhibited low basal level of Beclin 1 (Bec1, left panel) and LC3 expression (LC3, left panel); rarely TUNEL positive enterocytes (TUNEL, left panel) and almost no positive cleaved caspase-3 signal (Cas3, left panel). In NEC pups (with NEC score ≥2 and no Epo treatment), villus structure is damaged (H&E, right panel) associated with extensive Beclin 1 (Bec1, right panel) and LC3 signal (LC3, right panel) with punctuate staining of typical LC3II (indicated in the enlarged inset); TUNEL positive enterocytes (TUNEL, right panel, yellow dots) and cleaved caspase-3 positive enterocytes (Cas3, right panel) were observed. (n = 6 animals per group) Original magnification, ×20 and inset ×100. Scale bar = 50 µm.</p

Epo reduces apoptosis in IEC-6 cells by up regulating Bcl-2 through the MAPK/ERK pathway.

<p>Apoptosis in IEC-6 cells was induced by TNF-α<b>/</b>CHX for 6 hours. IEC-6 cells were either pre-incubated with 6 U/ml Epo for 24 hours (ETC) or without Epo pretreatment (TC). Control cells received no treatment (−). (A) Cells were lysed and assayed for DNA fragmentation at the end of the treatment. DNA fragmentation was measured by a cell death detection ELISA kit. Results are presented as the fold increase in treatment group over control group (−) and represent values of three independent experiments. 24 h pretreatment with Epo decreased TNF-α/CHX induced apoptosis in IEC-6 cells. (B) Assays in the presence of the inhibitors, AG490 (50 µM) (ETCAG) or PD98059 (20 µM) (ETCPD) or LY294002 (20 µM) (ETCLY) were carried out 15 minutes before the addition of Epo. The expression of Bcl-2 was detected by western blotting. Densitometric values were normalized to β-actin. Typical images from at least three independent experiments are shown. * depicts <i>p</i><0.05 by Student’s <i>t</i>-test. (C) The inhibitory effects of PD98059, LY294002 and AG490 were confirmed by western blotting analysis of phospho-p44/42 MAPK (p-p44 MAPK/p-p42MAPK), phospho-Akt (p-Akt) and total Akt (Akt), phospho-Jak2 (p-Jak2) and total Jak2 (Jak2) in IEC-6 cells after 15 minutes treatment with PD98059 (20 µM); LY294002 (20 µM) or AG490 (50 µM) respectively.</p