Fig 1.
A single-cell atlas of NEC and control human small intestinal samples.
(A) Experimental layout—human small intestinal tissues from neonates and NEC patients were harvested and used for scRNAseq, bulk RNAseq, smFISH, IMC, and NGS of the TCRβ. Samples used in various experiments listed on the right-hand side (S1 Table). (B) Single-cell atlas annotated by cell type. (C) Single-cell atlas annotated by condition. (D) Top 6 markers of the cell types in B. (E) Estimates of the proportion of enterocytes, fibroblasts, dendritic cells, macrophages, B cells, T-NK cells, enteroendocrine cells, and vascular/lymphatic endothelial cells based on computational deconvolution of the bulk RNAseq using the atlas single-cell populations (Methods). Each dot is a sample, fractions of enterocytes and fibroblasts normalized to the sum of cell fractions, remaining fractions normalized to the sum of all cells after excluding fibroblasts and enterocytes; q-values are computed based on FDR correction for all cell populations in the full atlas (Methods). Gray lines are medians, black/pink boxes are 25–75 percentiles. Only samples with Spearman correlations >0.3 between the mixture data and the synthetic mixtures are shown (neonatal: n = 4, NEC: n = 6). The data underlying this figure is available at the Zenodo repository under the following: https://doi.org/10.5281/zenodo.5813397 and in S10 Table. FDR, false discovery rate; IMC, imaging mass cytometry; NEC, necrotizing enterocolitis; NGS, next-generation sequencing; RNAseq, RNA sequencing; scRNAseq, single-cell RNA sequencing; smFISH, single-molecule fluorescence in situ hybridization; TCRβ, T-cell receptor beta.
Fig 2.
Inflammatory macrophages are increased in NEC.
(A) Re-clustered atlas of myeloid lineages. Mϕ–macrophages. (B) Single-cell atlas annotated by condition. (C) Top 8 markers of the myeloid cell subtypes. (D) Differential gene expression between NEC and neonatal macrophages. (E) Differential gene expression between NEC and neonatal DCs. Red dots (D, E) are selected differentially expressed genes among the genes with q-value <0.02 and fold change above 3 or below 1/3. Included are all genes with sum-normalized expression above 10−4. (F) GSEA of pathways enriched (red) or depleted (blue) in NEC samples compared to neonatal samples for macrophages and DCs (q-value <0.2). (K) = Kegg pathways, (H) = Hallmark pathways. (G) Estimates of the proportions of distinct myeloid cell subsets based on computational deconvolution of bulk sequencing data. Each dot is a sample, proportions were renormalized over all myeloid cells, q-values are computed based on FDR correction for myeloid cells only (Methods). Gray lines are medians, black/pink boxes are 25–75 percentiles. Only samples with Spearman correlations >0.3 between the mixture data and the synthetic mixtures are shown (neonatal: n = 4, NEC: n = 6). The data underlying this figure is available at the Zenodo repository under the following: https://doi.org/10.5281/zenodo.5813397 and in S3 and S10 Tables. DC, dendritic cell; FDR, false discovery rate; GSEA, gene set enrichment analysis; NEC, necrotizing enterocolitis.
Fig 3.
Landscape and transcriptional signatures of T/NK/ILCs populations in NEC.
(A) Re-clustered atlas of the T/NK cluster. (B) Single-cell atlas annotated by condition. (C) NGS of TCRβ candy plots where each small square represents 1 clone with the squares proportional to the number of T cells with a particular clone with quantification on the right. Each dot represents 1 tissue sample (neonatal: n = 7, NEC: n = 6, from 7 neonatal and 4 NEC patients). (D) PCA plot of variable (V), differential (D), and joining (J) regions use in NEC and neonatal cases (neonatal: n = 7, NEC: n = 6, from 7 neonatal and 4 NEC patients). Shaded areas are 95% confidence intervals. (E) Differential gene expression between NEC and neonatal T-cell populations. Red dots are selected differentially expressed genes among the genes with q-value <0.02 and fold change above 2 or below 1/2. Included are all genes with normalized expression above 10−4. The data underlying this figure is available at the Zenodo repository under the following: https://doi.org/10.5281/zenodo.5813397 and in S3 Table. ILC, innate lymphoid cell; NEC, necrotizing enterocolitis; NGS, next-generation sequencing; PCA, principal component analysis; TCRβ, T-cell receptor beta.
Fig 4.
Lymphatic and vascular endothelial cells in NEC exhibit proinflammatory signatures.
(A) Re-clustered atlas of the lymphatic and vascular endothelial cluster. (B) Single-cell atlas annotated by condition. (C) Top 8 markers for the cell types in A. (D, E) Differential gene expression between NEC and neonatal cells for vascular endothelial cells (D) and lymphatic endothelial cells (E). Included are all genes with sum-normalized expression above 10−4. Red genes are selected differentially expressed genes among the genes with q-value <0.02 and fold change above 2 or below 1/2. (F, G) GSEA of pathways enriched (red) or depleted (blue) in NEC samples compared to neonatal samples for vascular endothelial (F) and lymphatic endothelial cells (G) with q-value <0.1. (K) = Kegg pathways, (H) = Hallmark pathways. (H) Representative immunofluorescence images from neonatal and NEC samples stained with LYVE-1 (red) and TUNEL staining (white). Gray asterisk (*) represents apoptotic endothelial cells (Tunel+LYVE-1+). Scale bar: 90 μm. (I) Quantification of Tunel+LYVE-1+ cells. Each dot represents 1 image, 2 images/sample (neonatal: n = 4, NEC: n = 4). (J, K) smFISH demonstrating increase in SELE+ endothelial cells in NEC. Red dots are individual mRNAs of SELE; cyan dots are individual mRNAs of CLDN5, a marker of vascular/lymphatic endothelial cells. Blue are DAPI-stained nuclei; scale bar: 10 μm. J and K are representative images from n = 2 subjects per group. The data underlying this figure is available at the Zenodo repository under the following: https://doi.org/10.5281/zenodo.5813397 and in S10 Table. GSEA, gene set enrichment analysis; NEC, necrotizing enterocolitis; smFISH, single-molecule fluorescence in situ hybridization.
Fig 5.
Changes in enterocyte gene expression and zonal representation in NEC.
(A) Re-clustered atlas of the enterocyte cluster colored by crypt-villus zone. (B) Single-cell atlas annotated by condition. (C) UMAPs colored by top villus marker–APOA4, proliferation marker–MKI67, and stem cell marker–LGR5. Color bar is log10 (normalized expression). (D) smFISH of epithelial cells demonstrates increase in APOA4+ (white) epithelial cells towards the top of the villus. (E) smFISH of crypt cells, magenta dots are individual mRNAs of MKI67, green dots are individual mRNAs of LGR5 in the crypts. Blue are DAPI-stained nuclei, scale bars: 20 μm. (F) Estimates of the proportions of villus-crypt zones subsets based on computational deconvolution of bulk sequencing data. Each dot is a sample, proportions were renormalized over all villus-crypt zones, q-values are computed based on FDR correction for enterocytes only (Methods). Only samples with Spearman correlations >0.3 between the mixture data and the synthetic mixtures are shown (neonatal: n = 4, NEC: n = 6). (G) Representative images from Histocat 1.7.6.1 showing villus blunting in NEC compared to neonatal tissue. DNA– 191/193-intercolator (blue), SMA- smooth muscle actin (red), panCK- pancytokeratin (green). (H) Differential gene expression between NEC and neonatal cells for the mid-bottom villus zone. Included are all genes with sum-normalized expression above 5 × 10−5. Red dots are the top 20 most differentially expressed genes among the genes with q-value <0.02 and fold change above 2 or below 1/2. (I) TLR4 gene signature in NEC and neonatal samples. P-value calculated using two-sided Wilcoxon rank-sum test. The data underlying this figure is available at the Zenodo repository under the following: https://doi.org/10.5281/zenodo.5813397 and in S3 and S10 Tables. FDR, false discovery rate; NEC, necrotizing enterocolitis; smFISH, single-molecule fluorescence in situ hybridization.
Fig 6.
Altered cellular adjacencies and protein–ligand interactions in NEC.
(A) Dot plot showing the 20 interaction types that have the highest increase (red) or decrease (blue) interaction values between neonatal (n = 3) and NEC (n = 6) samples (Methods). Interactions values (S6 Table) were computed by IMC analysis using Histocat 1.7.6.1 and 999 permutations and a p-value <0.01 [50]. Dot size corresponds to the interaction values in NEC. (B–D) Significantly elevated molecular interactions between vascular endothelial cells and macrophages (B), dendritic cells (C), naïve T cells (D). (E–G) Significantly elevated interactions between lymphatic endothelial cells and macrophages (E), dendritic cells (F), and naïve T cells (G). (H) Interactions between mid-bottom villus cells and macrophages. (I) Interactions between NK cells and mid-bottom villus cells. (J) Interactions between fibroblasts and macrophages. Shown are 16–25 significant interactions (q-value <0.01) with highest fold change (Methods). In all interaction, maps sender population is on the y-axis, receiver population is on the x-axis. (B–J) Neo = Neonatal. The data underlying this figure is available at the Zenodo repository under the following: https://doi.org/10.5281/zenodo.5813397 and in S6 and S7 Tables. IMC, imaging mass cytometry; NEC, necrotizing enterocolitis.