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CSF1 is expressed by the intestinal epithelial cells to regulate Mφ macrophages and maintain epithelial homeostasis and is downregulated in neonates with necrotizing enterocolitis

BMC Pediatrics volume 24, Article number: 608 (2024) Cite this article

Abstract

Background

Colony stimulating factor 1 (CSF1) is generally expressed by immune cells in response to pro-inflammatory stimuli. The CSF1 receptor (CSFR) is activated by CSF1, and plays a key role in macrophage homeostasis. Furthermore, the CSF1R+ macrophages maintain homeostasis in the intestinal epithelium. The aim of this study was to explore the functions of CSF1-expressing and CSF1R+ macrophages in necrotizing enterocolitis (NEC), which commonly affects the ileum of neonates.

Methods

In-situ CSF1 expression in the intestines of neonates with NEC or intestinal atresia (n = 4 each) was detected by immunofluorescence staining. The CSF1 levels in the intestinal crypt-derived organoid cultures were measured by ELISA. Peripheral blood monocyte-derived Mφ macrophages were co-cultured with the organoids and stimulated with lipopolysaccharide (LPS) to mimic the inflamed state of the ileum in NEC patients.

Results

CSF1 was expressed in the intestinal epithelial cells of the fetal and neonatal samples, but suppressed in the NEC samples. Furthermore, CSF1 expression was downregulated in the intestinal crypt-derived organoids by LPS. CSF1R+ macrophages were detected near the intestinal crypts in the non-inflamed intestines but were absent in tissues obtained from pediatric NEC patients. Peripheral blood monocyte-derived macrophages promoted intestinal organoid proliferation in vitro following CSF1 stimulation. Finally, low concentrations of LPS slightly enhanced the proliferation of organoids co-cultured with the macrophages, whereas higher doses had a significant inhibitory effect.

Conclusions

Intestinal epithelial cells express CSF1 to regulate the resident macrophages, maintain epithelial homeostasis, and resist infection. The abundant CSF1R+ macrophages in the fetal intestine may overexpress TNF-α upon activation of the TLR4/NF-κB pathway, resulting in epithelial damage and NEC induction.

Peer Review reports

Background

Necrotizing enterocolitis (NEC) is a common devastating neonatal disease characterized by gastrointestinal inflammation. Despite recent advances in therapeutic strategies, the morbidity rate in low-birth-weight premature infants remains high at 9% [1], and the mortality rate is approximately 18.5–28.8% [2].

Although the exact pathological basis of NEC remains unknown, there is evidence supporting that the immune response is an underlying mechanism [3]. Studies show that the pro-inflammatory response can damage the intestinal epithelium [4]. The secretion of pro-inflammatory cytokines in the intestinal tract is mainly regulated through the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway [5, 6], which can be activated by bacteria and bacterial products such as lipopolysaccharide (LPS) [3, 7,8,9]. However, given that the intestinal cavity is not sterile, the pathogenesis of NEC cannot be solely explained by inflammation induced by bacterial infection. Therefore, it is reasonable to surmise that any disturbance in the balance between pro-inflammatory and anti-inflammatory responses may also play a significant role in the development of NEC.

Macrophages play a key role in the maintenance of Paneth cells and the homeostasis of the intestinal epithelium [10]. The intestinal stem cells (ISCs) characteristically express the leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), and mediate the self-renewal and maintenance of the epithelial layer [11] by differentiating into various epithelial cell populations. The differentiation of ISCs is regulated by the Wnt/β-catenin pathway [12]. The Paneth cells near the ISCs produce Wnt3a and epidermal growth factor (EGF) to maintain the ISC population [13].

Previous studies have shown that the colony stimulating factor 1 receptor (CSF1R) is a key factor in fashioning the ISC niche [14]. However, CSF1R is expressed on the intestinal macrophages rather than the ISCs and Paneth cells. Knocking out the CSF1R gene in mice decreased the intestinal Paneth cell population and disrupted the intestinal epithelium [10]. Furthermore, CSF1R is a master regulator of macrophage homeostasis and can be activated by colony stimulating factor 1 (CSF1) [15, 16]. The latter is generally expressed by the intestinal immune cells following stimulation by tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) [17]. The fulminant infection in the intestines of NEC patients triggers production of several pro-inflammatory cytokines, which in turn upregulate the expression of CSF1 and promote epithelial repair by the CSF1R+ macrophages. However, the intestinal epithelium is seriously damaged in the pediatric NEC patients, resulting in an impaired ISC niche. Therefore, we hypothesized that the CSF1 produced by non-immune cells also regulates CSF1R+ macrophages.

In this study, we confirmed CSF1 expression in the intestinal epithelial cells through bioinformatics analysis and in vitro intestinal crypt-derived organoid cultures. LPS stimulation decreased the number of organoids, which led to a corresponding decrease in CSF1 production. We also detected CSF1R+ macrophages near the intestinal crypts in the non-inflamed intestines, which were absent in the inflamed intestines of NEC patients. Furthermore, peripheral blood monocyte-derived macrophages promoted the growth of co-cultured intestinal organoids due to CSF1 stimulation. Therefore, CSF1 secretion by intestinal epithelial cells, rather than the pro-inflammatory effects of immune cells, may be critical for the self-renewal and maintenance of the intestinal epithelium.

Methods

Extraction and analysis of single-cell RNA (scRNA)-seq data

The GSE178088 [18] dataset (GPL16791 Illumina HiSeq 2500, Homo sapiens) including the intestinal scRNA-seq data of two NEC patients, two newborns, and two fetuses was downloaded from the Gene Expression Omnibus database (GEO; http://www.ncbi.nlm.nih.gov/geo). The scRNA-seq data were transferred into the Seurat Object and then analyzed using the R software (Seurat 4.1). The specific markers and subclusters of cells were classified, and their relative functions were predicted.

Patients

This study was approved by the ethics committee of Soochow University and conformed to the ethical norms and standards outlined in the Declaration of Helsinki (1983). Intestinal tissue samples and peripheral blood were obtained from four full-term neonates with congenital intestinal atresia (collected within 4 h after surgery), and only samples from the same patient were co-cultured for the in vitro experiments.

Intestinal organoid culture

Intestinal tissue samples were washed with ice-cold PBS, cut into small pieces (< 1 mm), and homogenized in a gentle cell dissociation reagent (Stemcell, Canada) for 30 min on a shaker. The samples were then centrifuged at 300 g for 5 min, and the supernatant was removed. The cell pellet was dissociated in 1% BSA in DMEM-12 medium by pipetting several times and then filtered through a 70-µm nylon mesh. The suspension was centrifuged, and the pellet was resuspended in Matrigel (Corning, USA), and seeded in a 24-well plate with Human IntestiCult™ Organoid Growth Medium (Stemcell, Canada). The culture medium was replaced every two days, and the cells were passaged after one week.

Human peripheral blood monocyte culture and stimulation

Around 2–3 mL peripheral blood was collected from each patient, and the monocytes were isolated within 4 h using the Easysep™ Human CD11b Positive Selection Kit (Stemcell, Canada) according to the manufacturer’s instructions. The monocytes were seeded in 24-well plates at the density of 1 × 105 per well in ImmunoCult™-SF Macrophage Medium (Stemcell, Canada) containing 50 ng/mL hr-CSF1 (Stemcell, Canada). After four days of culture, half volume of fresh medium was added. The Mφ macrophages were harvested two days later.

Co-culture of intestinal organoids and Mφ macrophages

Intestinal organoids were seeded with 1 × 105 Mφ macrophages per well in 24-well plates and cultured in Human IntestiCult™ Organoid Growth Medium (Stemcell, Canada) supplemented with different concentrations of LPS.

Enzyme-linked immunosorbent assay (ELISA)

The organoid culture medium was aspirated and centrifuged at 300 g for 5 min at room temperature. CSF1 and TNF-α levels in the supernatants were evaluated using the corresponding human ELISA kits (SAB, Maryland, USA). The absorbance of each well was measured at 450 nm using a microplate reader (Themo, USA) and analyzed using the GraphPad software (version 8.0).

RNA isolation and quantitation

Total RNA was extracted from the harvested organoids using the RNAfast200 isolation kit (FASTAGEN, Shanghai, China) and reverse transcribed into cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a LightCycler® 480 System (Roche Diagnostics, Basel, Switzerland). The volume of the reaction mix was 20 µL (10 µL 2 × 480 SYBR Green I Master, 1 µL forward and reverse primer, 4 µL water, and 4 µL cDNA). The reaction conditions were as follows: denaturation at 95°C for 10 min, and 50 cycles of denaturation at 95°C for 20 s, annealing at 65°C for 20 s, and extension at 72°C for 20 s. The primer sequences (CSF1 forward: 5’-CGCTACAGCGACGTGAAGAA-3’, reverse: 5’-GTTCCAGGCGTTGTACCAC-3’; β-actin forward: 5’-AGAGGGAAATCGTGCGTGAC-3’, reverse: 5’-CAATAGTGATGACCTGGCCGT-3’) were synthesized by Genewiz (Suzhou, China). The relative expression level of CSF1 was normalized to β-actin, and calculated using the 2−△△Ct method.

Immunofluorescence staining

Immunostaining was performed on the intestinal tissue samples from NEC and non-NEC pediatric patients (n = 4 each; mentioned in Sect. 2.2). Eight tissue samples were retrieved from each patient. The guardians of all pediatric donors provided informed consent for the use of tissues. The tissue specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 4 μm-thick sections. After dewaxing and hydration, the tissue sections were UV-irradiated to quench autofluorescence, and then blocked with 10% goat serum at room temperature. The serum was aspirated, and the sections were incubated overnight with appropriately diluted fluorochrome-conjugated primary antibodies at 4 °C. The specimens were rinsed thrice with PBS (5 min each time) in the dark, and incubated with 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI) to stain the nuclei. The slides were observed under a microscope, and images were captured using the integrated camera. The following primary antibodies were used: anti-CSF1, anti-CD68, anti-TNF-α (Abcam, Cambridge, UK), and anti-CSF1R (Cell Signaling Technology, MA, USA).

Results

CSF1 was downregulated in the intestinal epithelial cells from pediatric NEC patients

According to the annotation method used in the GSE178088 dataset [18] (Supplementary Figure S1), the cells were divided into the following six groups: T cells, B cells, natural killer/natural killer T (NK/NKT) cells, dendritic cells (DCs), macrophages, and non-immune cells (Fig. 1A). CSF1R was mainly expressed in the macrophages, and its expression levels were significantly higher in the fetal group. Nevertheless, the proportion of macrophages was the highest in the pediatric NEC patients (Fig. 1B and C).

Fig. 1
figure 1

ScRNA-seq data confirmed CSF1 expression in the intestinal epithelial cells. (A) UMAP of the small intestine cell populations. The different cell types are color-coded. (B) Median percentage of the major populations with the same color code as in A. (C) Violin plots showing the gene expression level within each cell cluster shown in A. (D) UMAP embedding showing rerun of cells from non-immune cell types colored by cluster. (E) Plot showing the average expression and percentage expression of genes used to identify clusters in D. scRNA, single-cell RNA; CSF1, colony stimulating factor 1

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We performed a post hoc cluster analysis to further characterize the non-immune cells, and found that all CSF1+ clusters also expressed the epithelial cell markers (ANPEP, AOC1, EPCAM), stem cell markers (LGR5, DCLK1, SOX9, MEX3A, and CD24), the Paneth cell marker LYZ, goblet cell marker ERN2, or endocrine cell marker CHGA (Fig. 1D and E). However, the total CSF1 expression was the lowest in the NEC group compared to the others, while the fetal group showed the highest expression (Fig. 1C).

CSF1R+ macrophages were abundant near the crypts of non-inflamed intestines but not in the intestines of pediatric NEC patients

The location of CSF1R+ macrophages in the intestine was determined by immunostaining the intestinal tissues – obtained from pediatric NEC patients and neonates with intestinal atresia – using the macrophage marker CD68. The macrophages were mainly localized near the intestinal epithelium, and showed a significant increase in the NEC groups compared to the non-NEC group. This result was consistent with the scRNA-seq data from GSE178088 (Fig. 2A and B).

Fig. 2
figure 2

CSF1R + macrophages were abundant near the intestinal crypts of non-inflamed but not inflamed intestines. (A) Representative immunofluorescence micrographs of intestine tissue sections obtained from pediatric subjects with intestinal atresia and NEC (the region between the double white dotted line indicates the area near the intestinal crypt). (B) Quantification of CD68+ macrophages per image in the experimental groups. Relative gene expression levels of (C) CSF1R and (D) CSF1. Samples were taken from the terminal ileum of each patients. *p < 0.05, using one-way ANOVA. CSF1, colony stimulating factor 1; CSF1R, CSF1 receptor; NEC, necrotizing enterocolitis

Full size image

Intriguingly, quantification of CSF1R expression did not show a significant difference between the two groups (Fig. 2C and D). Nevertheless, a distinct “CSF1R+ layer” co-expressing CD68 was detected in the intestinal crypts in the non-NEC tissues, whereas the NEC tissues had abundant CSF1R+CD68+ macrophages in the intestinal stromal layer. The CSF1R+ macrophage population was markedly decreased in the intestinal crypts of the NEC group. In addition, the activating molecule CSF1 was mainly distributed in the intestinal epithelium of the non-NEC group, especially near the intestinal crypt and the CSF1R+ layer. On the other hand, the CSF1 signal was scattered in the NEC group and not concentrated near the intestinal crypts. Taken together, CSF1R+ macrophages were abundant near the crypts of non-inflamed intestines but decreased significantly in the intestines of pediatric NEC patients.

CSF1 was downregulated in intestinal organoids following LPS stimulation

The CSF1-expressing cell types in the intestinal epithelium could not be identified on the basis of the GSE178088 annotation data. To this end, we established intestinal crypt-derived organoids as an in vitro model of intestinal epithelial cells. As shown in Fig. 3A, LPS stimulation significantly decreased the population of intestinal organoids in a concentration-dependent manner, which was consistent with another study showing that the gastric epithelium is refractory to LPS-induced activation of the TLR4 pathway [19]. Consistent with this, the CSF1 levels in the supernatants of intestinal organoid cultures showed a gradual decrease with increasing LPS concentration (Fig. 3C). In contrast, CSF1 mRNA expression in the stimulated organoids was not significantly affected by the different concentrations of LPS (Fig. 3D). These findings suggest that LPS-induced CSF1 downregulation in the intestinal epithelium is the result of a decrease in the number of epithelial cells as opposed to transcriptional deactivation.

Fig. 3
figure 3

LPS impeded intestinal organoid proliferation but did not inhibit CSF1 expression. (A) Representative images of intestinal organoid cultures exposed to a concentration gradient of LPS. (B) Median number (± SD) of organoids per image in the indicated groups. (C) CSF1 levels in the supernatants of organoid cultures from the indicated groups. (D) Expression of CSF1 mRNA in the organoids of the indicated groups. Data are presented as the mean ± SD. **p < 0.01; ***p < 0.001, using one-way ANOVA. LPS, lipopolysaccharide; CSF1, colony stimulating factor 1

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Peripheral blood monocyte-derived Mφ macrophages promoted the proliferation of intestinal organoids

To demonstrate the effect of CSF1R+ macrophages on intestinal epithelium cells, the peripheral monocytes were stimulated with CSF1 and differentiated into Mφ macrophages, and the latter were then co-cultured with intestinal organoids. After five days of co-culture, the Mφ macrophages significantly enhanced organoid proliferation (Fig. 4A and B). However, the CSF1 levels in the supernatants of the monocultured intestinal organoids were similar to that of the co-cultured organoids (Fig. 4C).

Fig. 4
figure 4

LPS suppressed the pro-growth effect of the Mφ macrophages on intestinal organoids by upregulating TNF-α expression. (A) Representative images of intestinal organoids cultured with or without Mφ macrophages (1 × 105 cells per well) in the presence of LPS concentration gradient. (B) Median number (± SD) of organoids per image in the indicated groups. CSF1 (C) and TNF-α (D) levels in the culture supernatants from the indicated groups. (E) TNF-α levels in the supernatants of organoids co-cultured with Mφ macrophages in the presence of LPS concentration gradient. Data are presented as the mean ± SD. *p < 0.05; ****p < 0.0001, using one-way ANOVA. LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; CSF1, colony stimulating factor 1

Full size image

Mφ macrophages protected intestinal organoids against TLR4 activation

To determine whether Mφ macrophages can protect intestinal organoids against the activation of the TLR4/NF-κB pathway, we stimulated the co-culture system with an LPS concentration gradient as described previously. Compared to the intestinal organoids cultured without Mφ macrophages (control), the co-cultured organoids showed a slight increase in number following stimulation with low doses (1–10 ng/mL) of LPS, although the size of these co-cultured organoids was significantly larger compared to that of the control group (Fig. 4A and B). However, at higher LPS concentrations ranging from 10 to 1000 ng/mL, the number of co-cultured organoids deceased significantly, and showed extensive differentiation or apoptosis. Furthermore, the LPS-induced decrease in the organoid number was more rapid in the co-cultured group as opposed to the milder decline observed in the monocultured organoids (Figs. 3B and 4B).

CSF1R+ Mφ macrophages expressed TNF-α after LPS stimulation

To further clarify the mechanism underlying the inhibitory effect of LPS on the intestinal organoids co-cultured with Mφ macrophages, we analyzed CSF1 levels in the supernatant. As shown in Fig. 4C, the level of CSF1 rose consistently with increasing concentrations of LPS. This suggested that Mφ macrophages also express CSF1 upon LPS stimulation, and the inhibitory effect of LPS on the organoids may be independent of CSF1. Given that LPS stimulation led to a decrease in CSF1 expression in the epithelial cells, the increase in secreted CSF1 levels in the co-culture system may be associated with the pro-inflammatory effect of Mφ macrophages. Bunders et al. showed that pro-inflammatory cytokines such as TNF-α are associated with intestinal epithelial injury [20]. Indeed, LPS stimulation significantly increased TNF-α expression in the co-culture system in a concentration-dependent manner (Fig. 4D). Furthermore, Mφ macrophages stimulated with the same concentration gradient of LPS showed greater TNF-α upregulation compared to the co-cultured cells (Fig. 4E). Taken together, CSF1R+ Mφ macrophages initiate a pro-inflammatory response in response to high concentrations of LPS and might have the ability to protect intestinal epithelial cells.

TNF-α was abundantly expressed in the intestinal stroma of pediatric NEC patients

To ascertain the possible role of TNF-α in NEC development, we analyzed its expression in the intestinal tissue samples obtained from pediatric NEC patients and neonates with intestinal atresia (control). Compared to the control group, TNF-α expression was significantly upregulated in the intestines of NEC patients, especially in the stroma layer, and co-expressed with CD68 (Fig. 5A and B). Bartfeld et al. had shown that the human gastric epithelium is refractory to TNF-α stimulation only from the basal and not the apical side. Therefore, we can surmise that the high expression of TNF-α in the intestinal stroma could be a critical factor underlying the epithelial injury observed in NEC.

Fig. 5
figure 5

TNF-α expression was upregulated in the intestines of pediatric NEC patients. (A) Representative immunofluorescence micrographs of the intestine tissue sections obtained from pediatric subjects with intestinal atresia and NEC. (B) Quantification of TNF-α expression in the indicated groups. Data are presented as the mean ± SD. ****p < 0.0001, using one-way ANOVA. TNF-α, tumor necrosis factor-α; NEC, necrotizing enterocolitis

Full size image

In summary, intestinal epithelial cells enhance the proliferation of CSF1R+ Mφ macrophages in the sub-epithelial region by secreting CSF1. These macrophages maintain intestinal homeostasis and prevent pathogens from invading the intestinal cavity during normal conditions. However, when intestinal inflammation exceeds a certain threshold, the abundant TNF-α in the intestinal stroma layer causes epithelial injury and induces NEC.

Discussion

Resident macrophages in the intestine not only regulate immune responses and clear pathogens [21], but also maintain homeostasis in the intestinal epithelium [21,22,23,24]. These macrophages usually reside at the bottom of the intestinal crypts and are spatially close to the ISCs. We detected a population of CSF1R+ macrophages under the intestinal crypts in the tissues of non-NEC neonates. However, these cells were absent in the intestines of pediatric NEC patients. Neil et al. confirmed that CSF1R+ macrophages maintain homeostasis of the ISCs and the intestinal epithelial layer [10]. Based on our findings, we hypothesize that CSF1R+ macrophages might play an important role in promoting intestinal epithelial cell proliferation.

Merad et al. showed that the renewal and proliferation of tissue-resident macrophages are largely CSF1-dependent [25]. Based on the scRNA-seq data from the GSE178088 dataset, we confirmed that fetal and neonatal intestinal epithelial cells express CSF1. Furthermore, the macrophages derived from peripheral blood monocytes following CSF1 stimulation promoted the growth of intestinal organoids in vitro. Therefore, we surmised that Mφ macrophages in the non-inflamed intestine are regulated by CSF1 secreted by the epithelial cells. The Mφ macrophages mainly reside near the intestinal crypt, and promote self-renewal of ISCs and maintain epithelial homeostasis.

To simulate the pro-inflammatory environment in the NEC intestine, we cultured intestinal organoids in the presence of LPS concentration gradient with or without Mφ macrophages. The number of monocultured organoids gradually decreased with increasing LPS concentration. In contrast, while lower doses of LPS slightly promoted the proliferation of the co-cultured organoids, higher doses led to a significant decline in organoid numbers. This “switching” phenomenon suggests that the pro-growth effect of Mφ macrophages on the intestinal epithelial cells has limitations.

Tissue-resident macrophages in the intestine arise from both bone marrow-derived monocytes and embryonic precursors [23]. Moreover, the GSE178088 data showed that CSF1R expression was increased in the fetal intestines. This suggests that intestinal macrophages should be more abundant in the neonates, especially premature infants. Although these resident macrophages improve the ability of intestinal epithelial cells to maintain homeostasis, activation of the TLR4/NF-κB pathway in response to LPS or other stimuli can trigger an inflammatory response. Once inflammation exceeds a certain threshold, it can cause severe damage to the intestinal epithelium. Therefore, the abundance of intestinal CSF1R+ macrophages in premature infants may be one of the factors responsible for their susceptibility to NEC.

Conclusions

The fetal and neonatal intestinal epithelial cells express CSF1 to regulate the macrophages residing near the crypts, maintain epithelial homeostasis, and resist infections. However, activation of the TLR4/NF-κB pathway in the resident macrophages triggers production of pro-inflammatory cytokines, such as TNF-α. The abundance of CSF1R+ macrophages in the fetal intestine may increase the risk of epithelial damage via the TLR4/NF-κB/TNF-α pathway, and is one of the factors responsible for the higher susceptibility of neonates to NEC. This pathological axis can be potentially targeted for the prevention and treatment of NEC, and warrants further investigation.

Data availability

The datasets GSE178088 for this study can be found in the Gene Expression Omnibus database (GEO) http://www.ncbi.nlm.nih.gov/geo.

Abbreviations

CSF1:

Colony stimulating factor 1

CSF1R:

Colony stimulating factor 1 receptor

NEC:

Necrotizing enterocolitis

LPS:

Lipopolysaccharide

ISC:

Intestinal stem cells

EGF:

Epidermal growth factor

Lgr5:

Leucine-rich repeat-containing G-protein coupled receptor 5

TNF-α:

Tumor necrosis factor-α

NK/NKT cells:

Natural killer/natural killer T cells

DC:

Dendritic cells

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Acknowledgements

This work was grateful to all the Institute of Pediatric Research members affiliated with the Children’s Hospital of Soochow University for supporting every step of this study. And we appreciate the linguistic assistance provided by Medlive (www.medlive.cn) during the preparation of this manuscript.

Funding

This work was supported by the grants from the Natural Science Foundation of Jiangsu Province (Grant BK20190053), the Science and Technology Program of Suzhou (Grant SYS2018067) and the Graduate Research and Practice Innovation Program of Jiangsu Province (Grant KYCX19-1996).

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Authors and Affiliations

  1. The Affiliated Hospital of Guiyang Medical University, Guiyang, 550004, China

    Xu Sun, Jun Du & Huajian Gu

  2. Children’s Hospital of Soochow University, Suzhou, 215025, China

    Lingqi Xu, Shurong Ma & Jian Wang

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  1. Xu Sun
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  2. Lingqi Xu
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Contributions

Jian Wang and Xu Sun designed the experiments and research project. Xu Sun and Lingqi Xu and Lulu Chen performed the experiments and analyzed the data. Jun Du and Huajian Gu perform surgery and obtain intestine tissue samples from clinical patients. Shurong Ma participated in the discussion. Xu Sun wrote the paper.

Corresponding authors

Correspondence to Xu Sun or Jian Wang.

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Ethics approval and consent to participate

This study was approved by the ethical committee of Soochow University and conformed to the ethical norms and standards in the Declaration of Helsinki (1983). All the pediatric donors’ guardians provided informed consent for the use of materials in this study.

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Not applicable.

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The authors declare no competing interests.

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Sun, X., Xu, L., Ma, S. et al. CSF1 is expressed by the intestinal epithelial cells to regulate Mφ macrophages and maintain epithelial homeostasis and is downregulated in neonates with necrotizing enterocolitis. BMC Pediatr 24, 608 (2024). https://doi.org/10.1186/s12887-024-05047-9

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  • DOI: https://doi.org/10.1186/s12887-024-05047-9

Keywords

BMC Pediatrics

ISSN: 1471-2431

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