Dataset Information

NLRP3 activation in macrophages promotes acute intestinal injury in neonatal necrotizing enterocolitis.

ABSTRACT:

Background

Macrophages are involved in various immune inflammatory disease conditions. This study aimed to investigate the role and mechanism of macrophages in regulating acute intestinal injury in neonatal necrotizing enterocolitis (NEC).

Methods

CD68, nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3), cysteine aspartate-specific protease-1 (caspase-1), and interleukin-1β (IL-1β) in paraffin sections of intestinal tissues from NEC and control patients were detected with immunohistochemistry, immunofluorescence, and western blot. Hypertonic pet milk, hypoxia and cold stimulation were used to establish a mouse (wild type and Nlrp3^-/-) model of NEC. The mouse macrophage (RAW 264.7) and rat intestinal epithelial cell-6 lines were also cultured followed by various treatments. Macrophages, intestinal epithelial cell injuries, and IL-1β release were determined.

Results

Compared to the gut "healthy" patients, the intestinal lamina propria of NEC patients had high macrophage infiltration and high NLRP3, caspase-1, and IL-1β levels. Furthermore, in vivo, the survival rate of Nlrp3^-/- NEC mice was dramatically improved, the proportion of intestinal macrophages was reduced, and intestinal injury was decreased compared to those of wild-type NEC mice. NLRP3, caspase-1, and IL-1β derived from macrophages or supernatant from cocultures of macrophages and intestinal epithelial cells also caused intestinal epithelial cell injuries.

Conclusions

Macrophage activation may be essential for NEC development. NLRP3/caspase-1/IL-1β cellular signals derived from macrophages may be the underlying mechanism of NEC development, and all these may be therapeutic targets for developing treatments for NEC.

SUBMITTER: Shi B

PROVIDER: S-EPMC10884143 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Publications

NLRP3 activation in macrophages promotes acute intestinal injury in neonatal necrotizing enterocolitis.

Shi Bo B Lyu Cheng-Jie CJ Le Zhen-Kai ZK Ji Hao-Sen HS Xiao Yi Y Zhang Yuan-Yuan YY Huang Shou-Jiang SJ Yu Lin-Jun LJ Shu Qiang Q Tou Jin-Fa JF Lai Deng-Ming DM

World journal of pediatrics : WJP 20230630 2

<h4>Background</h4>Macrophages are involved in various immune inflammatory disease conditions. This study aimed to investigate the role and mechanism of macrophages in regulating acute intestinal injury in neonatal necrotizing enterocolitis (NEC).<h4>Methods</h4>CD68, nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3), cysteine aspartate-specific protease-1 (caspase-1), and interleukin-1β (IL-1β) in paraffin sections of intestinal tissues from N ...[more]

PMID: 37389784

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Project description:BackgroundNecrotizing enterocolitis (NEC) is the most prevalent gastrointestinal disorder that predominantly threatens preterm newborns. Succinate is an emerging metabolic signaling molecule that was recently studied in relation to the regulation of intestinal immunity and homeostasis. We aimed to investigate the relationship between NEC and gut luminal succinate and preliminarily explored the effect of succinate on NEC pathogenesis.MethodsFecal samples from human neonates and mouse pups were analyzed by HPLC - MS/MS and 16S rRNA gene sequencing. C57BL/6 mice were randomly divided into four groups: control, NEC, Lsuc, and Hsuc. The mortality, weight gain, and intestinal pathological changes in four mouse groups were observed. Inflammatory cytokines and markers of macrophages were identified by quantitative real-time PCR. Succinate receptor 1 (SUCNR1) localization was visualized by immunohistochemistry. The protein levels of SUCNR1 and hypoxia-inducible factor 1a (HIF-1a) were quantified by western blotting.ResultsThe levels of succinate in feces from NEC patients were higher than those in feces from non-NEC patients (P <0.05). In the murine models, succinate levels in intestinal content samples were also higher in the NEC group than in the control group (P <0.05). The change in succinate level was closely related to intestinal flora composition. In samples from human neonates, relative to the control group, the NEC group showed a higher abundance of Enterobacteriaceae and a lower abundance of Lactobacillaceae and Lactobacillus (P <0.05). In the murine models, relative to the control group, increased abundance was observed for Clostridiaceae, Enterococcaceae, Clostridium_sensu_stricto_1, and Enterococcus, whereas decreased abundance was observed for Lactobacillaceae and Lactobacillus (P <0.05). Increased succinate levels prevented mice from gaining weight, damaged their intestines, and increased their mortality; upregulated the gene expression of interleukin-1β (IL-1β), IL-6, IL-18 and tumor necrosis factor (TNF); and downregulated the gene expression of IL-10 and transforming growth factor (TGF)-β. Exogenous succinic acid increased inducible nitric oxide synthase (iNOS) gene expression but decreased Arginase-1 (Arg1) gene expression; and increased the protein expression of SUCNR1 and HIF-1a.ConclusionSuccinate plays an important role in the development of necrotizing enterocolitis severity, and the activation of the HIF-1a signaling pathway may lead to disease progression.

Project description:BackgroundNecrotizing enterocolitis (NEC) remains a life-threatening disease in neonates. Numerous studies have shown a correlation between the intestinal microbiota and NEC, but the causal link remains unclear. This study aimed to demonstrate the causal role of gut microbiota in NEC and explore potential mechanisms involved.MethodsEighty-one fecal samples from patients with NEC and eighty-one matched controls (matched to the NEC infants by gestational age, birth weight, date of birth, mode of delivery and feeding patterns) were collected. To explore if altered gut microbiota contributes to the pathogenesis of NEC, fecal microbiota transplantation (FMT) was carried out in germ-free (GF) mice prior to a NEC-induction protocol that included exposure to hypoxia and cold stress. Butyric acid was also administered to demonstrate its role in NEC. The fecal microbiota from patients and mice were analyzed by 16S rRNA gene sequencing analysis. Short chain fatty acid (SCFA) levels were measured by gas chromatography-mass spectrometry (GC-MS). The ontogeny of T cells and regulatory T cells (Tregs) in lamina propria mononuclear cells (LPMC) from the ileum of patients and mice were isolated and analyzed by flow cytometry.The transcription of inflammatory cytokines was quantified by qRT-PCR.ResultsNEC patients had increased Proteobacteria and decreased Firmicutes and Bacteroidetes compared to fecal control samples, and the level of butyric acid in the NEC group was lower than the control group. FMT in GF mice with samples from NEC patients achieved a higher histological injury scores when compared to mice that received FMT with control samples. Alterations in microbiota and butyrate levels were maintained in mice following FMT. The ratio of Treg/CD4+T (Thelper) cells was reduced in both NEC patients and mice modeling NEC following FMT.ConclusionsThe microbiota was found to have NEC and the microbial butyrate-Treg axis was identified as a potential mechanism for the observed effects.

Dataset Information

NLRP3 activation in macrophages promotes acute intestinal injury in neonatal necrotizing enterocolitis.

Background

Methods

Results

Conclusions

Publications

NLRP3 activation in macrophages promotes acute intestinal injury in neonatal necrotizing enterocolitis.

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