The incidence and severity of NEC are strongly associated with preterm infants. Immature intestinal function, particularly immune defense, intestinal motility, and circulatory regulation, is a significant risk factor for NEC susceptibility in these infants (1). IMφs exhibit significant developmental plasticity and are instrumental in shaping neonatal intestinal immunity, motility, and circulation.

The interaction between IMφs and intestinal epithelial cells (IEC) is essential for regulating homeostasis of intestinal barrier function (54). Human IECs produce IL-10 through Mφ-epithelial crosstalk mediated by TLR4 signaling in Mφs to maintain epithelial cell integrity (55). Destruction of the neonatal intestinal barrier is a key step in the occurrence and development of NEC (1), which shows not only a decline in the immature intestinal epithelial barrier structure to allow the invasion of pathogens, but also the lack of barrier repair ability after intestinal epithelial injury (56). Intestinal epithelial injury needs to be repaired by a series of events that coordinate IECs with intestinal resident and permeable Mφs (57). In the damaged intestinal mucosa, epithelial cells in the crypt near inflammation migrate collectively to the damaged site, while cytokines, growth factors, Wnt ligands, specialized pro-resolving mediators, and matrix metalloproteinases (MMPs) released by Mφs transmit regenerative signals to damaged IEC to promote epithelial cell proliferation and migration to restore the homeostasis and function of the epithelial barrier (58). Impaired Wnt/β-catenin pathway can affect IEC homeostasis and lead to intestinal regeneration dysfunction during NEC (59). Although TNF-α is generally considered a pro-inflammatory cytokine, recent evidence has shown that it promotes the repair of damaged mucosa by activating WNT/β-catenin signaling transduction, promoting IEC proliferation, and upregulating the expression of intestinal healing receptors (60). Nitric oxide (NO) is the earliest and most effective cytokine released by Mφs. In the inflammatory state of NEC, the sustained release of NO from the injured intestinal mucosa not only impairs gap junction communication between adjacent IEC mediated by connexin 43 (Cx43), but also inhibits IEC migration and mucosal repair by activating RhoA-GTPase (61, 62). IFN-γ also reduces gap junction communication by inhibiting the dephosphorylation and internalization of Cx43, thus inhibiting the migration ability of IEC and affecting intestinal healing during NEC (63). Improving our understanding of Mφ-IEC crosstalk is very promising for developing better strategies to treat NEC while maintaining barrier function.

In NEC, multiple types of regulatory cell death (RCD) of IEC lead to intestinal barrier dysfunction and induce local intestinal inflammation, including apoptosis, autophagy, pyroptosis, and ferroptosis (64–67). Several studies have shown that Mφs have a certain extent of association with the RCD occurrence of IEC in NEC. Interferon regulatory factor 5 (IRF5) is a master regulator for M1 macrophages and plays an important role in the induction of apoptosis (68, 69). Wei et al. found that IRF5 is upregulated in infiltrated macrophages in human neonates with NEC, and IRF5 deficiency in myeloid cells inhibits IEC apoptosis and prevents the destruction of the intestinal barrier in experimental NEC (70). In a rat model of NEC, heparin-binding epidermal growth factor (HB-EGF) inhibited M1 macrophage polarization and promoted M2 macrophage polarization, thereby reducing IEC apoptosis to protect the intestine barrier (71). Recent experimental data have shown that ferroptosis is an important way in which intestinal epithelial RCD appear in the pathogenesis of NEC. Bioinformatics analysis based on microarray data showed that ferroptosis in NEC is related to activated Mφs, and ACSL4, as a key regulator of ferroptosis, may regulate the immune function and inflammatory response of NEC by activating TLR (67).

The interaction between immune cells and gut microbiota plays a crucial role in the maturation of the neonatal immune system (72). Gut microbial colonization drives CCR2-expressing monocytes to constantly replenish Mφs in intestinal mucosa, and the continuous sampling of intestinal microbes by Mφs helps to maintain intestinal immune balance (73). Gut microbiota dysbiosis occurs before NEC in preterm infants (74), alterations in gut microbiota can be perceived by antigen presenting cells, and Mφs directly interact with gut microbiota (75) and regulate Paneth cell differentiation (76). After birth, intestinal stem cells located at the base of the crypt begin to differentiate into enterocytes and secretory cells. Paneth cells are secretory cells, and their disruption is involved in the development of NEC (77). Recent studies have found that postnatal gut microbial exposure promotes Mφs to differentiate into M2-type non-inflammatory states and secrete the epithelial Wnt signaling required for the differentiation of stem cells into Paneth cells, thus maintaining mesenchymal niche cell (MNC) proliferation, a key part of the intestinal stem cell niche (78). However, antibiotic treatment in early life can lead to gut microbial disorders, affect Mφ differentiation and Wnt secretion, lead to proliferation defects of MNCs, and promote the pathogenesis of NEC, while Lactobacillus treatment or supplementation with exogenous CD206⁺ M2 macrophages can partially rescue Paneth cell differentiation deficiency and NEC-like phenotype (78). Maike et al. found that neonatal intestinal Mφ-derived S100A8-A9 can promote the co-development of host gut microbiota and mucosal immune system, while the loss of fecal S100A8-A9 is associated with diseases related to microbial disorders, such as NEC, and supplementation with these proteins may contribute to the development of preterm infants (79).

With changes in the gut microbiota of NEC, the metabolites change accordingly. The affected metabolites are usually regarded as a bridge between the microbiota and the host, which may regulate the function of IMφs through pattern recognition receptor (PRR) signaling and participate in the pathogenesis of NEC (80). Short-chain fatty acids are products of the bacterial fermentation of carbohydrates in the gut, which butyric acid (BT) induces a more effective immunomodulatory effect (81, 82). BT-treated mouse Mφs can down-regulate LPS-induced pro-inflammatory mediators by inhibiting the activity of histone deacetylase (83). BT has shown potential for early prediction and disease identification of NEC (84, 85), which is related to the immunomodulatory mechanism of BT in the immature intestine of preterm infants. BT inhibits IL-1β-induced inflammation in fetal intestinal organoids or fetal mouse intestines and significantly reduces the release of IL-8 or Mφ inflammatory protein 2 (86). Studies have shown that exogenous BT can alleviate intestinal pathological damage in NEC by inhibiting the expression of high-mobility group box 1 (HMGB1) and increase the proportion of beneficial intestinal bacteria (87). Therefore, the intestinal protective effect of BT on NEC may be achieved by exerting the anti-inflammatory effect of Mφs and inhibiting the downstream inflammatory pathway. Succinic acid is a metabolic signaling molecule that maintains intestinal homeostasis and immune regulation, and its specific surface receptor, succinate receptor 1 (SUCNR1), activates immune cells and participates in the response to intestinal inflammation (88). In the fecal samples of NEC children and NEC model mice, increased levels of succinate are related to changes in gut microbiota, and succinic acid activated Mφs by activating the SUCNR1-mediated HIF-1α signaling pathway, disrupting the balance of pro-inflammatory and anti-inflammatory mediators and leading to the progression of NEC (89).

The development of NEC also involves some other common clinical risk factors, such as severe anemia and blood transfusion exposure, and the exploration of its pathogenesis has confirmed the importance of IMφs. Evidence supports the association between RBC transfusion and adverse clinical outcomes, such as NEC, after anemia in preterm infants (90). However, there is no consensus on whether transfusion-related intestinal injury is caused by severe anemia or RBC transfusion (91, 92), which was recently investigated using a neonatal murine model of transfusion-associated NEC. Studies have found that severe anemia can independently promote the secretion of pro-inflammatory cytokines by Mφs, becoming a key initiation event that causes intestinal inflammation and barrier dysfunction (93). RBC transfusion activates these Mφs, and free hemoglobin, the RBC degradation product, mediates the production of ROS and inflammatory cytokines through TLR4 in Mφs and the NF-κB pathway downstream, resulting in a second blow to the intestinal tract (94). The occurrence of NEC requires continuous exposure to anemia and RBC transfusion, neither of which is an independent risk factor for intestinal injury (95). Selective depletion of inflammatory Mφs with diphtheria toxin or clodronate liposomes not only reversed the effect of anemia on intestinal barrier function, but also weakened the intestinal injury caused by RBC transfusion, and the inhibition of Mφ activation by anti-NF-κNPS also had the same effect (94). Early prevention and treatment of anemia and adoption of restrictive transfusion guidelines are key elements in the management of preterm infants, and intervention in the number and activity of Mφs may be a new target for the treatment of transfusion-related NEC. In addition, platelet transfusion increases the risk of NEC (96), and activated platelets may cause intestinal injury by releasing preformed vasoconstrictor factors and inflammatory mediators (97). Kopperuncholan et al. found that whatever medical NEC or surgical NEC in human both show increased the level of plasma tissue factor and the expression of tissue factor in IMφs. Neonatal IMφs release their unique tissue factor, which promotes platelet activation and aggregation by activating thrombin, and depleting platelets in microthrombosis formed in intestinal microvessels (98). Inhibition of TLR4 signaling in Mφs or targeted inhibition of thrombin by antithrombin nanoparticles can reverse NEC intestinal injury (98).

Therefore, IMφs act the multi-function role in NEC pathogenesis, and maintaining the homeostasis of IMφs may protect from NEC ( Table 1 ).

Mφs could be polarized into different subtypes to play different biological functions under the action of different induction factors. Generally, two subtypes of polarized Mφs are classical macrophages (M1) and alternative macrophages (M2), at the same time the polarization is reversible (99). As the initiator of inflammation, M1 macrophages activate TLR4 and up-regulate NF-κB signaling when stimulated by LPS and release pro-inflammatory cytokines such as IL-1β, inducible nitric oxide synthase) and TNF-α to form an inflammatory microenvironment. In contrast, IL-4/IL-13 induce Mφs to polarize M2 macrophages and up-regulated the expression of IL-10, TGF-β, IGF, and other molecules to exert the functions of anti-inflammation and tissue repair (100). Preterm infants are vulnerable to a large amount of LPS, and the increased levels of IFN-γ and TNF-α in the intestines of children with NEC, that can provide sufficient stimulation for the intestinal mucosa and lamina propria Mφs, and provide a prerequisite for M1-type polarization.

Many studies have shown that M1 macrophages promote the development of NEC, and inhibiting M1 polarization and promoting M2 polarization reduces the incidence of NEC (70, 71, 101). HB-EGF prevents M1 and promotes M2 polarization by activators of transcription 3, thereby protecting the intestine from NEC (71). Recent studies have shown that IRF5 is a key regulator of neonatal M1 macrophages (102). Myeloid-specific deficiency of IRF5, which is associated with reduced M1 macrophage polarization and systematic inflammation, dramatically prevents experimental NEC (70). Glutaredoxin-1 deficiency promotes the inactivation of NF-κB, which attenuates the recruitment of monocytes and M1 macrophage polarization and protects against NEC-like intestinal injury (103). Hydrogen promotes the polarization of Mφs from M1 to M2 by inhibiting the expression of NF-κB p65 in the nucleus, thus reducing the severity of NEC (104). Therefore, polarization of Mφs may be a key factor in determining the regression or progression of NEC.

In the process of pathogen invading the intestinal mucosa, IMφs can recognise and bind pathogen-related molecular patterns and damage-related molecular patterns (DAMPs), because they express PRRs of cellular surface and intracellular, such as TLRs, nod-like receptors (NLRs), and C-type lectin receptors (10, 105). Therefore, pathogens and tissue destruction can be detected rapidly and efficiently. The repair and homeostatic functions of IMφs can be destroyed by persistent damage, resulting in a causal relationship between Mφs and the inflammatory state of disease (106).

During NEC, Mφ-related inflammatory signaling pathways are involved in pathogenesis ( Figure 2 ). The functional PRRs in Mφs includes TLRs and NLRs. The neonatal intestine is sensitive to LPS derived from gram-negative bacteria (107). In Mφs, LPS binds to CD14, which in turn binds to the TLR4/MD-2 complex, recruiting MyD88 and IRAK1/IRAK4. These molecules activate TRAF6 which in turn activates the IKK complex and MAPK kinase. MAPK kinase subsequently phosphorylates JNK and p38 MAPK, which activate the transcription factor AP-1. The IKK complex allows phosphorylation of IκB and subsequently degraded following ubiquitination, resulting in the activation of NF-κB and the release of pro-inflammatory cytokines (108). The importance of TLR4 signaling in IMφs has been confirmed in different mouse models of NEC. Inhibition of TLR4 signaling in Mφs can reverse the occurrence of NEC and NEC-related thrombocytopenia (98). In myeloid TLR4^-/- mice, after anemia-transfusion treatment, only newly recruited blood monocyte-derived Mφs were detected, but there was no intestinal injury (94). An MD2 inhibitor alleviated intestinal mucosal injury caused by inflammation in NEC by blocking TLR4-MD2/NF-κB signal axis (109). NLRP3 is an important member of the NLR-like receptor family. Under the stimulation of exogenous microbiota or endogenous danger signals, the activation of NLRP3 mediates the assembly of the inflammasome complex, triggering activation of caspase-1 and secretion of IL-1β and IL-18, and this process is called pyroptosis. Activation of NLRP3/caspase-1 pyroptosis pathway can destroy the intestinal barrier, guide neutrophils to the injured site, and promote the activation of Mφs, resulting in the recruitment of more inflammatory cells and the expansion of inflammatory response (110). In an in vitro experiment, CS induced TLR4/MyD88 signaling in Mφs and up-regulated the expression of NF-κB, which triggered NEC through the NLRP3/caspase-1 pyroptosis pathway (111). Melatonin and miR-146a-5p, which inhibit the activation NLRP3, are promising therapeutic targets in NEC (112, 113).

The function of Mφs is regulated by signaling pathways, such as NF-κB and MAPK activation (114, 115). NF-κB plays a central role in regulating the immune response of NEC (116, 117). Once TLR and other receptors are activated, IKKβ is activated, which leads to the nuclear translocation of NF-κB and the transcription of NF-κB target genes, including cytokines, chemokines, adhesion molecules, and cell surface receptors (118). In experimental NEC mice, the recruitment and differentiation of Ly6c⁺ monocytes into the intestine mediated by NF-κB signaling occurred within 24 h of induction, that is, the early stage of NEC. IKKβ is a key kinase mediating the activation of NF-κB. Pups with IKKβ deletion in Lysm⁺ cells prevented the NF-κB activation in monocytes and reduced NEC occurrence, whereas NEC was unabated in pups with IKKβ deletion in IECs (119). Krishnan et al. found that bacterial products specifically induced high Smad7 expression in IMφs of preterm infants, thus blocking normal autocrine induction of TGF-β₂ in epithelial cells (120) and promoting NF-κB-mediated inflammatory signaling transduction by increasing the expression of IKKβ, ultimately activating inflammation (121). Another pathway involved in regulating Mφ activation in NEC is the MAPK signaling pathway, which is activated by pro-inflammatory cytokines and chemokines released by Mφs (122). Previous studies have found that the activation of p38 MAPK is differentially expressed and localized between normal neonatal intestinal segments and NEC intestinal segments (123) and that endotoxins cause enterocytes to release p38-dependent pro-inflammatory molecule COX-2, which potentiates the systemic inflammatory response during NEC (124).

The development of NEC is closely related to the cytokines released by activated Mφs, such as the pro-inflammatory factors TNF-a, IL-1, IL-12, IL-18, and CXCL5, and the anti-inflammatory factors IL-10 and TGF-β (27). In the process of NEC, intestinal injury is partly believed to be the result of TNF-α-induced activation of MMP pathway that can degrade the mucosal extracellular matrix (125). IL-1 binding to its receptor can trigger a series of signaling cascade reactions, leading to the activation of pro-inflammatory transcription factors, such as NF-κB and AP-1, which in turn induce the secretion of pro-inflammatory cytokines, such as IL-6, TNF, and IL-1 (126). The secretion of IFN-γ triggers the accumulation of IL-12 and IL-18 positive monocytes/Mφs, and these pro-inflammatory cytokines contribute a correlation with the progression of tissue injury in NEC (127). A significant decrease in the number of intestinal Mφs and mild NEC damage were detected in IL-18-deficient mice induced by NEC (128). Chemokine CXCL5, as the main initiator, recruited Mφ precursors from the circulation to the inflammatory intestinal mucosa, and this chemotaxis can be blocked by anti-CXCR2 (cognate receptor of CXCL5) antibodies (8). IL-10 inhibits intestinal inflammation because the specific knockout of IL-10R signaling in Mφs residing in the intestinal lamina propria causes severe spontaneous colitis (18). IL-10 also blocks the activation of NF-κB by inhibiting IκB kinase activity and NF-κB DNA binding activity to reduce intestinal mucosal inflammation (129). The inflammatory downregulation of TGF-β on Mφs inhibited the occurrence of intestinal mucosal inflammation in NEC (25).

HMGB1 is an important late inflammatory factor, which is actively secreted by activated Mφs or passively released from necrotic or damaged cells and plays a role as a DAMP (130, 131). Clinical data have confirmed that increased HMGB1 levels in fecal and serum assist in the early identification and prognosis of NEC (132, 133). The Mφ deactivator semapimod inhibited the secretion of HMGB1 and reduced the expression of its receptor for advanced glycation end products (RAGE), thereby reducing intestinal cell death and inflammatory response in a rat model of NEC (134). TLR and RAGE are important receptors for HMGB1, which itself not only affects NEC mucosal repair by inhibiting the migration of IECs in a TLR4-dependent manner (135), but also promotes intestinal inflammation in NEC by activating NLRP3 through TLR4 and NF-κB signaling pathways (136). In addition, HMGB1 promotes chemotaxis through RAGE pathway and activates NF-κB signaling pathway to induce inflammation; therefore, targeting HMGB1/RAGE/NF-κB pathway may be one of the measures to reduce intestinal inflammation (137). Lactate in circulating is the key regulator of HMGB1 secreted by Mφs, and the increase of neonatal lactate levels and the occurrence of sepsis are closely related to the mortality and prognosis of NEC (138, 139). During sepsis, Mφs uptake extracellular lactate via monocarboxylate transporters (MCT) to promote HMGB1 lactylation via a p300/CBP-dependent mechanism. Extracellular lactate is also recruited to the nucleus through G protein-coupled receptor 81 (GPR81) to stimulate the acetylation of HMGB1. Lactylated/acetylated HMGB1 is released into extracellular through exosomes secreted by Mφs to induce endothelial dysfunction (140). Specificity protein-1 was found to promote histone deacetylase 4-mediated deacetylation of HMGB1, thereby reducing intestinal barrier dysfunction, oxidative stress, and the inflammatory response induced by sepsis (141).