Necrotizing enterocolitis (NEC) is the most common devastating gastrointestinal emergency that causes significant morbidity and mortality rates in preterm infants (1, 2). Classical NEC occurs in 7%–12% of preterm infants most of whom are born before 32 weeks of gestation or with very low birth weight (VLBW, birthweight less than 1,500 g) (3, 4). Meanwhile, NEC can occur in late preterm or term infants with other comorbidities, including those with congenital cardiac defects (5). The precise cause of NEC remains elusive, although several risk factors have been identified, including prematurity, immune dysregulation, formula feeding, abnormal bacterial colonization of the gut, and intestinal ischemia (4). NEC symptoms can be insidious at first, but can quickly progress with hallmark signs on radiographic images such as pneumatosis intestinalis (air within the bowel wall), portal venous gas, and free air in the abdomen (6). The severity is characterized using modified Bell's staging system to classify infants into 1 of 3 stages of NEC severity based on systemic, radiographic and gastrointestinal findings (7). While some patients with NEC can be treated conservatively with antibiotics and withholding feeds, thirty to fifty percent of infants with NEC must undergo surgical intervention to remove compromised intestine (1). NEC is associated with multiple immediate and long-term complications, such as death, sepsis, intestinal failure, growth delay, and adverse neurodevelopmental outcomes (4) despite advances in neonatal care. Thus, highlighting the need for continued research to better understand its pathophysiology, improve diagnostic capabilities, and develop targeted therapeutic strategies.

Premature infants are particularly susceptible to ischemic insults, leading to mucosal damage, barrier dysfunction, and bacterial translocation, initiating the inflammatory cascade characteristic of NEC (8). Furthermore, proinflammatory mediators and chemo attractants trigger an infiltration of macrophages, neutrophils and myeloid-derived suppressor cells (MDSCs) that migrate from the blood into intestinal mucosa resulting in mucosal injury, barrier dysfunction, and systemic inflammation (9). MDSCs are immature myeloid cells with potent immunosuppressive functions that serve as a key component of neonatal immune defense and are essential for preventing excessive intestinal inflammation, including NEC (10). Reduced MDSC frequency and suppressive activity in preterm and very low birth weight infants correlate with increased NEC susceptibility and disease severity, highlighting impaired MDSC-mediated immune regulation as a contributor to NEC pathogenesis (10).

In healthy intestinal mucosa, macrophages are one of the first responders in infection, which are crucial for cytokine production, antigen presentation to other immune cells, and maintain homeostasis through the phagocytosis of debris and pathogens (11). Macrophages exist along a functional spectrum, broadly categorized as pro-inflammatory M1 cells that activate Toll-like receptor-4 (TLR4) signaling and drive intestinal injury, and anti-inflammatory M2 cells that promote immune regulation, epithelial repair, and resolution of inflammation (11). Monocytes circulate within the peripheral blood, while resident macrophages are localized to tissues including the intestine (11). Intestinal monocytes are broadly categorized into classical and non-classical subsets based on their surface marker expression and functional profiles. Classical monocytes, which express high levels of CD14 and low or no CD16 (CD14++CD16−), are primarily involved in phagocytosis and the initiation of inflammatory responses (12, 13). In contrast, non-classical monocytes, defined by lower CD14 and high CD16 expression (CD14 + CD16++), migrate along the endothelium and are typically involved in tissue surveillance and inflammation resolution under normal conditions (12, 13).

Neutrophils are a type of white blood cell that play a key role in the body's innate immune response, acting as the first line of defense against infection by rapidly migrating to sites of tissue injury or microbial invasion. In healthy individuals, they circulate in the blood and help maintain immune surveillance by identifying and neutralizing pathogens through phagocytosis, cytokine secretion, and the release of inflammatory mediators. They can also function as antigen-presenting cells to modulate downstream adaptive immune responses. In the context of NEC, a decline in circulating neutrophil count is often observed and is a critical indicator of disease severity and predictor of rapid progression (1, 8). Emerging evidence supports a role for neutrophils in maintaining intestinal immune homeostasis, suggesting that disruption of these protective functions may further contribute to intestinal injury and inflammation in NEC (8). Understanding the intricate roles of neutrophils, monocytes, macrophages, and MDSCs in both health and disease states is essential for unraveling the mechanisms underlying NEC. In this review, we delve into the multifaceted roles of these myeloid cells in NEC pathogenesis and intestinal homeostasis, while aiming to shed light on potential targeted preventive strategies and therapeutic interventions to mitigate the impact of this condition on premature infants.

For this scoping review of literature on the role of monocytes and neutrophils in the pathogenesis of NEC, we performed a search using PubMED with a timeframe spanning January 2011 through December 31, 2025 (60). The search was performed in separate attempts to capture unique research articles. We used the following strings in separate searches: “monocytes necrotizing enterocolitis” (82 articles), “neutrophils necrotizing enterocolitis” (199 articles), and “MDSC necrotizing enterocolitis” (10 articles). To highlight relevant studies capturing patients with congenital heart disease (CHD) who develop NEC we searched: “monocytes cardiac necrotizing enterocolitis” (2 articles), “neutrophils cardiac necrotizing enterocolitis” (8 articles), and “MDSC cardiac necrotizing enterocolitis” (0 articles), yielding a total of 109 unique articles. After all authors reviewed the abstracts, we excluded 154 articles that lacked relevance to the question, were outside the publication year range, or were review articles (Figure 1). Upon review of all articles, primary research findings that elucidate the role of neutrophils, monocytes, and MDSCs in the pathogenesis of NEC from 2011 to 2025 were summarized. We included 38 articles in total.

Thus far, this review has focused on “classical” inflammatory NEC which is primarily a disease of prematurity, intestinal dysbiosis, and inflammation, recent studies have distinguished cardiac NEC as a disease with its own clinical and molecular pathogenesis (5, 39–41). Infants with cardiac NEC, which arises in the setting of medical or surgical treatment for cardiac disease, are typically chronologically younger at the time of diagnosis but of greater gestational age, with higher birth weights compared to infants with classical NEC (5). Cardiac NEC is primarily driven by vascular (ischemia/reperfusion) injury rather than the dysbiosis-induced inflammation and tissue injury seen in classical NEC (39) as such neutrophils, monocytes and macrophages may play a different role and have a distinct phenotype in these patients. However, studies exploring the role of these cell types in infants with congenital heart disease who develop NEC remain limited.

Emerging data suggests neutrophil activation may be a defining feature of cardiac NEC. Compared with infants with classical inflammatory NEC, those with cardiac NEC demonstrate higher circulating leukocyte and neutrophil counts (5). One proposed mechanism is increased formation of NETs leading to increased neutrophil activation and eventual hyperinflammation (5, 40). A study by O'Connor et al. validated fecal calprotectin as a noninvasive biomarker of intestinal inflammation in neonates with congenital heart disease, demonstrating significantly higher levels in infants with definitive NEC compared with those with suspected disease (41). Similarly, MacQueen et al. reported elevated fecal calprotectin concentrations at the onset of bowel symptoms in neonates with NEC and further showed that calprotectin is actively exported from neutrophils through NET formation (40). Together, these findings support a neutrophil-driven inflammatory signature in cardiac NEC, in contrast to classical NEC, which is more commonly associated with sepsis-related neutropenia (40, 41). The distinct clinical and molecular profiles of classical and cardiac NEC indicate that they are separate classes driven by unique inflammatory and ischemic pathways, respectively. Considering these differences is necessary to develop diagnostic tools and therapeutic strategies to improve outcomes for neonates with each disease type.

Other hematologic abnormalities are associated with NEC severity and progression. We briefly describe some here but acknowledge that including the extensive body of work on anemia-transfusion associated NEC is beyond the scope of this review (42–44). Thrombocytopenia particularly in infants <28 weeks' gestation strongly associated with S-NEC and increased mortality (33, 45–47). Similarly, severe anemia (hematocrit <25%) at presentation was linked to a higher risk of mortality across all gestations (43, 45, 47). Likewise, infants born at gestational ages between 28 and 32 weeks are more likely to present with pancytopenia following NEC onset (47). Hematological markers based on gestational age are critical for assessing NEC severity, mortality risk, and the need for surgical intervention though universally accepted values are currently lacking.

To reduce the incidence and severity of NEC, future investigations should focus on incorporating therapeutics that target neutrophils, monocytes/macrophages, and MDSCs. Ideally, these therapies should be safe, effective, easily administered, widely available, and have a known mechanism of action. Several approaches have been explored to date, including modulation of macrophage phenotype, administration of bioactive milk-derived factors, probiotics, and anti-inflammatory or anti-cytokine therapies.

Macrophages play a central role in NEC pathogenesis and exist along a functional spectrum, broadly categorized as classically activated pro-inflammatory M1 macrophages and alternatively activated anti-inflammatory M2 macrophages (48). M1 macrophages activate TLR4 signaling and produce pro-inflammatory cytokines that contribute to intestinal injury, whereas M2 macrophages promote immune regulation, epithelial repair, and resolution of inflammation (11, 48). Dysregulation of this balance, with predominance of M1 polarization, has been implicated in NEC development (48). Additionally, inhibition of TLR4 signaling has been shown to alleviate NEC-associated inflammation, thrombocytopenia, and intestinal damage (11). Consequently, therapeutic strategies aimed at suppressing M1 polarization and/or promoting M2 polarization represent a promising avenue for intervention.

Several experimental therapies have been developed to directly target macrophage activity. Semapimod, a macrophage deactivator, suppresses pro-inflammatory cytokine production (49). In neonatal rat models of NEC, administration of semapimod reduced macrophage activation, limited intestinal injury, and partially protected against intestinal epithelial cell death through suppression of pro-inflammatory cytokine signaling both in vitro and in vivo (49).

Beyond macrophage depletion or deactivation, therapies that actively promote M2 polarization have also shown benefit. Heparin-binding EGF-like growth factor (HB-EGF) suppresses M1 polarization while promoting M2 transition, thereby enhancing mucosal healing (11). Hydrogen therapy has similarly been shown to inhibit NF-κB activation and favor M2 polarization (11). Moreover, glutaredoxin-1 deficiency and deletion of interferon regulatory factor 5 (IRF5) in myeloid cells reduce M1 polarization and systemic inflammation in experimental NEC (11). Collectively, these findings support targeted modulation of macrophage phenotype as a viable therapeutic strategy.

Anti-cytokine therapy offers a promising approach for treating NEC by targeting key inflammatory pathways. Blocking TNF-α signaling with antibodies or inhibitors reduces intestinal inflammation and tissue damage, while tocilizumab, an IL-6 receptor inhibitor, alleviates NEC by blocking IL-6 signaling (11). TNF-α and TGF-β, released by activated intestinal macrophages, play critical roles in NEC pathogenesis. Supplementing TGF-β2 encourages the differentiation of immature, pro-inflammatory macrophages into mature, non-inflammatory ones, reducing NEC incidence (11). In parallel, supplementation with IL-10 and TGF-β enhances FOXP3 expression, suggesting that monocyte dysfunction in NEC may impair regulatory T-cell development and contribute to disease progression (21). Together, these findings elucidate the role of monocytes and macrophages in intestinal inflammation in S-NEC and highlight the therapeutic potential of IL-10 and TGF-β–based strategies (13, 21). Overall, these strategies highlight the therapeutic potential of modulating cytokine activity to prevent intestinal injury and inflammation in NEC.

Similarly, IL-37 modulates immune homeostasis, TLR repertoires, and microbial diversity (50). Both IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, with lower IL-37 levels in blood monocytes of infants with NEC (50). Transgenic IL-37 mice were effectively protected from NEC-related complications, whereas exogenous IL-37 showed only moderate efficacy in preventing NEC (50). The study suggests that targeting TLRs and IL-37 could offer new therapeutic options for NEC. IL-37 counteracts inflammation, particularly in the small intestine, and restores intestinal TGF-β1 levels. While boosting the IL-37 pathway shows promise, further optimization is needed for effective exogenous IL-37 treatment (50).

Recent experimental studies support therapeutic strategies aimed at restoring MDSC recruitment and function. Inhibition of GIT2, along with modulation of other upstream regulators such as TFF3 and adenosine signaling, enhances intestinal MDSC accumulation resulting in reduced intestinal inflammation, improved barrier integrity, and restoration of adaptive immune balance (10, 31, 32). In murine models, these approaches consistently increase regulatory T-cell responses, suppress Th17-driven inflammation, and improve survival, underscoring the translational potential of targeting MDSC-mediated immune regulation to mitigate NEC severity (10, 31, 32).

Among biologic therapies, recombinant TGF-β2 has attracted interest. TGF-β2 is readily synthesized in large quantities, is naturally present at high concentrations in amniotic fluid and human milk, and is a known suppressor of macrophage cytokine production in NEC affected intestine (51). However, further studies are required to clarify its role as a prophylactic or therapeutic intervention in infants at risk for or affected by NEC.

Maximizing human milk feeding remains one of the most effective strategies for NEC prevention (11). Bioactive components of human milk, including lactoferrin and human milk oligosaccharides, enhance intestinal macrophage immune function and significantly reduce NEC incidence (11). Human milk oligosaccharides promote M2 macrophage activation, leading to reduced intestinal injury in preterm infants (11). Lactoferrin, in particular, plays a critical role in inducing a suppressive phenotype in MDSCs, which regulate inflammatory responses during early microbial gut colonization. This activity counteracts Th17 cell upregulation in the small intestine during NEC while increasing regulatory T cells in animal models (28).

A key limitation of lactoferrin as a therapeutic agent is that it is administered enterally, and infants receiving standard treatment for NEC are often not receiving enteral nutrition (52). Although the use of donor human milk in conjunction with maternal milk is increasing among extremely premature infants at risk for NEC, lactoferrin concentrations vary in maternal milk and are reduced by pasteurization of donor human milk (52). As such, establishing a therapeutic threshold for lactoferrin in human milk and providing targeted supplementation to infants with inadequate exposure may represent a useful direction for future studies.

Adding to this protective evidence, Vakhal et al. demonstrated that human milk derived extracellular vesicles (EVs) carry bioactive micro-RNA molecules that may shield the newborn intestines from inflammation (53). The study found that term-derived EVs were shown to inhibit secretion of IL-6 and reduced the expression of IL-1β in macrophages, two primary drivers of the hyperinflammatory response. Furthermore, both term and preterm milk derived EVs were shown to reduce pyroptosis which is often triggered by the cytokine storm seen in NEC (53).

Probiotics have been extensively researched as a preventative strategy for infants at elevated risk of developing NEC (11, 54, 55). Sajankila et al. highlight how probiotics may help protect against NEC by restoring balance to the gut microbiota and modulating the neonatal immune response (54). Specific strains, such as Lactobacillus, have been shown to promote anti-inflammatory M2 macrophage differentiation, potentially reducing intestinal injury associated with NEC (55). Moreover, byproducts of probiotic activity, like butyric acid, contribute to inflammation control by encouraging macrophage maturation and immune regulation (11). Evidence suggests that multi-strain probiotic combinations, particularly those containing both Lactobacillus and Bifidobacterium, are more effective than single-strain formulations in decreasing NEC incidence and related mortality (54). The protective effects of probiotics may be further amplified when given alongside human milk, which supports more effective gut colonization (54). In addition to probiotics, other strategies such as prebiotics (e.g., human milk oligosaccharides), synbiotics (combinations of probiotics and prebiotics), and postbiotics (bioactive microbial metabolites) are under investigation for their potential to prevent NEC. However, despite encouraging data, routine probiotic use remains controversial especially in the U.S. Concerns persist regarding inconsistent clinical outcomes, the risk of probiotic-associated sepsis, and the absence of formulations approved by the Food and Drug Administration (FDA). While the NEC Society (56) advocates for their cautious use in very low birth weight infants, the American Academy of Pediatrics (57) calls for more robust evidence to determine standardized, safe, and effective probiotic use in this population.

Understanding the interplay between intestinal macrophages and other cells within the intestine including the enteric nervous system (ENS), and intestinal epithelial cells (IECs) is crucial for innovative NEC treatments. Disruptions in intestinal motility, regulated by the ENS and interstitial cells of Cajal (ICCs), increase NEC risk in preterm infants (11). Muscularis macrophages (MMs) and an immature ENS contribute to dysregulated motility, which might be restored by enhancing gut microbiota (9). Additionally, intestinal macrophages influence IECs to produce IL-10 via TLR4 signaling, maintaining epithelial integrity. While TNF-α supports mucosal repair, sustained nitric oxide release during NEC hinders IEC migrate and repair (11). Exploring these interactions could improve NEC therapies while preserving intestinal barrier function.

Early broad spectrum antibiotic exposure can result in intestinal dysbiosis which is known to increase the incidence of NEC (58). On the contrary, Nguyen et al. highlight the therapeutic potential of oral antibiotic administration during the early postnatal days in preterm neonates (59). In this study, preterm pigs received oral broad-spectrum antibiotics, ampicillin (30 mg/kg three times daily), gentamicin (2.5 mg/kg twice daily), and metronidazole (10 mg/kg three times daily), for five days after birth. Oral administration was superior to systemic antibiotic delivery in preventing NEC, as it more effectively enhanced blood neutrophil maturation, reduced gut colonization and permeability, and prevented bacterial translocation, thereby protecting formula-fed preterm infants from bacteremia and NEC-like lesions. While concerns about increased microbial resistance and adverse long-term effects may limit the widespread use of neonatal short-term antibiotics, particularly for preterm newborns, it is crucial to explore novel preventive and therapeutic approaches for these vulnerable pediatric patients. Antimicrobial proteins and peptides in human milk may replicate the effects of oral antibiotics, and there is a need to comprehend the combined effects of early milk and microbiota on the developing gut and immune systems to support the health of preterm infants in both the short and long term (59).

Myeloid cells play a pivotal role in the pathogenesis of NEC by driving inflammatory processes that contribute to intestinal injury. Excessive activation of neutrophils and monocytes coupled with impaired MDSC suppressive function is prevalent in NEC affected mucosa. Clinical biomarkers can be obtained from a CBC, as the AMC, NLR, and ANC within 1 week of NEC onset are associated with disease severity. Profound declines in peripheral monocyte and neutrophil counts are correlated with an increase in intestinal neutrophil and monocyte counts in patients with S-NEC. Monocytes and neutrophils are implicated in inflammation-driven intestinal damage and support the exploration of strategies to modulate their activation and inflammatory responses for therapeutic benefit (Supplementary Table S1, Figures 2A,B).

Several therapeutic interventions have shown potential in mitigating monocyte and neutrophil-driven inflammation in NEC. Blocking TLR4 signaling or modulating cytokine responses, such as with TNF-α or IL-6 inhibitors, has been shown to reduce intestinal injury in experimental models. Similarly, human milk EVs can inhibit inflammatory cytokines IL-6 and IL1β. Human milk derived TGF-β2 and lactoferrin promote anti-inflammatory macrophage differentiation and enhance the generation of immunosuppressive myeloid-derived suppressor cells (MDSCs) respectively (Figure 2C). In animal models, lactoferrin-induced MDSCs were more effective than lactoferrin alone, suggesting cell-based therapies may hold future promise. Multi-strain probiotics when administered with breast milk, offer additional protection against NEC by modulating immune responses, suppressing TLR4 signaling, and enhancing Treg activity. These diverse therapeutic strategies highlight a multifaceted approach to NEC prevention and treatment that combines immune regulation, microbial support, and nutritional interventions.

Despite these promising developments, several limitations remain in current research. Many studies are retrospective or rely only on animal models, which may not fully reflect the complexity of NEC in human neonates. Additionally, gaps in understanding the precise molecular interactions between immune cells, the intestinal epithelium, and the microbiota limit the development of targeted therapies. Future research should prioritize prospective, multi-center studies in infants with NEC, employing advanced immunologic and microbiome profiling techniques to uncover precise cellular pathways. Ultimately, a better understanding of the immune mechanisms underlying NEC will guide the development of safe, effective therapies that reduce disease burden while preserving critical immune functions in preterm infants.