Prevention of Inflammatory disorders in the preterm neonate: an update with a special focus on BPD

Abstract

Background:

The rates of major neonatal morbidities, such as bronchopulmonary dysplasia, necrotizing enterocolitis, preterm white matter disease, and retinopathy of prematurity remain high among surviving preterm infants. Exposure to inflammatory stimuli and the subsequent host innate immune response contribute to the risk of developing these complications of prematurity. Notably, the burden of inflammation and associated neonatal morbidity is inversely related to gestational age – leaving primarily but not exclusively the tiniest babies at highest risk.

Summary:

Avoidance, prevention and treatment of inflammation to reduce this burden remains a major goal for neonatologists worldwide. In this review, we discuss the link between the host response to inflammatory stimuli and the disease state. We argue that inflammatory exposures play a key role in the pathobiology of preterm birth, and that preterm neonates hereafter are highly susceptible to immune stimulation not only from their surrounding environment but also from therapeutic interventions employed in clinical care. Using bronchopulmonary dysplasia as an example, we report clinical studies demonstrating the potential utility of targeting inflammation to prevent this neonatal morbidity. On the contrary, we highlight limitations in our current understanding of how inflammation contributes to disease prevention and treatment.

Key Message:

To be successful in preventing and treating inflammation-driven morbidity in neonatal intensive care, it may be necessary to better identify at-risk patients and pair therapeutic interventions to key pathways and mediators of inflammation-associated neonatal morbidity identified in pre-clinical and translational studies.

I. Neonatology and Inflammation: a complex relationship

Therapeutic advances in neonatal intensive care have led to steady improvements in survival of preterm infants and lowering of the generally accepted “limits of viability.” However, despite these gains, rates of major complications of prematurity, such as bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC), preterm white matter disease (WMD) and retinopathy of prematurity (ROP) remain high [1]. Observational and pre-clinical studies have established that various exposures in a multiple-hit sequence initiate a pro-inflammatory host response that adversely affects parenchymal development and contributes to the pathogenesis of these major morbidities [2–5]. Thus, most of the common neonatal morbidities could be classified as inflammatory disorders. Broadly and effectively targeting “inflammation” is a major therapeutic focus aiming to reduce the burden of common morbidities associated with preterm birth.

II. The host response to inflammatory exposures: thematic links in the development of major neonatal morbidities

Unique Aspects of the Innate Immune Response

Through anatomic barriers, physiologic responses, endocytic and phagocytic mechanisms, and pro-inflammatory responses the innate immune system represents the first line of defense against potentially injurious stimuli [6]. A central feature of the innate immune inflammatory process is a conserved set of pattern recognition receptors (PRR) that allow cells to respond to a diverse set of potentially detrimental stimuli [7]. This paradigm, first proposed by Janeway in 1989 [8] is beautifully sophisticated in its simplicity: through the recognition of conserved molecular structures among pathogens, a relatively small number of receptors serve to active multiple signaling pathways. Beyond these pathogen-associated molecular patterns (PAMPS), molecules from damaged cells termed damage-associated molecular patterns (DAMPS) signal through PRRs. The latter include Toll-like receptors (TLRs) [7, 9], RIG-I-like receptors, NOD-like receptors, cyclic guanosine monophosphate adenosine monophosphate synthase like receptors, and C-type lectin receptors [7, 10]. Notably, shared signaling pathways converge to activate conserved transcription factors including NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), AP-1 (activator protein 1) and the interferon response factors (IRFs) [7] culminating in cell-type specific expression of cytokines, chemokines, lipid mediators, antimicrobial peptides and complement [10]. Importantly, this release of pro-inflammatory mediators is paralleled by the activation of innate immune signaling driving anti-inflammatory as well as pro- and anti-apoptotic responses both being critical for the limitation and resolution of inflammation [11, 12]. The wide-ranging implications of innate immune activation must be recognized when considering therapeutic interventions to dampen inflammation.

Evidence of Innate Immune Activation in Common Neonatal Morbidities

It is widely recognized that the detection of pro-inflammatory biomarkers in tracheal aspirates, cord blood, infant blood, and urine correlates with an increased risk of developing BPD [13]. Pro-inflammatory biomarkers have also been shown to predict development and disease progression of NEC [14–16]. Similar observations exist for WMD [3, 17] and ROP [18].

The interleukin-1 (IL-1) family provides a useful example of how shared innate immune pathways contribute to the pathogenesis of multiple neonatal morbidities. To date, eleven IL-1 family ligands have been identified and their unique roles continue to be defined [19]. Importantly, both pre-clinical and clinical studies have shown that IL-1β plays a central role in BPD, NEC, WMD and ROP. This potent pro-inflammatory cytokine is thought to be secreted locally by immune cells (monocytes, macrophages, dendritic cells) and mediate downstream systemic effects through the activation of the transcription factors NFκB, mitogen activated protein kinases (MAPK), and AP-1. Elevated levels of IL-1β can be detected in neonates exposed to chorioamnionitis, and in tracheal aspirates and blood samples of those preterm infants who subsequently develop BPD [13]. In infants with NEC, elevated levels of IL-1β are present in the blood and intestinal tissue [20–22]. Additionally, increased levels of IL-1β correlate with a neurologic insult in preterm infants [23]. Preclinical studies strongly implicate a role of IL-1β in the pathogenesis of ROP [19], although more clinical data are needed to establish this specific link [24].

III. Inflammatory exposures are inevitable for the preterm infant

It could be argued that exposure to inflammatory stimuli is a “normal condition” following preterm delivery, with exposures ranging from the mechanisms driving preterm delivery to many therapies employed by neonatologists. Understanding this paradigm helps identify therapies to prevent inflammatory disorders specific to the perinatal period.

Inflammation is associated with the majority of preterm births

It is increasingly recognized that normal labor itself is an “inflammatory process”, with PRRs, proinflammatory cytokines/chemokines and proteases/enzymes contributing to uterine contractility, cervical ripening and membrane activation and rupture [25]. These inflammatory sequences become even more relevant when considering the ~10% of births worldwide that occur preterm [26, 27]. Notably, a significant proportion of those born in the setting of spontaneous preterm labor with intact membranes or premature preterm rupture of membranes followed by labor have been exposed to intrauterine infection and associated inflammation. Intrauterine infection accounts for 25–40% of preterm births, disproportionately affecting those born at lower GA [28]. The current paradigm suggests that infectious exposures accelerate and potentiate the innate immune signaling contributing to the initiation of labor. This hypothesis is further supported by the finding that systemic maternal infectious exposures, including asymptomatic bacteriuria, pneumonia, appendicitis, and periodontal disease, are associated with preterm delivery [26]. Pathogens associated with intrauterine infection and inflammation comprise aerobic, anaerobic, and atypical bacteria – many of those generally considered of low virulence [29]. It deserves special consideration, that Ureaplasma species are some of the pathogens which are most frequently detected in the amniotic fluid and placental tissues of histologically confirmed chorioamnionitis associated with preterm birth – most recent data coming from investigations using metagenomic approaches [30, 29]. Isolation of Ureaplasma from the amniotic fluid is strongly associated with an intrauterine inflammatory response and adverse pregnancy and neonatal outcomes, especially BPD [30–32].

There is a growing recognition that a significant percentage of deliveries for maternal or fetal indications has associated innate immune signaling [33]. While multiple mechanisms are under active investigation, here we will highlight the role played by TLR9. The ligand for the intracellular PRR TLR9 is the DAMP CpG-rich DNA, commonly found in viruses, bacteria and protozoa [7]. However, as these CpG-rich motifs are found in mammalian mitochondrial DNA, TLR9 allows for an innate immune response to self in the setting of cellular injury and has been implicated in multiple disease states in adults [34]. The presence of cell free fetal DNA (cff-DNA) in the maternal circulation following placental transfer is well accepted and clinically utilized [35]. Importantly, fetal DNA-TLR9 activation has been implicated spontaneous labor both in the term and preterm population [36, 37]. In addition, both IUGR and preeclampsia have been associated with increased cff-DNA [37, 38] and placental TLR9 expression [38]. In this paradigm, rather than initiating labor, TLR9-mediated innate immune activation leads to vascular disturbances and placental dysfunction [39, 38].

Inflammation occurs with multiple clinically indicated interventions: the clinician’s role in maintaining a pro-inflammatory state

Supplemental Oxygen

First proposed by Saugstad in 1988 [40], oxygen radical disease of the newborn links the etiology of common neonatal morbidities, including BPD, NEC, WMD and ROP, to oxidative stress [40–42]. New evidence demonstrate that prenatal hypoxia is likely another source of oxidative stress-related diseases, including vascular remodeling and pulmonary hypertension [43]. Oxidative stress following hypoxia, ischemia/reperfusion, and/or hyperoxia is a major contributor and an additive injurious hit to inflammation, both potentiating subsequent organ injury in a complex interplay [41, 44, 42]. Notably, supplemental oxygen is one of the most prescribed therapies for preterm neonates that may cause oxidative stress through inadvertent hyperoxia [41, 45]. Hyperoxia-associated inflammation has been clearly associated with the development of BPD, NEC, WMD and ROP [46–49].

Mechanical Ventilation

Of the nearly 10,000 babies born at 22–28 weeks’ GA at National Institute of Child Health and Human Development (NICHD) Neonatal Research Network centres in the US between 2013–18, ~85% were exposed to mechanical ventilation (MV) during their hospitalization [1]. The latter is a major and often prolonged stimulus for pulmonary and systemic inflammation in preterm infants. Any type of MV, even strategies designed to minimize preterm lung injury, may be injurious to developing airways and lung tissue, provoking a cascade characterized by epithelial and endothelial injury, release of pro-inflammatory mediators, and activation and recruitment of inflammatory cells [50]. In addition, exposure to MV and associated inflammation has been implicated in the pathogenesis of WMD [50]. Of note, the duration of MV exposure is associated with elevated levels of circulating pro-inflammatory cytokines (IL-1β, TNF-α) and chemokines (IL-8, MCP-1) [51].

Nutrition, and transfusions

Nutritional support can cause inflammation in the preterm infant. Formula feeding is a known risk factor for developing NEC [52]. A pro-inflammatory response to cow’s milk-based formula has been implicated in NEC development. Newborn mouse pups exposed to preterm formula demonstrate intestinal NFκB activation and upregulation of pro-inflammatory gene expression [53]. Furthermore, peripheral blood mononuclear cells isolated from NEC patients exposed to cow’s milk protein showed increased expression of immunomodulatory cytokines including IL-4 and IFN-γ [54]. It has been hypothesized that the lack of anti-inflammatory factors abundantly found in maternal milk contributes to the increased risk of NEC in formula fed infants [55]. Parenteral nutrition is an additional potential pro-inflammatory stimulus [56]. Recent evidence has shed light on the unique contribution of the fatty acid and phytosterol component of soybean oil rich intravenous lipid emulsions on the pro-inflammatory milieu [56].

Administration of blood products is an additional source of pro-inflammatory stimuli [57]. Although rare, the potential for immunologic reactions to red blood cell (RBC) transfusion, including transfusion-related acute lung injury and graft-versus-host disease, have long been recognized [58]. Importantly, there is a growing recognition that transfusion-related immunomodulation (TRIM) may be more common that currently appreciated and have meaningful affects upon neonatal short- and long-term outcomes [58]. TRIM encompasses the pro-inflammatory and immunosuppressive impact following administration of blood products. In neonates, RBC transfusions increase circulating levels of proinflammatory Il-1β, Il-8, IFN-γ, and IP-10 [59]. The immunomodulatory impact of RBC transfusions on neonates may be sex-specific [60]. Platelets are also known to have a central role in the pathogenesis of immune-mediated inflammatory diseases [61] being a potent source of proinflammatory cytokines and chemokines, including Il-1β, CXCL1 and CCL5 [57]. Whether platelet transfusions induce expression of pro-inflammatory mediators or result in inflammatory injury is unknown, and remains to be studied [57].

IV. Current approaches to mitigate neonatal inflammatory disease: with special focus on preventing BPD

The onus is upon the neonatologist to recognize and accept the ubiquitous nature of inflammatory exposures and either prevent or minimize the effect of these injurious events. Here, we used the example of targeting inflammation to prevent BPD to demonstrate what we have learned about preventing neonatal inflammatory disorders. This is not meant to be an exhaustive list of therapies, but rather an exercise in understanding the current challenges with targeting inflammation.

Preventing pro-inflammatory exposures: supplemental oxygen, mechanical ventilation, formula, transfusions

Interventions aimed at avoiding pro-inflammatory exposures have been shown to decrease the risk of BPD. The five RCT (SUPPORT, COT, BOOST-AUS, BOOST-UK, BOOST-NZ) comprising NeOProM (Neonatal Oxygenation Prospective Meta-analysis Collaboration) asked whether targeting saturations of 85–89% compared to 90–95% could decrease death or disability at 2 years corrected age [62]. Limiting supplemental oxygen exposure through targeting saturations of 85–89% led to decreased rates of BPD [63, 64]. However, given the observed increase in mortality in this arm, more refined approaches to limit supplemental oxygen exposure are needed. Randomized controlled trials comparing routine use of CPAP in very high-risk neonates (24 0/7 to 28 6/7 wks’ GA) to intubation and prophylactic surfactant found decreased exposure to MV and a reduction in the incidence of BPD or death with a NNT of 17.7 [65]. Importantly, a recent meta-analysis demonstrated the protective effect of breast milk and donor breast milk compared to formula in preventing BPD [66]. Meta-analysis of trials interrogating restrictive versus liberal transfusion criteria for RBCs in preterm infants did not demonstrate a protective effect, however the point estimate favored less exposure [(RR .95, (.89–1.02)] [67]. Individual trials interrogating restrictive versus liberal transfusion criteria (PLANET-2) [68] demonstrate a protective effect with less exposure. Collectively, these studies demonstrate the clinical utility of limiting pro-inflammatory exposures with the goal of decreasing BPD risk.

Blunting the inflammatory response: the evolution of the role of glucocorticoids and other antiinflammatory strategies to prevent BPD

The promise of glucocorticoids – and the risk

The first RCTs evaluating the use of glucocorticoids to prevent BPD were published in the 1980s [69, 70]. The rationale behind these early studies was that BPD was common, associated with high morbidity and mortality, no effective therapies existed, and targeting inflammation with a potent anti-inflammatory treatment was pathophysiologically sound. Given their mechanism of action, corticosteroids would appear to be a near perfect pharmacologic approach to prevent or dampen inflammation in the developing lung through genomic and non-genomic mechanisms [71]. Through interactions with the glucocorticoid receptor (GR), the glucocorticoid-GR complex acts on the DNA at glucocorticoid response elements (GRE) to both repress the translation of pro-inflammatory mediators and induce the expression of anti-inflammatory mediators. Of important note, repression occurs through inhibition of transcription factors central to the innate immune response, including NFκB and AP-1. In addition, specific and non-specific non-genomic mechanisms provide additional anti-inflammatory effect.

Early reports confirmed the effectiveness of glucocorticoids, specifically dexamethasone, in preventing BPD in those extremely high-risk neonates requiring prolonged MV and supplemental oxygen [72, 73]. The robust effectiveness in preventing BPD led to trials evaluating earlier treatment of infants requiring MV for respiratory distress syndrome. While these trials confirmed the effectiveness of this approach in preventing BPD, early (within the first week) glucocorticoid exposure was found to increase the risk of cerebral palsy and poor neuromotor and cognitive outcomes [74–76]. Based on these findings, the American Academy of Pediatrics issued a moratorium in 2002 (since revised [77]) on the routine use of postnatal steroids to prevent BPD [78] and subsequently use precipitously fell [79]. Since this time, the allure of the powerful treatment effect [80] continues to motivate investigators to attempt to fine-tune steroid administration to minimize associated risk.

Decreasing the risk by changing the drug or route of delivery

One approach to minimize risk associated with anti-inflammatory therapy is to change the pharmacologic agent. Pre-clinical evidence demonstrates that dexamethasone, a pure glucocorticoid, starves neurons of mineralocorticoid stimulation resulting in apoptosis [70]. Thus, hydrocortisone, despite less glucocorticoid activity, may still confer benefit while preventing toxicity through its mineralocorticoid activity. This hypothesis was tested by Watterberg and colleagues in the early noughties [81]. In this study, babies with BW 500–999 grams who were mechanically ventilated in the first two days of life were given a 15-day course of hydrocortisone. The trial was stopped early due to more frequent intestinal perforation in the treatment arm; the concerning signal for intestinal perforation has been subsequently linked to co-administration of indomethacin for treatment of PDA. No difference in the incidence of BPD was found between groups; however, in those exposed to chorioamnionitis, treatment resulted in a significant decrease in BPD. A larger and more recent trial published in 2016 by Baud and colleagues demonstrated a significant increase in survival without BPD [OR 1.48, 1.02–2.16 p=0.04] (PREMILOC) [82]. Experts noted that this trial also had limitations, including early stoppage for logistical reasons, a wide confidence interval (e.g., number needed to treat 95% confidence interval ranged 6 to 200), and inclusion of only inborn infants [83]. However, these tantalizing data reinforce the conclusion that attenuating inflammation is beneficial, but the appropriate approach remains to be determined.

An alternative approach to minimize risk of exposure is to target a population that is highly likely to develop BPD and thus benefit from exposure to therapy. Two recent large RCTs interrogated whether hydrocortisone could prevent BPD in a high-risk cohort of preterm infants – those being born at <30 wks’ GA and requiring MV later in their NICU course. Watterberg and colleagues targeted intubated babies at 14 to 28 days of age with a 10-day course of hydrocortisone [84]. Onland and colleagues selected intubated babies between 7 and 14 days with a 22-day course of hydrocortisone [85]. Neither trial found a significant reduction in BPD rates following hydrocortisone.

Yet another approach to minimize risk of exposure is tissue or organ specific delivery using inhaled corticosteroids or surfactant combined with corticosteroid. Meta-analysis confirms that early administration of inhaled corticosteroids prevents BPD [86], however both the largest trial as well as the meta-analysis demonstrate an increased risk of mortality in exposed infants [87, 70]. This finding has muted enthusiasm for this approach. A protective effect with late administration of inhaled corticosteroids is not observed [88]. Meta-analysis of surfactant combined with corticosteroid demonstrates a robust treatment effect on death or BPD (31.3% vs 52.8%; RR, 0.59; 95% CI, 0.50–0.70; NNT, 5; 95% CI, 4–6) [89]. Ongoing and recently completed RCTs will further inform these results [54] https://clinicaltrials.gov/study/NCT04545866).

The inability to identify the right approach to safely blunt the immune response with corticosteroids and prevent BPD has recently been summarized by Dr. Lex Doyle: “With greater than 80 randomized controlled trials of corticosteroids to prevent or treat bronchopulmonary dysplasia, enrolling greater than 9,000 infants, corticosteroids are one of the most investigated medications in newborn medicine. After such intensive investigation, one might be forgiven for thinking that the answers about who and when to treat, and with what medication might be clear cut. Alas, that is not the case.” [69]. The right agent, the right dose and the target population remain elusive.

V. What’s next in preventing inflammation: better targets and better therapies

It has been shown that when the risk of BPD exceeds ~50%, the benefit of steroids begins to outweigh possible long-term neurologic harm (e.g., cerebral palsy) [90]. Given the relationship between MV and BPD [91], previous trials interrogating the ability of steroids to prevent BPD have used need for MV to risk stratify subjects. However, as demonstrated above, this approach has not revealed the appropriate therapeutic approach. Perhaps the use of MV as a proxy for BPD risk is flawed, not allowing for optimal selection of the target population. Recent work supports the hypothesis that risk assessment can be improved and allow more targeted therapy. Jensen et al reported that among babies at National Research Network centers and exposed to steroids (between day 8 and 42 after birth), treatment conferred benefit that correlated with increasing pretreatment risk of death or grade 2 or 3 BPD [92]. Just as importantly, treatment was associated with harm as pretreatment risk fell. Similarly, by adjusting for baseline risk greater evidence of treatment advantage was found for those enrolled the PREMILOC study [93]. These findings support the premise that targeting a population with higher baseline risk will decrease the number needed to treat and minimize exposure to potential harm [94].

Assessment of the individual pretreatment risk is reflected by presence of variables known to be associated with developing BPD, and in that way limited to our current understanding of the disease pathogenesis. Artificial intelligence, not limited by clinician bias and comprising clinical and biomarkers, genomics, proteomics, and metabolomics, has been employed to help identify BPD risk [95–99]. In addition, these approaches might help to predict other neonatal inflammatory disorders including NEC [95, 98, 100] and ROP [95, 98, 101]. However, to date, artificial intelligence has not been used to select patients and target anti-inflammatory strategies to prevent BPD.

Finally, it is likely that more targeted therapies are needed to harness the full clinical benefit of preventing inflammation. Glucocorticoids act at the genomic level to interrupt the activity of key transcription factors, including NFκB [71]. While this is favorable in terms of preventing inflammation, there are likely detrimental effects of global inhibition of these key transcriptional regulators. For example, NFκB controls the expression of anti-oxidative and anti-apoptotic genes, growth factors, cell cycle regulators and other transcription factors [102]. Blunted expression of these protective factors may delay or prevent resolution of inflammation [103, 11, 104, 105]. Thus, global inhibition of “pro-inflammatory” pathways may impair recovery or result in abnormal development. Pre-clinical studies show that in the absence of any injurious stimuli, complete NFκB inhibition disrupts normal lung development and impairs resolution of inflammation [103, 106, 107]. Whether inhibition of target genes critical for normal development contributes to harm seen clinically with systemic delivery of glucocorticoids is likely impossible to determine. However, this potential complication argues for targeted therapies that don’t inhibit the immune response but specifically attenuate the effects of pro-inflammatory mediators. Stem cell therapy is one area of active investigation that may help resolve the inflammatory imbalance contributing to inflammatory disorders associated with prematurity, including BPD, WMD, ROP and NEC [108–110].

As given above, pro-inflammatory IL-1β is one potential target downstream of transcriptional repression. Clinically, elevated levels of IL-1β have consistently been identified as a risk factor for developing BPD [19]. Preclinical models demonstrate that blocking IL-1β activity at its receptor attenuates experimental BPD caused by hyperoxia or hyperoxia and systemic inflammation [111, 112]. A phase I/II trial is underway to first determine the safety of exposing 24–27+6 weeks gestation neonates to an interleukin-1 receptor antagonist (Anakinra) (https://clinicaltrials.gov/study/NCT05280340) [113]. Future studies will determine if more targeted therapies like this will prevent the development of BPD while minimizing previously identified side effects associated with more broad inhibition of the immune response.

Finally, macrolides, particularly azithromycin, have been discussed as potential agents for prevention of adverse neonatal lung inflammation due to anti-infective and anti-inflammatory properties and beneficial effects seen in children and adults with inflammatory lung diseases [114, 115]. However, the very recently published AZTEC trial (Azithromycin Therapy for Chronic Lung Disease of Prematurity) did not find reduced rates of BPD in preterm infants < 30 weeks’ gestation prophylactically treated with azithromycin in the first days of life independent of the Ureaplasma colonization status [115].

Conclusion

Inflammatory exposures contribute to the pathogenesis of BPD, NEC, WMD and ROP. Prenatal and postnatal inflammatory exposures promote a host innate immune response initiating a multi-hit cascade of adverse events culminating in tissue injury. Prevention and early treatment of fetal and neonatal inflammation is essential in limiting neonatal morbidity. Unfortunately, current strategies are limited. Further understanding of the thematic links between exposures and host immune response and how these contribute to disease pathogenesis is needed to identify targetable pathogenic mechanisms, prevent adverse off-target effects, and maintain the beneficial effects of a functioning innate immune system. Finally, it is necessary to better identify preterm infants at-risk and tailor therapeutic interventions.