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Acute respiratory distress syndrome (ARDS) results in decreased quality of life, including increased risk of pulmonary hypertension (PH). In animal models, ARDS can be induced by lipopolysaccharide (LPS), which can disrupt the pulmonary endothelium and epithelium and induce inflammation. We tested whether in vivo administration or ex vivo treatment with LPS alters the reactivity of intrapulmonary arteries and airways to constrictors relevant to both ARDS and PH, using the precision cut lung slice (PCLS) technique. Mice were administered LPS (10μg/50μl, intranasal) or saline daily for 4d before collection of bronchoalveolar lavage fluid (BALF) or preparation of PCLS. Alternatively, PCLS from naïve mice were left untreated or treated ex vivo with LPS (10μg/mL) or tumour necrosis factor (TNF, 10ng/mL) for 18h. Contraction to endothelin-1 (ET-1), U46619 (a stable mimetic of TXA 2 ) or serotonin (5HT) were quantified. In vivo LPS administration increased BAL total inflammatory cells 5-fold, neutrophils 125-fold and protein 2-fold, as well as the thickness of the pulmonary arterial smooth muscle layer. After in vivo LPS, contraction of intrapulmonary arteries in PCLS to ET-1 and U46619, but not 5HT, increased, while bronchoconstrictor responses were unchanged. In PCLS treated with LPS ex vivo , these differential effects on pulmonary artery and airway contraction were maintained. While LPS increased TNF secretion from PCLS, TNF treatment only increased U46619-induced vasoconstriction. This study demonstrates the potential contributions of LPS-induced inflammation and vascular remodelling to altered intrapulmonary artery reactivity to specific agonists with implications for ARDS-associated PH.

Background and Purpose: Pulmonary arterial hypertension (PAH), a rare fatal disorder characterised by inflammation, vascular remodelling and vasoconstriction. Current vasodilator therapies reduce pulmonary arterial pressure but not mortality. The G-protein coupled formyl peptide receptors (FPRs) mediates vasodilatation and resolution of inflammation, actions possibly beneficial in PAH. We investigated dilator and anti-inflammatory effects of the FPR biased agonist compound 17b in pulmonary vasculature using mouse precision-cut lung slices (PCLS)., Experimental Approach: PCLS from 8-week-old male and female C57BL/6 mice, intrapulmonary arteries were pre-contracted with 5-HT for concentration-response curves to compound 17b and 43, and standard-of-care drugs, sildenafil, iloprost and riociguat. Compound 17b-mediated relaxation was assessed with FPR antagonists or inhibitors and in PCLS treated with TNF-α or LPS. Cytokine release from TNF-α- or LPS-treated PCLS ± compound 17b was measured., Key Results: Compound 17b elicited concentration-dependent vasodilation, with potencies of iloprost > compound 17b = riociguat > compound 43 = sildenafil. Compound 17b was inhibited by the FPR1 antagonist cyclosporin H but not by soluble guanylate cyclase, nitric oxide synthase or cyclooxygenase inhibitors. Under inflammatory conditions, the efficacy and potency of compound 17b were maintained, while iloprost and sildenafil were less effective. Additionally, compound 17b inhibited secretion of PAH-relevant cytokines via FPR2., Conclusions and Implications: Vasodilation to compound 17b but not standard-of-care vasodilators, is maintained under inflammatory conditions, with additional inhibition of PAH-relevant cytokine release. This provides the first evidence that targeting FPR, with biased agonist, simultaneously targets vascular function and inflammation, supporting the development of FPR-based pharmacotherapy to treat PAH., Linked Articles: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Introduction : The World Health Organization (WHO) recommends vaccination against hepatitis B as soon as possible following birth for all infants, regardless of prematurity. Hepatitis B vaccination at birth is clearly justified, represents a crucial step in the global control of perinatally acquired hepatitis B and there are no safety concerns in infants born at term. However, there is limited information on the safety of the hepatitis B vaccine in preterm infants, whose immune responses and morbidity risk differ from those in infants born at term. Objectives : The objectives of this paper are to systematically review the literature regarding the safety and risk of adverse events following immunisation (AEFIs) associated with the administration of the hepatitis B vaccine (monovalent or as part of a combination vaccine) to preterm infants. Methods : We performed a search for relevant papers published between 1 January 2002 and 30 March 2023 in the Ovid MEDLINE, Ovid Embase, Cochrane Central Register of Controlled Trials and CINAHL Plus databases. Two authors independently reviewed and analysed each article to include in the systematic review. Narrative synthesis is presented. Results : Twenty-one relevant papers were identified and included in this systematic review. The vast majority of data pertained to multi-antigen (combination) vaccine preparations and vaccination episodes from 6 weeks of age onwards. We found no publications investigating the timing of the birth dose of the hepatitis B vaccine, and AEFI reporting was exclusively short-term (hours to days following administration). There was substantial variability in the reported rate of AEFIs between studies, ranging from 0% to 96%. Regardless of frequency, AEFIs were mostly minor and included injection site reactions, temperature instability and self-limiting cardiorespiratory events. Six studies reported serious adverse events (SAEs) such as the requirement for escalation of respiratory support. However, these occurred predominantly in high-risk infant populations and were rare (~1%). Using the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) approach, the certainty of evidence was assessed as very low. Conclusions : Despite substantial variability between the relatively small number of published studies in terms of cohort selection, definitions, vaccine preparations and reporting, hepatitis B-containing vaccines (mostly as combination vaccines) appear to be relatively well tolerated in preterm infants from 6 weeks of age. Research focusing on the safety of hepatitis B vaccine in preterm infants specifically within 7 days of birth is lacking, particularly regarding long-term morbidity risk. Further research in this area is required.

Background: Perinatal infection/inflammation is associated with a high risk for neurological injury and neurodevelopmental impairment after birth. Despite a growing preclinical evidence base, anti-inflammatory interventions have not been established in clinical practice, partly because of the range of potential targets. We therefore systematically reviewed preclinical studies of immunomodulation to improve neurological outcomes in the perinatal brain and assessed their therapeutic potential., Methods: We reviewed relevant studies published from January 2012 to July 2023 using PubMed, Medline (OvidSP) and EMBASE databases. Studies were assessed for risk of bias using the SYRCLE risk of bias assessment tool (PROSPERO; registration number CRD42023395690)., Results: Forty preclinical publications using 12 models of perinatal neuroinflammation were identified and divided into 59 individual studies. Twenty-seven anti-inflammatory agents in 19 categories were investigated. Forty-five (76%) of 59 studies reported neuroprotection, from all 19 categories of therapeutics. Notably, 10/10 (100%) studies investigating anti-interleukin (IL)-1 therapies reported improved outcome, whereas half of the studies using corticosteroids (5/10; 50%) reported no improvement or worse outcomes with treatment. Most studies (49/59, 83%) did not control core body temperature (a known potential confounder), and 25 of 59 studies (42%) did not report the sex of subjects. Many studies did not clearly state whether they controlled for potential study bias., Conclusion: Anti-inflammatory therapies are promising candidates for treatment or even prevention of perinatal brain injury. Our analysis highlights key knowledge gaps and opportunities to improve preclinical study design that must be addressed to support clinical translation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Bronchopulmonary dysplasia (BPD) is the most common sequela of prematurity. Although multifactorial in etiology, there is increasing evidence that fetal growth restriction (FGR) and antenatal exposure of the fetus to inflammation play important roles in the postnatal pathophysiology of BPD. Recent studies have focused on disrupted angiogenesis and its influence on alveolarization. Although there are multiple mechanistic links, inflammation is known to be a key driver of this disruption, affecting pulmonary arterial circulation. Although postnatal corticosteroids are commonly used in extremely premature infants to treat inflammation, aimed at obviating the need for intubation and mechanical ventilation or to facilitate extubation, the use of dexamethasone has not reduced the incidence of BPD. Here, we summarize current knowledge on alternative anti-inflammatory treatment options, which have shown promising outcomes either preclinically or clinically. These include supplementation with vitamins C and E (antioxidants), ω-3 polyunsaturated fatty acids, pentoxifylline, anti-inflammatory cytokines of the IL (interleukin)-1 family, namely IL-1 receptor antagonist and IL-37, and the beneficial properties of breast milk. Evaluating these alternative treatments, either individually or as combination therapies in randomized controlled trials stands to immensely benefit the clinical outlook, particularly regarding BPD, for extremely premature infants.

Preterm birth is defined as delivery at <37 weeks of gestational age (GA) and exposes 15 million infants worldwide to serious early life diseases. Lowering the age of viability to 22 weeks GA entailed provision of intensive care to a greater number of extremely premature infants. Moreover, improved survival, especially at extremes of prematurity, comes with a rising incidence of early life diseases with short- and long-term sequelae. The transition from fetal to neonatal circulation is a substantial and complex physiologic adaptation, which normally happens rapidly and in an orderly sequence. Maternal chorioamnionitis or fetal growth restriction (FGR) are two common causes of preterm birth that are associated with impaired circulatory transition. Among many cytokines contributing to the pathogenesis of chorioamnionitis-related perinatal inflammatory diseases, the potent pro-inflammatory interleukin (IL)-1 has been shown to play a central role. The effects of utero-placental insufficiency-related FGR and in-utero hypoxia may also be mediated, in part, via the inflammatory cascade. In preclinical studies, blocking such inflammation, early and effectively, holds great promise for improving the transition of circulation. In this mini-review, we outline the mechanistic pathways leading to abnormalities in transitional circulation in chorioamnionitis and FGR. In addition, we explore the therapeutic potential of targeting IL-1 and its influence on perinatal transition in the context of chorioamnionitis and FGR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (© 2023 Owen, Garrick, Peterson, Berger, Nold, Sehgal and Nold-Petry.)

Preterm birth is a major contributor to neonatal morbidity and mortality. Complications of prematurity such as bronchopulmonary dysplasia (BPD, affecting the lung), pulmonary hypertension associated with BPD (BPD-PH, heart), white matter injury (WMI, brain), retinopathy of prematurity (ROP, eyes), necrotizing enterocolitis (NEC, gut) and sepsis are among the major causes of long-term morbidity in infants born prematurely. Though the origins are multifactorial, inflammation and in particular the imbalance of pro- and anti-inflammatory mediators is now recognized as a key driver of the pathophysiology underlying these illnesses. Here, we review the involvement of the interleukin (IL)-1 family in perinatal inflammation and its clinical implications, with a focus on the potential of these cytokines as therapeutic targets for the development of safe and effective treatments for early life inflammatory diseases.

Interactions between the developing microbiome and maturing immune system in early life are critical for establishment of a homeostasis beneficial to both host and commensals. The lung harbors a diverse community of microbes associated with health and local or systemic disease. We discuss how early life colonization and community changes correlate with immune development and health and disease throughout infancy, childhood, and adult life. We highlight key advances in microbiology, immunology, and computational biology that allow investigation of the functional relevance of interactions between the respiratory microbiome and host immune system, which may unlock the potential for microbiome-based therapeutics.

Perinatal inflammation is a significant risk factor for lifelong neurodevelopmental impairments such as cerebral palsy. Extensive clinical and preclinical evidence links the severity and pattern of perinatal inflammation to impaired maturation of white and grey matters and reduced brain growth. Multiple pathways are involved in the pathogenesis of perinatal inflammation. However, studies of human and experimental perinatal encephalopathy have demonstrated a strong causative link between perinatal encephalopathy and excessive production of the pro-inflammatory effector cytokine interleukin-1. In this review, we summarize clinical and preclinical evidence that underpins interleukin-1 as a critical factor in initiating and perpatuating systemic and central nervous system inflammation and subsequent perinatal brain injury. We also highlight the important role of endogenous interleukin-1 receptor antagonist in mitigating interleukin-1-driven neuroinflammation and tissue damage, and summarize outcomes from clinical and mechanistic animal studies that establish the commercially available interleukin-1 receptor antagonist, anakinra, as a safe and effective therapeutic intervention. We reflect on the evidence supporting clinical translation of interleukin-1 receptor antagonist for infants at the greatest risk of perinatal inflammation and impaired neurodevelopment, and suggest a path to advance interleukin-1 receptor antagonist along the translational path for perinatal neuroprotection.

Autoimmune and inflammatory disorders, including inflammatory bowel disease (IBD), commonly affect women of childbearing age, warranting the use of immunomodulatory agents at a time where pregnancy may be desired. In utero exposure to pro-inflammatory mediators from maternal IBD, IBD-associated intestinal dysbiosis, and immunomodulatory drug use may impact neonatal immune system development during what is considered to be a critical period, with potential long-lasting impacts on susceptibility to disease. Both the innate and adaptative immune systems of the neonatal differ to that of the adult in terms of both cellular composition and sensitivity to antigenic and innate stimulation. The infant immune system gradually develops to more closely resemble that of the adult. Exposure to maternal inflammation in utero may aberrantly impact this period of infant immune system development, with maternal autoimmune and inflammatory disorders shown to affect the physiologic changes in serum cytokine abundance observed during pregnancy. The maternal and neonatal intestinal microbiome greatly influence infant mucosal and peripheral immune system development, and thereby impact the susceptibility to short-term inflammatory diseases, the adequacy of vaccine response, and later life risk of atopic and inflammatory disorders. Maternal disease, mode of delivery, method of feeding, time of weaning to include solid foods in the diet, and neonatal antibiotic exposure all influence the composition of the infant microbiome, and thereby infant immune system maturation. How exposure to specific immunosuppressive medications in utero alters infant immune cell phenotype and response to stimulation has been explored, but with existing studies limited by the time at which samples are performed, heterogenicity in methods, and small sample size. Furthermore, the impact of more recently introduced biologic agents have not been explored. Evolving knowledge in this field may influence therapeutic preferences for individuals with IBD planning to conceive, particularly if substantive differences in the risk of infant infection and childhood immune disease are identified., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Research has established inflammation in the pathogenesis of brain injury and the risk of developing cerebral palsy (CP). However, it is unclear if inflammation is solely pathogenic and primarily contributes to the acute phase of injury, or if inflammation persists with consequence in CP and may therefore be considered a comorbidity. We conducted a scoping review to identify studies that analyzed inflammatory biomarkers in CP and discuss the role of inflammation in the pathogenesis of CP and/or as a comorbidity. Twelve included studies reported a range of analytes, methods and biomarkers, including indicators of inflammatory status, immune function and genetic changes. The majority of controlled studies concluded that one or more systemic biomarkers of inflammation were significantly different in CP versus controls; most commonly serum or plasma cytokines such as tumor necrosis factor, Interleukin (IL)-6 and IL-10. In addition, differences in inflammation were noted in distinct subgroups of CP (e.g., those with varying severity). The available evidence supports the pathogenic role of inflammation and its ongoing role as a comorbidity of CP. This review shows that inflammation may persist for decades, driving functional impairment across development and into adulthood. However, inflammation is complex, thus further research will increase our understanding.

Bronchopulmonary dysplasia (BPD) is the most common respiratory sequela of prematurity, and infants born with fetal growth restriction (FGR) are disproportionately represented in BPD statistics, as factors which affect somatic growth may also affect pulmonary growth. Effects of in-utero hypoxia underlying FGR on lung parenchymal architecture predisposing to BPD are well documented, but the pulmonary vascular constructs are not well appreciated. Disruption of angiogenesis during critical periods of lung growth impairs alveolarization, contributing to BPD pathogenesis. Pulmonary artery thickness/stiffness has been noted in FGR in the initial postnatal weeks, and also in well-grown infants with established BPD. The lack of waveform cushioning by the major arteries exposes the pulmonary resistance vessels to higher pulsatile stress, thereby accelerating microvascular disease. Reactive oxygen species, increased sympathetic activity and endothelial dysfunction are common mediators in FGR and BPD; each putative targets for prevention and/or therapeutics using interleukin (IL)-1 receptor antagonist (IL-1Ra), melatonin or inhibition of renin-angiotensin-aldosterone system. While BPD is the archetypal respiratory disease of infancy, effects of FGR on pulmonary function are long-term, extending well into childhood. This narrative links FGR in very/extremely preterm infants with BPD through the vascular affliction as a mechanistic and potentially, therapeutic pathway. Our objectives were to depict the burden of disease for FGR and BPD amongst preterm infants, portray vascular involvement in the placenta in FGR and BPD cohorts, provide high resolution vascular ultrasound information in both cohorts with a view to address therapeutic relevance, and lastly, link this information with paediatric age-group lung diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bronchopulmonary dysplasia (BPD) and pulmonary hypertension associated with BPD (BPD-PH) are of multifactorial origin and share common risk factors. Most murine models of BPD expose newborn pups to only one of these risk factors-more commonly postnatal hyperoxia-thereby mimicking the vital increased fraction of inspired oxygen (FiO2) that preterm infants in neonatal intensive care units often require. To improve representation of the multifactorial origins of BPD and BPD-PH, we established a double hit model, combining antenatal systemic inflammation followed by postnatal hyperoxia. On embryonic day 14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg of lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized and exposed to gas with either an FiO2 of 0.21 (room air) or 0.65 (hyperoxia 65%). In our BPD and BPD-PH double hit model, we can obtain multiple readouts from individual pups that include echocardiography, lung histology and immunohistochemistry, ex vivo X-ray micro computed tomography, and pulmonary and plasmatic immunity by RNA, protein, or flow cytometry. This protocol was validated in: Sci Transl Med (2022), DOI: 10.1126/scitranslmed.aaz8454 Graphical abstract Figure 1. Murine double hit model of cardiopulmonary disease. On embryonic day (E)14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized to be exposed to gas with either a fraction of inspired oxygen (FiO 2 ) of 0.21 (air; 21% O 2 ) or 0.65 (hyperoxia; 65% O 2 ) for a maximum of 28 days. According to the murine stage of lung development ( Schittny, 2017 ), experimental endpoints include postnatal day (D)3, D5, D14, D28, and D60., Competing Interests: Competing interests The authors declare no competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Background: Bronchopulmonary dysplasia (BPD), its complication pulmonary hypertension (BPD-PH) and preterm brain and gut injury lead to significant morbidity and mortality in infants born extremely prematurely. There is extensive evidence that the pro-inflammatory cytokine interleukin 1 (IL-1) plays a key role in the pathophysiology of these illnesses. Two decades of clinical use in paediatric and adult medicine have established an excellent safety and efficacy record for IL-1 blockade with IL-1 receptor antagonist (IL-1Ra, medication name anakinra). Building on robust pre-clinical evidence, the Anakinra Pilot trial aims to demonstrate safety and feasibility of administering anakinra to preterm infants, and to establish pharmacokinetics in this population. Its ultimate goal is to facilitate large studies that will test whether anakinra can ameliorate early-life inflammation, thus alleviating multiple complications of prematurity., Methods and Analysis: Anakinra Pilot is an investigator-initiated, single arm, safety and feasibility dose-escalation trial in extremely preterm infants born between 24 weeks 0 days (24 0 ) and 27 6 weeks of gestational age (GA). Enrolled infants will receive anakinra intravenously over the first 21 days after birth, starting in the first 24 h after birth. In the first phase, dosing is 1 mg/kg every 48 h, and dosage will increase to 1.5 mg/kg every 24 h in the second phase. Initial anakinra dosing was determined through population pharmacokinetic model simulations. During the study, there will be a interim analysis to confirm predictions before undertaking dose assessment. Anakinra therapy will be considered safe if the frequency of adverse outcomes/events does not exceed that expected in infants born at 24 0 -27 6 weeks GA., Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05280340., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor GT declared a shared parent affiliation with the author AG at the time of review., (Copyright © 2022 Green, Metz, Galinsky, Atkinson, Skuza, Clark, Gunn, Kirkpatrick, Hunt, Berger, Nold-Petry and Nold.)

Bacille-Calmette Guérin (BCG) modulates atherosclerosis development in experimental animals, but it remains unclear whether neonatal BCG vaccination is pro- or anti-atherogenic. Many animal models differ fundamentally from BCG administration to human infants in terms of age, vaccine preparation, dosing schedule, and route of administration. We aimed to elucidate the effect of neonatal subcutaneous BCG vaccination—analogous to human BCG vaccination—on atherosclerosis development in ApoE−/− mice. At 2 days of age, a total of 40 ApoE−/− mice received either a weight-equivalent human dose of BCG, or saline, subcutaneously. From 4 weeks onwards, the mice were fed a Western-type diet containing 22% fat. At 16 weeks of age, mice were sacrificed for the assessment of atherosclerosis. Body weight, plasma lipids, atherosclerosis lesion size and collagen content were similar in both groups. Atherosclerosis lesion number was lower in mice that received BCG. Macrophage content was 20% lower in the BCG-vaccinated mice (p < 0.05), whereas plaque lipid content was increased by 25% (p < 0.01). In conclusion, neonatal BCG vaccination reduces atherosclerosis plaque number and macrophage content but increases lipid content in a murine model of atherosclerosis. Human epidemiological and mechanistic studies are warranted to investigate whether neonatal BCG vaccination is potentially atheroprotective.

Introduction: The World Health Organization (WHO) recommends all newborn infants receive the first dose of the hepatitis B vaccine within 24 h of birth irrespective of maternal hepatitis B carrier status. However, the physiological immaturity of the immune system in preterm infants may influence the immune responses to the vaccine particularly in the first few days and weeks of life, and adverse events may occur following vaccination that are not observed in infants born at term. Objectives: To review existing published guidelines surrounding timing of the first dose of the hepatitis B vaccine in preterm infants born to hepatitis B surface antigen negative (HBsAg-negative) mothers. Methods: A search was performed for relevant papers and guidelines published between January 2002 and July 2022 on the Ovid MEDLINE and Embase databases and through targeted searches. Two authors independently reviewed the search results to identify relevant sources, which were then analysed and described through narrative synthesis. Results: Twenty-seven relevant papers and guidelines regarding 15 countries and regions were included. Of these, 13.3% of guidelines, which represented 16.8% of the overall population of 4.1 billion people covered by the identified guidelines, recommended a nationwide birth dose of the hepatitis B vaccine to all preterm infants. In 40.0% of guidelines (77.9% of the overall population), the birth dose was only recommended for infants with a birth weight of more than 2000-2200 g. Another 33.3% of countries and regions (covering 4.4% of the population) recommended no universal birth dose for all infants, including preterm infants, whilst 13.3% (1.0% of the population) had guidelines that varied between jurisdictions and hospitals within their country/region. Conclusions: Existing guidelines surrounding the timing of the first dose of the hepatitis B vaccine in preterm infants vary substantially between countries and regions. Further research comparing the immunogenicity and safety of different hepatitis B vaccine schedules is needed to provide concrete evidence to provide guidance regarding the timing of vaccination against hepatitis B in preterm infants.

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics., Competing Interests: Declaration of interests Monash University, Hudson Institute (J.C.W., M.F.N., C.A.N.-P., and A.M.E.), and F. Hoffmann-La Roche (A.B., F.S., G.T., and L.L.) hold two patent families on IL-37, namely PCT/AU2016/050495 (Monash and Hudson only) and PCT/EP2020/087031 (Monash, Hudson, and Roche). There are no other conflicts of interest for these authors. All other authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Postnatal maturation of the immune system is poorly understood, as is its impact on illnesses afflicting term or preterm infants, such as bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension. These are both cardiopulmonary inflammatory diseases that cause substantial mortality and morbidity with high treatment costs. Here, we characterized blood samples collected from 51 preterm infants longitudinally at five time points, 20 healthy term infants at birth and age 3 to 16 weeks, and 5 healthy adults. We observed strong associations between type 2 immune polarization in circulating CD3 + CD4 + T cells and cardiopulmonary illness, with odds ratios up to 24. Maternal magnesium sulfate therapy, delayed hepatitis B vaccination, and increasing fetal, but not maternal, chorioamnionitis severity were associated with attenuated type 2 polarization. Blocking type 2 mediators such as interleukin-4 (IL-4), IL-5, IL-13, or signal transducer and activator of transcription 6 (STAT6) in murine neonatal cardiopulmonary disease in vivo prevented changes in cell type composition, increases in IL-1β and IL-13, and losses of pulmonary capillaries, but not gains in larger vessels. Thereby, type 2 blockade ameliorated lung inflammation, protected alveolar and vascular integrity, and confirmed the pathological impact of type 2 cytokines and STAT6. In-depth flow cytometry and single-cell transcriptomics of mouse lungs further revealed complex associations between immune polarization and cardiopulmonary disease. Thus, this work advances knowledge on developmental immunology and its impact on early life disease and identifies multiple therapeutic approaches that may relieve inflammation-driven suffering in the youngest patients.

In humans, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is highly infective, often causes severe acute and/or long-term illness, and elicits a high rate of mortality, even in countries with sophisticated medical systems. Detailed knowledge on the immune responses underpinning COVID-19 (coronavirus disease 2019), and on strategies SARS-CoV-2 uses to evade them, can provide pivotal guidance to researchers and clinicians developing and administering potentially life-saving immunomodulatory therapies. The need for such therapies in COVID-19 is unlikely to abate soon given the emergence of variants of concern that may pose new challenges for some vaccines and neutralizing antibodies. Here, we summarize current knowledge on COVID-19 immunopathogenesis in relation to three clinical disease stages and focus on immune evasion strategies used by pathogenic coronaviruses such as skewing type I, II, and III interferon responses and inhibiting detection via pattern recognition and antigen presentation. Insights gained from bats, which exhibit minimal disease in response to SARS-CoV-2 infection, offer an informative perspective and may guide future development of new therapies. We also discuss how knowledge of immunopathology may inform therapeutic decisions, for example, on selecting the most appropriate immunotherapeutic agents and timing their administration, to reduce morbidity and mortality of COVID-19.

Background: Increased systemic and tissue levels of interleukin (IL)-1β are associated with greater risk of impaired neurodevelopment after birth. In this study, we tested the hypothesis that systemic IL-1 receptor antagonist (Ra) administration would attenuate brain inflammation and injury in near-term fetal sheep exposed to lipopolysaccharide (LPS)., Methods: Chronically instrumented near-term fetal sheep at 0.85 of gestation were randomly assigned to saline infusion (control, n = 9), repeated LPS infusions (0 h = 300 ng, 24 h = 600 ng, 48 h = 1200 ng, n = 8) or repeated LPS plus IL-1Ra infusions (13 mg/kg infused over 4 h) started 1 h after each LPS infusion (n = 9). Sheep were euthanized 4 days after starting infusions for histology., Results: LPS infusions increased circulating cytokines and were associated with electroencephalogram (EEG) suppression with transiently reduced mean arterial blood pressure, and increased carotid artery perfusion and fetal heart rate (P < 0.05 vs. control for all). In the periventricular and intragyral white matter, LPS-exposure increased IL-1β immunoreactivity, numbers of caspase 3+ cells and microglia, reduced astrocyte and olig-2+ oligodendrocyte survival but did not change numbers of mature CC1+ oligodendrocytes, myelin expression or numbers of neurons in the cortex and subcortical regions. IL-1Ra infusions reduced circulating cytokines and improved recovery of EEG activity and carotid artery perfusion. Histologically, IL-1Ra reduced microgliosis, IL-1β expression and caspase-3+ cells, and improved olig-2+ oligodendrocyte survival., Conclusion: IL-1Ra improved EEG activity and markedly attenuated systemic inflammation, microgliosis and oligodendrocyte loss following LPS exposure in near-term fetal sheep. Further studies examining the long-term effects on brain maturation are now needed., (© 2021. The Author(s).)

Interleukin (IL)-37 has an important function in limiting excessive inflammation. Its expression is increased in numerous inflammatory and autoimmune conditions and correlates with disease activity, suggesting it could have potential as a disease biomarker. Nevertheless, a reference range has yet to be determined. Our aim was to establish the first reference range of circulating IL-37 levels in healthy adult humans. PubMed was searched for studies reporting blood IL-37 concentrations in healthy adult subjects as measured by enzyme-linked immunosorbent assay. Nineteen studies were included in the analysis. Mean IL-37 levels were weighted by sample sizes, and weighted mean lower and upper levels ( ± 2SD of means) were calculated to provide a weighted mean and reference range. IL-37 levels were quantified in either serum or plasma from a total of 1035 (647 serum; 388 plasma) healthy subjects. The serum, plasma and combined matrix weighted means (reference ranges) were 72.9 (41.5 - 104.4) pg/mL, 83.9 (41.1 - 126.8) pg/mL, and 77.1 (41.4 - 112.8) pg/mL, respectively. There were no significant differences between serum and plasma means and upper and lower limits. Study means and upper IL-37 levels were significantly higher in Chinese population studies. From our analysis, a preliminary reference range for circulating IL-37 levels in healthy human adults has been established. In order to determine a reliable reference range for clinical application, large, prospective, multi-ethnic, healthy population studies are necessary. In addition, demographics, sample matrix, collection, processing and storage methods potentially affecting IL-37 detection levels should be thoroughly investigated., Competing Interests: Monash University, Hudson Institute (CN-P and MN) hold two patent families on IL-37, namely PCT/AU2016/050495 and EP19218657.5. No other conflicts of interest exist for these authors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Santarelli, Vincent, Rudloff, Nold-Petry, Nold and Russo.)

Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/T H 17 polarization. In murine NEC, pro-inflammatory type 3 NKp46 - RORγt + Tbet + innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46 + RORγt + ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2'-methoxyethyl (2'MOE) modifications. Through a screen of 192 2'OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2'OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Interleukin (IL)-37 is a member of the IL-1 family of cytokines. Although its broad anti-inflammatory properties are well described, the effects of IL-37 on inflammasome function remain poorly understood. Performing gene expression analyses, ASC oligomerization/speck assays and caspase-1 assays in bone marrow-derived macrophages (BMDM), and employing an in vivo endotoxemia model, we studied how IL-37 affects the expression and maturation of IL-1β and IL-18, inflammasome activation, and pyroptosis in detail. IL-37 inhibited IL-1β production by NLRP3 and AIM2 inflammasomes, and IL-18 production by the NLRP3 inflammasome. This inhibition was partially attributable to effects on gene expression: whereas IL-37 did not affect lipopolysaccharide (LPS)-induced mRNA expression of Il18 or inflammasome components, IL-37-transgenic BMDM displayed an up to 83% inhibition of baseline and LPS-stimulated Il1b compared to their wild-type counterparts. Importantly, we observed that IL-37 suppresses nigericin- and silica-induced ASC oligomerization/speck formation (a step in inflammasome activation and subsequent caspase-1 activation), and pyroptosis (-50%). In mice subjected to endotoxemia, IL-37 inhibited plasma IL-1β (-78% compared to wild-type animals) and IL-18 (-61%). Thus, our study adds suppression of inflammasome activity to the portfolio of anti-inflammatory pathways employed by IL-37, highlighting this cytokine as a potential tool for treating inflammasome-driven diseases.

Pulmonary hypertension secondary to bronchopulmonary dysplasia (BPD-PH) represents a major complication of BPD in extremely preterm infants for which there are currently no safe and effective interventions. The abundance of interleukin-1 (IL-1) is strongly correlated with the severity and long-term outcome of BPD infants and we have previously shown that IL-1 receptor antagonist (IL-1Ra) protects against murine BPD; therefore, we hypothesized that IL-1Ra may also be effective against BPD-PH. We employed daily injections of IL-1Ra in a murine model in which BPD/BPD-PH was induced by antenatal LPS and postnatal hyperoxia of 65% O 2 . Pups reared in hyperoxia for 28 days exhibited a BPD-PH-like disease accompanied by significant changes in pulmonary vascular morphology: micro-CT revealed an 84% reduction in small vessels (4-5 μm diameter) compared to room air controls; this change was prevented by IL-1Ra. Pulmonary vascular resistance, assessed at day 28 of life by echocardiography using the inversely-related surrogate marker time-to-peak-velocity/right ventricular ejection time (TPV/RVET), increased in hyperoxic mice (0.27 compared to 0.32 in air controls), and fell significantly with daily IL-1Ra treatment (0.31). Importantly, in vivo cine-angiography revealed that this protection afforded by IL-1Ra treatment for 28 days is maintained at day 60 of life. Despite an increased abundance of mediators of pulmonary angiogenesis in day 5 lung lysates, namely vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), no difference was detected in ex vivo pulmonary vascular reactivity between air and hyperoxia mice as measured in precision cut lung slices, or by immunohistochemistry in alpha-smooth muscle actin (α-SMA) and endothelin receptor type-A (ET A ) at day 28. Further, on day 28 of life we observed cardiac fibrosis by Sirius Red staining, which was accompanied by an increase in mRNA expression of galectin-3 and CCL2 (chemokine (C-C motif) ligand 2) in whole hearts of hyperoxic pups, which improved with IL-1Ra. In summary, our findings suggest that daily administration of the anti-inflammatory IL-1Ra prevents the increase in pulmonary vascular resistance and the pulmonary dysangiogenesis of murine BPD-PH, thus pointing to IL-1Ra as a promising candidate for the treatment of both BPD and BPD-PH.

Background: The expression of heat shock protein gp96 is strongly correlated with the degree of tissue inflammation in ulcerative colitis and Crohn's disease, thereby leading us to the hypothesis that inhibition of expression via gp96-II peptide prevents intestinal inflammation., Methods: We employed daily injections of gp96-II peptide in two murine models of intestinal inflammation, the first resulting from five daily injections of IL-12/IL-18, the second via a single intrarectal application of TNBS (2,4,6-trinitrobenzenesulfonic acid). We also assessed the effectiveness of gp96-II peptide in murine and human primary cell culture., Results: In the IL-12/IL-18 model, all gp96-II peptide-treated animals survived until day 5, whereas 80% of placebo-injected animals died. gp96-II peptide reduced IL-12/IL-18-induced plasma IFNγ by 89%, IL-1β by 63%, IL-6 by 43% and tumor necrosis factor (TNF) by 70% compared to controls. The clinical assessment Disease Activity Index of intestinal inflammation severity was found to be significantly lower in the gp96-II-treated animals when compared to vehicle-injected mice. gp96-II peptide treatment in the TNBS model limited weight loss to 5% on day 7 compared with prednisolone treatment, whereas placebo-treated animals suffered a 20% weight loss. Histological disease severity was reduced equally by prednisolone (by 40%) and gp96-II peptide (35%). Mice treated with either gp96-II peptide or prednisolone exhibited improved endoscopic scores compared with vehicle-treated control mice: vascularity, fibrin, granularity, and translucency scores were reduced by up to 49% by prednisolone and by up to 30% by gp96-II peptide. In vitro , gp96-II peptide reduced TLR2-, TLR4- and IL-12/IL-18-induced cytokine expression in murine splenocytes, with declines in constitutive IL-6 (54%), lipopolysaccharide-induced TNF (48%), IL-6 (81%) and in Staphylococcus epidermidis -induced TNF (67%) and IL-6 (81%), as well as IL-12/IL-18-induced IFNγ (75%). gp96-II peptide reduced IL-1β, IL-6, TNF and GM-CSF in human peripheral blood mononuclear cells to a similar degree without affecting cell viability, whereas RANTES, IL-25 and MIF were twofold to threefold increased., Conclusion: gp96-II peptide protects against murine intestinal inflammation by regulating inflammation in vivo and in vitro , pointing to its promise as a novel treatment for inflammatory bowel disease.

Bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension (BPD-PH) are chronic inflammatory cardiopulmonary diseases with devastating short- and long-term consequences for infants born prematurely. The immature lungs of preterm infants are ill-prepared to achieve sufficient gas exchange, thus usually necessitating immediate commencement of respiratory support and oxygen supplementation. These therapies are life-saving, but they exacerbate the tissue damage that is inevitably inflicted on a preterm lung forced to perform gas exchange. Together, air-breathing and necessary therapeutic interventions disrupt normal lung development by aggravating pulmonary inflammation and vascular remodelling, thus frequently precipitating BPD and PH via an incompletely understood pathogenic cascade. BPD and BPD-PH share common risk factors, such as low gestational age at birth, fetal growth restriction and perinatal maternal inflammation; however, these risk factors are not unique to BPD or BPD-PH. Occurring in 17-24% of BPD patients, BPD-PH substantially worsens the morbidity and mortality attributable to BPD alone, thus darkening their outlook; for example, BPD-PH entails a mortality of up to 50%. The absence of a safe and effective therapy for BPD and BPD-PH renders neonatal cardiopulmonary disease an area of urgent unmet medical need. Besides the need to develop new therapeutic strategies, a major challenge for clinicians is the lack of a reliable method for identifying babies at risk of developing BPD and BPD-PH. In addition to discussing current knowledge on pathophysiology, diagnosis and treatment of BPD-PH, we highlight emerging biomarkers that could enable clinicians to predict disease-risk and also optimise treatment of BPD-PH in our tiniest patients., (Copyright © 2017 Hudson Institute of Medical Research. Published by Elsevier B.V. All rights reserved.)

Bronchopulmonary dysplasia (BPD) is a severe lung disease of preterm infants, which is characterized by fewer, enlarged alveoli and increased inflammation. BPD has grave consequences for affected infants, but no effective and safe therapy exists. We previously showed that prophylactic treatment with interleukin-1 receptor antagonist (IL-1Ra) prevents murine BPD induced by perinatal inflammation and hyperoxia. Here, we used the same BPD model to assess whether an alternative anti-inflammatory agent, protein C (PC), is as effective as IL-1Ra against BPD. We also tested whether delayed administration or a higher dose of IL-1Ra affects its ability to ameliorate BPD and investigated aspects of drug safety. Pups were reared in room air (21% O 2 ) or hyperoxia (65% or 85% O 2 ) and received daily injections with vehicle, 1200 IU/kg PC, 10 mg/kg IL-1Ra (early or late onset) or 100 mg/kg IL-1Ra. After 3 or 28 days, lung and brain histology were assessed and pulmonary cytokines were analysed using ELISA and cytokine arrays. We found that PC only moderately reduced the severe impact of BPD on lung structure (e.g. 18% increased alveolar number by PC versus 34% by IL-1Ra); however, PC significantly reduced IL-1β, IL-1Ra, IL-6 and macrophage inflammatory protein (MIP)-2 by up to 89%. IL-1Ra at 10 mg/kg prevented BPD more effectively than 100 mg/kg IL-1Ra, but only if treatment commenced at day 1 of life. We conclude that prophylactic low-dose IL-1Ra and PC ameliorate BPD and have potential as the first remedy for one of the most devastating diseases preterm babies face., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The interleukin (IL)-1 family member IL-37 is one of few anti-inflammatory cytokines, and it is capable of countering a broad spectrum of proinflammatory assaults. Although it is known that leukocytes are a major source of IL-37, knowledge on IL-37 production and secretion in specific immune cell types remains limited. Thus, we investigated IL-37 mRNA expression as well as protein production and secretion in human PBMCs. In PBMCs stimulated with agonists of Toll-like receptors (TLRs) 1-6 and 9, IL1F7 (the IL-37-encoding gene) was induced up to 9-fold, peaked at 6-8 h and returned to steady-state at 72 h. LPS-induced IL1F7 expression comprised isoforms b and c but not a and e Flow cytometry revealed that among IL-37 + PBMCs, monocytes predominated (81-91%), but T cells (6-8%) and myeloid dendritic cells (mDCs, 1-2%) also contributed to the IL-37 + leukocyte pool. Monocytes and mDCs, but not T cells, were capable of secreting IL-37. Whereas monocytes and mDCs secreted IL-37 upon LPS stimulation, only mDCs also released IL-37 at steady-state. Among monocyte subsets, IL-37 was LPS inducible and secreted only in classical and, although less pronounced, in intermediate monocytes; secretion was observed as early as 3 h after stimulation. Overall, our data suggest that constitutive IL-37 secretion by mDCs may serve to maintain an anti-inflammatory milieu at steady state, whereas IL-37 is stored in monocytes to be available for rapid release upon inflammatory encounters, thus acting as a novel anti-inflammatory alarmin. These insights may prove important to advancing towards clinical use the protective functions of one of the most powerful anti-inflammatory mediators so far discovered., (© Society for Leukocyte Biology.)

Dysregulation of the inflammatory response underlies numerous diseases. Although most interleukin-1 family cytokines are proinflammatory, human interleukin-37 (IL-37) is a powerful, broad-spectrum inhibitor of inflammation and immunity. We determined the crystal structure of IL-37 to establish the anti-inflammatory mechanism of this key cytokine in view of developing IL-37-based therapies. We found that two β-trefoil fold IL-37 molecules form a head-to-head dimer that is stable in solution. IL-37 variants mutated to convert the cytokine into an obligate monomer were up to 13-fold more effective than the dimer in suppressing proinflammatory events both in primary human blood cells and in vivo in murine endotoxic shock. Therapeutic exploitation of the powerful anti-inflammatory properties of monomeric IL-37 may prove beneficial in treating a wide range of inflammatory and autoimmune disorders., (Copyright © 2017, American Association for the Advancement of Science.)

Streptococcus pneumoniae infections can lead to severe complications with excessive immune activation and tissue damage. Interleukin-37 (IL-37) has gained importance as a suppressor of innate and acquired immunity, and its effects have been therapeutic as they prevent tissue damage in autoimmune and inflammatory diseases. By using RAW macrophages, stably transfected with human IL-37, we showed a 70% decrease in the cytokine levels of IL-6, TNF-α, and IL-1β, and a 2.2-fold reduction of the intracellular killing capacity of internalized pneumococci in response to pneumococcal infection. In a murine model of infection with S. pneumoniae, using mice transgenic for human IL-37b (IL-37tg), we observed an initial decrease in cytokine expression of IL-6, TNF-α, and IL-1β in the lungs, followed by a late-phase enhancement of pneumococcal burden and subsequent increase of proinflammatory cytokine levels. Additionally, a marked increase in recruitment of alveolar macrophages and neutrophils was noted, while TRAIL mRNA was reduced 3-fold in lungs of IL-37tg mice, resulting in necrotizing pneumonia with augmented death of infiltrating neutrophils, enhanced bacteremic spread, and increased mortality. In conclusion, we have identified that IL-37 modulates several core components of a successful inflammatory response to pneumococcal pneumonia, which lead to increased inflammation, tissue damage, and mortality., (© 2017 S. Karger AG, Basel.)

Bronchopulmonary dysplasia (BPD) is often complicated by pulmonary hypertension (PH). We investigated three biomarkers potentially suitable as screening markers for extremely preterm infants at risk of BPD-associated PH. In this prospective observational cohort study conducted in a tertiary neonatal intensive care unit, 83 preterm infants with BPD born <28-wk gestation and still inpatients at 36-wk corrected age received an echocardiogram and blood tests of B-type natriuretic peptide (BNP), troponin I, and YKL-40. Infants were analyzed according to echocardiographic evidence of tricuspid regurgitation (TR). Thirty infants had evidence of TR on echocardiogram at 36-wk corrected age. Infants with or without TR had similar baseline demographics: mean ± SD gestational age 26 1 ± 1 2 vs. 26 1 ± 1 1 wk and birth weight 830 ± 206 vs. 815 ± 187 g, respectively. There was no difference in duration of respiratory support. The right ventricular systolic pressure of infants with evidence of TR was 40 ± 16 mmHg. BNP was the only biomarker that proved to be significantly higher in infants with evidence of TR: median (interquartile range) serum level 54.5 (35-105) vs. 41.5 (30-59) pg/ml, P = 0.043. Subgroup analysis of infants with severe BPD requiring discharge on home oxygen or BPD-related mortality revealed similar results. There was no difference between groups for troponin I and YKL-40. In conclusion, increased serum levels of BNP were associated with evidence of TR at 36-wk corrected gestational age in extremely preterm infants, suggesting a potential role as a screening biomarker for BPD-associated PH., (Copyright © 2016 the American Physiological Society.)

Bronchopulmonary dysplasia (BPD) is a chronic disease of extreme prematurity that has serious long-term consequences including increased asthma risk. We earlier identified IL-1 receptor antagonist (IL-1Ra) as a potent inhibitor of murine BPD induced by combining perinatal inflammation (intraperitoneal LPS to pregnant dams) and exposure of pups to hyperoxia (fraction of inspired oxygen = 0.65). In this study, we determined whether airway remodeling and hyperresponsiveness similar to asthma are evident in this model, and whether IL-1Ra is protective. During 28-day exposure to air or hyperoxia, pups received vehicle or 10 mg/kg IL-1Ra by daily subcutaneous injection. Lungs were then prepared for histology and morphometry of alveoli and airways, or for real-time PCR, or inflated with agarose to prepare precision-cut lung slices to visualize ex vivo intrapulmonary airway contraction and relaxation by phase-contrast microscopy. In pups reared under normoxic conditions, IL-1Ra treatment did not affect alveolar or airway structure or airway responses. Pups reared in hyperoxia developed a severe BPD-like lung disease, with fewer, larger alveoli, increased subepithelial collagen, and increased expression of α-smooth muscle actin and cyclin D1. After hyperoxia, methacholine elicited contraction with similar potency but with an increased maximum reduction in lumen area (air, 44%; hyperoxia, 89%), whereas dilator responses to salbutamol were maintained. IL-1Ra treatment prevented hyperoxia-induced alveolar disruption and airway fibrosis but, surprisingly, not the increase in methacholine-induced airway contraction. The current study is the first to demonstrate ex vivo airway hyperreactivity caused by systemic maternal inflammation and postnatal hyperoxia, and it reveals further preclinical mechanistic insights into IL-1Ra as a treatment targeting key pathophysiological features of BPD.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Necrotising enterocolitis (NEC) is an uncommon, but devastating intestinal inflammatory disease that predominantly affects preterm infants. NEC is sometimes dubbed the spectre of neonatal intensive care units, as its onset is insidiously non-specific, and once the disease manifests, the damage inflicted on the baby's intestine is already disastrous. Subsequent sepsis and multi-organ failure entail a mortality of up to 65%. Development of effective treatments for NEC has stagnated, largely because of our lack of understanding of NEC pathogenesis. It is clear, however, that NEC is driven by a profoundly dysregulated immune system. NEC is associated with local increases in pro-inflammatory mediators, e.g. Toll-like receptor (TLR) 4, nuclear factor-κB, tumour necrosis factor, platelet-activating factor (PAF), interleukin (IL)-18, interferon-gamma, IL-6, IL-8 and IL-1β. Deficiencies in counter-regulatory mechanisms, including IL-1 receptor antagonist (IL-1Ra), TLR9, PAF-acetylhydrolase, transforming growth factor beta (TGF-β)1&2, IL-10 and regulatory T cells likely facilitate a pro-inflammatory milieu in the NEC-afflicted intestine. There is insufficient evidence to conclude a predominance of an adaptive Th1-, Th2- or Th17-response in the disease. Our understanding of the accompanying regulation of systemic immunity remains poor; however, IL-1Ra, IL-6, IL-8 and TGF-β1 show promise as biomarkers. Here, we chart the emerging immunological landscape that underpins NEC by reviewing the involvement and potential clinical implications of innate and adaptive immune mediators and their regulation in NEC.

Objective: Knowledge of interleukin-38 (IL-38), formerly IL-1 family member 10, is sparse, but Il1f10 polymorphisms are associated with inflammatory diseases, and recombinant IL-38 inhibits inflammatory responses similar to those reported in the context of systemic lupus erythematosus (SLE). We undertook this study to explore the function of endogenous IL-38 in human peripheral blood mononuclear cells (PBMCs) as well as its abundance in serum in a well-characterized cohort of SLE patients., Methods: Serum IL-38 and IL-10 levels were quantified by enzyme-linked immunosorbent assay in 142 SLE patients at ≤3 consecutive visits and in 28 healthy volunteers. To assess IL-38 function, we silenced IL-38 in PBMCs from healthy donors using IL-38 small interfering RNA (siRNA)., Results: IL-38 (63-5,928 pg/ml) was detectable in 16% of 372 serum samples. IL-38 abundance was significantly higher in samples from SLE patients than in samples from healthy controls (P = 0.004) and 11-fold higher in patients with active disease (SLE Disease Activity Index 2000 [SLEDAI-2K] score of ≥4) than in those with inactive disease (SLEDAI-2K score of <4) (P = 0.044). Importantly, IL-38 detection was associated with increased risk of renal lupus (relative risk [RR] 1.6, P = 0.027) and central nervous system lupus (RR 2.3, P = 0.034), and detectable baseline IL-38 entailed a 1.6-fold increased risk of subsequently meeting criteria for persistently active disease (P = 0.0097). Longitudinal time-adjusted mean IL-38 concentration was also 6-fold higher in patients with persistently active disease than in those without (P = 0.023). Remarkably, PBMCs treated with IL-38 siRNA produced up to 28-fold more of the proinflammatory mediators IL-6, CCL2, and APRIL than did control siRNA-transfected cells upon stimulation with Toll-like receptor agonists. Similarly, in SLE patients, the antiinflammatory cytokine IL-10 was 5-fold more abundant when IL-38 was detectable., Conclusion: This is the first study of the function of endogenous IL-38, and the data suggest that IL-38 may be protective in SLE. A strong association between IL-38 and SLE severity suggests that IL-38 expression is driven by processes linked to SLE pathogenesis. Exploitation of the regulatory effects of IL-38 may represent a promising therapeutic strategy in SLE., (© 2015, American College of Rheumatology.)

Systemic maternal inflammation is implicated in preterm birth and bronchopulmonary dysplasia (BPD) and may induce morbidities including reduced pulmonary function, sleep-disordered breathing, and cardiovascular disorders. Here we test the hypothesis that antenatal maternal inflammation per se causes altered alveolar development and increased chemoreflex sensitivity that persists beyond infancy. Pregnant C57BL/6 mice were administered lipopolysaccharide (LPS) (150 μg/kg ip) to induce maternal inflammation or saline (SHAM) at embryonic day 16 (randomized). Pups were weighed daily. On days 7, 28, and 60 (D07, D28, and D60), unrestrained wholebody plethysmography quantified ventilation and chemoreflex responses to hypoxia (10%), hypercapnia (7%), and asphyxia (hypoxic hypercapnia). Lungs were harvested to quantify alveolar number, size, and septal thickness. LPS pups had reduced baseline ventilation per unit bodyweight (∼40%, P < 0.001) vs. SHAM. LPS increased ventilatory responses to hypoxia (D07: 66% vs. 28% increase in ventilation; P < 0.001) hypercapnia (170% vs. 88%; P < 0.001), and asphyxia (249% vs. 154%; P < 0.001); hypersensitive hypoxic responsiveness persisted until D60 (P < 0.001). LPS also increased apnea frequency (P < 0.01). LPS caused thicker alveolar septae (D07, P < 0.001), diminished alveolar number (D28, P < 0.001) vs. SHAM, but effects were minimal by D60. Pups delivered from mothers exposed to antenatal inflammation exhibit deficits in lung structure and hypersensitive responses to respiratory stimuli that persist beyond the newborn period. Antenatal inflammation may contribute to impaired gas exchange and unstable breathing in newborn infants and adversely affect long-term health., (Copyright © 2015 the American Physiological Society.)

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.