Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Oxidative Stress by the Mitochondrial Monoamine Oxidase B Mediates Calcium Pyrophosphate Crystal-Induced Arthritis.

OBJECTIVE: Calcium pyrophosphate (CPP) crystal deposition in the joints is associated with a heterogeneous set of debilitating syndromes characterized by inflammation and pain, for which no effective therapies are currently available. Because we found that the mitochondrial enzyme monoamine oxidase B (MAO-B) plays a fundamental role in promoting inflammatory pathways, this study aims at assessing the efficacy of two clinical-grade inhibitors (iMAO-Bs) in preclinical models of this disease to pave the way for a novel treatment. METHODS: We tested our hypothesis in two murine models of CPP-induced arthritis, by measuring cytokine and chemokine levels, along with immune cell recruitment. iMAO-Bs (rasagiline and safinamide) were administered either before or after crystal injection. To elucidate the molecular mechanism, we challenged in vitro primed macrophages with CPP crystals and assessed the impact of iMAO-Bs in dampening proinflammatory cytokines and in preserving mitochondrial function. RESULTS: Both in preventive and therapeutic in vivo protocols, iMAO-Bs blunted the release of proinflammatory cytokines (interleukin [IL]-6 and IL1-ß) and chemokines (CXCL10, CXCL1, CCL2 and CCL5) (n > 6 mice/group). Importantly, they also significantly reduced ankle swelling (50.3% vs 17.1%; P < 0.001 and 23.1%; P = 0.005 for rasagiline and safinamide, respectively). Mechanistically, iMAO-Bs dampened the burst of reactive oxygen species and the mitochondrial dysfunction triggered by CPP crystals in isolated macrophages. Moreover, iMAO-Bs blunted cytokine secretion and NLRP3 inflammasome activation through inhibition of the NF-κB and STAT3 pathways. CONCLUSION: iMAO-Bs dampen inflammation in murine models of crystal-induced arthropathy, thereby uncovering MAO-B as a promising target to treat these diseases.

High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Myonecrosis Induction by Intramuscular Injection of CTX.

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract.

OPA1 drives macrophage metabolism and functional commitment via p65 signaling.

Macrophages are essential players for the host response against pathogens, regulation of inflammation and tissue regeneration. The wide range of macrophage functions rely on their heterogeneity and plasticity that enable a dynamic adaptation of their responses according to the surrounding environmental cues. Recent studies suggest that metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the metabolic pathways orchestrating macrophage activation are still under scrutiny. Optic atrophy 1 (OPA1) is a mitochondria-shaping protein controlling mitochondrial fusion, cristae biogenesis and respiration; clear evidence shows that the lack or dysfunctional activity of this protein triggers the accumulation of metabolic intermediates of the TCA cycle. In this study, we show that OPA1 has a crucial role in macrophage activation. Selective Opa1 deletion in myeloid cells impairs M1-macrophage commitment. Mechanistically, Opa1 deletion leads to TCA cycle metabolite accumulation and defective NF-κB signaling activation. In an in vivo model of muscle regeneration upon injury, Opa1 knockout macrophages persist within the damaged tissue, leading to excess collagen deposition and impairment in muscle regeneration. Collectively, our data indicate that OPA1 is a key metabolic driver of macrophage functions.

CK2ß-regulated signaling controls B cell differentiation and function.

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the ß regulatory subunit of CK2. CK2ßKO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2ßKO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2ßKO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2ß have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2ßKO mice suggesting the importance of the ß subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.

The dominant-negative mitochondrial calcium uniporter subunit MCUb drives macrophage polarization during skeletal muscle regeneration.

Damaged skeletal muscle can regenerate because of the coordinated action of immune cells with muscle stem cells, called satellite cells. Proinflammatory macrophages infiltrate skeletal muscle soon after injury to sustain the proliferation of satellite cells. These macrophages later acquire the anti-inflammatory phenotype and promote the differentiation and fusion of satellite cells. Here, we showed that MCUb, the dominant-negative subunit of the mitochondrial calcium uniporter (MCU) complex, promotes muscle regeneration by controlling macrophage responses. Macrophages lacking MCUb lost the ability to efficiently acquire the anti-inflammatory profile, and mice with MCUb-deficient macrophages showed delayed regeneration through exhaustion of the satellite cell pool. MCUb ablation altered macrophage metabolism by promoting glycolysis and the accumulation of TCA cycle intermediates, which was accompanied by the stabilization of HIF-1α, the master transcriptional regulator of the macrophage proinflammatory program. Together, these data demonstrate that MCUb abundance is tightly controlled in macrophages to enable satellite cell functional differentiation and recovery of tissue homeostasis after damage.

GM-CSF Nitration Is a New Driver of Myeloid Suppressor Cell Activity in Tumors.

Reactive oxygen species, including RNS, contribute to the control of multiple immune cell functions within the tumor microenvironment (TME). Tumor-infiltrating myeloid cells (TIMs) represent the archetype of tolerogenic cells that actively contribute to dismantle effective immunity against cancer. TIMs inhibit T cell functions and promote tumor progression by several mechanisms including the amplification of the oxidative/nitrosative stress within the TME. In tumors, TIM expansion and differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced by cancer and immune cells. Nevertheless, the role of GM-CSF in tumors has not yet been fully elucidated. In this study, we show that GM-CSF activity is significantly affected by RNS-triggered post-translational modifications. The nitration of a single tryptophan residue in the sequence of GM-CSF nourishes the expansion of highly immunosuppressive myeloid subsets in tumor-bearing hosts. Importantly, tumors from colorectal cancer patients express higher levels of nitrated tryptophan compared to non-neoplastic tissues. Collectively, our data identify a novel and selective target that can be exploited to remodel the TME and foster protective immunity against cancer.

Reactive Oxygen Species in Macrophages: Sources and Targets.

Reactive oxygen species (ROS) are fundamental for macrophages to eliminate invasive microorganisms. However, as observed in nonphagocytic cells, ROS play essential roles in processes that are different from pathogen killing, as signal transduction, differentiation, and gene expression. The different outcomes of these events are likely to depend on the specific subcellular site of ROS formation, as well as the duration and extent of ROS production. While excessive accumulation of ROS has long been appreciated for its detrimental effects, there is now a deeper understanding of their roles as signaling molecules. This could explain the failure of the "all or none" pharmacologic approach with global antioxidants to treat several diseases. NADPH oxidase is the first source of ROS that has been identified in macrophages. However, growing evidence highlights mitochondria as a crucial site of ROS formation in these cells, mainly due to electron leakage of the respiratory chain or to enzymes, such as monoamine oxidases. Their role in redox signaling, together with their exact site of formation is only partially elucidated. Hence, it is essential to identify the specific intracellular sources of ROS and how they influence cellular processes in both physiological and pathological conditions to develop therapies targeting oxidative signaling networks. In this review, we will focus on the different sites of ROS formation in macrophages and how they impact on metabolic processes and inflammatory signaling, highlighting the role of mitochondrial as compared to non-mitochondrial ROS sources.

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis.

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1ß, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.

TGF-ß in Cancer: Metabolic Driver of the Tolerogenic Crosstalk in the Tumor Microenvironment.

Overcoming tumor immunosuppression still represents one ambitious achievement for cancer immunotherapy. Of note, the cytokine TGF-ß contributes to immune evasion in multiple cancer types, by feeding the establishment of a tolerogenic environment in the host. Indeed, it fosters the expansion and accumulation of immunosuppressive regulatory cell populations within the tumor microenvironment (TME), where it also activates resident stromal cells and enhances angiogenesis programs. More recently, TGF-ß has also turned out as a key metabolic adjuster in tumors orchestrating metabolic pathways in the TME. In this review, we will scrutinize TGF-ß-mediated immune and stromal cell crosstalk within the TME, with a primary focus on metabolic programs.

Administration of Human MSC-Derived Extracellular Vesicles for the Treatment of Primary Sclerosing Cholangitis: Preclinical Data in MDR2 Knockout Mice.

Primary Sclerosing Cholangitis (PSC) is a progressive liver disease for which there is no effective medical therapy. PSC belongs to the family of immune-mediated biliary disorders and it is characterized by persistent biliary inflammation and fibrosis. Here, we explored the possibility of using extracellular vesicles (EVs) derived from human, bone marrow mesenchymal stromal cells (MSCs) to target liver inflammation and reduce fibrosis in a mouse model of PSC. Five-week-old male FVB.129P2-Abcb4tm1Bor mice were intraperitoneally injected with either 100 µL of EVs (± 9.1 × 109 particles/mL) or PBS, once a week, for three consecutive weeks. One week after the last injection, mice were sacrificed and liver and blood collected for flow cytometry analysis and transaminase quantification. In FVB.129P2-Abcb4tm1Bor mice, EV administration resulted in reduced serum levels of alkaline phosphatase (ALP), bile acid (BA), and alanine aminotransferase (ALT), as well as in decreased liver fibrosis. Mechanistically, we observed that EVs reduce liver accumulation of both granulocytes and T cells and dampen VCAM-1 expression. Further analysis revealed that the therapeutic effect of EVs is accompanied by the inhibition of NFkB activation in proximity of the portal triad. Our pre-clinical experiments suggest that EVs isolated from MSCs may represent an effective therapeutic strategy to treat patients suffering from PSC.

Age-severity matched cytokine profiling reveals specific signatures in Covid-19 patients.

A global effort is currently undertaken to restrain the COVID-19 pandemic. Host immunity has come out as a determinant for COVID-19 clinical outcomes, and several studies investigated the immune profiling of SARS-CoV-2 infected people to properly direct the clinical management of the disease. Thus, lymphopenia, T-cell exhaustion, and the increased levels of inflammatory mediators have been described in COVID-19 patients, in particular in severe cases1. Age represents a key factor in COVID-19 morbidity and mortality2. Understanding age-associated immune signatures of patients are therefore important to identify preventive and therapeutic strategies. In this study, we investigated the immune profile of COVID-19 hospitalized patients identifying a distinctive age-dependent immune signature associated with disease severity. Indeed, defined circulating factors - CXCL8, IL-10, IL-15, IL-27, and TNF-α - positively correlate with older age, longer hospitalization, and a more severe form of the disease and may thus represent the leading signature in critical COVID-19 patients.

CD73+ extracellular vesicles inhibit angiogenesis through adenosine A2B receptor signalling.

Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A2B adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression in vivo. Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis.

Cell-free DNA screening for aneuploidies in 7113 pregnancies: single Italian centre study.

INTRODUCTION: Non-invasive prenatal testing (NIPT) using cell-free foetal DNA has been widely accepted in recent years for detecting common foetal chromosome aneuploidies, such as trisomies 13, 18 and 21, and sex chromosome aneuploidies. In this study, the practical clinical performance of our foetal DNA testing was evaluated for analysing all chromosome aberrations among 7113 pregnancies in Italy. METHODS: This study was a retrospective analysis of collected NIPT data from the Ion S5 next-generation sequencing platform obtained from Altamedica Medical Centre in Rome, Italy. RESULTS: In this study, NIPT showed 100% sensitivity and 99.9% specificity for trisomies 13, 18 and 21. Out of the 7113 samples analysed, 74 cases (1%) were positive by NIPT testing; foetal karyotyping and follow-up results validated 2 trisomy 13 cases, 5 trisomy 18 cases, 58 trisomy 21 cases and 10 sex chromosome aneuploidy cases. There were no false-negative results. CONCLUSION: In our hands, NIPT had high sensitivity and specificity for common chromosomal aneuploidies such as trisomies 13, 18 and 21.

Developmental and Tumor Angiogenesis Requires the Mitochondria-Shaping Protein Opa1.

While endothelial cell (EC) function is influenced by mitochondrial metabolism, the role of mitochondrial dynamics in angiogenesis, the formation of new blood vessels from existing vasculature, is unknown. Here we show that the inner mitochondrial membrane mitochondrial fusion protein optic atrophy 1 (OPA1) is required for angiogenesis. In response to angiogenic stimuli, OPA1 levels rapidly increase to limit nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling, ultimately allowing angiogenic genes expression and angiogenesis. Endothelial Opa1 is indeed required in an NFκB-dependent pathway essential for developmental and tumor angiogenesis, impacting tumor growth and metastatization. A first-in-class small molecule-specific OPA1 inhibitor confirms that EC Opa1 can be pharmacologically targeted to curtail tumor growth. Our data identify Opa1 as a crucial component of physiological and tumor angiogenesis.

Pharmacological targets of metabolism in disease: Opportunities from macrophages.

From advances in the knowledge of the immune system, it is emerging that the specialized functions displayed by macrophages during the course of an immune response are supported by specific and dynamically-connected metabolic programs. The study of immunometabolism is demonstrating that metabolic adaptations play a critical role in modulating inflammation and, conversely, inflammation deeply influences the acquisition of specific metabolic settings.This strict connection has been proven to be crucial for the execution of defined immune functional programs and it is now under investigation with respect to several human disorders, such as diabetes, sepsis, cancer, and autoimmunity. The abnormal remodelling of the metabolic pathways in macrophages is now emerging as both marker of disease and potential target of therapeutic intervention. By focusing on key pathological conditions, namely obesity and diabetes, rheumatoid arthritis, atherosclerosis and cancer, we will review the metabolic targets suitable for therapeutic intervention in macrophages. In addition, we will discuss the major obstacles and challenges related to the development of therapeutic strategies for a pharmacological targeting of macrophage's metabolism.

Dietary Daily Sodium Intake Lower than 1500 mg Is Associated with Inadequately Low Intake of Calorie, Protein, Iron, Zinc and Vitamin B1 in Patients on Chronic Hemodialysis.

Background: To measure daily sodium intake in patients on chronic hemodialysis and to compare the intake of nutrients, minerals, trace elements, and vitamins in patients who had a daily sodium intake below or above the value of 1500 mg recommended by the American Heart Association. Methods: Dietary intake was recorded for 3 days by means of 3-day diet diaries in prevalent patients on chronic hemodialysis. Each patient was instructed by a dietitian on how to fill the diary, which was subsequently signed by a next of kin. Results: We studied 127 patients. Mean sodium intake (mg) was 1295.9 ± 812.3. Eighty-seven (68.5%) patients had a daily sodium intake <1500 mg (group 1) and 40 (31.5%) ≥ 1500 mg (group 2). Correlation between daily sodium intake and daily calorie intake was significant (r = 0.474 [0.327 to 0.599]; p < 0.0001). Daily calorie intake (kcal/kg/day) was lower in group 1 (21.1 ± 6.6; p = 0.0001) than in group 2 (27.1 ± 10.4). Correlation between daily sodium intake and daily protein intake was significant (r = 0.530[0.392 to 0.644]; p < 0.0001). The daily protein intake (grams/kg/day) was lower in group 1 (0.823 ± 0.275; p = 0.0003) than in group 2 (1.061 ± 0.419). Daily intake of magnesium, copper, iron, zinc, and selenium was significantly lower in group 1 than in group 2. Daily intake of vitamin A, B2, B3, and C did not differ significantly between group 1 and group 2. Daily intake of vitamin B1 was significantly lower in group 1 than in group 2. Significantly lower was, in group 1 than in group 2, the percentage of patients within the target value with regard to intake of calories (11.5% vs. 37.5%; p = 0.001) and proteins (9.2% vs. 27.5%; p = 0.015) as well as of iron (23% vs. 45%; p = 0.020), zinc (13.8% vs. 53.8%; p = 0.008) and vitamin B1 (8.1% vs. 50%; p < 0.001). Conclusion: A low daily intake of sodium is associated with an inadequately low intake of calorie, proteins, minerals, trace elements, and vitamin B1. Nutritional counselling aimed to reduce the intake of sodium in patients on chronic hemodialysis should not disregard an adequate intake of macro- and micronutrients, otherwise the risk of malnutrition is high.

Innate immunity ascertained from blood and tracheal aspirates of preterm newborn provides new clues for assessing bronchopulmonary dysplasia.

AIM: The study aimed to establish how granulocytes, monocytes and macrophages contribute to the development of bronchopulmonary dysplasia (BPD). MATERIALS AND METHODS: Study A: samples of blood and tracheal aspirates (TAs) collected from preterm newborn infants during the first 3 days of life were investigated by flow cytometry, and testing for white blood cells (WBCs), neutrophils and neutrophil extracellular traps (NETs). Maternal blood samples were also collected. Study B: data from previously-tested samples of TAs collected from preterm newborn infants were re-analyzed in the light of the findings in the new cohort. RESULTS: Study A: 39 preterm newborn infants were studied. A moderate correlation emerged between maternal WBCs and neutrophils and those of their newborn in the first 3 days of life. WBCs and neutrophils correlated in the newborn during the first 8 days of life. Decision rules based on birth weight (BW) and gestational age (GA) can be used to predict bronchopulmonary dysplasia (BPD). Neutrophil levels were lower in the TAs from the newborn with the lowest GAs and BWs. Study B: after removing the effect of GA on BPD development, previously-tested newborn were matched by GA. Monocyte phenotype 1 (Mon1) levels were lower in the blood of newborn with BPD, associated with a higher ratio of Monocyte phenotype 3 (Mon3) to Mon1. Newborn infants from mothers with histological chorioamnionitis (HCA) had lower levels of classically-activated macrophages (M1) and higher levels of alternatively-activated macrophages (M2) in their TAs than newborn infants from healthy mothers. CONCLUSION: Immune cell behavior in preterm newborn infants was examined in detail. Surprisingly, neutrophil levels were lower in TAs from the newborn with the lowest GA and BW, and no correlation emerged between the neutrophil and NET levels in TAs and the other variables measured. Interestingly, monocyte phenotype seemed to influence the onset of BPD. The rise in the ratio of Mon 3 to Mon 1 could contribute to endothelial dysfunction in BPD.