Bifidobacterium and Lactobacillus Probiotics and Gut Dysbiosis in Preterm Infants: The PRIMAL Randomized Clinical Trial.

Importance: The effects of probiotic interventions on colonization with resistant bacteria and early microbiome development in preterm infants remain to be clarified. Objective: To examine the efficacy of Bifidobacterium longum subsp infantis, Bifidobacterium animalis subsp lactis (BB-12), and Lactobacillus acidophilus (La-5) probiotics to prevent colonization with multidrug-resistant organisms or highly epidemic bacteria (MDRO+) and to shape the microbiome of preterm infants toward the eubiotic state of healthy full-term infants. Design, Setting, and Participants: The multicenter, double-blinded, placebo-controlled, group sequential, phase 3 Priming Immunity at the Beginning of Life (PRIMAL) randomized clinical trial, conducted from April 2018 to June 2020, included infants with gestational age of 28 to 32 weeks at 18 German neonatal units. Data analyses were conducted from March 2020 to August 2023. Intervention: A total of 28 days of multistrain probiotics diluted in human milk/formula starting within the first 72 hours of life. Main Outcomes and Measures: Colonization with MDRO+ at day 30 of life (primary end point), late-onset sepsis and severe gastrointestinal complication (safety end points), and gut dysbiosis, ie, deviations from the microbiome of healthy, term infants (eubiosis score) based on 16-subunit ribosomal RNA and metagenomic sequencing. Results: Among the 643 infants randomized until the stop of recruitment based on interim results, 618 (median [IQR] gestational age, 31.0 [29.7-32.1] weeks; 333 male [53.9%]; mean [SD] birth weight, 1502 [369] g) had follow-up at day 30. The interim analysis with all available data from 219 infants revealed MDRO+ colonization in 43 of 115 infants (37.4%) in the probiotics group and in 39 of 104 infants (37.5%) in the control group (adjusted risk ratio, 0.99; 95% CI, 0.54-1.81; P = .97). Safety outcomes were similar in both groups, ie, late-onset sepsis (probiotics group: 8 of 316 infants [2.5%]; control group: 12 of 322 infants [3.7%]) and severe gastrointestinal complications (probiotics group: 6 of 316 infants [1.9%]; control group: 7 of 322 infants [2.2%]). The probiotics group had higher eubiosis scores than the control group at the genus level (254 vs 258 infants; median scores, 0.47 vs 0.41; odds ratio [OR], 1.07; 95% CI, 1.02-1.13) and species level (96 vs 83 infants; median scores, 0.87 vs 0.59; OR, 1.28; 95% CI, 1.19-1.38). Environmental uptake of the B infantis probiotic strain in the control group was common (41 of 84 [49%]), which was highly variable across sites and particularly occurred in infants with a sibling who was treated with probiotics. Conclusions and Relevance: Multistrain probiotics did not reduce the incidence of MDRO+ colonization at day 30 of life in preterm infants but modulated their microbiome toward eubiosis. Trial Registration: German Clinical Trials Register: DRKS00013197.

HIF-1α targeted deletion in myeloid cells decreases MDSC accumulation and alters microbiome in neonatal mice.

The newborn's immune system is faced with the challenge of having to learn quickly to fight off infectious agents, but tolerating the colonization of the body surfaces with commensals without reacting with an excessive inflammatory response. Myeloid-derived suppressor cells (MDSC) are innate immune cells with suppressive activity on other immune cells that regulate fetal-maternal tolerance during pregnancy and control intestinal inflammation in neonates. Until now, nothing is known about the role of MDSC in microbiome establishment. One of the transcription factors regulating MDSC homeostasis is the hypoxia-inducible factor 1α (HIF-1α). We investigated the impact of HIF-1α on MDSC accumulation and microbiome establishment during the neonatal period in a mouse model with targeted deletion of HIF-1α in myeloid cells (Hif1a loxP/loxP LysMCre+). We show that in contrast to wildtype mice, where an extensive expansion of MDSC was observed, MDSC expansion in neonatal Hif1a loxP/loxP LysMCre+ mice was dramatically reduced both systemically and locally in the intestine. This was accompanied by an altered microbiome composition and intestinal T-cell homeostasis. Our results point toward a role of MDSC in inflammation regulation in the context of microbiome establishment and thus reveal a new aspect of the biological role of MDSC during the neonatal period.

Training vs. Tolerance: The Yin/Yang of the Innate Immune System.

For almost nearly a century, memory functions have been attributed only to acquired immune cells. Lately, this paradigm has been challenged by an increasing number of studies revealing that innate immune cells are capable of exhibiting memory-like features resulting in increased responsiveness to subsequent challenges, a process known as trained immunity (known also as innate memory). In contrast, the refractory state of endotoxin tolerance has been defined as an immunosuppressive state of myeloid cells portrayed by a significant reduction in the inflammatory capacity. Both training as well tolerance as adaptive features are reported to be accompanied by epigenetic and metabolic alterations occurring in cells. While training conveys proper protection against secondary infections, the induction of endotoxin tolerance promotes repairing mechanisms in the cells. Consequently, the inappropriate induction of these adaptive cues may trigger maladaptive effects, promoting an increased susceptibility to secondary infections-tolerance, or contribute to the progression of the inflammatory disorder-trained immunity. This review aims at the discussion of these opposing manners of innate immune and non-immune cells, describing the molecular, metabolic and epigenetic mechanisms involved and interpreting the clinical implications in various inflammatory pathologies.

Decreased expression of hypoxia-inducible factor 1α (HIF-1α) in cord blood monocytes under anoxia.

BACKGROUND: Infections are a major cause for morbidity and mortality in neonates; however, the underling mechanisms for increased infection susceptibility are incompletely understood. Hypoxia, which is present in inflamed tissues, has been identified as an important activation signal for innate immune cells in adults and is mainly mediated by hypoxia-inducible factor 1α (HIF-1α). Fetal tissue pO2 physiologically is low but rises immediately after birth. METHODS: In this study, the effect of low oxygen partial pressure (pO2) on HIF-1α expression and its targets phagocytosis, reactive oxygen species (ROS) production and vascular endothelial growth factor (VEGF) secretion was compared in vitro between immune cells from adult peripheral blood and cord blood using anoxia, HIF-1α stabilizer desferroxamin (DFO) and E. coli as stimuli. RESULTS: We show that anoxia-induced HIF-1α protein accumulation, phagocytosis, ROS-production and VEGF-expression were greatly diminished in cord blood compared to adult cells. E. coli led to HIF-1α gene expression in adult and cord blood immune cells; however, cord blood cells failed to accumulate HIF-1α protein and VEGF upon E. coli stimulation. CONCLUSIONS: Taken together, our results show a diminished activation of cord blood immune cells by low pO2, which might contribute to impaired reactivity in the context of infection. IMPACT: Neonatal immune cells do not accumulate HIF-1α under low oxygen partial pressure leading to decreased phagocytosis and decreased ROS production. We demonstrate a previously unknown mechanism of reduced activation of neonatal immune cells in the context of an inflammatory response. This could contribute to the increased susceptibility of newborns and preterm infants to infection.

Gram-positive Staphylococcus aureus LTA promotes distinct memory-like effects in murine bone marrow neutrophils.

Neutrophils primarily act as first responders in acute infection and directly maintain inflammatory responses. However, a growing body of evidence suggests that neutrophils also bear the potential to mediate chronic inflammation by exhibiting memory-like features. We now asked whether bone marrow-derived murine neutrophils can be primed by lipoteichoic acid (LTA) from gram-positive S. aureus. We found that low-dose (1 ng/mL) LTA-priming promoted increased production of pro-inflammatory mediators (TNF-α, IL-6, ROS), whereas high-dose (10 µg/mL) priming resulted in opposing reactions marked by increased IL-10 and suppressed pro-inflammatory mediators upon a second stimulus. A similar pattern of pro-inflammatory activation (trained sensitivity) and anti-inflammatory properties (tolerance) was recapitulated in cellular functional in vitro assays (transmigration and phagocytosis). Priming by LTA correlated with TLR2/MyD88-mediated regulation of NFκB-p65 through intermediate PI3Ks/MAPK. Collectively, our data suggest a previously unknown capacity of neutrophils to be differentially primed by varying doses of LTA, endorsing memory-like features in neutrophils.

Gut Microbiota-Derived Small Extracellular Vesicles Endorse Memory-like Inflammatory Responses in Murine Neutrophils.

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune-plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram-negative bacteria) or LTA (Gram-positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow-derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low-dose priming with gut microbiota-derived small EVs enhanced pro-inflammatory sensitivity as indicated by elevated levels of TNF-α, IL-6, ROS and MCP-1 and increased migratory and phagocytic activity. In contrast, high-dose priming resulted in a tolerant phenotype, marked by increased IL-10 and decreased transmigration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were correlated with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut-immune axis.

Meconium Microbiome of Very Preterm Infants across Germany.

Meconium constitutes infants' first bowel movements postnatally. The consistency and microbial load of meconium are different from infant and adult stool. While recent evidence suggests that meconium is sterile in utero, rapid colonization occurs after birth. The meconium microbiome has been associated with negative health outcomes, but its composition is not well described, especially in preterm infants. Here, we characterized the meconium microbiomes from 330 very preterm infants (gestational ages 28 to 32 weeks) from 15 hospitals in Germany and in fecal samples from a subset of their mothers (N = 217). Microbiome profiles were compiled using 16S rRNA gene sequencing with negative and positive controls. The meconium microbiome was dominated by Bifidobacterium, Staphylococcus, and Enterococcus spp. and was associated with gestational age at birth and age at sample collection. Bifidobacterial abundance was negatively correlated with potentially pathogenic genera. The amount of bacterial DNA in meconium samples varied greatly across samples and was associated with the time since birth but not with gestational age or hospital site. In samples with low bacterial load, human mitochondrial sequences were highly amplified using commonly used, bacterial-targeted 16S rRNA primers. Only half of the meconium samples contained sufficient bacterial material to study the microbiome using a standard approach. To facilitate future meconium studies, we present a five-level scoring system ("MecBac") that predicts the success of 16S rRNA bacterial sequencing for meconium samples. These findings provide a foundational characterization of an understudied portion of the human microbiome and will aid the design of future meconium microbiome studies. IMPORTANCE Meconium is present in the intestines of infants before and after birth and constitutes their first bowel movements postnatally. The consistency, composition and microbial load of meconium is largely different from infant and adult stool. While recent evidence suggests that meconium is sterile in utero, rapid colonization occurs after birth. The meconium microbiome has been associated with short-term and long-term negative health outcomes, but its composition is not yet well described, especially in preterm infants. We provide a characterization of the microbiome structure and composition of infant meconium and maternal feces from a large study cohort and propose a method to evaluate meconium samples for bacterial sequencing suitability. These findings provide a foundational characterization of an understudied portion of the human microbiome and will aid the design of future meconium microbiome studies.

LPS Induces Opposing Memory-like Inflammatory Responses in Mouse Bone Marrow Neutrophils.

A growing body of evidence suggests that innate immune cells can respond in a memory-like (adaptive) fashion, which is referred to as trained immunity. Only few in vivo studies have shown training effects in neutrophils; however, no in vitro setup has been established to study the induction of trained immunity or tolerance in neutrophils by microbial agents. In light of their short lifespan (up to 48 h), we suggest to use the term trained sensitivity for neutrophils in an in vitro setting. Here, we firstly describe a feasible two-hit model, using different doses of lipopolysaccharide (LPS) in bone marrow neutrophils. We found that low doses (10 pg/mL) induce pro-inflammatory activation (trained sensitivity), whereas priming with high doses (100 ng/mL) leads to suppression of pro-inflammatory mediators such as TNF-α or IL-6 (tolerance) (p < 0.05). On a functional level, trained neutrophils displayed increased phagocytic activity and LFA-1 expression as well as migrational capacity and CD11a expression, whereas tolerant neutrophils show contrasting effects in vitro. Mechanistically, TLR4/MyD88/PI3Ks regulate the activation of p65, which controls memory-like responses in mouse bone marrow neutrophils (p < 0.05). Our results open a new window for further in vitro studies on memory-like inflammatory responses of short-lived innate immune cells such as neutrophils.

The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

Maternal smoking as an independent risk factor for the development of severe retinopathy of prematurity in very preterm infants.

BACKGROUND/OBJECTIVES: Retinopathy of prematurity (ROP) is a severe neonatal complication potentially leading to visual impairment and blindness. Known risk factors include preterm birth, low birth weight and respiratory support. Limited and contradictory data exist on the risk of maternal smoking during pregnancy on the development of ROP. This study aims to investigate smoking as an independent risk factor for the development of severe ROP (≥stage 3). SUBJECTS/METHODS: This is a single centre retrospective case-control study of prospectively collected clinical data of infants born before 32 weeks of gestation between 2001 and 2012 at a tertiary care university hospital. The association between maternal smoking during pregnancy and the development of severe ROP was analyzed by multivariate logistic regression. RESULTS: In total, n = 751 infants born < 32 weeks of gestation were included in this study. In total, 52.9% (n = 397) were diagnosed with ROP and 10.8% (n = 81) developed ROP ≥ stage 3. In total, 8.4% (n = 63) mothers presented with a history of smoking during pregnancy, which was associated to a higher rate of ROP (OR 2.59, 95% CI 1.10-6.12). Low gestational age, low birth weight and prolonged respiratory support were confirmed as independent risk factors for the development of severe ROP. CONCLUSIONS: To date, this is the largest study evaluating the effect of maternal smoking on the development of ROP. Maternal smoking during pregnancy is identified as an independent risk factor for the development of severe ROP in preterm infants born < 32 weeks of gestation.

The Role of Microbiota in Neutrophil Regulation and Adaptation in Newborns.

Newborns are highly susceptible to infections and mainly rely on innate immune functions. Reduced reactivity, delayed activation and subsequent failure to resolve inflammation however makes the neonatal immune system a very volatile line of defense. Perinatal microbiota, nutrition and different extra-uterine factors are critical elements that define long-term outcomes and shape the immune system during the neonatal period. Neutrophils are first responders and represent a vital component of the immune system in newborns. They have long been regarded as merely executive immune cells, however this notion is beginning to shift. Neutrophils are shaped by their surrounding and adaptive elements have been described. The role of "innate immune memory" and the main triangle connection microbiome-neutrophil-adaptation will be discussed in this review.

Challenges in Acceptance and Compliance in Digital Health Assessments During Pregnancy: Prospective Cohort Study.

BACKGROUND: Pregnant women are increasingly using mobile apps to access health information during the antenatal period. Therefore, digital health solutions can potentially be used as monitoring instruments during pregnancy. However, a main factor of success is high user engagement. OBJECTIVE: The aim of this study was to analyze engagement and factors influencing compliance in a longitudinal study targeting pregnant women using a digital health app with self-tracking. METHODS: Digitally collected data concerning demographics, medical history, technical aspects, and mental health from 585 pregnant women were analyzed. Patients filling out ≥80% of items at every study visit were considered to be highly compliant. Factors associated with high compliance were identified using logistic regression. The effect of a change in mental and physical well-being on compliance was assessed using a one-sample t test. RESULTS: Only 25% of patients could be considered compliant. Overall, 63% left at least one visit blank. Influential variables for higher engagement included higher education, higher income, private health insurance, nonsmoking, and German origin. There was no relationship between a change in the number of physical complaints or depressive symptoms and study dropout. CONCLUSIONS: Maintaining high engagement with digital monitoring devices over a long time remains challenging. As cultural and socioeconomic background factors had the strongest influence, more effort needs to be directed toward understanding the needs of patients from different demographic backgrounds to ensure high-quality care for all patients. More studies need to report on compliance to disclose potential demographic bias.

Memory-Like Responses of Brain Microglia Are Controlled by Developmental State and Pathogen Dose.

Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1ß, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p < 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGF- ß, MSR1, and IL-4 in newborn microglia (p < 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation.

L-Arginine Modulates Neonatal Leukocyte Recruitment in a Gestational Age-Dependent Manner.

(1) Background: L-arginine is a complex modulator of immune functions, and its levels are known to decrease under septic conditions. L-arginine may suppress leukocyte recruitment in vivo; however, little is known about the gestational age-specific effects of L-arginine on leukocyte recruitment in preterm infants. We now asked whether L-arginine alters leukocyte recruitment in preterm and term neonates. (2) Methods: Leukocytes were isolated from preterm (28 + 0 to 32 + 6 weeks of gestation) and term (>37 weeks of gestation) newborns as well as from healthy adults. After incubation with 10 µg/mL L-arginine, we assessed leukocyte rolling and adhesion in dynamic microflow chamber experiments and leukocyte transmigration in fluorescence assays. In addition, we measured the expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) in neutrophils by flow cytometry. (3) Results: Leukocyte rolling, adhesion, and transmigration increased with gestational age. Leukocyte rolling, adhesion, and transmigration were decreased by L-arginine in term-born infants and adults. Preterm leukocytes showed no change in recruitment upon L-arginine exposure. Leukocyte adhesion after L-arginine exposure reached similar levels among all groups. In line, the expression of iNOS and Arg-1 was similar in all three age groups. (4) Conclusion: L-arginine dampens the ex vivo recruitment capacity of leukocytes from term-born infants, whereas no effect was seen in premature infants. As levels of iNOS and Arg-1 in neutrophils remain ontogenetically unchanged, the anti-inflammatory effect of L-arginine on the leukocyte recruitment cascade needs further investigation. These results add to the controversial debate of L-arginine supplementation in premature infants in sepsis.

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease. We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section. IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Antenatal exposure to fenoterol is not associated with the development of retinopathy of prematurity in infants born before 32 weeks of gestation.

PURPOSE: Despite safety concerns, ß2-sympathomimetics are still widely used as tocolytic agents. ß-Blockers in turn are used to treat vasculo-proliferative diseases of the newborn such as retinopathy of prematurity (ROP), which may lead to visual impairment and blindness. The scope of this study was to investigate whether antenatal exposure to the ß2-sympathomimetic fenoterol contributes to the development of ROP. METHODS: For this single-center retrospective case-control study of prospectively collected clinical data, all infants born before 32 weeks of gestation between 2001 and 2012 were included. The association of prenatal exposure to fenoterol and the development of ROP were analyzed by multivariate logistic regression. RESULTS: n = 1134 infants < 32 weeks of gestation were screened for eligibility, out of which n = 722 met the inclusion criteria. Exposure to fenoterol (n = 505) was not associated with a higher rate of ROP (OR 0.721, 95% CI 0.463-1.122). Further, duration of exposure (days) did not alter the incidence of ROP (OR 1.001, 95% CI 0.986-1.016). Frequency distribution of different ROP stages and the need for therapeutic intervention was also not affected by prenatal exposure to fenoterol. Risk factors for the development of ROP like low birth weight, low gestational age, prolonged respiratory support and multiple gestation were confirmed in our large study cohort. CONCLUSION: ß2-Sympathomimetic tocolysis does not increase the rate of ROP in premature infants born < 32 weeks of gestation. Our results render fenoterol a safe tocolytic agent regarding neonatal ROP development.

Efficacy of Bifidobacterium longum, B. infantis and Lactobacillus acidophilus probiotics to prevent gut dysbiosis in preterm infants of 28+0-32+6 weeks of gestation: a randomised, placebo-controlled, double-blind, multicentre trial: the PRIMAL Clinical Study protocol.

INTRODUCTION: The healthy 'eubiosis' microbiome in infancy is regarded as the microbiome derived from term, vaginally delivered, antibiotic free, breastfed infants at 4-6 months. Dysbiosis is regarded as a deviation from a healthy state with reduced microbial diversity and deficient capacity to control drug-resistant organisms. Preterm infants are highly sensitive to early gut dysbiosis. Latter has been associated with sepsis and necrotising enterocolitis, but may also contribute to long-term health problems. Probiotics hold promise to reduce the risk for adverse short-term outcomes but the evidence from clinical trials remains inconclusive and none has directly assessed the effects of probiotics on the microbiome at high resolution. METHODS AND ANALYSIS: A randomised, double blind, placebo-controlled study has been designed to assess the safety and efficacy of the probiotic mix of Bifidobacterium longum and infantis and Lactobacillus acidophilus in the prevention of gut dysbiosis in preterm infants between 28+0 and 32+6 weeks of gestation. The study is conducted in 18 German neonatal intensive care units. Between April 2018 and March 2020, 654 preterm infants of 28+0-32+6 weeks of gestation will be randomised in the first 48 hours of life to 28 days of once daily treatment with either probiotics or placebo. The efficacy endpoint is the prevention of gut dysbiosis at day 30 of life. A compound definition of gut dysbosis is used: (1) colonisation with multidrug-resistant organisms or gram-negative bacteria with high epidemic potential or (2) a significant deviation of the gut microbiota composition as compared with healthy term infants. Dysbiosis is determined by (1) conventional microbiological culture and (2) phylogenetic microbiome analysis by high-throughput 16S rRNA and metagenome sequencing. Persistence of dysbiosis will be assessed at 12-month follow-up visits. Side effects and adverse events related to the intervention will be recorded. Key secondary endpoint(s) are putative consequences of dysbiosis. A subgroup of infants will be thoroughly phenotyped for immune parameters using chipcytometry. ETHICS AND DISSEMINATION: Ethics approval was obtained in all participating sites. Results of the trial will be published in peer-review journals, at scientific meetings, on the website (www.primal-study.de) and via social media of parent organisations. TRIAL REGISTRATION NUMBER: DRKS00013197; Pre-results.

Memory-Like Inflammatory Responses of Microglia to Rising Doses of LPS: Key Role of PI3Kγ.

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions-trained immunity or immune tolerance-appears to be shaped by pathogen dose.

Dystrophin deficiency promotes leukocyte recruitment in mdx mice.

BACKGROUND: A growing body of evidence defines inflammation as a hallmark feature of disease pathogenesis of Duchenne muscular dystrophy. To tailor potential immune modulatory interventions, a better understanding of immune dysregulation in Duchenne muscular dystrophy is needed. We now asked whether dystrophin deficiency affects the cascade of leukocyte recruitment. METHODS: We performed intravital microscopy on the cremaster muscle of wild-type and dystrophin-deficient mdx mice. Recruitment was triggered by preparation alone (traumatic inflammation) or in combination with scrotal TNFα injections. Neutrophilic infiltration of the cremaster muscle was assessed on tissue sections. Integrin expression on circulating neutrophils and serum levels of pro-inflammatory cytokines were measured by flow cytometry. RESULTS: Mdx mice show increased rolling and adhesion at baseline (traumatic inflammation) and a more profound response upon TNFα injection compared with wild-type animals. In both models, neutrophilic infiltration of the cremaster muscle is increased. Upregulation of the integrins LFA-1 and Mac-1 on circulating leukocytes and pro-inflammatory cytokines IL-6 and CCL2 in the serum points toward systemically altered immune regulation in mdx mice. CONCLUSION: We are the first to show exaggerated activation of the leukocyte recruitment cascade in a dystrophin-deficient organism in vivo.