Multisystem inflammatory syndrome in children (MIS-C): A nationwide collaborative study in the Greek population.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but severe hyperinflammatory condition that may occur following SARS-CoV-2 infection. This retrospective, descriptive study of children hospitalized with multisystem inflammatory syndrome in children (MIS-C) in 12 tertiary care centers from 3/11/2020 to 12/31/2021. Demographics, clinical and laboratory characteristics, treatment and outcomes are described. Among 145 patients (95 males, median age 8.2 years) included, 123 met the WHO criteria for MIS-C, while 112 (77%) had serological evidence of SARS-CoV-2 infection. Fever was present in 99%, gastrointestinal symptoms in 77%, mucocutaneous involvement in 68% and respiratory symptoms in 28%. Fifty-five patients (38%) developed myocarditis, 29 (20%) pericarditis and 19 (13%) coronary aneurysms. Among the above cases 11/55 (20%), 1/29 (3.4%) and 5/19 (26.3%), respectively, cardiac complications had not fully resolved at discharge. Underlying comorbidities were reported in 18%. Median CRP value was 155 mg/l, ferritin 535 ng/ml, PCT 1.6 ng/ml and WBC 14.2 × 109/mm3. Most patients had elevated troponin (41.3%) and/or NT-pro-BNP (49.6%). Intravenous immunoglobulin plus corticosteroids were used in 117/145 (80.6%), monotherapy with IVIG alone in 13/145 (8.9%) and with corticosteroids alone in 2/145 (1.3%). Anti-IL1 treatment was added in 15 patients (10.3%). Thirty-three patients (23%) were admitted to the PICU, 14% developed shock and 1 required ECMO. Mortality rate was 0.68%. The incidence of MIS-C was estimated at 0.69/1000 SARS-CoV-2 infections. Patients who presented with shock had higher levels of NT-pro-BNP compared to those who did not (p < 0.001). Acute kidney injury and/or myocarditis were associated with higher risk of developing shock. CONCLUSION: MIS-C is a novel, infrequent but serious disease entity. Cardiac manifestations included myocarditis and pericarditis, which resolved in most patients before discharge. Timely initiation of immunomodulatory therapy was shown to be effective. NT-pro-BNP levels may provide a better prediction and monitoring of the disease course. Further research is required to elucidate the pathogenesis, risk factors and optimal management, and long-term outcomes of this clinical entity. WHAT IS KNOWN: • MIS-C is an infrequent but serious disease entity. • Patients with MIS-C present with multi-organ dysfunction, primarily involving the gastrointestinal and cardiovascular systems. WHAT IS NEW: • NT-pro-BNP levels may provide a better prediction and monitoring of the disease course. • Acute kidney injury and/or myocarditis were associated with higher risk of developing shock.

SARS-CoV-2 seroprevalence among children in Greece during Omicron variant period.

The Omicron variant is associated with increased transmissibility, but evidence about the impact of Omicron in seropositivity of children is limited. This study aims to evaluate SARS-CoV-2 seroprevalence in children during the different variants' subperiods. A prospective multicenter seroprevalence study was conducted in 7 University public hospitals in Greece from November 2021 to August 2022 (3 subperiods: November 2021-February 2022, March 2022-May 2022, June 2022-August 2022). Children from different age groups, admitted to the hospital or examined in outpatient clinics for reasons other than COVID-19 were enrolled. Neutralizing antibodies (Nabs), anti-Spike (anti-S) and anti-nucleocapsid (anti-N) SARS-CoV-2 IgG in serum were evaluated. A total of 2127 children (males:57,2%; median age:4,8years) were enrolled. Anti-N IgG seropositivity increased from 17,8% in the first sub-period to 40,7% in the second sub-period and then decreased in the third sub-period (36,7%). Anti-S IgG seropositivity appeared to have an increasing trend over the study period, starting from 34,8% and reaching 80,7%. Children aged 1-4 years old have significantly higher anti-N IgG titers compared to children aged 0-1 years old (p < 0,001). Infants have significantly lower anti-S IgG titers compared to all other age groups (p < 0,001). Immunocompromised children and infants have the lowest seropositivity for NAbs.Conclusions During the Omicron period, seropositivity significantly increased, as a result of higher transmissibility. Neonates and infants have lower antibody titers compared to other age groups, while young children aged 1-4 years old present higher antibody titers, suggesting that this age group may mount a higher antibody response. Continuous surveillance seroprevalence studies are needed in children, in order to identify the true extent of SARS-CoV-2 and guide the planning of adequate public health measures.

WHAT IS KNOWN: • Seroprevalence surveys among children may be extremely useful, in order to properly monitor the immunity, either natural or acquired, through the quantification of IgG antibodies and to plan further immunization policies. • There are variations in the seroprevalence of COVID-19 between the different periods, according to the vaccination rates, the type of circulating variant and the transmissibility of the virus. • The Omicron variant is associated with increased transmissibility, but evidence about the impact of Omicron in seropositivity of children is limited. WHAT IS NEW: • In this large multicenter seroepidemiological study, SARS-CoV-2 seroprevalence rate in children is higher during the Omicron period in comparison to the previous pandemic waves, due to the high transmissibility of the virus and the increased rates of reinfection. • Neonates and infants have lower antibody titers compared to other age groups, while young children aged 1­4 years old present higher antibody titers, indicating that the children of this age group mount a higher antibody response. • This study provides essential information about immunity and the level of protection in the pediatric population, as neutralizing antibodies were evaluated, in addition to the anti-N and anti-S IgG antibodies.

AKT Isoforms in Macrophage Activation, Polarization, and Survival.

Macrophages display an array of activation phenotypes depending on the activation signal and the cellular microenvironment. The type and magnitude of the response depend on signaling molecules as well as on the epigenetic and metabolic status of the cells at the time of activation. The AKT family of kinases consists of three isoforms encoded by independent genes possessing similar functions and structures. Generation of research tools such as isoform-specific gene deletion mutant mice and cells and isoform-specific antibodies has allowed us to understand the role of each kinase isoform in macrophage activation and homeostasis. This chapter discusses the current evidence on the role of AKT kinases in macrophage activation, polarization, and homeostasis, highlighting the gaps in knowledge and future challenges in the field.

Seroprevalence of anti-SARS-CoV-2 antibodies among children and their parents in Greece.

School closures were enforced as measures to restrain the COVID-19 pandemic, based on the assumption that young children may play a key role in SARS-CoV-2 spread. This study aims to determine the prevalence of SARS-CoV-2 IgG antibodies in children and corresponding parents, in order to improve surveillance and estimate the prevalence of asymptomatic or subclinical COVID-19 cases. A prospective multicenter study was conducted between March and June 2021 in Greece. Children admitted to the hospital or examined in outpatient clinics for reasons other than COVID-19 and their parents were tested for anti-Spike SARS-CoV-2 IgG in serum. A questionnaire about clinical and demographic data was completed. The study included 823 participants: 427 children and 396 corresponding parents. The overall seroprevalence was 16.4% in parents and 13.8% in children. Among families with ≥ 1 seropositive child or parent, the combination of a seropositive parent and a corresponding seronegative child was 29.6%, a seronegative parent and a corresponding seropositive child was 24.7%, and a seropositive child with a corresponding seropositive parent was 45.7%. Age, level of education, and school or work attendance were not significantly associated with increased seropositivity. On the contrary, ethnic minority of Roma, close contact with known COVID-19 case, previous symptoms consistent with COVID-19, and mass gatherings were risk factors for seropositivity. CONCLUSION: The spread of SARS-CoV-2 during a period of lockdown in Greece was low in children and comparable to adults most likely due to intrafamilial transmission. Accordingly, it is unlikely that children have boosted virus transmission. WHAT IS KNOWN: • In the earliest months of the pandemic, it was demonstrated that children had significantly lower seroprevalence rates than the older age groups, due to the fact that children had decreased exposure to the virus, because of early public health interventions, such as school and day care closure. • Later, further studies reported that children have similar incidence rate of SARS-CoV-2 infection compared to adults in households and community settings. WHAT IS NEW: • In this seroprevalence study, the spread of SARS-CoV-2 infection during a period of lockdown in Greece with the predominance of the Alpha-variant was particularly low in children and comparable to adults, most likely due to intrafamilial transmission. • These study findings will be useful for decisions regarding non-pharmaceutical interventions during the pandemic, and especially, to guide in designing and implementing appropriate containment measures for schools and social gatherings.

Mild coronary artery dilatation developed in some children with mild COVID-19 but completely regressed within 3 months.

AIM: We studied the incidence and time course of any coronary artery changes in children up to 2 years of age who were hospitalised with mild COVID-19. METHODS: This was a single-centre prospective study of 29 children (19 males) with a median age of 3 months and interquartile range (IQR) of 1.6-4.3 months. They were admitted to a Greek University hospital for mild COVID-19 from 1 March to 30 December 2021. Three echocardiographic evaluations were performed at a median (IQR) of 19 (16-24) days, 82 (75-89) days and 172 (163-197) after the first symptoms. The prevalence of coronary artery dilation, regression, and changes was documented. RESULTS: Coronary artery dilation was present in 3 (10.3%) cases at the first evaluation, with complete regression at the second. Regression was observed in 18/24 (75%) cases with follow-up data and 9 (31%) demonstrated significant z-score changes of >2. Coronary artery changes in any segment at any time were documented in 18/29 (62%) of the patients. CONCLUSION: Cases of transient and very mild coronary artery dilatation following mild COVID-19 completely regressed within 3 months. Large-scale studies are needed to document the extent and time course of coronary artery dilation following paediatric COVID-19.

Acute abdomen in multisystem inflammatory syndrome in children: A systematic review.

AIM: Multisystem inflammatory syndrome in children (MIS-C), a rare severe complication of SARS-CoV-2 infection, has been recently reported to mimic acute abdomen and lead to surgical interventions, posing challenges for clinicians. In this systematic review, we evaluated the rate of acute abdomen and abdominal surgical emergencies in children with MIS-C. METHODS: Systematic review of all MIS-C cases presented with acute abdomen. RESULTS: A total of 385 patients with MIS-C, from 38 studies, were included. Gastrointestinal manifestations were prominent in 233/385 (60.5%) children. Acute abdomen was noted in 72/385 (18.7%) of MIS-C cases and in 72/233 (30.9%) of MIS-C cases with gastrointestinal symptoms. Final diagnoses were mostly non-surgical (55/72, 76.4%), such as mesenteric lymphadenitis (23/72, 31.9%), terminal ileitis/ileocolitis (19/72, 26.4%), free abdominal fluid/ascites (8/72, 11.1%) and paralytic ileus (3/72, 4.2%). Laparotomy was performed in 35/72 (48.6%) of children with MIS-C, and acute abdomen and was proven unnecessary in 18/35 (51.4%) cases. True abdominal surgical emergencies, such as appendicitis and obstructive ileus, were confirmed in 17/72 (23.6%) cases. CONCLUSION: MIS-C often presents with acute abdomen, mostly due to non-surgical intestinal inflammatory pathology. However, surgical complications occur in patients with MIS-C; therefore, a high index of suspicion should remain.

Insulin Resistance in Macrophages Alters Their Metabolism and Promotes an M2-Like Phenotype.

Obesity and insulin resistance influences metabolic processes, but whether it affects macrophage metabolism is not known. In this study, we demonstrate that chronic exposure of macrophages to insulin either in culture or in vivo in diet-induced, glucose-intolerant mice rendered them resistant to insulin signals marked by failure to induce Akt2 phosphorylation. Similarly, macrophages lacking Akt2 or IGF1 receptor were also resistant to insulin signals. Insulin-resistant macrophages had increased basal mTORC1 activity, possessed an M2-like phenotype, and reduced LPS responses. Moreover, they exhibited increased glycolysis and increased expression of key glycolytic enzymes. Inhibition of mTORC1 reversed the M2-like phenotype and suppressed glycolysis in insulin-resistant macrophages. In the context of polymicrobial sepsis, mice harboring insulin-resistant macrophages exhibited reduced sepsis-induced lung injury. Thus, macrophages obtain resistance to insulin characterized by increased glycolysis and a unique M2-like phenotype, termed M-insulin resistant, which accounts for obesity-related changes in macrophage responses and a state of trained immunity.

Polymicrobial gastroenteritis in children.

AIM: Co-infections with viral and bacterial enteropathogens often augment severity of diarrhoea, however, there is limited evidence on the clinical importance of bacterial enteric co-infections. We investigated the rate, type and impact of bacterial enteropathogens and their associations in children with gastroenteritis. METHODS: Retrospective cohort study that included children 0-18 years old with acute bacterial diarrhoea during a 27-year period (1993-2019), in Crete, Greece. Differences in clinical characteristics and pathogen associations were investigated between single and multiple infections. RESULTS: Two or more bacteria were isolated in stool culture in 53 out of 1932 children (2.74%). Patients with co-infections were younger (p 0.0001) and had higher hospitalisation rates (p 0.03). Escherichia coli (E. coli) was the most prevalent pathogen associated with co-infections, in particular the E. coli enteropathogenic strains O127 and O111 (p 0.001), and Salmonella spp the least (p 0.001). Co-occurrence analysis revealed two positively associated pathogen pairs, E. coli with Campylobacter spp and E. coli (p 0.001) with Salmonella spp (p 0.04). CONCLUSION: Bacterial enteropathogen co-infection was most common with E. coli strains and related to higher hospitalisation rates and younger age.

Short-term antibiotic exposure affected the type and resistance of uropathogens similar to long-term antibiotic prophylaxis in children hospitalised for urinary tract infections.

AIM: We investigated the impact of any antibiotic exposure on unusual and resistant pathogens in children with a urinary tract infection (UTI). METHODS: This was a retrospective cohort study of 695 children (54% female) hospitalised with 711 UTI episodes at Heraklion University Hospital, Greece, from January 2007 to December 2017. Three groups were studied: no previous antibiotic exposure, ongoing prophylaxis and short-term exposure to antibiotics in the last six months. RESULTS: The median age at hospitalisation was 0.8 years (range 25 days to 15.9 years). Previous short-term exposure and prophylaxis were important determinants of non-Escherichia coli (E. coli) (OR 2.05, 95% CI 1.25-3.36, P = .0017 and OR 3.84, 95% CI 2.32-6.34, P < .0001, respectively) and extended-spectrum beta-lactamase-positive uropathogens (OR 2.43, 95% CI 1.36-4.32, P = .0025 and OR 2.63, 95% CI 1.31-5.33, P = .0070, respectively). Short-term antibiotics in the last 30 days or intravenous antibiotics were mostly associated with non-E. coli pathogens (OR 6.71 and OR 2.55, respectively). The most important determinants of E. coli resistance were short-term antibiotics for ampicillin (OR 2.58) and prophylaxis for cotrimoxazole (OR 2.64). CONCLUSION: Recent short-term exposure to antibiotics and ongoing prophylaxis both had a significant impact on the type and resistance of uropathogens.

Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization.

Macrophages become activated initiating innate immune responses. Depending on the signals, macrophages obtain an array of activation phenotypes, described by the broad terms of M1 or M2 phenotype. The PI3K/Akt/mTOR pathway mediates signals from multiple receptors including insulin receptors, pathogen-associated molecular pattern receptors, cytokine receptors, adipokine receptors, and hormones. As a result, the Akt pathway converges inflammatory and metabolic signals to regulate macrophage responses modulating their activation phenotype. Akt is a family of three serine-threonine kinases, Akt1, Akt2, and Akt3. Generation of mice lacking individual Akt, PI3K, or mTOR isoforms and utilization of RNA interference technology have revealed that Akt signaling pathway components have distinct and isoform-specific roles in macrophage biology and inflammatory disease regulation, by controlling inflammatory cytokines, miRNAs, and functions including phagocytosis, autophagy, and cell metabolism. Herein, we review the current knowledge on the role of the Akt signaling pathway in macrophages, focusing on M1/M2 polarization and highlighting Akt isoform-specific functions.

Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice.

Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2(-/-) mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2(-/-) mice compared with WT mice. Alveolar macrophages from acid-injured Akt2(-/-) mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2(-/-) mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2(-/-) mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2(-/-) macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization.

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2(-/-) mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1(-/-) mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2(-/-) macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2(-/-) macrophages, and its target C/EBPß appear to play a key role in this process. C/EBPß, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Title: Repeated implantation failure is associated with increased Th17/Treg cell ratio, during the secretory phase of the human endometrium.

Repeated implantation failure (RIF) is a significant limiting factor in assisted reproduction. Chronic endometrial inflammation has been noted in RIF women, therefore we sought to investigate the potential association of endometrial Th17/Treg ratio and endometrial inflammation in these cases. Endometrial pipelle biopsies were obtained from volunteers, 29 women with RIF (failure to achieve pregnancy following at least 3 transfers of high-grade embryos in IVF-cycles) and 27 fertile women (at least one child) in total, at the secretory phase of the menstrual cycle. Using tissues from 17 fertile and 18 RIF endometrial samples, stromal and immune cells were isolated and flow cytometry analysis was performed to determine Th17 and CD4+ CD25high FOXP3+ cell populations in endometrial stromal cell suspensions. Another group of tissues from 10 fertile and 11 RIF samples were used for mRNA expression levels of Treg and Th17-cell transcription factors, FOXP3 and RORγt respectively. Endometrial inflammatory mediators' mRNA expression was also analyzed. A statistically significant increase in protein flow cytometry analysis of Th17/Treg ratio (p ≤ 0.05) as well as a reduction in absolute Treg cells in the endometrium (p ≤ 0.05) was noted in women with RIF. Additionally, RNA analysis on the same set of women indicated RORγt/FOXP3 significantly increased in women with RIF compared to fertile ones (p ≤ 0.05). Finally, women with RIF exhibited significantly (p ≤ 0.05) elevated mRNA levels of pro-inflammatory mediators (ΤΝF-a, ΙL-6, IL-8 and CCl2). Women with RIF exhibit elevated Th17/Treg ratio, mostly due to endometrial Treg depletion, as well as a pro-inflammatory state in the endometrium.

Effectiveness of COVID-19 vaccination against school absenteeism in children and adolescents hospitalized with COVID-19.

BACKGROUND: COVID-19 vaccination has been recommended for children to protect them and to enable in-person educational and social activities. METHODS: We estimated COVID-19 vaccination effectiveness (VE) against school absenteeism in children 5-17 years old hospitalized from September 1, 2021 through May 31, 2023. Full vaccination was defined as two vaccine doses. RESULTS: We studied 231 children admitted to hospital with COVID-19, including 206 (89.2 %) unvaccinated/partially vaccinated and 25 (10.8 %) fully vaccinated. Unvaccinated/partially vaccinated children were absent from school for longer periods compared to fully vaccinated children (median absence: 14 versus 10 days; p-value = 0.05). Multivariable regression showed that full COVID-19 vaccination was associated with fewer days of absence compared to no/partial vaccination on average (adjusted relative risk: 0.77; 95 % CI: 0.61 to 0.98). COVID-19 VE was 50.7 % (95 % CI: -11.3 % to 78.2 %) for school absenteeism above the median duration of absenteeism. CONCLUSIONS: Full COVID-19 vaccination conferred protection against school absenteeism in hospitalized school-aged children with COVID-19.

An IL-10/DEL-1 axis supports granulopoiesis and survival from sepsis in early life.

The limited reserves of neutrophils are implicated in the susceptibility to infection in neonates, however the regulation of neutrophil kinetics in infections in early life remains poorly understood. Here we show that the developmental endothelial locus (DEL-1) is elevated in neonates and is critical for survival from neonatal polymicrobial sepsis, by supporting emergency granulopoiesis. Septic DEL-1 deficient neonate mice display low numbers of myeloid-biased multipotent and granulocyte-macrophage progenitors in the bone marrow, resulting in neutropenia, exaggerated bacteremia, and increased mortality; defects that are rescued by DEL-1 administration. A high IL-10/IL-17A ratio, observed in newborn sepsis, sustains tissue DEL-1 expression, as IL-10 upregulates while IL-17 downregulates DEL-1. Consistently, serum DEL-1 and blood neutrophils are elevated in septic adult and neonate patients with high serum IL-10/IL-17A ratio, and mortality is lower in septic patients with high serum DEL-1. Therefore, IL-10/DEL-1 axis supports emergency granulopoiesis, prevents neutropenia and promotes sepsis survival in early life.

Antifungal Drug Usage in European Neonatal Units: A Multicenter Weekly Point Prevalence Study.

BACKGROUND: Data on antifungal prescribing in neonatal patients are limited to either single-center or single-country studies or to 1-day recording. Therefore, we assessed antifungal longitudinal usage in neonatal units (NUs) within Europe. METHODS: CALYPSO, a prospective weekly point prevalence study on antifungal drug usage in NUs in 18 hospitals (8 European countries), was conducted in 2020 during a 12-week period. All patients receiving systemic antifungals were included. Ward demographics were collected at the beginning; ward and patient data including indication, risk factors and antifungal regimen were weekly collected prospectively. RESULTS: Among 27 participating NUs, 15 (56%) practiced antifungal prophylaxis for neonates with birth weight <1000 g or <1500 g and additional risk factors. In total, 174 patients received antifungals with a median frequency per week of 10.5% ranging from 6.9% to 12.6%. Indication for antifungal prescribing was prophylaxis in 135/174 (78%) courses and treatment in 22% [39 courses (69% empirical, 10% preemptive, 21% targeted)]. Fluconazole was the most frequent systemic agent used both for prophylaxis (133/135) and treatment (15/39, 39%). Among neonates receiving prophylaxis, the most common risk factors were prematurity (119/135, 88%), mechanical ventilation (109/135, 81%) and central vascular catheters (89/135, 66%). However, gestational age <28 weeks was only recorded in 55/135 (41%) courses and birth weight <1000 g in 48/135 (35%). Most common reason for empirical treatment was late-onset sepsis; all 8 targeted courses were prescribed for invasive candidiasis. CONCLUSION: Antifungal usage in European NUs is driven by prophylaxis and empirical treatment with fluconazole being the most prescribed agent for both indications.

Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension.

BACKGROUND: Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension. Mesenchymal stromal cell transplantation inhibits lung inflammation, vascular remodeling, and right heart failure and reverses hypoxic pulmonary hypertension in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which mesenchymal stromal cells are protective in hypoxic pulmonary hypertension. METHODS AND RESULTS: We fractionated mouse mesenchymal stromal cell-conditioned media to identify the biologically active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of hypoxic pulmonary hypertension. Intravenous delivery of mesenchymal stromal cell-derived exosomes (MEX) inhibited vascular remodeling and hypoxic pulmonary hypertension, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA, the expression of which is decreased in human pulmonary hypertension. MEX produced by human umbilical cord mesenchymal stromal cells inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells, demonstrating a direct effect of MEX on hypoxic vascular cells. CONCLUSION: This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit pulmonary hypertension through suppression of hyperproliferative pathways, including STAT3-mediated signaling induced by hypoxia.

Early macrophage recruitment and alternative activation are critical for the later development of hypoxia-induced pulmonary hypertension.

BACKGROUND: Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however, its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and to elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1, an anti-inflammatory enzyme, is protective in HPH. METHODS AND RESULTS: We generated bitransgenic mice that overexpress human heme oxygenase-1 under doxycycline control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of doxycycline resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of found in inflammatory zone-1, arginase-1, and chitinase-3-like-3. A brief 2-day pulse of doxycycline delayed, but did not prevent, the peak of hypoxic inflammation, and could not protect against HPH. In contrast, a 7-day doxycycline treatment sustained high heme oxygenase-1 levels during the entire period of hypoxic inflammation, inhibited macrophage accumulation and activation, induced macrophage interleukin-10 expression, and prevented the development of HPH. Supernatants from hypoxic M2 macrophages promoted the proliferation of pulmonary artery smooth muscle cells, whereas treatment with carbon monoxide, a heme oxygenase-1 enzymatic product, abrogated this effect. CONCLUSIONS: Early recruitment and alternative activation of macrophages in hypoxic lungs are critical for the later development of HPH. Heme oxygenase-1 may confer protection from HPH by effectively modifying the macrophage activation state in hypoxia.

Pulmonary microRNA profiling in a mouse model of ventilator-induced lung injury.

The aim of this study was to investigate the changes induced by high tidal volume ventilation (HVTV) in pulmonary expression of micro-RNAs (miRNAs) and identify potential target genes and corresponding miRNA-gene networks. Using a real-time RT-PCR-based array in RNA samples from lungs of mice subjected to HVTV for 1 or 4 h and control mice, we identified 65 miRNAs whose expression changed more than twofold upon HVTV. An inflammatory and a TGF-ß-signaling miRNA-gene network were identified by in silico pathway analysis being at highest statistical significance (P = 10(-43) and P = 10(-28), respectively). In the inflammatory network, IL-6 and SOCS-1, regulated by miRNAs let-7 and miR-155, respectively, appeared as central nodes. In TGF-ß-signaling network, SMAD-4, regulated by miR-146, appeared as a central node. The contribution of miRNAs to the development of lung injury was evaluated in mice subjected to HVTV treated with a precursor or antagonist of miR-21, a miRNA highly upregulated by HVTV. Lung compliance was preserved only in mice treated with anti-miR-21 but not in mice treated with pre-miR-21 or negative-control miRNA. Both alveolar-arterial oxygen difference and protein levels in bronchoalveolar lavage were lower in mice treated with anti-miR-21 than in mice treated with pre-miR-21 or negative-control miRNA (D(A-a): 66 ± 27 vs. 131 ± 22, 144 ± 10 mmHg, respectively, P < 0.001; protein concentration: 1.1 ± 0.2 vs. 2.3 ± 1, 2.1 ± 0.4 mg/ml, respectively, P < 0.01). Our results show that HVTV induces changes in miRNA expression in mouse lungs. Modulation of miRNA expression can affect the development of HVTV-induced lung injury.