Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion.

Analysis of Chronic Granulomatous Disease in the Kavkazi Population in Israel Reveals Phenotypic Heterogeneity in Patients with the Same NCF1 mutation (c.579G>A).

PURPOSE: Chronic granulomatous disease (CGD) is an innate immune deficiency disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex that impair the synthesis of oxygen radicals, thus rendering patients susceptible to recurrent infections and excessive hyperinflammatory responses. The most common autosomal recessive form of CGD is p47phox deficiency, which is often clinically milder than the more common X-linked recessive form. Here, we report data on genetics, clinical and biochemical findings in 17 CGD patients of Kavkazi origin with the nonsense mutation c.579G>A in the NCF1 gene, leading to p47phox deficiency. METHODS: Diagnosis was based on detailed clinical evaluation, respiratory burst activity by cytochrome c reduction and dihydrorhodamine-1,2,3 (DHR) assay by flow cytometry, expression of p47phox by immunoblotting and molecular confirmation by DNA sequence analysis. RESULTS: Twelve male and five female patients with median age at onset of 2.5 years (range 1 day to 9 years) were included in the study. The present cohort displays an encouraging 88% overall long-term survival, with median follow-up of 17 years. Clinical manifestations varied from mild to severe expression of the disease. Correlation between genotype and phenotype is unpredictable, although the Kavkazi patients were more severely affected than other patients with p47phox deficiency. CONCLUSIONS: Kavkazi CGD patients harbor a common genetic mutation that is associated with a heterogeneous clinical phenotype. Early diagnosis and proper clinical management in an experienced phagocytic leukocyte center is imperative to ensure favorable patient outcome. New treatment strategies are ongoing, but results are not yet conclusive.

Quantitative proteomic analysis of gingival crevicular fluids from deciduous and permanent teeth.

AIM: Gingivitis commonly progresses to periodontitis in permanent dentition but rarely in deciduous teeth. Little is known about the biochemical differences between gingiva of deciduous and permanent teeth. Here, we compared the protein profiles of gingival crevicular fluids (GCF) from the gingiva of deciduous and permanent teeth. MATERIALS AND METHODS: Forty children with mixed dentition (Hellman's dental age IIIA) were selected and GCF samples were collected from deciduous cuspids and central incisors in the maxilla. Pairs of GCF samples were labelled using isobaric tags to permit quantitative comparison of protein abundance in the samples using liquid chromatography-electron spray ionization-tandem mass spectrometry. RESULTS: Sixty-two proteins were upregulated in deciduous teeth GCF and 54 in permanent teeth GCF. In particular, neutrophil-derived proteins, including myeloperoxidase and lactoferrin, were repeatedly higher in deciduous teeth GCF than in permanent teeth GCF. These differences were verified using ELISA (p < 0.01). In contrast, immunoglobulin components were upregulated in permanent teeth GCF. CONCLUSIONS: Neutrophil-related proteins were enriched in deciduous teeth GCF and immunoglobulins in permanent teeth GCF. This suggests that neutrophil accumulation plays a protective role in innate immunity against bacterial infection in gingival tissue of deciduous teeth.

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

RATIONALE: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. OBJECTIVES: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. METHODS: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. MEASUREMENTS AND MAIN RESULTS: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. CONCLUSIONS: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

Macrolide Use and Neutrophil Function/Cytokine Levels in Hospitalized Patients with Community-Acquired Pneumonia: A Pilot Study.

PURPOSE: The objective of this study was to measure plasma cytokine levels and blood neutrophil functions as well as clinical outcomes in hospitalized patients with community-acquired pneumonia (CAP) treated with or without macrolide use--a known modulator of inflammatory response. METHODS: Subjects with CAP had peripheral blood analyzed for some neutrophil functions (degranulation of secretory vesicles and specific granules, respiratory burst response and phagocytosis) and ten cytokine levels measured in serum and sputum supernatants. Neutrophil function in healthy volunteers was also measured for reference. Values were measured on the day of enrollment, days 2-4 and 5-7, depending on a patient's length of stay. Early and late clinical outcomes were also evaluated. All values were compared between those treated with or without a macrolide. RESULTS: A total of 40 subjects were in this study; 14 received macrolide treatment, and 26 did not. Neutrophil function in the macrolide group was not significantly different compared to the non-macrolide group. None of the median cytokine levels or IQRs were statistically significant between the groups. However, a trend toward decreased IL-6, IL-8, and IFN-γ levels, and favorable clinical outcomes were present in the macrolide group. CONCLUSIONS: This pilot study showed no statistical difference between cytokine levels or neutrophil activity for CAP patients prescribed a macrolide containing regimen. Considering the trend of lower cytokine levels in the macrolide group when comparing the 5- to 7-day time period with the non-macrolide group, a full study with an appropriate sample size may be warranted.

Bereavement reduces neutrophil oxidative burst only in older adults: role of the HPA axis and immunesenescence.

BACKGROUND: The effect of the chronic stress of bereavement on immunity is poorly understood. Previous studies have demonstrated negative effects on immunity in older adults, and those who report higher depressive symptoms. The aim of the present study was to compare the effect of bereavement on neutrophil function in healthy young and old adults, also assessing serum levels of the stress hormones, cortisol and dehydroepiandrosterone sulphate (DHEAS). 41 young (mean age 32 years) and 52 older adults (mean age 72 years), bereaved and non-bereaved, took part in the study. They completed questionnaires on socio-demographic and health behaviour characteristics, as well as psychosocial variables, and provided a blood sample for analysis of neutrophil function (phagocytosis and reactive oxygen species (ROS) production) and stress hormone analysis. RESULTS: Bereaved participants in both age groups reported more symptoms of depression and anxiety than controls and scored moderately highly on bereavement-specific questionnaires for these symptoms. Despite this, young bereaved participants showed robust neutrophil function when compared to age-matched non-bereaved controls, and comparable stress hormone levels, while reduced neutrophil ROS production and raised stress hormone levels (cortisol:DHEAS ratio) were seen in the older bereaved group compared to their age-matched controls. CONCLUSIONS: Reduced neutrophil function among older bereaved participants may be the result of the inability to maintain stress hormone balance, specifically the cortisol:DHEAS ratio.

Effect of an injectable trace mineral supplement containing selenium, copper, zinc, and manganese on immunity, health, and growth of dairy calves.

The objective of this study was to evaluate the effect of 2 subcutaneous injections of a multimineral preparation, each containing 60 mg of zinc, 10mg of manganese, 5mg of selenium, and 15 mg of copper at 3 and 30 d after birth on immunity, health, and growth of dairy calves during the preweaning period. The study was conducted in upstate New York in 2 commercial dairy farms. A total of 790 Holstein heifer calves were randomly allocated at birth into 1 of 2 treatments: trace mineral supplement (TMS) treated or control. Blood samples were collected at 3, 14, and 35 d after birth to evaluate glutathione peroxidase (GPx) activity, superoxide dismutase (SOD) activity, haptoglobin, and neutrophil and monocyte function. Incidence of diseases and average daily gain was evaluated in the first 50 d of life. At 14 d of life, TMS-treated calves had increased neutrophil activity compared with control calves. Moreover, TMS-treated calves had greater GPx activity on d 14 after birth than control calves. The TMS treatment reduced the incidence of diarrhea (TMS=41.7% vs. control=49.7%) and combined incidence of pneumonia or otitis or both (TMS=41.7% vs. control=49.1%). Additionally, GPx was greater for calves diagnosed with otitis at d 35 after birth. However, calves diagnosed with pneumonia had decreased GPx activity at d 35 after birth. Serum SOD and haptoglobin concentrations were not affected by treatment or disease. Moreover, no effects were observed on average daily gain and survivability between TMS-treated and control calves during the preweaning period. Supplementation with trace minerals at 3 and 30 d of life increased neutrophil function and GPx activity and reduced the incidence of health disorders.

The Role of Neutrophil in COVID-19: Positive or Negative.

BACKGROUND: Neutrophils are the first line of defense against pathogens. They are divided into multiple subpopulations during development and kill pathogens through various mechanisms. Neutrophils are considered one of the markers of severe COVID-19. SUMMARY: In-depth research has revealed that neutrophil subpopulations have multiple complex functions. Different subsets of neutrophils play an important role in the progression of COVID-19. KEY MESSAGES: In this review, we provide a detailed overview of the developmental processes of neutrophils at different stages and their recruitment and activation after SARS-CoV-2 infection, aiming to elucidate the changes in neutrophil subpopulations, characteristics, and functions after infection and provide a reference for mechanistic research on neutrophil subpopulations in the context of SARS-CoV-2 infection. In addition, we have also summarized research progress on potential targeted drugs for neutrophil immunotherapy, hoping to provide information that aids the development of therapeutic drugs for the clinical treatment of critically ill COVID-19 patients.

Effect of albumin infusion on oxidative albumin modification and albumin binding capacity in chronic liver failure.

Oxidative albumin modification and impaired albumin binding function have been described both in chronic liver failure and for therapeutic albumin solutions. The aim of the present study was to evaluate the effect of albumin infusion on redox state and binding function of circulating albumin. We studied 20 patients with cirrhosis who routinely received albumin infusions for prevention of post-paracentesis circulatory dysfunction or treatment of hepatorenal syndrome. We measured albumin fractions by redox state of cysteine-34 and albumin binding properties using dansylsarcosine as site II ligand. Therapeutic albumin solutions showed high contents of human nonmercaptalbumin-1 and human nonmercaptalbumin-2, exceeding the respective values in our patients with decompensated cirrhosis. An initial protocol for the first nine patients sampled at baseline, 24 h and 48 h after albumin infusion revealed no significant changes of oxidized albumin species or albumin binding properties. However, a modified protocol for the remaining 11 patients sampled at baseline, <1 h after and 24 h after albumin infusion revealed short-lived changes of oxidized albumin species while no changes in albumin binding properties were observed. In conclusion, therapeutic albumin infusion transiently changed albumin redox state but did not improve binding function of circulating albumin in chronic liver failure.

Depressive symptoms are associated with reduced neutrophil function in hip fracture patients.

Hip fracture is a common trauma in older adults with a high incidence of depression, which relates to poorer prognosis including increased risk of infection. Ageing is accompanied by reduced immunity, termed immunesenescence, resulting in increased susceptibility to infection. We examined whether physical trauma (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system that might contribute to poor outcomes after injury. Neutrophil function was assessed in 101 hip fracture patients (81 female) 6 weeks and 6 months after injury and 43 healthy age-matched controls (28 female). Thirty eight fracture patients had depressive symptoms at 6 weeks. No difference in neutrophil phagocytosis of Escherichia coli was observed between controls and hip fracture patients, but superoxide production was significantly reduced in hip fracture patients with depressive symptoms compared with patients without symptoms (p=.001) or controls (p=.004) at 6 weeks. Superoxide production improved 6 months following fracture to the level seen in controls. We detected elevated serum cortisol, reduced dehydroepiandrosterone sulphate (DHEAS) and an increased cortisol:DHEAS ratio in fracture patients with depressive symptoms compared with patients without depressive symptoms or controls at 6 weeks and 6 months after injury. Serum IL6, TNFα and IL10 were higher among patients with depressive symptoms at 6 weeks. The cortisol:DHEAS ratio and IL6 levels related to depressive symptom scores but not to neutrophil function. In conclusion, depressive symptoms related to poorer neutrophil function after hip fracture, but this was not driven by changes in stress hormone or cytokine levels.

Effect of glutamine supplementation on neutrophil function in male judoists.

Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty-six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre-ULE), then 1 and 2 weeks after ULE (post-ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre-ULE and post-ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period.

Changes in immune functions during a peaking period in male university soccer players.

The concept of peaking ensures that athletes have trained to attain their absolute peak performance levels prior to a competition. This study investigates the effects of peaking on the functions of neutrophils and lymphocytes in university soccer players during a five-day soccer training camp followed by two weeks of tapering. The study subjects were 22 soccer players who were members of a university soccer club. We carried out our investigation during a five-day training camp and two weeks after the training camp (i.e. the tapering period). We measured body composition, immune-related parameters (leukocyte count, neutrophil count, lymphocyte count, immunoglobulins and complements), myogenic enzymes, superoxide dismutase activity (SOD) and neutrophil functions [reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and lymphocyte subtypes]. Leukocyte and neutrophil counts tend to increase after the training camp compared with values before the training camp, and recovered during the conditioning period, although the final values were still lower than those before the training camp. The amount of ROS production per neutrophil and level of SOD decreased significantly during the conditioning period compared with before the training camp (p < 0.05, p < 0.01, respectively). Levels of Th1 cells decreased significantly during the conditioning period compared with the training camp (p < 0.05). In conclusion, the period of two weeks for the tapering period was considered insufficient to allow the athletes' immune function to recover completely after an intensive training camp.

Cytomegalovirus durably primes neutrophil oxidative burst.

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects most humans, thereafter persisting lifelong in tissues of the host. It is a known pathogen in immunosuppressed patients, but its impact on immunocompetent hosts remains less understood. Recent data have shown that CMV leaves a significant and long-lasting imprint in host immunity that may confer some protection against subsequent bacterial infection. Such innate immune activation may come at a cost, however, with potential to cause immunopathology. Neutrophils are central to many models of immunopathology, and while acute CMV infection is known to influence neutrophil biology, the impact of chronic CMV infection on neutrophil function remains unreported. Using our murine model of CMV infection and latency, we show that chronic CMV causes persistent enhancement of neutrophil oxidative burst well after resolution of acute infection. Moreover, this in vivo priming of marrow neutrophils is associated with enhanced formyl peptide receptor expression, and ultimately constitutive c-Jun N-terminal kinase phosphorylation and enhanced CD14 expression in/on circulating neutrophils. Finally, we show that neutrophil priming is dependent on viral load, suggesting that naturally infected human hosts will show variability in CMV-related neutrophil priming. Altogether, these findings represent a previously unrecognized and potentially important impact of chronic CMV infection on neutrophil responsiveness in immunocompetent hosts.

How neutrophil metabolism affects bacterial killing.

Neutrophils are front line cells in immunity that quickly recognize and eliminate pathogens, relying mainly on glycolysis to exert their killing functions. Even though investigations into the influence of metabolic pathways in neutrophil function started in the 1930s, the knowledge of how neutrophils metabolically adapt during a bacterial infection remains poorly understood. In this review, we discuss the current knowledge about the metabolic regulation underlying neutrophils response to bacterial infection. Glycogen metabolism has been shown to be important for multiple neutrophil functions. The potential contribution of metabolic pathways other than glycolysis, such as mitochondrial metabolism, for neutrophil function has recently been explored, including fatty acid oxidation in neutrophil differentiation. Complex III in the mitochondria might also control glycolysis via glycerol-3-phosphate oxidation. Future studies should yield new insights into the role of metabolic change in the anti-bacterial response in neutrophils.

Neonatal sepsis and transient immunodeficiency: Potential for novel immunoglobulin therapies?

Neonates, especially preterm neonates, have the highest risk of sepsis of all age groups. Transient immaturity of the neonatal immune system is an important risk factor. Neonates suffer from hypogammaglobulinemia as nor IgA nor IgM is transferred over the placenta and IgG is only transferred over the placenta late in gestation. In addition, neutrophil numbers and complement function are also decreased. This mini-review focuses on strategies to improve neonatal host-defense. Both clinical and preclinical studies have attempted to boost neonatal immunity to lower the incidence of sepsis and improve outcome. Recent advances in the development of (monoclonal) antibodies show promising results in preclinical studies but have yet to be tested in clinical trials. Strategies to increase complement activity seem efficient in vitro but potential disadvantages such as hyperinflammation have held back further clinical development. Increase of neutrophil numbers has been tested extensively in clinical trials but failed to show improvement in mortality. Future research should focus on clinical applicability of promising new prevention strategies for neonatal sepsis.

Investigation and assessment of neutrophil dysfunction early after severe burn injury.

BACKGROUND: Extensive burn injury results in a complex immune response that is associated with mortality and prognosis. Studies on acquired immune and the development of sepsis in burn patients have been reported. However, one of the main cells in innate immune, neutrophil dysfunction in the burn shock stage has not been thoroughly characterized. METHODS: Neutrophil chemotaxis, expression of neutrophil surface markers (P2X1 receptor, [P2RX1]), degranulation (myeloperoxidase [MPO], heparin-binding protein [HBP], matrix metalloproteinase-9 [MMP-9] and neutrophil elastase [NE]), oxidative burst capacity, neutrophil extracellular trap (NET) generation, phagocytosis and apoptosis were measured in 18 patients with major burns (≥30% total body surface area [TBSA]) within 48 h after burn injury. In addition, circulating neutrophils and vascular permeability in mice model with 30% TBSA third-degree burns were also observed and investigated. RESULTS: Neutrophil functions were reduced considerably in burn shock stage, which was characterized by decreased chemotaxis, phagocytosis and abnormal bactericidal function. Increased release of heparin-binding protein (HBP) and the expression of P2RX1 on the neutrophil surface are related to fluid leakage and decreased chemotaxis during burn shock stage, respectively. The combination of HBP concentration in plasma and P2RX1 expression on neutrophils gives a better prediction of neutrophil dysfunction in burn-injured patients. CONCLUSION: Neutrophil dysfunction plays a key role in the development of burn injury. Targeting the restoration of neutrophil function may be a feasible therapeutic intervention to help reduce fluid loss during shock and the severity of subsequent infection.

The influence of extracellular tissue on neutrophil function and its possible linkage to inflammatory diseases.

BACKGROUND: Migration, production of reactive oxygen species (ROS), release of myeloperoxidase (MPO), and NETosis are functional immunological reactions of elementary importance for polymorphonuclear neutrophils (PMN). Unregulated inflammatory response of PMN within tissues plays a key role in the pathophysiology of several diseases. However, little is known about the behavior of PMN after migration through blood vessel walls. Therefore, we investigated the influence of the extracellular matrix (ECM) on PMN function. MATERIALS AND METHODS: We established an in vitro chemotaxis model of type I and III collagen, fibrin, and herbal agarose tissues using µ-slide chemotaxis devices and N-formylmethionine-leucyl-phenylalanine (fMLP). PMN within the matrices were assessed with a fluorescent time-lapse microscope for live-cell imaging. RESULTS: PMN function was obviously influenced by the ECM. Type III collagen had an inhibitory effect on PMN migration regarding track length, direction, and targeting. Type III collagen also had an accelerating effect on neutrophil ROS production. Agarose had an inhibitory effect on MPO release and fibrin a retarding effect on NETosis. CONCLUSION: Because of the high abundance of type III collagen in lung and skin matrices, the interaction of PMN with the respective matrix could be an important mechanism in the pathophysiology of acute respiratory distress syndrome and pyoderma gangrenosum.

Mesenchymal stromal cells expressing a dominant-negative high mobility group A1 transgene exhibit improved function during sepsis.

High mobility group (HMG)A proteins are nonhistone chromatin proteins that bind to the minor groove of DNA, interact with transcriptional machinery, and facilitate DNA-directed nuclear processes. HMGA1 has been shown to regulate genes involved with systemic inflammatory processes. We hypothesized that HMGA1 is important in the function of mesenchymal stromal cells (MSCs), which are known to modulate inflammatory responses due to sepsis. To study this process, we harvested MSCs from transgenic (Tg) mice expressing a dominant-negative (dn) form of HMGA1 in mesenchymal cells. MSCs harvested from Tg mice contained the dnHMGA1 transgene, and transgene expression did not change endogenous HMGA1 levels. Immunophenotyping of the cells, along with trilineage differentiation revealed no striking differences between Tg and wild-type (WT) MSCs. However, Tg MSCs growth was decreased compared with WT MSCs, although Tg MSCs were more resistant to oxidative stress-induced death and expressed less IL-6. Tg MSCs administered after the onset of Escherichia coli-induced sepsis maintained their ability to improve survival when given in a single dose, in contrast with WT MSCs. This survival benefit of Tg MSCs was associated with less tissue cell death, and also a reduction in tissue neutrophil infiltration and expression of neutrophil chemokines. Finally, Tg MSCs promoted bacterial clearance and enhanced neutrophil phagocytosis, in part through their increased expression of stromal cell-derived factor-1 compared with WT MSCs. Taken together, these data demonstrate that expression of dnHMGA1 in MSCs provides a functional advantage of the cells when administered during bacterial sepsis.

Molecular and Cellular Biomarkers of COVID-19 Prognosis: Protocol for the Prospective Cohort TARGET Study.

BACKGROUND: Since the beginning of the COVID-19 pandemic, the world's attention has been focused on better understanding the relation between the human host and the SARS-CoV-2 virus, as its action has led to hundreds of thousands of deaths. OBJECTIVE: In this context, we decided to study certain consequences of the abundant cytokine release over the innate and adaptive immune systems, inflammation, and hemostasis, comparing mild and severe forms of COVID-19. METHODS: To accomplish these aims, we will analyze demographic characteristics, biochemical tests, immune biomarkers, leukocyte phenotyping, immunoglobulin profile, hormonal release (cortisol and prolactin), gene expression, thromboelastometry, neutralizing antibodies, metabolic profile, and neutrophil function (reactive oxygen species production, neutrophil extracellular trap production, phagocytosis, migration, gene expression, and proteomics). A total of 200 reverse transcription polymerase chain reaction-confirmed patients will be enrolled and divided into two groups: mild/moderate or severe/critical forms of COVID-19. Blood samples will be collected at different times: at inclusion and after 9 and 18 days, with an additional 3-day sample for severe patients. We believe that this information will provide more knowledge for future studies that will provide more robust and useful clinical information that may allow for better decisions at the front lines of health care. RESULTS: The recruitment began in June 2020 and is still in progress. It is expected to continue until February 2021. Data analysis is scheduled to start after all data have been collected. The coagulation study branch is complete and is already in the analysis phase. CONCLUSIONS: This study is original in terms of the different parameters analyzed in the same sample of patients with COVID-19. The project, which is currently in the data collection phase, was approved by the Brazilian Committee of Ethics in Human Research (CAAE 30846920.7.0000.0008). TRIAL REGISTRATION: Brazilian Registry of Clinical Trials RBR-62zdkk; https://ensaiosclinicos.gov.br/rg/RBR-62zdkk. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/24211.