Hemin regulates platelet clearance in hemolytic disease by binding to GPIbα.

Hemolysis is associated with thrombosis and vascular dysfunction, which are the pathological components of many diseases. Hemolytic products, including hemoglobin and hemin, activate platelets (PLT). Despite its activation, the effect of hemolysis on platelet clearance remains unclear, It is critical to maintain a normal platelet count and ensure that circulating platelets are functionally viable. In this study, we used hemin, a degradation product of hemoglobin, as a potent agonist to treat platelets and simulate changes in vivo in mice. Hemin treatment induced activation and morphological changes in platelets, including an increase in intracellular Ca2+ levels, phosphatidylserine (PS) exposure, and cytoskeletal rearrangement. Fewer hemin-treated platelets were cleared by macrophages in the liver after transfusion than untreated platelets. Hemin bound to glycoprotein Ibα (GPIbα), the surface receptor in hemin-induced platelet activation and aggregation. Furthermore, hemin decreased GPIbα desialylation, as evidenced by reduced Ricinus communis agglutinin I (RCA- I) binding, which likely extended the lifetime of such platelets in vivo. These data provided new insight into the mechanisms of GPIbα-mediated platelet activation and clearance in hemolytic disease.

What is the context? Hemolysis is a primary hematological disease. Hemolysis is a pathological complication of several diseases.Hemin, a degradation product of cell-free hemoglobin, has been proven to be a more potent agonist than hemoglobin for directly activating platelets.Platelet membrane glycoproteins (GP), including GPIb-IX and GPIIb/IIIa complexes, play crucial roles in platelet hemostasis.Desialylation (loss of sialic acid residues) of GPIbα, is believed to regulate physiological platelet clearance through liver macrophages and hepatocytes.What is new? In this study, we evaluated the effects of hemolysis on platelet clearance. We first analyzed the influence of hemin at 0-50 µM on platelets in vitro before exploring the mechanism underlying hemin-induced platelet activation and its role in platelet clearance in vitro and in vivo.Our analyses suggest that: Hemin bound to GPIbα on the platelet surface with high affinity.Platelet clearance occurred slowly in the liver and spleen after hemin treatment.Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.What is the impact? This study provides new insights into the role of hemin in the mechanisms of GPIbα-mediated platelets activation and clearance in diseases associated with hemolysis.

Current Advances in Nanomaterials Affecting Functions and Morphology of Platelets.

Nanomaterials have been extensively used in the biomedical field due to their unique physical and chemical properties. They promise wide applications in the diagnosis, prevention, and treatment of diseases. Nanodrugs are generally transported to target tissues or organs by coupling targeting molecules or enhanced permeability and retention effect (EPR) passively. As intravenous injection is the most common means of administration of nanomedicine, the transport process inevitably involves the interactions between nanoparticles (NPs) and blood cells. Platelets are known to not only play a critical role in normal coagulation by performing adhesion, aggregation, release, and contraction functions, but also be associated with pathological thrombosis, tumor metastasis, inflammation, and immune reactions, making it necessary to investigate the effects of NPs on platelet function during transport, particularly the way in which their physical and chemical properties determine their interaction with platelets and the underlying mechanisms by which they activate and induce platelet aggregation. However, such data are lacking. This review is intended to summarize the effects of NPs on platelet activation, aggregation, release, and apoptosis, as well as their effects on membrane proteins and morphology in order to shed light on such key issues as how to reduce their adverse reactions in the blood system, which should be taken into consideration in NP engineering.

Rebalancing TGF-ß/PGE2 breaks RT-induced immunosuppressive barriers by enhancing tumor-infiltrated dendritic cell homing.

A better understanding of how tumor microenvironments shape immune responses after radiotherapy (RT) is required to improve patient outcomes. This study focuses on the observation that dendritic cells (DCs) infiltrating irradiated cervical tumors are retained in transforming growth factor (TGF)-ß-abundant regions. We report that TGF-ß secretion from cervical cancer cells was increased by irradiation in a dose-dependent manner and that this significantly suppressed the expression of allostimulatory markers and Th1 cytokines in DCs. To investigate further, we blocked the TGF-ß signal in DCs and observed that RT had a dose-dependent immune-promoting effect, improving DC maturation. This suggested that proinflammatory mediators may also be induced by RT, but their effects were being counteracted by the simultaneously increased levels of TGF-ß. Prostaglandin E2 (PGE2), a proinflammatory molecule, was shown to be one such mediator. Adjusting the TGF-ß/PGE2 ratio by inhibiting TGF-ß rebooted RT-induced DC cytoskeletal organization by stimulating myosin light chain (MLC) phosphorylation. Consequently, the homing of intra-tumorally infiltrated DCs to tumor-draining lymph nodes was enhanced, leading to the induction of more robust cytotoxic T cells. Ultimately, rebalancing the TGF-ß/PGE2 ratio amplified the therapeutic effects of RT, resulting in increased intra-tumoral infiltration and activation of CD8+ T cells, and improved tumor control and overall survival rate in mice. DC depletion experiments verified that the improvement in tumor control is directly correlated with the involvement of DCs via the PGE2-MLC pathway. This study emphasizes the importance of maintaining a balanced cytokine environment during RT, particularly hypofractionated RT; and it is advisable to block TGF-ß while preserving PGE2 in the tumor microenvironment in order to better stimulate DC homing and DC -T priming.

X-ray irradiation effectively inactivated lymphocytes in transfusion in vivo monitored by the bioluminescence transfusion-associated graft-versus-host disease model.

BACKGROUND AND OBJECTIVES: Transfusion of cold-stored whole blood is the preferred resuscitation method for trauma patients but may cause transfusion-associated graft-versus-host disease (TA-GVHD). Standard clinical practice to prevent this is to irradiate blood components with gamma-rays. X-ray irradiations are also a safe and effective alternative to gamma-ray irradiation. We established a visual mouse model of TA-GVHD to compare the viability and function of lymphocytes exposed to gamma- and x-ray irradiation. MATERIALS AND METHODS: A haploidentical transplantation mouse model was established to simulate TA-GVHD with Balb/c mice as donors and hybrid F1 CB6 mice (Balb/c × C57) as recipients. Spleen cells from Tg-Fluc+ Balb/c mice were isolated and irradiated with gamma-rays and x-rays. Lymphocyte activation, apoptosis and proliferation post phorbol 1 2-myristate 1 3-acetate (PMA) stimulation were evaluated. After transfusion, we monitored Fluc+ lymphocytes daily by bioluminescence imaging. Recipients were euthanized on day 21, and tissues were examined pathologically and for inflammatory cytokines. RESULTS: The viability of gamma- or x-ray irradiated lymphocytes decreased significantly with slight changes in proliferation in vivo after transfusion. Compared with the non-irradiated group, both the gamma- and x-ray irradiated groups showed significantly decreased clinical scoring and inflammatory cytokine levels. The fluorescence intensity of the body and target organs was reduced after irradiation. CONCLUSION: No recipients acquired TA-GVHD after lymphocyte transfusion subjected to gamma- or x-rays, showing that x-rays inactivate as well as gamma rays and are suitable for irradiating whole blood.

Laponite Lights Calcium Flickers by Reprogramming Lysosomes to Steer DC Migration for An Effective Antiviral CD8+ T-Cell Response.

Immunotherapy using dendritic cell (DC)-based vaccination is an established approach for treating cancer and infectious diseases; however, its efficacy is limited. Therefore, targeting the restricted migratory capacity of the DCs may enhance their therapeutic efficacy. In this study, the effect of laponite (Lap) on DCs, which can be internalized into lysosomes and induce cytoskeletal reorganization via the lysosomal reprogramming-calcium flicker axis, is evaluated, and it is found that Lap dramatically improves the in vivo homing ability of these DCs to lymphoid tissues. In addition, Lap improves antigen cross-presentation by DCs and increases DC-T-cell synapse formation, resulting in enhanced antigen-specific CD8+ T-cell activation. Furthermore, a Lap-modified cocktail (Lap@cytokine cocktail [C-C]) is constructed based on the gold standard, C-C, as an adjuvant for DC vaccines. Lap@C-C-adjuvanted DCs initiated a robust cytotoxic T-cell immune response against hepatitis B infection, resulting in > 99.6% clearance of viral DNA and successful hepatitis B surface antigen seroconversion. These findings highlight the potential value of Lap as a DC vaccine adjuvant that can regulate DC homing, and provide a basis for the development of effective DC vaccines.

Stored whole blood transfusion initiates serum amyloid A activation monitored by real-time dynamic imaging.

BACKGROUND: Transfusion of stored whole blood (SWB) to resuscitate severe traumatic haemorrhage patients in military operations and civilian emergency centres is being increasingly used in routine practice. It has been well established that transfusion of red blood cells (RBCs) after prolonged storage has harmful effects, mainly mediated by inflammation. Whether the side effects of inflammation are brought about by SWB transfusion remains unclear. MATERIALS AND METHODS: A hepatocyte SAA (serum amyloid A) specific reporter mouse that facilitated non-invasive imaging of hepatocyte SAA expression was used to evaluate acute inflammation and acute-phase reaction after the transfusion of SWB or components separated from end-storage whole blood. The whole blood of C57BL/6 donor mouse was used to model an allogeneic transfusion to BALB/c recipient mouse. RESULTS: End-storage whole blood (14 days of storage) transfusion induced the most significant SAA expression, while 10-day storage resulted in a much weaker signal compared to their fresh and 5-day storage counterparts. RBCs rather than white blood cells and plasma-containing platelets are thought to be responsible for the systemic inflammatory and SAA activation during end-storage whole blood transfusion. Circulatory and hepatic pro-inflammatory cytokines secreted by M1-polarised macrophage initiated the SAA expression in hepatocytes through nuclear transcription factor NF-κB. DISCUSSION: Storage lesions will also occur during the storage of whole blood, which is related to the change in RBCs with prolonged storage. The side effect induced by systemic inflammation and acute-phase reaction should be considered before resuscitation with long-term storage whole blood transfusion.

Effects of urban particulate matter on the quality of erythrocytes.

With the acceleration of industrialisation and urbanisation, air pollution has become a serious global concern as a hazard to human health, with urban particulate matter (UPM) accounting for the largest share. UPM can rapidly pass into and persist within systemic circulation. However, few studies exist on whether UPM may have any impact on blood components. In this study, UPM standards (SRM1648a) were used to assess the influence of UPM on erythrocyte quality in terms of oxidative and metabolic damage as well as phagocytosis by macrophages in vitro and clearance in vivo. Our results showed that UPM had weak haemolytic properties. It can oxidise haemoglobin and influence the oxygen-carrying function, redox balance, and metabolism of erythrocytes. UPM increases the content of reactive oxygen species (ROS) and decreases antioxidant function according to the data of malonaldehyde (MDA), glutathione (GSH), and glucose 6 phosphate dehydrogenase (G6PDH). UPM can adhere to or be internalised by erythrocytes at higher concentrations, which can alter their morphology. Superoxide radicals produced in the co-incubation system further disrupted the structure of red blood cell membranes, thereby lowering the resistance to the hypotonic solution, as reflected by the osmotic fragility test. Moreover, UPM leads to an increase in phosphatidylserine exposure in erythrocytes and subsequent clearance by the mononuclear phagocytic system in vivo. Altogether, this study suggests that the primary function of erythrocytes may be affected by UPM, providing a warning for erythrocyte quality in severely polluted areas. For critically ill patients, transfusion of erythrocytes with lesions in morphology and function will have serious clinical consequences, suggesting that potential risks should be considered during blood donation screening. The current work expands the scope of blood safety studies.

A point-of-care microfluidic channel-based device for rapid and direct detection of fibrinogen in whole blood.

Hemorrhage is the leading cause of preventable death in civilian and battlefield traumatic injuries. Patients with severe traumatic hemorrhagic shock are more likely to be deficient in fibrinogen than those with other coagulation factors, and hypofibrinogenemia is an independent risk factor for mortality. Thus, rapid detection of fibrinogen levels is of great importance in these patients during damage control resuscitation. Plasma is used as an analyte in fibrinogen detection, which restricts the use of existing devices in emergencies. To meet the needs of on-site detection, we developed a point-of-care microfluidic channel-based device for direct measurement of fibrinogen concentration in whole blood. In our method, thrombin is dispersed on a reaction strip to initiate conversion of fibrinogen to fibrin. The permeability of the resulting blood clots depends on the fibrinogen level. A hydrophobic plastic protection flake between the reaction strip and a wicking strip is then removed to allow flow of unclotted blood. The rate of blood flow along the wicking strip was inversely related to the fibrinogen concentration. The whole process could be completed in as fast as 5 minutes for a whole blood sample size of 150 µL, and yielded accurate results ranging from 0 to 4 g L-1, which were unaffected by Ca2+, blood lipids, hematocrit, warfarin and tissue plasminogen activators (tPAs). Results using clinical whole blood samples were also highly consistent with those using an automatic coagulation analyzer, yielding a Pearson correlation coefficient of up to 0.919. This approach has potential for allowing rapid diagnosis of fibrinogen concentration in critically ill bleeding patients in different settings, thus helping to judge the suitability of fibrinogen replacement therapy (FRT) in cases of emergency bleeding and in patients at risk of thrombosis due to hyperfibrinogenemia.

Establishment of a Novel Mouse Hepatocellular Carcinoma Model for Dynamic Monitoring of Tumor Development by Bioluminescence Imaging.

In this study, a novel mouse model of hepatocellular carcinoma (HCC) was established by simultaneously knocking out Pten and p53 suppressor genes and overexpressing c-Met and △90-ß-catenin proto-oncogenes in the livers of mice via hydrodynamic injection (HDI). The mutations were introduced using the CRISPR/Cas9 and Sleeping Beauty transposon systems. In this way, a primary liver cancer model was established within six weeks. In addition, macrophages expressing arginase-1(Arg1) promoter coupled with firefly luciferase were engineered for bioluminescence imaging (BLI) of the tumor microenvironment. This novel, rapidly-generated model of primary hepatocellular carcinoma can be monitored noninvasively, which can facilitate not only applications of the model, but also the development of new drugs and treatment strategies of HCC.

Large-Sized Graphene Oxide Nanosheets Increase DC-T-Cell Synaptic Contact and the Efficacy of DC Vaccines against SARS-CoV-2.

Dendritic cell (DC) vaccines are used for cancer and infectious diseases, albeit with limited efficacy. Modulating the formation of DC-T-cell synapses may greatly increase their efficacy. The effects of graphene oxide (GO) nanosheets on DCs and DC-T-cell synapse formation are evaluated. In particular, size-dependent interactions are observed between GO nanosheets and DCs. GOs with diameters of >1 µm (L-GOs) demonstrate strong adherence to the DC surface, inducing cytoskeletal reorganization via the RhoA-ROCK-MLC pathway, while relatively small GOs (≈500 nm) are predominantly internalized by DCs. Furthermore, L-GO treatment enhances DC-T-cell synapse formation via cytoskeleton-dependent membrane positioning of integrin ICAM-1. L-GO acts as a "nanozipper," facilitating the aggregation of DC-T-cell clusters to produce a stable microenvironment for T cell activation. Importantly, L-GO-adjuvanted DCs promote robust cytotoxic T cell immune responses against SARS-CoV-2 spike 1, leading to >99.7% viral RNA clearance in mice infected with a clinically isolated SARS-CoV-2 strain. These findings highlight the potential value of nanomaterials as DC vaccine adjuvants for modulating DC-T-cell synapse formation and provide a basis for the development of effective COVID-19 vaccines.

Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation.

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.

Current advances in nanomaterials affecting morphology, structure, and function of erythrocytes.

In recent decades, nanomaterials have been widely used in the field of biomedicine due to their unique physical and chemical properties, and have shown good prospects for in vitro diagnosis, drug delivery, and imaging. With regard to transporting nanoparticles (NPs) to target tissues or organs in the body intravenously or otherwise, blood is the first tissue that NPs come into contact with and is also considered an important gateway for targeted transport. Erythrocytes are the most numerous cells in the blood, but previous studies based on interactions between erythrocytes and NPs mostly focused on the use of erythrocytes as drug carriers for nanomedicine which were chemically bound or physically adsorbed by NPs, so little is known about the effects of nanoparticles on the morphology, structure, function, and circulation time of erythrocytes in the body. Herein, this review focuses on the mechanisms by which nanoparticles affect the structure and function of erythrocyte membranes, involving the hemocompatibility of NPs, the way that NPs interact with erythrocyte membranes, effects of NPs on erythrocyte surface membrane proteins and their structural morphology and the effect of NPs on erythrocyte lifespan and function. The detailed analysis in this review is expected to shed light on the more advanced biocompatibility of nanomaterials and pave the way for the development of new nanodrugs.

Single High-Dose Radiation Enhances Dendritic Cell Homing and T Cell Priming by Promoting Reactive Oxygen Species-Induced Cytoskeletal Reorganization.

PURPOSE: Radiation therapy (RT) affects tumor-infiltrating immune cells, cooperatively driving tumor growth inhibition. However, there is still no absolute consensus on whether the homing ability of dendritic cells (DCs) is affected by direct x-ray irradiation. Most importantly, the underlying mechanisms are poorly understood. METHODS AND MATERIALS: Using noninvasive imaging, we systematically examined the dose effect of RT on the in vivo homing and distribution of bone marrow-derived DCs and elucidated the detailed mechanisms underlying these events. After exposure to 2, 5, 10, 15, and 20 Gy, DCs were analyzed for maturation, in vivo homing ability, and T cell priming. RESULTS: At ranges of 2 to 20 Gy, irradiation did not cause direct cellular apoptosis or necrosis, but it induced mitochondrial damage in DCs independent of dose. In addition, upregulation of CD40, CD80, CD86, CXCR4, and CCR7 were detected on irradiated DCs. Secretion of IL-1ß and IL-12p70 remained unchanged, whereas decreased secretion of IL-6 and promotion of tumor necrosis factor α secretion were observed. In particular, the homing ability of both the local residual and blood circulating DCs to lymphoid tissues was significantly higher in groups that received ≥5 Gy radiation than in the group that received 2 Gy. Furthermore, improved homing ability was associated with rearrangement of the cytoskeleton, which was regulated by reactive oxygen species accumulation through the RhoA/ROCK1 signaling pathway. Finally, more robust T cell activation was observed in mice inoculated with 20 Gy-treated DCs than in those inoculated with 2 Gy-irradiated DCs, and T cell activation also correlated with reactive oxygen species production. CONCLUSIONS: An RT dose ≥5 Gy has distinct advantages over 2 Gy in facilitating DC homing to lymph nodes and cross-priming T cells.

Immunostimulatory Potential of MoS2 Nanosheets: Enhancing Dendritic Cell Maturation, Migration and T Cell Elicitation.

BACKGROUND: Due to their extraordinary physical and chemical properties, MoS2 nanosheets (MSNs) are becoming more widely used in nanomedicine. However, their influence on immune systems remains unclear. MATERIALS AND METHODS: Two few-layered MSNs at sizes of 100-250 nm (S-MSNs) and 400-500 nm (L-MSNs) were used in this study. Bone marrow-derived dendritic cells (DCs) were exposed to both MSNs at different doses (0, 8, 16, 32, 64, 128 µg/mL) for 48 h and subjected to analyses of surface marker expression, cytokine secretion, lymphoid homing and in vivo T cell priming. RESULTS: Different-sized MSNs of all doses did not affect the viability of DCs. The expression of CD40, CD80, CD86 and CCR7 was significantly higher on both S-MSN- and L-MSN-treated DCs at a dose of 128 µg/mL. As the dose of MSN increased, the secretion of IL-12p70 remained unchanged, the secretion of IL-1ß decreased, and the production of TNF-α increased. A significant increase in IL-6 was observed in the 128 µg/mL L-MSN-treated DCs. In particular, MSN treatment dramatically improved the ex vivo movement and in vivo homing ability of both the local resident and blood circulating DCs. Furthermore, the cytoskeleton rearrangement regulated by ROS elevation was responsible for the enhanced homing ability of the MSNs. More robust CD4+ and CD8+ T cell proliferation and activation (characterized by high expression of CD107a, CD69 and ICOS) was observed in mice vaccinated with MSN-treated DCs. Importantly, exposure to MSNs did not interrupt LPS-induced DC activation, homing and T cell priming. CONCLUSION: Few-layered MSNs ranging from 100 to 500 nm in size could play an immunostimulatory role in enhancing DC maturation, migration and T cell elicitation, making them a good candidate for vaccine adjuvants. Investigation of this study will not only expand the applications of MSNs and other new transition metal dichalcogenides (TMDCs) but also shed light on the in vivo immune-risk evaluation of MSN-based nanomaterials.

Correction: Large-sized graphene oxide synergistically enhances parenchymal hepatocyte IL-6 expression monitored by dynamic imaging.

Correction for 'Large-sized graphene oxide synergistically enhances parenchymal hepatocyte IL-6 expression monitored by dynamic imaging' by Yulong Zhang et al., Nanoscale, 2020, DOI: 10.1039/C9NR10713D.

Large-sized graphene oxide synergistically enhances parenchymal hepatocyte IL-6 expression monitored by dynamic imaging.

Graphene oxides (GOs) have received significant attention as emerging biomedical materials due to their special properties. The application of GOs in biological systems has raised considerable concern about their hepatotoxicity, however their biological effects on parenchymal hepatocytes remain unclear, despite the fact that GOs have shown size-dependent interactions with immunocytes in the liver. Herein we chose pleiotropic cytokine IL-6 as the model parameter to investigate inflammation responses upon exposure to GOs. An early and sensitive reporter mouse model was constructed, allowing non-invasive and longitudinal imaging of parenchymal hepatocyte IL-6 expressions. GOs of various lateral dimensions were assessed by using the reporter mice. The results demonstrated that large-sized GOs (L-GO) induced much stronger IL-6 activation. A detailed analysis uncovered that L-GO induced ROS production and TLR-4 activation promoted macrophage polarization and secretion of pro-inflammatory cytokines IL-1ß and TNF-α, activated via> the NF-κB signaling pathway, which in turn initiated the expression of IL-6 in hepatocytes. These in-depth investigations are expected to help modulate the inflammatory responses involved in hepatotoxicity and provide extended information to design sub-hepatic distribution and cell subset targeting by controlling the nanoparticle sizes.

Impairment of mitochondrial dynamics involved in iron oxide nanoparticle-induced dysfunction of dendritic cells was alleviated by autophagy inhibitor 3-methyladenine.

Iron oxide nanoparticles are nanomaterials that are used extensively in the biomedical field, but they are associated with adverse effects, including mitochondrial toxicity. Mitochondrial homeostasis is achieved through dynamic stability based on two sets of antagonistic balanced processes: mitochondrial biogenesis and degradation as well as mitochondrial fission and fusion. In this study, we showed that PEG-COOH-coated Fe3 O4 (PEG-Fe3 O4 ) nanoparticles induced mitochondrial instability in dendritic cells (DCs) by impairing mitochondrial dynamics due to promotion of mitochondrial biogenesis through activation of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) pathway, inhibiting mitochondrial degradation via decreased autophagy, and facilitating mitochondrial fragmentation involving increased levels of DRP1 and MFN2. The resulting reduced levels of dextran uptake, CD80, CD86 and chemokine receptor 7 (CCR7) suggested that PEG-Fe3 O4 nanoparticles impaired the functionally immature state of DCs. Autophagy inhibitor 3-methyladenine (3-MA) alleviated PEG-Fe3 O4 nanoparticle-induced mitochondrial instability and impairment of the functionally immature state of DCs due to unexpected enhancement of PGC1α/MFN2-mediated coordination of mitochondrial biogenesis and fusion.

M1-polarized alveolar macrophages are crucial in a mouse model of transfusion-related acute lung injury.

BACKGROUND: The pathogenesis of transfusion-related acute lung injury (TRALI) progress is incompletely understood, and specific therapies for TRALI are lacking. Alveolar macrophages (AMs) are critical for initiation and resolution of lung inflammation. However, the role of AMs in the pathogenesis of TRALI-associated lung failure is poorly understood. STUDY DESIGN AND METHODS: Mouse model for in vivo imaging of interleukin (IL)-6 activation in AMs was established by intratracheal instillation of a lentiviral vector carrying the luciferase reporter gene. The TRALI mouse model was produced by intraperitoneal lipopolysaccharide plus intravenous major histocompatibility complex Class I monoclonal antibody treatment. We focused on the changes in AMs in the lung during TRALI and examined whether targeting AMs is an effective strategy to alleviate this condition. MEASUREMENTS AND MAIN RESULTS: We confirmed that TRALI progress is accompanied by IL-6 activation in AMs. Further study showed that AMs undergo M1 activation during TRALI progress. AM depletion protected mice from TRALI, and transfusion of M1-polarized AMs into 34-1-2 s-treated mice elevated acute lung injury, indicating that the severity of TRALI was able to be ameliorated by targeting AM polarization. Next, we showed that α1 -antitrypsin (AAT) expression improved lung injury by modulating the production of IL-6 in AMs and decreased polarization of AMs toward the M1 phenotype. CONCLUSIONS: M1-polarized AMs are crucial in a mouse model of TRALI, and AAT may serve as a future treatment for TRALI by regulating the polarization of AMs.

Noninvasive imaging of hepatocyte IL-6/STAT3 signaling pathway for evaluating inflammation responses induced by end-stage stored whole blood transfusion.

OBJECTIVE: To monitor the inflammatory storage lesions of end-stage stored whole blood (SWB) using a noninvasive STAT3 signal pathway mouse model. RESULTS: In this study, we present a hydrodynamic transfection-based STAT3-Luc mouse model in which hepatocyte STAT3 signal pathway activation can be monitored by measuring luciferase activity using a noninvasive imaging system. Such a mouse model may reflect systemic IL-6 and inflammation levels by monitoring the activation of STAT3. During end-stage SWB transfusion, in vivo imaging of STAT3-Luc mice showed obvious luciferase activity in the hepatic region, which was consistent with an increase in IL-6 levels in the liver homogenate and circulation. We also confirmed that the mononuclear phagocytic system contributed to the elevation of serum and liver IL-6 after end-stage SWB transfusion. CONCLUSION: The hepatocyte STAT3 signaling pathway, which is activated by end-stage SWB transfusion, is associated with the elevation of systemic IL-6 secreted by macrophages. The STAT3-Luc mouse may serve as a mouse model for monitoring inflammation responses after end-stage SWB transfusion.

Stored red blood cells enhance in vivo migration of dendritic cells by promoting reactive oxygen species-induced cytoskeletal rearrangement.

BACKGROUND: A complex array of physicochemical changes occurs in red blood cells (RBCs) during storage, leading to enhanced posttransfusion clearance. Dendritic cells (DCs) play crucial roles in the engulfment of aged RBCs; however, it is unclear how stored RBCs (sRBCs) modulate their responses to inflammatory stimuli and DC migration ability. STUDY DESIGN AND METHODS: In this study, we examined whether sRBCs affect the migration ability of DCs and elucidated the detailed mechanisms mediating this process. Murine RBCs were incubated with marrow DCs after removing the storage supernatant. The effects of sRBCs on cytokine secretion from DCs, surface marker expression, and homing ability were examined. RESULTS: More sRBCs were internalized by DCs than fresh RBCs (fRBCs), and RBC accumulation significantly promoted the expression of allostimulatory molecules and the secretion of Th1-type cytokines in the presence of lipopolysaccharide (LPS). In particular, the lymphoid-tissue homing ability of transfused DCs treated with sRBCs (sRBC-DCs) was also significantly greater than that of fRBCs. Up regulation of CCR7 and improved organization of the cytoskeleton were observed in sRBC-DCs, and blocking Rho/Rho-associated protein kinase (ROCK), PI3K/Akt, and NF-κB pathways greatly hindered cytoskeletal rearrangement. Moreover, high levels of reactive oxygen species (ROS) were detected in sRBC-DCs, and treatment with N-acetylcysteine simultaneously decreased the lymph node-homing ability of DCs and phosphorylation of RhoA, ROCK1, and cortactin. CONCLUSIONS: sRBCs initiated differential immune responses compared to fRBCs, and the presence of LPS augmented this phenomenon. Up regulation of CCR7 and ROS production promotes cytoskeletal reorganization and contributes to the increased homing of sRBCs-DCs.