scholarly journals Fc-Mediated Complement Activation Is Associated with Macrophage Trafficking and Induction of Neutrophil Extracellular Traps in Transfusion-Related Acute Lung Injury

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 354-354
Author(s):  
Saskia van der Velden ◽  
Thijs L.J. van Osch ◽  
Amina Seghier ◽  
Arthur Bentlage ◽  
Juk Yee Mok ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Complement Activation ◽  
Neutrophil Extracellular Traps ◽  
Fc Receptor ◽  
Healthy Controls ◽  
Plasma Samples ◽  
Extracellular Traps

Abstract Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-related fatalities without available therapies. The pulmonary endothelium is damaged in TRALI, through incompletely understood pathogenic mechanisms, resulting in pulmonary edema. Generally, anti-leukocyte antibodies or biological response modifiers in the transfusion product, in combination with predisposing risk factors in the transfused recipient (e.g. inflammation), are responsible for initiation of TRALI. Remarkably, not all anti-leukocyte antibodies cause TRALI. In a previous in vitro study, we identified increased Fc-mediated complement activation to be a key feature of murine TRALI-inducing antibody 34-1-2S (anti-H-2K d) compared to TRALI-resistant antibody SF1.1.10 (anti-H-2K d) (Zeeuw van der Laan et al, Blood Adv 2020). In the current study, we further explored antibody-mediated TRALI mechanisms in vivo using our previously established TRALI mouse model in which mice are pre-depleted of protective CD4+ T cells and primed with LPS, followed by infusion of antibody 34-1-2S (Kapur et al, Blood 2017, Blood Adv 2018, Blood 2019). A key read-out for TRALI was the lung wet/dry weight ratio (Lung W/D, measure for pulmonary edema). We found that in vitro antibody-mediated complement activation was associated with in vivo antibody-mediated TRALI. 34-1-2S caused severe TRALI (complement activation: +++, Lung W/D: 7.4 ± 0.21), while SF1.1.10 did not cause TRALI (complement activation: +, Lung W/D: 4.68 ± 0.16). Furthermore, Fc-deglycosylated 34-1-2S still caused significant TRALI (complement activation: ++, in vivo Lung W/D: 5.16 ± 0.52). TRALI development was fully Fc-dependent as 34-1-2S-Fab did not cause any TRALI (no complement activation, Lung W/D: 4.35 ± 0.18). Importantly, we found significantly increased levels of complement C1q-C4 complexes in plasma samples of TRALI patients (n=46) compared to healthy controls (n=25): 6.49 ± 5.27 vs 3.84 ± 2.27 AU/ml, respectively, P: 0.0005,***. Similarly, C5a levels were significantly elevated in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 2.52 ± 2.17 vs 1.37 ± 1.08 ng/ml, respectively, P: 0.0006,***. To further dissect the effect of the Fc-part of 34-1-2S in TRALI-induction, we aim to investigate the contribution of Fc-mediated complement activation vs Fc-receptor interaction. We therefore successfully generated chimeric variants of 34-1-2S with a humanized IgG1 Fc-domain containing mutations making them functionally complement dead (K322A), Fc-receptor dead (dG236) or both complement and Fc-receptor dead (PG LALA). We are currently investigating the effects of these variants on in vivo TRALI induction. We next investigated how the in vivo murine TRALI reaction was related to numbers of macrophages, monocytes and neutrophils in blood and lungs. We found that 34-1-2S-TRALI was associated with significantly decreased levels of macrophages in the lungs and increased levels in blood, compared to mice infused with PBS, SF1.1.10 or 34-1-2S-Fab, suggesting that Fc-mediated complement activation and TRALI induction is related to macrophage trafficking from lungs to blood. We did not observe any significant differences between blood and lung neutrophil levels of 34-1-2S-TRALI mice compared to SF1.1.10 TRALI-resistant mice. We hypothesized that in TRALI neutrophils undergo formation of neutrophil extracellular traps (NETs) induced by complement. We observed that C5a enabled potent neutrophil-chemotaxis in vitro (P: 0.0048,**). In addition, using direct immunofluorescence staining of extracellular DNA and Citrullinated histone H3, we observed that both LPS and C5a on their own could induce NET formation in vitro, which was synergistically increased with a combination of both LPS and C5a (P: 0.0417,*), as occurring during TRALI. Furthermore, we found increased levels of NETs to be present in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 1.64 ± 0.97 vs 0.80 ± 0.34 MPO-DNA OD, respectively, P: 0.0002,***. Finally, we targeted the C5a-receptor (C5aR) using a C5aR-antagonist in our TRALI mouse model. Surprisingly, this did not prevent but even worsened TRALI (P: 0.0398,*), with elevated levels of blood monocytes and macrophages. This suggests that an approach directly targeting complement components may be a more promising therapeutic strategy to explore in combatting TRALI. Disclosures No relevant conflicts of interest to declare.

scholarly journals ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250265
Author(s):  
Hubert Hayden ◽  
Nahla Ibrahim ◽  
Johannes Klopf ◽  
Branislav Zagrapan ◽  
Lisa-Marie Mauracher ◽  
...  
Keyword(s):  
Human Plasma ◽  
Dynamic Range ◽  
Neutrophil Extracellular Traps ◽  
Plasma Samples ◽  
Dna Complexes ◽  
High Background ◽  
Isotype Control ◽  
Extracellular Traps

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

scholarly journals In vitro and in vivo assessment of the protective effect of sufentanil in acute lung injury

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098635
Author(s):  
Qi Gao ◽  
Ningqing Chang ◽  
Donglian Liu
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Protective Effect ◽  
High Mobility ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Hmgb1 Protein

Objectives To investigate the mechanisms underlying the protective effect of sufentanil against acute lung injury (ALI). Material and Methods Rats were administered lipopolysaccharide (LPS) by endotracheal instillation to establish a model of ALI. LPS was used to stimulate BEAS-2B cells. The targets and promoter activities of IκB were assessed using a luciferase reporter assay. Apoptosis of BEAS-2B cells was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Results Sufentanil treatment markedly reduced pathological changes in lung tissue, pulmonary edema and secretion of inflammatory factors associated with ALI in vivo and in vitro. In addition, sufentanil suppressed apoptosis induced by LPS and activated NF-κB both in vivo and in vitro. Furthermore, upregulation of high mobility group box protein 1 (HMGB1) protein levels and downregulation of miR-129-5p levels were observed in vivo and in vitro following sufentanil treatment. miR-129-5p targeted the 3ʹ untranslated region and its inhibition decreased promoter activities of IκB-α. miR-129-5p inhibition significantly weakened the protective effect of sufentanil on LPS-treated BEAS-2B cells. Conclusion Sufentanil regulated the miR-129-5p/HMGB1 axis to enhance IκB-α expression, suggesting that sufentanil represents a candidate drug for ALI protection and providing avenues for clinical treatment.

scholarly journals Group V Phospholipase A2 Mediates Endothelial Dysfunction and Acute Lung Injury Caused by Methicillin-Resistant Staphylococcus aureus

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1731
Author(s):  
Yu Maw Htwe ◽  
Huashan Wang ◽  
Patrick Belvitch ◽  
Lucille Meliton ◽  
Mounica Bandela ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Acute Lung Injury ◽  
Endothelial Dysfunction ◽  
Lung Injury ◽  
Phospholipase A2 ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Group V ◽  
Methicillin Resistant

Lung endothelial dysfunction is a key feature of acute lung injury (ALI) and clinical acute respiratory distress syndrome (ARDS). Previous studies have identified the lipid-generating enzyme, group V phospholipase A2 (gVPLA2), as a mediator of lung endothelial barrier disruption and inflammation. The current study aimed to determine the role of gVPLA2 in mediating lung endothelial responses to methicillin-resistant Staphylococcus aureus (MRSA, USA300 strain), a major cause of ALI/ARDS. In vitro studies assessed the effects of gVPLA2 inhibition on lung endothelial cell (EC) permeability after exposure to heat-killed (HK) MRSA. In vivo studies assessed the effects of intratracheal live or HK-MRSA on multiple indices of ALI in wild-type (WT) and gVPLA2-deficient (KO) mice. In vitro, HK-MRSA increased gVPLA2 expression and permeability in human lung EC. Inhibition of gVPLA2 with either the PLA2 inhibitor, LY311727, or with a specific monoclonal antibody, attenuated the barrier disruption caused by HK-MRSA. LY311727 also reduced HK-MRSA-induced permeability in mouse lung EC isolated from WT but not gVPLA2-KO mice. In vivo, live MRSA caused significantly less ALI in gVPLA2 KO mice compared to WT, findings confirmed by intravital microscopy assessment in HK-MRSA-treated mice. After targeted delivery of gVPLA2 plasmid to lung endothelium using ACE antibody-conjugated liposomes, MRSA-induced ALI was significantly increased in gVPLA2-KO mice, indicating that lung endothelial expression of gVPLA2 is critical in vivo. In summary, these results demonstrate an important role for gVPLA2 in mediating MRSA-induced lung EC permeability and ALI. Thus, gVPLA2 may represent a novel therapeutic target in ALI/ARDS caused by bacterial infection.

scholarly journals H2S in acute lung injury: a therapeutic dead end(?)

2020 ◽  
Vol 8 (S1) ◽  
Author(s):  
Tamara Merz ◽  
Nicole Denoix ◽  
Martin Wepler ◽  
Holger Gäßler ◽  
David A. C. Messerer ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Circulatory Shock ◽  
Therapeutic Window ◽  
Pharmacokinetics And Pharmacodynamics ◽  
Clinical Reality ◽  
Dead End

AbstractThis review addresses the plausibility of hydrogen sulfide (H2S) therapy for acute lung injury (ALI) and circulatory shock, by contrasting the promising preclinical results to the present clinical reality. The review discusses how the narrow therapeutic window and width, and potentially toxic effects, the route, dosing, and timing of administration all have to be balanced out very carefully. The development of standardized methods to determine in vitro and in vivo H2S concentrations, and the pharmacokinetics and pharmacodynamics of H2S-releasing compounds is a necessity to facilitate the safety of H2S-based therapies. We suggest the potential of exploiting already clinically approved compounds, which are known or unknown H2S donors, as a surrogate strategy.

scholarly journals Down-regulation of SNHG16 alleviates the acute lung injury in sepsis rats through miR-128-3p/HMGB3 axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Junli Sun ◽  
Keke Xin ◽  
Chenghui Leng ◽  
Jianlin Ge
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Western Blot ◽  
Cell Viability ◽  
Inflammatory Diseases ◽  
Cecal Ligation ◽  
Inflammatory Factors ◽  
Lung Epithelial

Abstract Background Long noncoding RNAs contribute to various inflammatory diseases, including sepsis. We explore the role of small nucleolar RNA host gene 16 (SNHG16) in sepsis-mediated acute lung injury (ALI) and inflammation. Methods A sepsis-induced ALI rat model was constructed by the cecal ligation and perforation method. The profiles of SNHG16, miR-128-3p, and high-mobility group box 3 (HMGB3) were monitored by quantitative reverse transcription PCR and Western blot. The pathologic changes of lung tissues were evaluated by Hematoxylin–Eosin staining, immunohistochemistry, and dry and wet method. Meanwhile, the pro-inflammatory factors and proteins were determined by ELISA and Western blot. In contrast, a sepsis model in BEAS-2B was induced with lipopolysaccharide (LPS) to verify the effects of SNHG16/miR-128-3p/HMGB3 on lung epithelial cell viability and apoptosis. Results As a result, SNHG16 and HMGB3 were up-regulated, while miR-128-3p was down-regulated in sepsis-induced ALI both in vivo and in vitro. Inhibiting SNHG16 reduced the apoptosis and inflammation in the sepsis-induced ALI model. Overexpressing SNHG16 promoted LPS-mediated lung epithelial apoptosis and inhibited cell viability and inflammation, while miR-128-3p had the opposite effects. Mechanistically, SNHG16 targeted miR-128-3p and attenuated its expression, while miR-128-3p targeted the 3′ untranslated region of HMGB3. Conclusions Overall, down-regulating SNHG16 alleviated the sepsis-mediated ALI by regulating miR-128-3p/HMGB3.

Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Thomas Helbing ◽  
Elena Ketterer ◽  
Bianca Engert ◽  
Jennifer Heinke ◽  
Sebastian Grundmann ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Bmp Signaling ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

scholarly journals Adrenomedullin expression in a rat model of acute lung injury induced by hypoxia and LPS

2005 ◽  
Vol 288 (3) ◽  
pp. L536-L545 ◽  
Author(s):  
Jackeline Agorreta ◽  
Javier J. Zulueta ◽  
Luis M. Montuenga ◽  
Mercedes Garayoa
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Cell Lines ◽  
Rat Model ◽  
In Vitro Model ◽  
Rat Lung ◽  
Vitro Model

Adrenomedullin (ADM) is upregulated independently by hypoxia and LPS, two key factors in the pathogenesis of acute lung injury (ALI). This study evaluates the expression of ADM in ALI using experimental models combining both stimuli: an in vivo model of rats treated with LPS and acute normobaric hypoxia (9% O2) and an in vitro model of rat lung cell lines cultured with LPS and exposed to hypoxia (1% O2). ADM expression was analyzed by in situ hybridization, Northern blot, Western blot, and RIA analyses. In the rat lung, combination of hypoxia and LPS treatments overcomes ADM induction occurring after each treatment alone. With in situ techniques, the synergistic effect of both stimuli mainly correlates with ADM expression in inflammatory cells within blood vessels and, to a lesser extent, to cells in the lung parenchyma and bronchiolar epithelial cells. In the in vitro model, hypoxia and hypoxia + LPS treatments caused a similar strong induction of ADM expression and secretion in epithelial and endothelial cell lines. In alveolar macrophages, however, LPS-induced ADM expression and secretion were further increased by the concomitant exposure to hypoxia, thus paralleling the in vivo response. In conclusion, ADM expression is highly induced in a variety of key lung cell types in this rat model of ALI by combination of hypoxia and LPS, suggesting an essential role for this mediator in this syndrome.

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