An Activation-Specific Anti-Mac-1 Designed-Ankyrin-Repeat-Protein Attenuates Colitis in Mice.

(1) Background: Inflammatory bowel diseases are complex and multifactorial disorders of unknown etiology. The extravasation of activated leukocytes is a critical step in the pathogenesis of these diseases. Leukocyte integrin Mac-1 (αMß2; CD11b/CD18) is crucial for the extravasation of myeloid cells, and a novel activation-specific anti-Mac-1 Designed Ankyrin Repeat protein (DARPin F7) is a promising therapeutic agent for inflammatory diseases. In its activated conformation, Mac-1 expresses the high-affinity binding site I-domain, which the DARPin F7 selectively targets. In our study, we aimed to explore the therapeutic potential of anti-Mac-1 DARPin F7 in murine dextrane sodium sulfate (DSS)-induced colitis. (2) Methods: C57BL/6J mice received 3% DSS drinking water for five days, followed by normal drinking water for one week. The mice were treated with DARPin F7 or a control substance daily via intraperitoneal injections. Disease activity index (DAI), colon length, myeloperoxidase (MPO) activity measurements, H&E staining, and qRT-PCR were conducted after euthanizing the mice on day 12. (3) Results: Treatment with DARPin F7 resulted in less pronounced colon shortening and significantly lower histological scores. The DARPin F7-treated animals experienced substantially less disease and myeloperoxidase (MPO) activity. Animals that received DARPin F7 treatment suffered less weight loss and recovered from the weight loss more efficiently. Treatment with DARPin F7 also led to significantly reduced mRNA expression of inflammatory cytokines. (4) Conclusion: Anti-Mac-1 treatment markedly reduced disease activity and inflammatory reaction accompanying DSS-induced colitis in mice.

Decreased level of serum NT-proCNP associates with disease severity in COVID-19.

BACKGROUND: C-type natriuretic peptide (CNP) is an endothelium-derived paracrine molecule with an important role in vascular homeostasis. In septic patients, the serum level of the amino-terminal propeptide of CNP (NT-proCNP) shows a strong positive correlation with inflammatory biomarkers and, if elevated, correlates with disease severity and indicates a poor outcome. It is not yet known whether NT-proCNP also correlates with the clinical outcome of patients suffering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the current study, we aimed to determine possible changes in the NT-proCNP levels of patients with coronavirus disease 2019 (COVID-19), with special regard to disease severity and outcome. METHODS: In this retrospective analysis, we determined the serum level of NT-proCNP in hospitalized patients with symptoms of upper respiratory tract infection, using their blood samples taken on admission, stored in a biobank. The NT-proCNP levels of 32 SARS-CoV-2 positive and 35 SARS-CoV-2 negative patients were measured to investigate possible correlation with disease outcome. SARS-CoV-2 positive patients were then divided into two groups based on their need for intensive care unit treatment (severe and mild COVID-19). RESULTS: The NT-proCNP was significantly different in the study groups (e.g. severe and mild COVID-19 and non-COVID-19 patients), but showed inverse changes compared to previous observations in septic patients: lowest levels were detected in critically ill COVID-19 patients, while highest levels in the non-COVID-19 group. A low level of NT-proCNP on admission was significantly associated with severe disease outcome. CONCLUSIONS: Low-level NT-proCNP on hospital admission is associated with a severe COVID-19 disease course. The pathomechanism underlying this observation remains to be elucidated, while future studies in larger patient cohorts are necessary to confirm these observations and reveal therapeutic importance. Trial registration DRKS00026655 Registered 26. November 2021.

An activation specific anti-Mac-1 designed ankyrin repeat protein improves survival in a mouse model of acute lung injury.

The acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition. The number of ARDS cases has risen dramatically recently but specific treatment options are limited. ARDS is associated with an overshooting inflammatory response and neutrophils play a central role in its pathogenesis. Neutrophils express the integrin Mac-1 on their surface which adopts a resting and activated conformation depending on leukocyte activation. The aim of this study was to investigate the anti-inflammatory effects of the unique activation-specific anti-Mac-1 DARPin 'F7' in a mouse model of ARDS. ARDS was induced by intratracheal lipopolysaccharide (LPS) instillation and the acute (day 1-4) and chronic phase (day 5-10) were studied. After expression and purification, F7, a control DARPin and PBS, were applied daily via the intraperitoneal route. Survival and weight loss were recorded. Histological analysis of lung sections, flow cytometric leukocyte analysis of blood and bronchioalveolar lavage (BALF) were performed. Moreover, protein concentration and cytokine levels were determined in the BALF. Treatment with F7 improved survival and reduced weight loss significantly compared to treatment with the control DARPin or PBS. Neutrophil count in the BALF and peripheral blood were significantly reduced in mice treated with F7. Histology revealed significantly reduced pulmonary inflammation in the F7 treated group. Treatment with DARPin F7 inhibited neutrophil accumulation, reduced signs of local and systemic inflammation and improved survival in a mouse model of ARDS. F7 may be a novel anti-inflammatory drug candidate for the treatment of severe ARDS.

Early platelet dysfunction in patients receiving extracorporeal membrane oxygenation is associated with mortality.

Extracorporeal membrane oxygenation (ECMO) is used for patients with cardiopulmonary failure and is associated with severe bleeding and poor outcome. Platelet dysfunction may be a contributing factor. The aim of this prospective observational study was to characterize platelet dysfunction and its relation to outcome in ECMO patients. Blood was sampled from thirty ECMO patients at three timepoints. Expression of CD62P, CD63, activated GPIIb/IIIa, GPVI, GPIbα and formation platelet-leukocyte aggregates (PLA) were analyzed at rest and in response to stimulation. Delta granule storage-pool deficiency and secretion defects were also investigated. Fifteen healthy volunteers and ten patients with coronary artery disease served as controls. Results were also compared between survivors and non-survivors. Compared to controls, expression of platelet surface markers, delta granule secretion and formation of PLA was reduced, particularly in response to stimulation. Baseline CD63 expression was higher and activated GPIIb/IIIa expression in response to stimulation was lower in non-survivors on day 1 of ECMO. Logistic regression analysis revealed that these markers were associated with mortality. In conclusion, platelets from ECMO patients are severely dysfunctional predisposing patients to bleeding complications and poor outcome. Platelet dysfunction on day 1 of ECMO detected by the platelet surface markers CD63 and activated GPIIb/IIIa is associated with mortality. CD63 and activated GPIIb/IIIa may therefore serve as novel prognostic biomarkers, but future studies are required to determine their true potential.

Monocyte Dysfunction Detected by the Designed Ankyrin Repeat Protein F7 Predicts Mortality in Patients Receiving Veno-Arterial Extracorporeal Membrane Oxygenation.

Background: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used for critically ill patients requiring hemodynamic support but has been shown to induce an inflammatory response syndrome potentially leading to severe complications and poor outcome. Monocytes are comprised of different subsets and play a central role in the innate immune system. The unique small binding proteins, Designed Ankyrin Repeat Protein "F7" and single chain variable fragment "MAN-1," specifically detect the activated conformation of the leukocyte integrin Mac-1 enabling the highly sensitive detection of monocyte activation status. The aim of this study was to characterize monocyte function and heterogeneity and their association with outcome in VA-ECMO patients. Methods: VA-ECMO patients were recruited from the ICUs of the University Hospital in Freiburg, Germany. Blood was sampled on day 0 and day 3 after VA-ECMO placement, after VA-ECMO explantation and from healthy controls. Monocyte subset distribution, baseline activation and stimulability were analyzed by flow cytometry using the unique small binding proteins F7 and MAN-1 and the conventional activation markers CD163, CD86, CD69, and CX3CR1. Furthermore, expression of monocyte activation markers in survivors and non-survivors on day 0 was compared. Simple logistic regression was conducted to determine the association of monocyte activation markers with mortality. Results: Twenty two patients on VA-ECMO and 15 healthy controls were recruited. Eleven patients survived until discharge from the ICU. Compared to controls, baseline monocyte activation was significantly increased, whereas stimulability was decreased. The percentage of classical monocytes increased after explantation, while the percentage of intermediate monocytes decreased. Total, classical, and intermediate monocyte counts were significantly elevated compared to controls. On day 0, baseline binding of F7 was significantly lower in non-survivors than survivors. The area under the ROC curve associated with mortality on day 0 was 0.802 (p = 0.02). Conclusions: Distribution of monocyte subsets changes during VA-ECMO and absolute classical and intermediate monocyte counts are significantly elevated compared to controls. Monocytes from VA-ECMO patients showed signs of dysfunction. Monocyte dysfunction, as determined by the unique tool F7, could be valuable for predicting mortality in patients receiving VA-ECMO and may be used as a novel biomarker guiding early clinical decision making in the future.

A DARPin targeting activated Mac-1 is a novel diagnostic tool and potential anti-inflammatory agent in myocarditis, sepsis and myocardial infarction.

The monocyte ß2-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the αM I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.

Annexin V positive microvesicles are elevated and correlate with flow rate in patients receiving veno-arterial extracorporeal membrane oxygenation.

OBJECTIVES: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used in critically ill patients requiring haemodynamic support. Microvesicles (MV) are released by activated blood cells acting as mediators of intercellular communication. We aimed to determine MV count and composition over time in patients with VA-ECMO and explore what drives MV formation. METHODS: VA-ECMO patients and healthy controls were recruited prospectively, and blood was taken at different time points (day 0, 1, 3 after ECMO placement and after explantation) for MV analysis. RESULTS: Annexin V positive MV were increased in patients (n = 14, mean age = 61.4 ± 9.0 years, 11 males, 3 females) compared to healthy controls (n = 6, Annexin V positive MV count per millilitre day 1 versus healthy controls: 2.3 × 106 vs 1.3 × 105, P < 0.001). Furthermore, patients had higher proportions of endothelial and leukocyte MV [leukocyte MV day 1 versus healthy controls (%): 32.8 vs 17.5, P = 0.001; endothelial MV day 1 versus healthy controls (%): 10.5 vs 5.5, P = 0.01]. Annexin V positive and leucocyte MV correlated with the flow rate (r = 0.46, P = 0.01). CONCLUSIONS: Patients on VA-ECMO have increased levels of circulating MV and a changed MV composition. Our data support the hypothesis that MV release may be driven by higher flow rate and cellular activation in the extracorporeal circuit leading to poor outcomes in these patients. CLINICAL TRIAL REGISTRATION NUMBER: German Clinical Trials Register-ID: DRKS00011106.

Molecular magnetic resonance imaging of activated platelets allows noninvasive detection of early myocarditis in mice.

MRI sensitivity for diagnosis and localization of early myocarditis is limited, although it is of central clinical interest. The aim of this project was to test a contrast agent targeting activated platelets consisting of microparticles of iron oxide (MPIO) conjugated to a single-chain antibody directed against ligand-induced binding sites (LIBS) of activated glycoprotein IIb/IIIa (= LIBS-MPIO). Myocarditis was induced by subcutaneous injection of an emulsion of porcine cardiac myosin and complete Freund's adjuvant in mice. 3D 7 T in-vivo MRI showed focal signal effects in LIBS-MPIO injected mice 2 days after induction of myocarditis, whereas in control-MPIO injected mice no signal was detectable. Histology confirmed CD41-positive staining, indicating platelet involvement in myocarditis in mice as well as in human specimens with significantly higher LIBS-MPIO binding compared to control-MPIO in both species. Quantification of the myocardial MRI signal confirmed a signal decrease after LIBS-MPIO injection and significant less signal in comparison to control-MPIO injection. These data show, that platelets are involved in inflammation during the course of myocarditis in mice and humans. They can be imaged non-invasively with LIBS-MPIO by molecular MRI at an early time point of the inflammation in mice, which is a valuable approach for preclinical models and of interest for both diagnostic and prognostic purposes.

Cardiomyocyte microvesicles: proinflammatory mediators after myocardial ischemia?

Myocardial infarction is a frequent complication of cardiovascular disease leading to high morbidity and mortality worldwide. Elevated C-reactive protein (CRP) levels after myocardial infarction are associated with heart failure and poor prognosis. Cardiomyocyte microvesicles (CMV) are released during hypoxic conditions and can act as mediators of intercellular communication. MicroRNA (miRNA) are short non-coding RNA which can alter cellular mRNA-translation. Microvesicles (MV) have been shown to contain distinct patterns of miRNA from their parent cells which can affect protein expression in target cells. We hypothesized that miRNA containing CMV mediate hepatic CRP expression after cardiomyocyte hypoxia. H9c2-cells were cultured and murine cardiomyocytes were isolated from whole murine hearts. H9c2- and murine cardiomyocytes were exposed to hypoxic conditions using a hypoxia chamber. Microvesicles were isolated by differential centrifugation and analysed by flow cytometry. Next-generation-sequencing was performed to determine the miRNA-expression profile in H9c2 CMV compared to their parent cells. Microvesicles were incubated with a co-culture model of the liver consisting of THP-1 macrophages and HepG2 cells. IL-6 and CRP expression in the co-culture was assessed by qPCR and ELISA. CMV contain a distinct pattern of miRNA compared to their parent cells including many inflammation-related miRNA. CMV induced IL-6 expression in THP-1 macrophages alone and CRP expression in the hepatic co-culture model. MV from hypoxic cardiomyocytes can mediate CRP expression in a hepatic co-culture model. Further studies will have to show whether these effects are reproducible in-vivo.