Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.We thank M. Jaeger (Radboudumc) for kindly providing flourescein isothiocyanate-labelled Candida albicans. D. Williams (East Tennessee State University) provided the β-glucan we used in our initial experiments. H. Lemmers (Radboudumc) kindly prepared the purified lipopolysaccharide used for stimulation of primary human monocytes and macrophages. Part of the figures were prepared using (among other software) Biorender.com. B.N. is supported by a National Health and Medical Research Council (Australia) Investigator Grant (APP1173314). This work was supported by National Institutes of Health grants R01 HL144072, R01 CA220234 and P01 HL131478, as well as a Vici grant from the Dutch Research Council NWO and an ERC Advanced Grant (all to W.J.M.M.). M.G.N. was supported by a Spinoza grant from Dutch Research Council NWO and an ERC Advanced Grant (#833247).S

Regulating trained immunity with nanomedicine

Trained immunity refers to a hyperresponsive functional state of the innate immune system, which is induced by certain stimuli, such as infections or vaccination. Trained immunity plays a key part in a variety of diseases, including cancer and inflammation, and is regulated through epigenetic and metabolic reprogramming of haematopoietic stem and progenitor cells in the bone marrow, giving rise to hyperactive myeloid cells. Nanomaterials inherently interact with phagocytic myeloid cells and are thus ideal platforms with which to regulate trained immunity. In this Review, we discuss the key pathways of trained immunity and investigate nanomedicine strategies to therapeutically regulate trained immunity. Nanomedicine can be applied not only to induce trained immunity to treat cancer or to enhance resistance to infections, but also to manage hyperinflammation and maladaptive trained immunity in a variety of clinical scenarios. We conclude with an outlook to future possibilities and some remaining challenges for nanomedicine approaches in trained immunity regulation

Prosaposin mediates inflammation in atherosclerosis

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Nur77 variants solely comprising the amino-terminal domain activate hypoxia-inducible factor-1 α and affect bone marrow homeostasis in mice and humans

Gene targeting via homologous recombination can occasionally result in incomplete disruption of the targeted gene. Here, we show that a widely used Nur77-deficient transgenic mouse model expresses a truncated protein encoding for part of the N-terminal domain of nuclear receptor Nur77. This truncated Nur77 protein is absent in a newly developed Nur77-deficient mouse strain generated using Cre-Lox recombination. Comparison of these two mouse strains using immunohistochemistry, flow cytometry, and colony-forming assays shows that homologous recombination-derived Nur77-deficient mice, but not WTor Cre-Lox-derived Nur77-deficient mice, suffer from liver immune cell infiltrates, loss of splenic architecture, and increased numbers of bone marrow hematopoietic stem cells and splenic colony-forming cells with age. Mechanistically, we demonstrate that the truncated Nur77 N-terminal domain protein maintains the stability and activity of hypoxia-inducible factor (HIF)-1, a transcription factor known to regulate bone marrow homeostasis. Additionally, a previously discovered, but uncharacterized, human Nur77 transcript variant that encodes solely for its N-terminal domain, designated TR3β, can also stabilize and activate HIF-1α. Meta-analysis of publicly available microarray data sets shows that TR3β is highly expressed in human bone marrow cells and acute myeloid leukemia samples. In conclusion, our study provides evidence that a transgenic mouse model commonly used to study the biological function of Nur77 has several major drawbacks, while simultaneously identifying the importance of nongenomic Nur77 activity in the regulation of bone marrow homeostasis

Diverse ultrastructural landscape of atherosclerotic endothelium

Background and aims: The endothelium plays a major role in atherosclerosis, yet the endothelial plaque surface is a largely uncharted territory. Here we hypothesize that atherosclerosis-driven remodeling of the endothelium is a dynamic process, involving both damaging and regenerative mechanisms. Methods: Using scanning electron microscopy (SEM) and immuno-SEM, we studied endothelial junction ultrastructure, endothelial openings and immune cell-endothelium interactions in eight apoe−/− mice and two human carotid plaques. Results: The surface of early mouse plaques (n = 11) displayed a broad range of morphological alterations, including junctional disruptions and large transcellular endothelial pores with the average diameter between 0.6 and 3 μm. The shoulder region of advanced atherosclerotic lesions (n = 7) had a more aggravated morphology with 8 μm-size paracellular openings at two-fold higher density. In contrast, the central apical surface of advanced plaques, i.e., the plaque body (n = 7), displayed endothelial normalization, as shown by a significantly higher frequency of intact endothelial junctions and a lower incidence of paracellular pores. This normalized endothelial phenotype correlated with low immune cell density (only 5 cells/mm2). The human carotid plaque surface (n = 2) displayed both well-organized and disrupted endothelium with similar features as described above. In addition, they were accompanied by extensive thrombotic areas. Conclusions: Our study unveils the spectrum of endothelial abnormalities associated with the development of atherosclerosis. These were highly abundant in early lesions and in the shoulder region of advanced plaques, while normalized at the advanced plaque's body. Similar endothelial features were observed in human atherosclerotic plaques, underlining the versatility of endothelial transformations in atherosclerosis

Self-gated, dynamic contrast-enhanced magnetic resonance imaging with compressed-sensing reconstruction for evaluating endothelial permeability in the aortic root of atherosclerotic mice

High-risk atherosclerotic plaques are characterized by active inflammation and abundant leaky microvessels. We present a self-gated, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) acquisition with compressed sensing reconstruction and apply it to assess longitudinal changes in endothelial permeability in the aortic root of Apoe−/− atherosclerotic mice during natural disease progression. Twenty-four, 8-week-old, female Apoe−/− mice were divided into four groups (n = 6 each) and imaged with self-gated DCE-MRI at 4, 8, 12, and 16 weeks after high-fat diet initiation, and then euthanized for CD68 immunohistochemistry for macrophages. Eight additional mice were kept on a high-fat diet and imaged longitudinally at the same time points. Aortic-root pseudo-concentration curves were analyzed using a validated piecewise linear model. Contrast agent wash-in and washout slopes (b1 and b2) were measured as surrogates of aortic root endothelial permeability and compared with macrophage density by immunohistochemistry. b2, indicating contrast agent washout, was significantly higher in mice kept on an high-fat diet for longer periods of time (p = 0.03). Group comparison revealed significant differences between mice on a high-fat diet for 4 versus 16 weeks (p = 0.03). Macrophage density also significantly increased with diet duration (p = 0.009). Spearman correlation between b2 from DCE-MRI and macrophage density indicated a weak relationship between the two parameters (r = 0.28, p = 0.20). Validated piecewise linear modeling of the DCE-MRI data showed that the aortic root contrast agent washout rate is significantly different during disease progression. Further development of this technique from a single-slice to a 3D acquisition may enable better investigation of the relationship between in vivo imaging of endothelial permeability and atherosclerotic plaques' genetic, molecular, and cellular makeup in this important model of disease

Nanoengineering Apolipoprotein A1-Based Immunotherapeutics

In the slipstream of targeting the adaptive immune system, innate immunotherapy strategies are being developed. In this context, technologies based on natural carrier vehicles that inherently interact with the innate immune system, are increasingly being considered. Immunoregulatory nanotherapeutics based on natural apolipoprotein A1 (apoA1) are discussed here. This protein is a helical, amphipathic macromolecule and the main constituent of high-density lipoprotein. In that capacity, apoA1 interacts specifically with innate immune cells, such as monocytes and macrophages, to collect and transport lipophilicmolecules throughout the body. Exactly these unique features make apoA1 a compelling elementary constituent of biocompatible self-assembled nanotherapeutics. Such apoA1-based nanotherapeutics (A1-nanotherapeutics) can be engineered and functionalized to induce or mitigate an innate immune response or to orchestrate an adaptive immune response through antigen delivery to dendritic cells. The authors first discuss apoA1's properties and how these can be exploited to generate libraries of A1-nanotherapeutics using advanced manufacturing approaches such as microfluidics or continuous flow methods. Using high-throughput in vitro screening methods and in vivo imagingto identify promising formulations are then recommend. Finally, Three distinct immunotherapy strategies are proposed to effectively treat a variety of diseases—including cancer, infection, and cardiovascular disease—and promote allograft survival in transplantation

Nanoengineering Apolipoprotein A1-Based Immunotherapeutics

In the slipstream of targeting the adaptive immune system, innate immunotherapy strategies are being developed. In this context, technologies based on natural carrier vehicles that inherently interact with the innate immune system, are increasingly being considered. Immunoregulatory nanotherapeutics based on natural apolipoprotein A1 (apoA1) are discussed here. This protein is a helical, amphipathic macromolecule and the main constituent of high-density lipoprotein. In that capacity, apoA1 interacts specifically with innate immune cells, such as monocytes and macrophages, to collect and transport lipophilicmolecules throughout the body. Exactly these unique features make apoA1 a compelling elementary constituent of biocompatible self-assembled nanotherapeutics. Such apoA1-based nanotherapeutics (A1-nanotherapeutics) can be engineered and functionalized to induce or mitigate an innate immune response or to orchestrate an adaptive immune response through antigen delivery to dendritic cells. The authors first discuss apoA1's properties and how these can be exploited to generate libraries of A1-nanotherapeutics using advanced manufacturing approaches such as microfluidics or continuous flow methods. Using high-throughput in vitro screening methods and in vivo imagingto identify promising formulations are then recommend. Finally, Three distinct immunotherapy strategies are proposed to effectively treat a variety of diseases—including cancer, infection, and cardiovascular disease—and promote allograft survival in transplantation

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe(-/-) mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received Zr-89-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe(-/-) mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP immune cell interactions significantly decreased over the disease progression. Zr-89-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosi

Nanoparticle-Aided Characterization of Arterial Endothelial Architecture during Atherosclerosis Progression and Metabolic Therapy

Atherosclerosis is associated with a compromised endothelial barrier, facilitating the accumulation of immune cells and macromolecules in atherosclerotic lesions. In this study, we investigate endothelial barrier integrity and the enhanced permeability and retention (EPR) effect during atherosclerosis progression and therapy in Apoe-/- mice using hyaluronan nanoparticles (HA-NPs). Utilizing ultrastructural and en face plaque imaging, we uncover a significantly decreased junction continuity in the atherosclerotic plaque-covering endothelium compared to the normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly, the plaque advancement had a positive effect on junction stabilization, which correlated with a 3-fold lower accumulation of in vivo administrated HA-NPs in advanced plaques compared to early counterparts. Furthermore, by using super-resolution and correlative light and electron microscopy, we trace nanoparticles in the plaque microenvironment. We find nanoparticle-enriched endothelial junctions, containing 75% of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying extracellular matrix, which suggest an endothelial junctional traffic of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs in the context of metabolic therapy with a glycolysis inhibitor, 3PO, proposed as a vascular normalizing strategy. The observed trend of attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with the endothelial silencing activity of 3PO, demonstrated in vitro. Interestingly, the therapy also reduced the plaque inflammatory burden, while activating macrophage metabolism. Our findings shed light on natural limitations of nanoparticle accumulation in atherosclerotic plaques and provide mechanistic insight into nanoparticle trafficking across the atherosclerotic endothelium. Furthermore, our data contribute to the rising field of endothelial barrier modulation in atherosclerosis