Inhibiting Inflammation with Myeloid Cell-Specific Nanobiologics Promotes Organ Transplant Acceptance.

Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short-term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T (Treg) cell expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6-specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimmunotherapy resulted in indefinite allograft survival. Together, we show that HDL-based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.

High-Density Lipoprotein Nanobiologics for Precision Medicine.

Nature is an inspirational source for biomedical engineering developments. Particularly, numerous nanotechnological approaches have been derived from biological concepts. For example, among many different biological nanosized materials, viruses have been extensively studied and utilized, while exosome research has gained much traction in the 21st century. In our body, fat is transported by lipoproteins, intriguing supramolecular nanostructures that have important roles in cell function, lipid metabolism, and disease. Lipoproteins' main constituents are phospholipids and apolipoproteins, forming a corona that encloses a hydrophobic core of triglycerides and cholesterol esters. Within the lipoprotein family, high-density lipoprotein (HDL), primarily composed of apolipoprotein A1 (apoA-I) and phospholipids, measuring a mere 10 nm, is the smallest and densest particle. Its endogenous character makes HDL particularly suitable as a nanocarrier platform to target a range of inflammatory diseases. For a decade and a half, our laboratories have focused on HDL's exploitation, repurposing, and reengineering for diagnostic and therapeutic applications, generating versatile hybrid nanomaterials, referred to as nanobiologics, that are inherently biocompatible and biodegradable, efficiently cross different biological barriers, and intrinsically interact with immune cells. The latter is facilitated by HDL's intrinsic ability to interact with the ATP-binding cassette receptor A1 (ABCA1) and ABCG1, as well as scavenger receptor type B1 (SR-BI). In this Account, we will provide an up-to-date overview on the available methods for extraction, isolation, and purification of apoA-I from native HDL, as well as its recombinant production. ApoA-I's subsequent use for the reconstitution of HDL (rHDL) and other HDL-derived nanobiologics, including innovative microfluidic-based production methods, and their characterization will be discussed. The integration of different hydrophobic and amphiphilic imaging labels, including chelated radioisotopes and paramagnetic or fluorescent lipids, renders HDL nanobiologics suitable for diagnostic purposes. Nanoengineering also allows HDL reconstitution with core payloads, such as diagnostically active nanocrystals, as well as hydrophobic drugs or controlled release polymers for therapeutic purposes. The platform technology's specificity for inflammatory myeloid cells and methods to modulate specificity will be highlighted. This Account will build toward examples of in vivo studies in cardiovascular disease and cancer models, including diagnostic studies by magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). A translational success story about the escalation of zirconium-89 radiolabeled HDL (89Zr-HDL) PET imaging from atherosclerotic mice to rabbits and pigs and all the way to cardiovascular disease patients is highlighted. Finally, recent advances in nanobiologic-facilitated immunotherapy of inflammation are spotlighted. Lessons, success stories, and perspectives on the use of these nature-inspired HDL mimetics are an integral part of this Account.

Prosaposin mediates inflammation in atherosclerosis.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient (Apoe -/-) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified Psap, a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that Psap inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of Psap -/- bone marrow to low-density lipoprotein receptor knockout (Ldlr -/-) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between PSAP expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target.

Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates.

Macrophage accumulation in atherosclerosis is directly linked to the destabilization and rupture of plaque, causing acute atherothrombotic events. Circulating monocytes enter the plaque and differentiate into macrophages, where they are activated by CD4+ T lymphocytes through CD40-CD40 ligand signalling. Here, we report the development and multiparametric evaluation of a nanoimmunotherapy that moderates CD40-CD40 ligand signalling in monocytes and macrophages by blocking the interaction between CD40 and tumour necrosis factor receptor-associated factor 6 (TRAF6). We evaluated the biodistribution characteristics of the nanoimmunotherapy in apolipoprotein E-deficient (Apoe-/-) mice and in non-human primates by in vivo positron-emission tomography imaging. In Apoe-/- mice, a 1-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, which was due to the impaired migration capacity of monocytes, as established by a transcriptome analysis. The rapid reduction of plaque inflammation by the TRAF6-targeted nanoimmunotherapy and its favourable toxicity profiles in both mice and non-human primates highlights the translational potential of this strategy for the treatment of atherosclerosis.

Author Correction: Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates.

In the version of this Article originally published, the surname of the author Edward A. Fisher was spelt incorrectly as 'Fischer'. This has now been corrected.

Targeting CD40-Induced TRAF6 Signaling in Macrophages Reduces Atherosclerosis.

BACKGROUND: Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. OBJECTIVES: This study evaluates the potential of TRAF-STOP treatment in atherosclerosis. METHODS: The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe-/-) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages. RESULTS: TRAF-STOP treatment of young Apoe-/- mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe-/- mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair "classical" immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and ß2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe-/- mice. CONCLUSIONS: TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

Monocytes and macrophages as nanomedicinal targets for improved diagnosis and treatment of disease.

The important role of monocytes and macrophages in diseases like cancer and atherosclerosis has started to get uncovered in the last decade. In addition, subsets of these cell types are believed to participate in the initiation and aggravation of several diseases including cancer and cardiovascular disease. For this reason, monocytes and macrophages have recently been identified as interesting targets for both diagnosis and treatment of the aforementioned pathologies. Compared with free therapeutic or imaging agents, nanoparticle formulations provide several advantages that improve the pharmacokinetics and bioavailability of these agents. In addition, the possibility of surface functionalization creates numerous ways to optimize nanoparticle delivery. Recent advances in nanomedicine have led to the development of multifunctional nanoparticles that allow simultaneous diagnosis and treatment of monocytes and macrophages with high specificity. Relying on the inherent ability of monocytes and macrophages to easily take up foreign particles, the use of nanoparticles provides a precious opportunity for the management of several inflammatory diseases.