SUMMARY 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 cell (Treg) 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 nanoimunnotherapy 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.

BackgroundDisrupting 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.ObjectivesThis study evaluates the potential of TRAF-STOP treatment in atherosclerosis.MethodsThe 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.ResultsTRAF-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 β2-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.ConclusionsTRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

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.

In atherosclerosis, macrophage accumulation 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 PET imaging. In Apoe−/− mice, a one-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, caused by the impaired migration capacity of monocytes, as established by 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.