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

Abstract

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.

Keywords: CD40; TRAF6; immunotherapy; innate immune memory; mTOR; nanoimmunotherapy; trained immunity; transplantation.

Figure 1.. Vimentin and HMGB1 are upregulated following organ transplantation and promote training of graft infiltrating macrophages.

(A-C) Immunostaining, real-time PCR and western blot analysis of vimentin and HMGB1 expression in donor and non-transplanted hearts (n=3/mice per group of three independent experiments, t-test; **P<0.01). (D) Dectin-1 and TLR4 expression in graft infiltrating macrophages (n=3 mice/group of two independent experiments). (E) Ly-6C expression in graft infiltrating macrophages from WT, dectin1 KO and TLR4 KO untreated recipient mice (n=3 mice/group of two independent experiments). (F) Inflammatory cytokine production and chromatin immunoprecipitation of mouse monocytes trained with vimentin and HMGB1 (n=3 independent experiments, one-way ANOVA, **P<0.01; dashed line displays control non-trained conditions). (G) Cytokine and lactate production of graft-infiltrating macrophages (n=4 mice/group of 2 independent experiments, one-way ANOVA, **P<0.01). (H) Chromatin immunoprecipitation of graft-infiltrating macrophages (n=4 mice/group of 2 independent experiments, one-way ANOVA, *P<0.05; **P<0.01).

Figure 2.. mTORi-HDL nanoimmunotherapy prevents trained immunity in vitro and distributes systemically in vivo.

(A) Transmission electron microscopy (TEM) of mTORi-HDL nanobiologics. (B) Cytokine and lactate production of human macrophages trained in vitro (n=3 independent experiments, t-test, *P<0.05; dashed line displays control non-β-glucan trained condition). (C) Chromatin immunoprecipitation of human macrophages trained in vitro (n=3 independent experiments, t-test, *P<0.05; dashed line displays control non-β-glucan trained condition). (D) Labeling of mTORi-HDL with either the radioisotope ⁸⁹Zr or the fluorescent dyes DiO or DiR. (E) micro-PET/CT and cellular specificity of mTORi-HDL nanobiologics. (F) Representative micro-PET/CT 3D fusion image and PET maximum intensity projection (MIP) (mean ± SEM, n=3). (G) Uptake of fluorescently labeled DiO mTORi-HDL by myeloid and lymphoid cells (n=5 mice/group, one-way ANOVA, **P<0.01). (H) Uptake of fluorescently labeled DiO mTORi-HDL by bone marrow progenitors (mean ± SEM, n=5).

Figure 3.. mTORi-HDL nanoimmunotherapy targets myeloid cells in the allograft and prevents trained immunity.

(A) BALB/c donor hearts (H2d) were transplanted into fully allogeneic C57BL/6 recipients (H2b). (B) micro-PET/CT 3D fusion image 24 hours after intravenous administration of ⁸⁹Zr-mTORi-HDL (n=3 mice/group of 2 independent experiments). (C) Ex vivo autoradiography in native (N) and transplanted hearts (Tx) at 24 hours after intravenous ⁸⁹Zr-mTORi-HDL (n=3 mice/group of 2 independent experiments, t-test, *P<0.05). (D) Uptake of fluorescently labeled DiO mTORi-HDL by myeloid and lymphoid cells in the allograft (n=4 mice/group of 3 independent experiments; one-way ANOVA, *P<0.05; **P<0.01). (E) Ly-6C^hi / Ly-6C^lo MΦ ratio in the allograft from either placebo or mTORi-HDL-treated recipients at day 6 post-transplantation (n=4 mice/group of 3 independent experiments; one-way ANOVA, *P 0.05; **P <0.01). (F-G) GSEA gene array analysis for the mTOR and glycolysis pathways in intra-graft MΦ from placebo or mTORi-HDL-treated recipients (n=3 mice/group). (H) Cytokine and lactate production of graft-infiltrating macrophages from either placebo or mTORi-HDL-treated recipients (n=4 mice/group of 3 independent experiments, t-test, *P<0.05; **P<0.01). (I) Chromatin immunoprecipitation of graft-infiltrating macrophages from either placebo or mTORi-HDL-treated recipients (n=4 mice/group of 3 independent experiments, t-test, *P<0.05; **P<0.01).

Figure 4.. mTORi-HDL nanoimmunotherapy promotes organ transplant acceptance.

(A) Functional characterization of graft-infiltrating MΦ from placebo and mTORi-HDL-treated recipients using CD8 T cell suppressive and CD4 Treg expansion assays (n=4 mice/group of 3 independent experiments, t-test, **P≤0.01). (B) Percentage of graft-infiltrating CD4⁺CD25⁺ Treg cells from placebo and mTORi-HDL-treated recipients (n=4 mice/group of 3 independent experiments, t-test, **P≤0.01). (C) Depletion of CD169⁺ graft-infiltrating Mreg in placebo and mTORi-HDL-treated recipients (n= 5 mice/group of 3 independent experiments, t-test, **P<0.01). (D) Graft survival following depletion CD169⁺ graft-infiltrating Mreg (n= 5 mice/group; Kaplan-Meier **P≤0.01). (E) Graft survival following depletion of CD11c⁺ cells and in CCR2 deficient recipient mice (n=5 mice/group, Kaplan-Meier, **P<0.01). (F) Graft survival of mTORi-HDL-treated recipients receiving agonistic stimulatory CD40 mAb in vivo with or without TRAF6i-HDL nanoimmunotherapy (n=5 mice/group, Kaplan-Meier, **P<0.01). (G) Graft survival of placebo, vehicle HDL, mTORi-HDL, TRAF6i-HDL and mTORi-HDL/TRAF6i-HDL treated recipients (n=7–8 mice/group, Kaplan-Meier, **P<0.01). (H) Immunohistochemistry of heart allografts from mTORi-HDL/TRAF6i-HDL-treated recipients on day 100 after transplantation (n = 5 mice/group; magnification X200).