Aim: We previously reported the capacity of immune-modifying nanoparticles (IMPs) to bind to circulating inflammatory monocytes/macrophages (MΦ) via the specific scavenger receptor MARCO, thereby causing MΦ removal in the spleen with subsequent protection in models of infection, autoimmunity and ischemia-reperfusion injury (IRI). Here we investigated the therapeutic potential of IMPs to target MΦ in kidney allograft rejection in a murine model of kidney transplantation.
Methods: Kidney transplants were performed from BALB/c to C57BL/6 mice as WT allografts. A group of WT allograft mice received a daily intravenous injection of 300µl (1.46x1010particles/ml, 500nm in diameter) of negatively-charged fluorescein isothiocyanate (FITC)–labelled IMPs (FITC-IMPs) from day 1 post-transplant for two weeks (WT+IMP). Samples were collected at days 14 and 100 post-transplant to access acute and chronic allograft rejection.
Results and Discussion: WT+IMP allografts had prolonged survival compared to WT allografts (Figure 1, p<0.05). At day 14 post-transplant, WT+IMP allografts were protected from acute rejection with lower creatinine (23.8±1.4 vs. 48.1±18.7mmol/L, p<0.01) and less tubulitis (95.6±12.6 vs. 140.4±33.9scores, p<0.001)(Figure 2). Histologically, accumulation of CD4+ (38±13 vs. 73±32 cells/HPFs, p<0.01), CD8+ (39±18 vs. 76±39 cells/HPFs, p<0.05), CD68+ (12±5 vs. 22±7% field positive, p<0.01) and CD11c (2±1 vs. 13±5% field positive, p<0.001) cells were reduced in WT+IMP allografts, compared to WT allografts. High dimensional flow cytometry analysis of cells isolated from allografts showed significantly reduced T cells and myeloid cells in WT+IMP. By immunohistology double staining with F4/80, MARCO and caspase-3, FITC-IMPs were found to predominantly co-localize with F4/80-expressing red pulp macrophages that expressed MARCO and caspase-3, suggesting that IMPs divert MΦ to the spleen, undergoing caspase-3–mediated apoptosis (Figure 3). WT+IMP allografts expressed significantly less pro-inflammatory (IL6) and Th1 (IFNγ) cytokines and cytotoxic molecules (perforin, granzyme B & iNOS). At day 100 post-transplant, WT+IMP allografts exhibited chronic allograft injury with comparable levels of serum creatinine (49.8±34.6 vs. 48.8±26.8mmol/L, p>0.99) and interstitial fibrosis by collagen deposition compared to WT allografts.

Conclusion: IMP affords significant protection from acute allograft rejection. IMP infusion caused inflammatory MΦ sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Coupled with our earlier demonstration of IMP-induced protection in kidney IRI, the additional ability to retard acute rejection identifies IMP as a potential induction agent in kidney transplantation. As both delayed graft function and acute rejection remain unmet needs in the clinic, translational studies using IMP warrant further consideration.