Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.

Summary Regulatory B cells (Bregs) have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-TIM-1 and anti-CD45RB antibody treatment results in tolerance to full MHC-mismatched islet allografts in mice by generating Bregs that are necessary for tolerance. Bregs are antigen-specific and are capable of transferring tolerance to untreated, transplanted animals. Here we demonstrate that adoptively transferred Bregs require the presence of Tregs to establish tolerance, and that adoptive transfer of Bregs increases the number of Tregs. Interaction with Bregs in vivo induces significantly more Foxp3 expression in CD4+CD25− T cells than with naive B cells. We also show that Bregs express the TGF-β associated latency-associated peptide (LAP) and that Breg-mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-β activity. Regulatory B cells, like regulatory T cells, demonstrate preferential expression of both CCR6 and CXCR3. Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Bregs promote graft survival by promoting Treg development, possibly via TGF-β production.

The role of B cells in transplant tolerance remains unclear. Although B cell depletion often prolongs graft survival, sometimes it results in more rapid rejection, suggesting that B cells may have regulatory activity. We previously demonstrated that tolerance induction by anti-CD45RB antibody requires recipient B cells. Here we show that anti-CD45RB in combination with anti-TIM-1 antibody has a synergistic effect, inducing tolerance in all recipients in a mouse islet allograft model. This effect depends on the presence of recipient B cells, requires B cell IL-10 activity, and is antigen-specific. These data suggest the existence of a regulatory B cell population that promotes tolerance via an IL-10-dependent pathway.