Drug-loaded red blood cell-mediated clearance of HIV-1 macrophage reservoir by selective inhibition of STAT1 expression

Abstract

Current highly active antiretroviral therapy (HAART) cannot eliminate HIV-1 from infected persons, mainly because of the existence of refractory viral reservoir(s). Beyond latently-infected CD4+-T lymphocytes, macrophages (M/M) are important persistent reservoirs for HIV in vivo, that represent a major obstacle to HIV-1 eradication. Therefore, a rational therapeutic approach directed to the selective elimination of long-living HIV-infected M/M may be relevant in the therapy of HIV infection. Here we report that HIV-1 chronic infection of human macrophages results in the marked increase of expression and phosphorylation of STAT1, a protein involved in the regulation of many functions such as cell growth, differentiation, and maintenance of cellular homeostasis, thereby providing a new molecular target for drug development. A single and brief exposure to 9-(beta-D-arabinofuranosyl)-2-fluoroadenine 5'-monophosphate (FaraAMP, Fludarabine), a potent antileukemic nucleoside analog active against STAT1 expressing cells, selectively kills macrophage cultures infected by HIV-1 without affecting uninfected macrophages. Furthermore, encapsulation of Fludarabine into autologous erythrocytes (RBC) and targeting to macrophages through a single-18 h treatment with drug-loaded RBC, not only abolishes the Fludarabine-mediated toxic effect on non-phagocytic cells, but also enhances the selective killing of HIV-infected macrophages. As a final result, a potent (>98%) and long-lasting (at least 4 weeks without rebound) inhibition of virus release from drug-loaded RBC-treated chronically-infected macrophages was achieved. Taken together, the evidence of HIV-1-induced increase of STAT1, and the availability of a selective drug targeting system, may prove useful in the design of new pharmacological treatments to clear the HIV-1 macrophage reservoir.

Administration of Fludarabine-Loaded Autologous Red Blood Cells in Simian Immunodeficiency Virus-Infected Sooty Mangabeys Depletes pSTAT-1-Expressing Macrophages and Delays the Rebound of Viremia after Suspension of Antiretroviral Therapy

“…SM RBC were loaded with fludarabine by a procedure involving hypotonic dialysis, isotonic resealing, and reannealing, as previously described (28). Some modifications were performed to adapt the procedure for SM RBC.…”

“…Cytoplasmic STAT proteins are activated by tyrosine phosphorylation, a transient and tightly regulated process that results in dimerization, nuclear translocation, and transcriptional activation of genes that control the cellular response (2,27,41). We found that levels of phosphorylated STAT1 (pSTAT1) were three-to fivefold greater in in vitro HIV-infected M/M compared to uninfected M/M (28). Therefore, we proposed that, at least in this in vitro system, the enhanced expression and phosphorylation of STAT1 may play a role in the development of a persistent state of active HIV replication in M/M.…”

“…In previous studies, we found that activation of the JAK/ STAT (signal transducers and activators of transcription) pathways is associated with survival of HIV-infected M/M (28). STAT proteins comprise a family of transcription factors that are normally located in the cytoplasm as inactive forms and that upon binding of extracellular signaling proteins (i.e., cytokine and growth factors) to specific cell surface receptors enter the nucleus and regulate several cellular events, including activation, differentiation, proliferation, cell survival, and apoptosis.…”

“…To selectively deliver fludarabine to M/M we used red blood cells (RBC) as carriers by taking advantage of the peculiar phagocytic capacity of M/M. Our group has validated the use of RBC as specific carriers for M/M with various compounds and in several different in vitro and in vivo models (13,15,28,29). The fludarabine-loaded RBC technology allows us to overcome the nonspecific toxic effect of fludarabine on actively replicating cells (such as lymphocytes), which are more sensitive than resting cells (such as M/M) to the DNA polymerase inhibition induced by the drug (20,21).…”

“…Following its loading in human RBC, fludarabine is converted by the RBC kinases to the corresponding triphosphate derivative, which is the active form of the drug (14). Fludarabine loaded within RBC strongly and selectively contributes, in vitro, to the induction of cell death in HIV-infected M/M, without any cytopathic effect upon nonphagocytosing cells (28).…”