Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association.

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.

Immunotherapy with CpG oligonucleotides and antibodies to TNF-alpha rescues neonatal mice from lethal arenavirus-induced meningoencephalitis.

Viral encephalitides are life-threatening diseases in neonates partly due to the irreversible damage inflammation causes to the CNS. This study explored the role of proinflammatory cytokines in the balance between controlling viral replication and eliciting pathologic immune responses in nonlytic viral encephalitis. We show that neonatal mice challenged with arenavirus Tacaribe (TCRV) develop a meningoencephalitis characterized by high IFN-gamma and TNF-alpha levels and mild T cell infiltration. Neutralization of the TNF-alpha using mAb was associated with lower chemokine expression, reduced T cell infiltration, and lower levels of IFN-gamma, and TNF-alpha in the CNS and led to 100% survival. Moreover, treatment with Abs to TNF-alpha improved mobility and increased survival even after the mice developed bilateral hind limb paralysis. Of note, animals treated with anti-TNF-alpha Abs alone did not clear the virus despite generating Abs to TCRV. Direct activation of the innate immune response using CpG oligodeoxynucleotides in combination with anti-TNF-alpha Abs resulted in 100% survival and complete viral clearance. To our knowledge, this is the first demonstration of the use of innate immune modulators plus Abs to TNF-alpha as therapeutics for a lethal neurotropic viral infection.

CpG oligodeoxynucleotides protect newborn mice from a lethal challenge with the neurotropic Tacaribe arenavirus.

The innate immune system is key to limiting the early spread of most pathogens and directing the development of Ag-specific immunity. Recently, a number of synthetic molecules that activate the innate immune system by stimulating TLRs have been identified. Among them, synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) were shown to activate TLR9-bearing B cells, macrophages, and dendritic cells to induce a strong proinflammatory milieu and a type 1-biased immune response that protects mice from a variety of parasitic, bacterial, and viral infections. Although the protective effect of CpG ODN in adult mice was well established, its effectiveness in neonates, which have lower numbers of dendritic, B, and T cells and tend to favor Th2 responses, was unclear. This study uses the New World arenavirus Tacaribe, a neurotropic pathogen that is lethal in newborn mice, to explore the effectiveness of TLR-mediated innate immune responses. Neonatal BALB/c mice treated with CpG ODN at the time of infection had reduced viral load (p < 0.01) and increased survival (52%, p < 0.001 i.p.; 36%, p < 0.05 intranasally). Protection was achieved in mice treated no later than 3 days postchallenge and appears to be mediated by an increase in Ag-specific Abs (IgG and IgM) and to require inducible NO synthase expression and NO production. To our knowledge, this is the first study assessing the mechanisms by which CpG ODN can protect mice from a neurotropic viral infection.