Human mast cells as potential reservoirs for persistent HIV-1 infection: Role of Toll-like receptors in activation of viral replication.

J. Bruce Sundstrom, Dawn M. Little, Francois Villinger, Jane E. Ellis, and Aftab A. Ansari. Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (USA).

Background: Evidence that human progenitor mast cells are susceptible to infection with CCR5 tropic strains of HIV-1 and that circulating HIV-1 infected FcRI⁺ cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. Material and Methods: Progenitor mast cells, developed in vitro from CD34⁺ cord blood stem cells (CBMCs), were experimentally infected with the CXCR5 tropic strain HIV-Bal after 28 days in culture as they reached their susceptible progenitor stage, and then were carried to a non-susceptible (tryptase/chymase positive) mature stage for an additional 70 days post-infection (PI) and monitored for HIV replication and cell proliferation.  Human toll-like receptor (TLR) gene expression was determined by reverse transcriptase PCR and flow microfluorometry. HIV-1 infection of CBMCs was confirmed by ELISA measurement of HIV-1 p24 antigen levels and by confocal microscopic imaging. Quantitative measurements of integrated HIV proviral DNA was determined by real-time PCR.  Precursor frequency of productively infected mast cells was determined by limiting dilution analysis. Results: HIV p24 antigen levels were readily detectable by day 7 PI, peaked at 3 weeks PI then steadily declined to below detectable limits by 10 weeks PI.  Post-integration viral latency, confirmed by PCR analysis for integrated HIV proviral DNA, was stably maintained at ≥ 300 copies per 1000 mast cells.  Stimulation by TLR ligands for TLR2 (S. aureus peptidoglycan), TLR4 (Chlamydia heat shock protein 60 or  E. coli LPS) or TLR9 (bacterial CpG oligo-deoxynucleotides) as well as IgE cross-linking induced high levels of HIV replication in a dose dependent manner without inducing detectable levels of mast cell apoptosis or  proliferation.  Limiting dilution analysis of TLR activated latently infected mature mast cells indicated that one in four were capable of establishing productive infections in A301 sentinel cells. Conclusion: These results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of post-integration latency in persistent HIV infection.