HIV persistence in tissue macrophages of humanized myeloid-only mice during antiretroviral therapy

Journal name:
Nature Medicine
Volume:
23,
Pages:
638–643
Year published:
DOI:
doi:10.1038/nm.4319
Received
Accepted
Published online

Despite years of fully suppressive antiretroviral therapy (ART), HIV persists in its hosts and is never eradicated. One major barrier to eradication is that the virus infects multiple cell types that may individually contribute to HIV persistence. Tissue macrophages are critical contributors to HIV pathogenesis1, 2, 3; however, their specific role in HIV persistence during long-term suppressive ART has not been established4, 5, 6. Using humanized myeloid-only mice (MoM), we demonstrate that HIV infection of tissue macrophages is rapidly suppressed by ART, as reflected by a rapid drop in plasma viral load and a dramatic decrease in the levels of cell-associated viral RNA and DNA. No viral rebound was observed in the plasma of 67% of the ART-treated animals at 7 weeks after ART interruption, and no replication-competent virus was rescued from the tissue macrophages obtained from these animals. In contrast, in a subset of animals (~33%), a delayed viral rebound was observed that is consistent with the establishment of persistent infection in tissue macrophages. These observations represent the first direct evidence, to our knowledge, of HIV persistence in tissue macrophages in vivo.

At a glance

Figures

  1. Viral suppression, persistence, and rebound induced by structured ART interruption in BLT mice.
    Figure 1: Viral suppression, persistence, and rebound induced by structured ART interruption in BLT mice.

    (a) Plasma viral load (VL) was monitored over time in HIV-infected ART-treated (n = 13; solid black line) and untreated (n = 5; dashed gray line) BLT mice. Each point represents mean ± s.e.m. The time of exposure to ART for the treated animals is indicated with a shaded gray box. A Mann–Whitney test was used to compare the plasma viral loads of ART-treated and untreated mice at 1 and 2 weeks following ART initiation (P = 0.0049 and P = 0.0080, respectively); **P < 0.01. (b) The reduction in log10 plasma viral load was calculated 1 week after ART initiation for each treated BLT mouse (n = 13). (c) The half-life of productively infected cells was estimated from the change in viral load during ART (n = 13). (d) Schematic for the magnetic sorting and purification of human T cells and macrophages from tissues obtained from infected animals. Human T cells (CD3+) and non-T cells were separated from pooled tissue cells of individual ART-treated BLT mice (n = 4). Macrophages (Macs) were then isolated from the non–T cell fraction. (e,f) Analysis of HIV DNA (e) and RNA (f) levels was performed by real-time PCR using purified cells isolated from ART-treated (n = 4) and untreated (n = 3) BLT mice. HIV DNA and RNA levels were normalized and are reported per 100,000 human cells. Samples with values below the level of detection are represented by open diamonds in e and f and are shown at the average lower limit of detection (dashed line). (g) Time to viral rebound after ART interruption in BLT mice (n = 5). (h) Viral rebound was observed 1–2 weeks after ART interruption in all BLT mice. The time of exposure to ART for each treated animal is indicated with a shaded gray box. For b, c, and eg, data are represented as mean ± s.e.m.

  2. ART rapidly suppresses viral replication in MoM.
    Figure 2: ART rapidly suppresses viral replication in MoM.

    (a) Longitudinal analysis of the plasma viral load in HIV-infected ART-treated (n = 8; solid black line) and untreated (n = 6; dashed gray line) MoM. Each point represents mean ± s.e.m. A Mann–Whitney test was used to compare the plasma viral loads of treated and untreated mice at 1–5 weeks following ART initiation (P = 0.0019, P = 0.0008, P = 0.0008, P = 0.0011, and P = 0.0017, respectively). The time of exposure to ART for treated animals is indicated with a shaded gray box. (b) The reduction in log10 plasma viral load was calculated 1 week after ART initiation for each treated MoM (n = 8). (c) The half-life of productively infected cells was estimated from the change in viral load during ART (n = 8). A Mann–Whitney test was used to compare MoM and BLT mice in b and c. (d,e) Cell-associated HIV DNA (d) and RNA (e) levels were measured in the liver, lung, spleen, and bone marrow of ART-treated (n = 6; gray squares) and untreated (n = 8; black squares) MoM. Undetectable samples are represented by an empty black box shown at the limit of detection for that sample (dependent on the number of cells available for analysis). Viral DNA and RNA levels were normalized per 100,000 human macrophages and compared between treated and untreated mice. A log-rank test was used to account for censoring due to the limits of detection in d and e. (f) There was no difference in the total numbers of human macrophages present in the tissues of ART-treated and untreated MoM (P > 0.05 for all tissue types, Mann–Whitney test). In bf, horizontal lines represent mean ± s.e.m. P values for ae are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

  3. HIV persistence in tissue macrophages during ART.
    Figure 3: HIV persistence in tissue macrophages during ART.

    Viral rebound was absent in most infected MoM after structured ART interruption. (a,b) Viral load in no-rebound (n = 6) (a) and rebound (n = 3) (b) mice. The time of exposure to ART for each treated animal is indicated with a shaded gray box. (c,d) Higher plasma viral load at the start of treatment (c) and higher total viral burden (as demonstrated by area under the curve (AUC) analysis of pre-ART viremia) (d) were associated with viral rebound after ART interruption. (e) The total numbers of human macrophages in the tissues of MoM were similar for mice in which viral rebound was absent (a) and observed (b) (P > 0.05). Mann–Whitney tests were used to compare mice in ce. For ce, data are represented as mean ± s.e.m. *P < 0.05.

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Primary accessions

GenBank/EMBL/DDBJ

Referenced accessions

NCBI Reference Sequence

References

  1. Kumar, A. & Herbein, G. The macrophage: a therapeutic target in HIV-1 infection. Mol. Cell. Ther. 2, 10 (2014).
  2. Koppensteiner, H., Brack-Werner, R. & Schindler, M. Macrophages and their relevance in human immunodeficiency virus type I infection. Retrovirology 9, 82 (2012).
  3. Campbell, J.H., Hearps, A.C., Martin, G.E., Williams, K.C. & Crowe, S.M. The importance of monocytes and macrophages in HIV pathogenesis, treatment, and cure. AIDS 28, 21752187 (2014).
  4. Sattentau, Q.J. & Stevenson, M. Macrophages and HIV-1: an unhealthy constellation. Cell Host Microbe 19, 304310 (2016).
  5. Collman, R.G., Perno, C.F., Crowe, S.M., Stevenson, M. & Montaner, L.J. HIV and cells of macrophage/dendritic lineage and other non–T cell reservoirs: new answers yield new questions. J. Leukoc. Biol. 74, 631634 (2003).
  6. Gavegnano, C. & Schinazi, R.F. Antiretroviral therapy in macrophages: implication for HIV eradication. Antivir. Chem. Chemother. 20, 6378 (2009).
  7. Honeycutt, J.B. et al. Macrophages sustain HIV replication in vivo independently of T cells. J. Clin. Invest. 126, 13531366 (2016).
  8. Denton, P.W. et al. Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice. PLoS One 5, e8829 (2010).
  9. Wahl, A. et al. Human breast milk and antiretrovirals dramatically reduce oral HIV-1 transmission in BLT humanized mice. PLoS Pathog. 8, e1002732 (2012).
  10. Olesen, R., Wahl, A., Denton, P.W. & Garcia, J.V. Immune reconstitution of the female reproductive tract of humanized BLT mice and their susceptibility to human immunodeficiency virus infection. J. Reprod. Immunol. 88, 195203 (2011).
  11. Melkus, M.W. et al. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat. Med. 12, 13161322 (2006).
  12. Denton, P.W. et al. Generation of HIV latency in humanized BLT mice. J. Virol. 86, 630634 (2012).
  13. Denton, P.W. et al. Targeted cytotoxic therapy kills persisting HIV infected cells during ART. PLoS Pathog. 10, e1003872 (2014).
  14. Thompson, M.A. et al. Antiretroviral treatment of adult HIV infection: 2012 recommendations of the International Antiviral Society–USA panel. J. Am. Med. Assoc. 308, 387402 (2012).
  15. Perelson, A.S., Neumann, A.U., Markowitz, M., Leonard, J.M. & Ho, D.D. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271, 15821586 (1996).
  16. Ho, D.D. et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373, 123126 (1995).
  17. Wei, X. et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373, 117122 (1995).
  18. Calin, R. et al. Treatment interruption in chronically HIV-infected patients with an ultralow HIV reservoir. AIDS 30, 761769 (2016).
  19. Shultz, L.D., Ishikawa, F. & Greiner, D.L. Humanized mice in translational biomedical research. Nat. Rev. Immunol. 7, 118130 (2007).
  20. Igarashi, T. et al. Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): implications for HIV-1 infections of humans. Proc. Natl. Acad. Sci. USA 98, 658663 (2001).
  21. Micci, L. et al. CD4 depletion in SIV-infected macaques results in macrophage and microglia infection with rapid turnover of infected cells. PLoS Pathog. 10, e1004467 (2014).
  22. Avalos, C.R. et al. Quantitation of productively infected monocytes and macrophages of simian immunodeficiency virus–infected macaques. J. Virol. 90, 56435656 (2016).
  23. Hansen, S.G. et al. Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature 473, 523527 (2011).
  24. Hansen, S.G. et al. Immune clearance of highly pathogenic SIV infection. Nature 502, 100104 (2013).
  25. Sung, J.A. et al. Expanded cytotoxic T-cell lymphocytes target the latent HIV reservoir. J. Infect. Dis. 212, 258263 (2015).
  26. Archin, N.M. et al. Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection of resting CD4+ T cells. AIDS 22, 11311135 (2008).
  27. Spina, C.A. et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog. 9, e1003834 (2013).
  28. Prochazka, M., Gaskins, H.R., Shultz, L.D. & Leiter, E.H. The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency. Proc. Natl. Acad. Sci. USA 89, 32903294 (1992).
  29. Hill, A.L. et al. Real-time predictions of reservoir size and rebound time during antiretroviral therapy interruption trials for HIV. PLoS Pathog. 12, e1005535 (2016).
  30. Steingrover, R. et al. HIV-1 viral rebound dynamics after a single treatment interruption depends on time of initiation of highly active antiretroviral therapy. AIDS 22, 15831588 (2008).
  31. Persaud, D. et al. Absence of detectable HIV-1 viremia after treatment cessation in an infant. N. Engl. J. Med. 369, 18281835 (2013).
  32. Henrich, T.J. et al. Long-term reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation. J. Infect. Dis. 207, 16941702 (2013).
  33. Nath, A. & Clements, J.E. Eradication of HIV from the brain: reasons for pause. AIDS 25, 577580 (2011).
  34. Joseph, S.B., Arrildt, K.T., Sturdevant, C.B. & Swanstrom, R. HIV-1 target cells in the CNS. J. Neurovirol. 21, 276289 (2015).
  35. Strain, M.C. et al. Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One 8, e55943 (2013).
  36. Honeycutt, J.B. et al. HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell–only mouse (TOM) model. Retrovirology 10, 121 (2013).
  37. Zhang, D., Fan, C., Zhang, J. & Zhang, C.H. Nonparametric methods for measurements below detection limit. Stat. Med. 28, 700715 (2009).
  38. Lafleur, B. et al. Statistical methods for assays with limits of detection: serum bile acid as a differentiator between patients with normal colons, adenomas, and colorectal cancer. J. Carcinog. 10, 12 (2011).
  39. Gillespie, B.W. et al. Estimating population distributions when some data are below a limit of detection by using a reverse Kaplan–Meier estimator. Epidemiology 21 (Suppl. 4), S64S70 (2010).

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Author information

Affiliations

  1. Division of Infectious Diseases, Center for AIDS Research, University of North Carolina, School of Medicine, Chapel Hill, North Carolina, USA.

    • Jenna B Honeycutt,
    • William O Thayer,
    • Caroline E Baker,
    • Rachel A Cleary &
    • J Victor Garcia
  2. Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.

    • Ruy M Ribeiro &
    • Youfang Cao
  3. Veterans Affairs, San Diego Healthcare System, San Diego, California, USA.

    • Steven M Lada &
    • Douglas D Richman
  4. Department of Biostatistics, University of North Carolina, Chapel Hill, North Carolina, USA.

    • Michael G Hudgens
  5. Department of Medicine, University of California, San Diego, San Diego, California, USA.

    • Douglas D Richman
  6. Department of Pathology, University of California, San Diego, San Diego, California, USA.

    • Douglas D Richman

Contributions

J.B.H. provided the experimental design, collected and processed blood and tissue samples from mice, prepared viral stocks, performed viral inoculations, administered ART, and performed flow cytometric analyses. W.O.T. processed blood and tissue samples from mice, prepared the injectable ART formulation, and administered ART. C.E.B. provided RNA and DNA analysis, prepared the injectable ART formulation, and processed tissue samples. R.A.C. performed immunohistochemical analysis of tissues. S.M.L. and D.D.R. performed the integrated DNA analysis and analyzed the results. J.B.H., R.M.R., Y.C., and M.G.H. analyzed the data and wrote the manuscript. J.V.G. conceived the study, designed and coordinated the study, and wrote the manuscript.

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The authors declare no competing financial interests.

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