Critical events of normal and pathologic processes, including those caused by microbes, occur in tissues, where cells contact neighboring cells and are immersed in a heterogeneous signal-rich microenvironment. We study the pathogenesis of HIV-1 variants that use either CCR5 (R5) or CXCR4 (X4) for cell entry into ex vivo human lymphoid tissues. In vivo, the former transmit infection and dominate early stages of HIV disease, whereas later, in approximately half of HIV cases, there is a switch to X4 dominance, which is followed by a rapid progression to AIDS. The cause of the R5-to-X4 switch remains unclear and is possibly a function of both viral and host factors. In our first project, we studied the role of viral factors, namely, whether R5 variants dominating early stages of HIV disease in patients who eventually undergo an R5-to-X4 switch differ from those variants found in patients whose disease progresses without a switch. In our second project, we addressed potential host factors, namely, whether infection of hosts by other microbes differentially affects R5 and X4 HIV. In our third project, we applied the ex vivo tissue system that we developed to study the effects of potential antivirals on R5 and X4 HIV variants.
Differential pathogenesis of primary CCR5-using HIV-1 isolates in human lymphoid tissue ex vivo
Grivel, Margolis; in collaboration with Fenyö, Karlsson
In the course of HIV disease, R5 HIV variants, which typically transmit infection and dominate the disease’s early stages, persist in approximately half of infected individuals (non-switch virus patients) while, in the other half (switch virus patients), viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. In lymphoid tissues ex vivo, productive X4 HIV-1 infection is associated with immunosuppression and, given that the majority of cells express CXCR4, massive depletion of T cells. We compared the pathogenesis of sequential primary HIV-1 isolates all of the R5 phenotype from switch and non-switch virus patients in ex vivo lymphoid tissue. The non-switch virus group contained three patients; we studied two R5 isolates from each patient. In the switch virus group, we studied two patients, one with three and one with two R5 isolates.
By infecting lymphoid tissue from one donor with a panel of different isolates and by infecting a panel of lymphoid tissues from different donors with one particular HIV-1 isolate, we were able to distinguish, in HIV tissue pathogenesis, which parameters are controlled by host and which by viral factors. We found that, although the absolute levels of viral replication varied by as much as 30-fold between tissues obtained from different donors and differed for different isolates, the viral hierarchy among sequential isolates remained constant, indicating that viral factors including Nef, Vpu, Vpr, and Vif are major determinants of the relative replication ability of these isolates in human lymphoid tissues ex vivo. In contrast, the absolute replicative capacity of HIV-1 isolates is controlled by host (tissue) factors that seem to enhance or suppress all replicating HIV-1 variants. Thus, various host factors seem to enhance or inhibit replication of all viral isolates, whereas viral factors determine which isolate has a higher or lower relative capacity to replicate.
Replication of the isolates, as monitored from the release of p24, became evident at day six post-infection and continued to increase during the course of the experiment. Patients’ HIV-1 isolates efficiently replicated in ex vivo–infected human lymphoid tissue. Consistent with productive infection, HIV-1 isolates depleted CD4+ T cells. R5 HIV-1 variants depleted their natural targets, i.e., CCR5+CD4+ T cells. We found that depletion of the cells was accompanied not only by downregulation of CD4, observed earlier in other systems, but also by downregulation of CCR5. Depletion of CCR5+CD4+ T cells seems to be related to the modes of disease progression in the patients who harbored the cells. On average, infection of tissues from 14 donors with six R5 isolates from the three nonswitch virus patients resulted in the loss of just over half of the CD3+CD8–CCR5+ cells, whereas infection of tissues from nine donors with five R5 isolates from the two switch virus patients resulted in a significantly smaller loss of CD3+CD8–CCR5+ cells relative to matched uninfected controls. Thus, R5 HIV-1 isolates from nonswitch virus patients are more cytopathic than R5 variants from switch virus patients; the difference may explain the steady decline of CD4+ T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value reflecting whether, in an infected individual, X4 HIV-1 will eventually dominate.
Fitzgerald W, Sylwester AW, Grivel JC, Lifson JD, Margolis LB. Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo. J Virol 2004;78:7061-7068.
Karlsson I, Grivel JC, Chen SS, Karlsson A, Albert J, Feny EM, Margolis LB. Differential pathogenesis of primary CCR5-using human immunodeficiency virus type 1 isolates in ex vivo human lymphoid tissue. J Virol 2005;79:11151-11160.
Rücker E, Grivel JC, Münch J, Kirchhoff F, Margolis L. Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo. J Virol 2004;78:12689-12693.
Rücker E, Münch J, Wildum S, Brenner M, Eisemann J, Margolis L, Kirchhoff F. A naturally occurring variation in the proline-rich region does not attenuate human immunodeficiency virus type 1 nef function. J Virol 2004;78:10197-10201.
Measles virus inhibits HIV-1 replication in human lymphoid tissue ex vivo
Grivel, Margolis; in collaboration with García, Moss
Most experimental data on HIV-1 infection of human cells come from experiments in “clean” systems: cultures of cell lines or primary cells infected in vitro with HIV-1 but otherwise sterile. In vivo, however, the situation is dramatically different. Humans are continuously exposed to microbes, some of which establish infection before HIV-1, whereas other, opportunistic infections take advantage of the weakened immune system induced by HIV-1 infection. The microbes interact with each other and with HIV-1 in the context of human tissues. Various pathogens enhance HIV-1 replication and disease progression in co-infected individuals. However, unexpected observations in vivo have recently revealed several microbes that can inhibit HIV-1 replication. In particular, acute infection with measles virus (MV) has been shown to suppress HIV-1 replication in co-infected children. The mechanisms of this phenomenon and the contributions of local and systemic factors to MV-triggered HIV-1 inhibition remain largely unknown. We addressed these issues in a study that used a system of ex vivo–infected human lymphoid tissues, given that critical events of HIV-1 infection occur in these tissues in vivo.
Inoculation of human lymphoid tissue with a laboratory-passaged wild-type isolate (Chicago-1) or an attenuated (Edmonston) MV strain resulted in productive infection and the release of infectious virions. As shown by flow cytometric analysis of N protein–stained cells, both strains readily infected B and T lymphocytes in lymphoid tissues, including the CD4+ and CD8+ subsets. The cells were infected indiscriminately in proportion to their relative abundance in lymphoid tissue. Both MV strains indiscriminately depleted lymphocytes, but MV of the Edmonston strain was much less cytopathic than that of the Chicago-1 strain. Therefore, to minimize tissue deterioration, we chose Edmonston MV for the co-infection experiments. We investigated whether MV alters replication of HIV of different co-receptor specificities in co-infected ex vivo human lymphoid tissues. MV dramatically inhibited replication of R5SF162. On average, production of R5SF162 in MV–co-infected cultures declined to 13 percent of that in matched singly infected tissues. Replication of X4LAI.04 was also inhibited in MV–co-infected tissues, but to a lesser extent than that of R5 HIV-1. To investigate further the possible mechanisms of MV-induced HIV-1 inhibition, we analyzed, in infected tissues, levels of key chemokines and cytokines that may be induced by MV and inhibit HIV-1 infection. Analysis of a panel of 19 cytokines and chemokines revealed that RANTES, MIP-1alpha, and MIP-1beta, the well-known CCR5 ligands and inhibitors of R5 entry, were significantly upregulated in MV-infected tissues. We observed upregulation of these chemokines in tissues infected with MV alone but not in those infected with R5 HIV-1 alone. Surprisingly, MV and R5 HIV-1 co-infection upregulated RANTES about four-fold compared with uninfected tissue. The differential inhibition of R5 and X4 HIV-1 by MV in co-infected lymphoid tissues ex vivo may be relevant to what happens in vivo when acute MV infection diminishes the HIV-1 viral load in co-infected children. Deciphering the molecular mechanisms by which MV and other pathogens alter local cytokine/chemokine networks and cause tissue microenvironments to become detrimental to HIV-1 may significantly contribute to the development of effective anti–HIV therapies.
Grivel JC, Garcia M, Moss WJ, Margolis LB. Inhibition of HIV-1 replication in human lymphoid tissues ex vivo by measles virus. J Infect Dis 2005;192:71-78.
Herbeuval JP, Boasso A, Grivel JC, Hardy AW, Anderson SA, Dolan MJ, Chougnet C, Lifson JD, Shearer GM. TNF-related apoptosis-inducing ligand (TRAIL) in HIV-1–infected patients and its in vitro production by antigen-presenting cells. Blood 2005;105:2458-2464.
Herbeuval JP, Grivel JC, Boasso A, Hardy AW, Chougnet C, Dolan MJ, Yagita H, Lifson JD, Shearer GM. CD4+ T cell death induced by infectious and noninfectious HIV-1: role of type I interferon-dependent, TRAIL/DR5-mediated apoptosis. Blood 2005;105:1243-1271.
Ito Y, Grivel JC, Chen S, Kiselyeva Y, Reichelderfer P, Margolis L. CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue by selective induction of CC-chemokines. J Infect Dis 2004;189:506-514.
Immunostimulatory and HIV-inhibitory properties of pertussis toxin B oligomer
Grivel, Margolis; in collaboration with Alfano, Poli
We found that pertussis toxin B oligomer PTX-B and PT-9K/129G, the genetically modified variant used for vaccination, inhibited HIV-1 replication in ex vivo–infected human lymphoid tissue. In addition, the oligomers increased the number of cells that emigrated from the tissue blocks into the surrounding medium and stimulated the proliferation of emigrated cells, which were mostly CD4+ T lymphocytes. Furthermore, cells that had emigrated from PTX-B–stimulated tissues upregulated the expression of typical activation antigens such as CD25, HLA-DR, and CD69. X4 HIV infection of PTX-B–stimulated tissue prevented the upregulation of the activation markers in emigrated cells, whereas their upregulation in R5-infected PTX-B–stimulated tissues was similar to that observed in matched uninfected controls. X4 HIV-1 inhibited PTX-B-induced CD4+ T cell activation while R5 HIV variants had no effect on cell activation. The lack of activation in the case of R5 HIV-1 infection is likely to be a consequence of differential signaling by R5 and X4 gp120 Env interacting with CD4 and chemokine receptors.
For at least three weeks in culture, HIV did not replicate in cells that had emigrated from the tissue; furthermore, the treatment of these cells with mitogenic concentrations of PTX-B did not induce HIV replication, as evaluated from RT activity. Therefore, we investigated whether some of the cells were indeed latently infected or whether simply none was infected. For this purpose, we stimulated emigrated cells with either IL-2 or PHA. High levels of HIV replication, comparable to those in acutely infected mitogen-activated peripheral blood mononuclear cells, were readily induced, indicating that a fraction of the cells was latently infected with a replication-competent virus. Moreover, HIV DNA was found in cells that had emigrated from control and PTX-B–treated tissue. PTX-B stimulation of emigrated cells led to viral DNA dilution attributable to cell replication while, after IL-2 stimulation, which induced viral spreading in emigrated cells, the quantity of HIV DNA was much higher than in control or PTX-B–treated cells. Thus, in this ex vivo system, PTX-B maintains viral latency despite triggering lymphocyte activation and proliferation, representing a new experimental model of latent inducible R5 and X4 HIV-1 infection of CD4+ T lymphocytes.
In summary, our study shows that PTX-B inhibits viral replication in ex vivo–infected human lymphoid tissue; cells emigrated from human lymphoid tissue infected with HIV ex vivo include nonproductively infected ones; and PTX-B promotes activation and proliferation of cells that emigrate from lymphoid tissue but, unlike PHA or IL-2, does not induce viral replication, thus uncoupling T-cell activation from HIV replication. PTX-B and the clinically approved PT-9K/129G are potential antiretroviral agents endowed with immunostimulatory capacity. Our work confirms that ex vivo–infected blocks of human lymphoid tissue can be used to evaluate the antiviral potential of new drugs before animal and clinical trials.
Alfano M, Grivel JC, Ghezzi S, Corti D, Trimarchi M, Poli G, Margolis L. Pertussis toxin B-oligomer dissociates T cell activation and HIV replication in CD4 T cells released from infected lymphoid tissue. AIDS 2005;19:1007-1014.
Biancotto A, Grivel JC, Gondois-Rey F, Bettendroffer L, Vigne R, Brown S, Margolis LB, Hirsch I. Dual role of prostratin in inhibition of infection and reactivation of human immunodeficiency virus from latency in primary blood lymphocytes and lymphoid tissue. J Virol 2004;78:10507-10515.
Fletcher P, Kiselyeva Y, Wallace G, Romano J, Griffin G, Margolis L, Shattock R. The nonnucleoside reverse transcriptase inhibitor UC-781 inhibits human immunodeficiency virus type 1 infection of human cervical tissue and dissemination by migratory cells. J Virol 2005;79:11179-11786.
collaborators
Massimo Alfano, PhD, Fondazione San Raffaele del Monte Tabor, Milan, Italy
Eva-Maria Fenyö, MD, PhD, Lund University, Lund, Sweden
Mayra García, PhD, John Hopkins Bloomberg School of Public Health, Baltimore, MD
Ingrid Karlsson, PhD, Lunds Universitet, Lund, Sweden
William J. Moss, MD, John Hopkins Bloomberg School of Public Health, Baltimore, MD
Guido Poli, MD, Fondazione San Raffaele del Monte Tabor, Milan, Italy
For further information, contact margolis@helix.nih.gov.