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The inhibition of multilineage hematopoiesis which occurs in the severe combined immunodeficiency mouse with transplanted human fetal thymus and liver tissues (SCID-hu Thy/Liv) due to human immunodeficiency virus type 1 (HIV-1) infection is also accompanied by a severe loss of c-Mpl expression on these progenitor cells. Inhibition of colony-forming activity (CFA) of the CD34+progenitor cells is partially revived to about 40% of mock-infected Thy/Liv implants, following reconstitution of the CD34+cells that were exposed to HIV-1 infection, in a new Thy/Liv stromal microenvironment of irradiated secondary SCID-hu recipients at 3 weeks post-re-engraftment. In addition, in these reconstituted animals, the proportion of c-Mpl+CD34+cells relative to c-Mpl−CD34+cells increased by about 25%, to 35% of mock-infected implants, suggesting a reacquirement of c-Mpl phenotype by the c-Mpl−CD34+cells. These results suggest a correlation between c-Mpl expression and multilineage CFA of the human CD34+progenitor cells that have experienced the effects of HIV-1 infection. Treatment of the secondary-recipient animals with the c-Mpl ligand, thrombopoietin (Tpo), further increased c-Mpl expression and CFA of re-engrafted CD34+cells previously exposed to virus in the primary implants to about 50 to 70% over that of those re-engrafted CD34+cells derived from implants of untreated animals. Blocking of c-Mpl with anti-c-Mpl monoclonal antibody in vivo by injecting the SCID-hu animals resulted in the reduction or loss of CFA. Thus, inhibition, absence, or loss of c-Mpl expression as in the c-Mpl−CD34+subset of cells is the likely cause of CFA inhibition. Further, CFA of the CD34+cells segregates with their c-Mpl expression. Therefore, c-Mpl may play a role in hematopoietic inhibition during HIV-1 infection, and control of its expression levels may aid in hematopoietic recovery and thereby reduce the incidence of cytopenias occurring in infected individuals.

Hematopoietic abnormalities including anemia, cytopenias, and alterations of the stem cell plasticity in the bone marrow microenvironment commonly occur in HIV infected patients. These observations suggest that HIV-1 infection may affect processes important during early stages of hematopoiesis or stem cell differentiation. Hematopoietic abnormalities may be caused by altered stem cell differentiation possibly due to abnormal lineage specific expression of certain cellular genes such as cytokines relevant to hematopoiesis. These cytokines could affect regulatory signals important in hematopoiesis. However, in HIV infected individuals, it is not only the virus but also the highly active antiretroviral therapy (HAART) that both contribute to persistent hematopoietic suppression and ensuing cytopenias. Even if a lowering of HIV replication by HAART were to occur in infected individuals, prolonged HAART by itself and/or appearance of drug resistant mutants can contribute to hematopoietic suppression and resulting cytopenias. However, confounding factors such as opportunistic infections, immune mediated effects, or the consequences of prolonged physiological stress, which could contribute to decreased hematopoiesis in patients or other individuals, make the causative role of HIV in vivo, uncertain. The severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues (SCID-hu) is a small animal model which mimics HIV infection in humans, and is useful to determine the mechanisms of HIV-1 induced hematopoietic inhibition and development of drug therapies for interventions of stem cell differentiation. Further, SCID mouse serves as a useful small animal recipient of human progenitor cells and also allows us to study the differentiation of these cells in vivo. Results from our studies are expected to provide relief for HIV infected individuals from hematopoietic inhibition and ensuing cytopenias.

We and others have previously shown that HIV infection of human thymus/liver implants in severe combined immunodeficient (SCID-hu Thy/Liv) mice results in a loss of CD4(+) thymocytes and a decreased recovery of human myeloid and erythroid colony-forming activity. Furthermore, our previous studies have shown that this decrease in colony-forming potential is due to indirect effects, rather than to massive loss of CD34(+) hematopoietic progenitor cells, suggesting that HIV infection might alter expression of hematopoietic cytokines. Herein we have investigated potential HIV-1-induced perturbations of hematolymphoid microenvironments by determining the effect of HIV-1 infection on levels of cytokine mRNAs in human stroma and thymocytes, using the reverse transcription-polymerase chain reaction (RT-PCR). The levels of interleukin 6 (IL-6), interferon gamma (IFN-gamma), and IL-2 RNAs increased and macrophage inflammatory protein 1beta (MIP-1beta) RNA decreased significantly in infected thymocytes. IL-6 RNA levels in stroma also increased somewhat with infection; however, expression of stromal cell-derived factor 1 (SDF-1) by stromal elements was not affected. IL-4 RNA levels were unaffected by infection in both stroma and thymocytes. Antiretroviral drug treatment of the infected animals, which results in restoration of colony-forming potential, tends to restore the cytokine mRNA levels in HIV-1-infected implants to those of mock-infected implants. These results indicate that HIV-1 infection can greatly distort the cytokine profiles in Thy/Liv implants, and suggest that cytokine perturbation could be involved in virus-induced inhibition of hematopoiesis.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals may exhibit multiple hematopoietic abnormalities including anemia, granulocytopenia, thrombocytopenia, and myelodysplastic/hyperplastic alterations of the bone marrow, suggesting virus-induced abnormalities in the bone marrow microenvironment (7, 9, 11, 35). Evidence of alteration of fetal hematopoiesis including leukopenia, anemia, and thrombocytopenia has also been found in aborted fetuses from HIV-1-seropositive women (6, 32). These observations suggest that HIV-1 infection may affect processes important during early stages of hematopoiesis. However, several different factors, including direct intracellular effects of virus infection, interaction with viral proteins at the cell surface, perturbation of the cytokine network, or immune-mediated effects, may play a role. Following HIV-1 infection in vitro in long-term bone marrow cultures, inhibition of hematopoietic progenitor cell production occurs and alteration of production of cytokines relevant to hematopoiesis has been documented (14, 15, 27). These infected stromal cultures showed reduced production of the cytokines interleukin-6 (IL-6) and granulocyte colony-stimulating factor, which could affect regulatory signals important in hematopoiesis. Further in vitro studies suggested that HIV-1-induced suppression of hematopoiesis is mediated by the HIV-1-encoded envelope glycoprotein gp120 and the Nef regulatory protein, as well as by cellular proteins such as tumor necrosis factor alpha (8, 23). The p24 Gag protein of HIV-1 also was shown to inhibit myeloid colony formation of bone marrow cultures but had minor effects on erythroid colony formation (29). Purified CD34+ cells were reported to be susceptible to HIV-1 infection, as shown by the presence of proviral sequences in the ensuing colonies of erythroid and myeloid lineages generated from these cells (10). These effects could be influenced by the infection of microvascular endothelial cells of bone marrow stromal cultures from HIV-seropositive patients (27). Infection of these cells could affect the relevant neighboring microenvironment, by providing a continuing source of virus and by causing alteration of local cytokine levels. Therefore, HIV is likely to alter the stromal/progenitor cell microenvironment that supports hematopoiesis. However, these previous studies on in vitro consequences of virus infection could not determine how HIV-1 infection influences complex hematopoietic microenvironments in vivo. To investigate how HIV-1 might affect hematopoiesis in vivo, we used the SCID-hu (Thy/Liv) mouse model, in which human fetal thymus and liver tissue are coimplanted into severe combined immunodeficient mice, resulting in a functional human hematopoietic organ (Thy/Liv) (24, 28). This model allows maintenance and differentiation through thymopoiesis of human hematopoietic progenitor cells (28) and also recapitulates the effects of HIV-1 infection in the human thymus. Direct infection of various strains of HIV-1 into Thy/Liv implants results in severe depletion of CD4-bearing human thymocytes which mirrors that seen in infected individuals (2, 5, 17, 20, 21, 30). Morphologic alterations of the thymic stroma, possibly due to infection of thymic epithelial cells, have been seen (30). These effects are precipitated by a large proviral burden, which may be as high as nearly one copy of the viral genome per CD4+ cell (18). Both direct virus-induced killing (18) and indirect apoptotic effects (31) have been implicated in the observed depletion of CD4+ thymocytes. Furthermore, this model has been used to determine the viral accessory genes involved in pathogenesis (3, 16) and the response of HIV-1 to antiviral therapies (19, 25, 26, 33). Previously, McCune et al. (24) reported that human pluripotent hematopoietic progenitor cells capable of giving rise to myeloid and erythroid colonies ex vivo in response to cytokines are maintained for extended periods of time in this model. The SCID-hu system has thus proven relevant to the clinical situation, is amenable to manipulation and analyses, and offers an opportunity to investigate the effects of HIV-1 infection on multiple arms of hematopoiesis. Here we report that HIV-1 infection profoundly decreases the ability to recover hematopoietic colony-forming activity (CFA) from Thy/Liv implants. However, this effect is reversible following administration of combination antiretroviral therapy. Our studies thus establish a causal effect of viral replication on hematopoiesis of multiple lineages. In addition, the reversible nature of this inhibitory effect following therapy suggests that neither the very immature hematopoietic progenitor cell nor the differentiation-inducing microenvironment is destroyed by high levels of HIV-1. Furthermore, we identify a viral strain, isolated from a pediatric patient exhibiting severe hematologic abnormalities, which preferentially inhibits hematopoietic CFA rather than inducing CD4+ thymocyte depletion. Together our studies suggest that HIV-1 may be directly responsible for many of the hematopoietic perturbations seen in infected individuals.

Stem cell gene therapy strategies for AIDS require that differentiation-inducing stromal elements of HIV-infected individuals remain functionally intact to support the maturation of exogenous progenitor cells into mature CD4+ cells. To investigate the feasibility of stem cell reconstitution strategies in AIDS, we used the SCID-hu mouse to examine the ability of HIV-infected CD4+ cell-depleted human thymic implants to support renewed thymopoiesis. Here we report that following treatment of these implants with antiretroviral drugs, new thymopoiesis is initiated. This suggests that antiviral therapies might allow de novo production of T lymphocytes and provides support for the concept of therapeutic strategies aimed at reconstitution of the peripheral CD4+ T-cell compartment.

Cells of the immune and central nervous systems communicate or interact with each other through proinflammatory cytokines (Benveniste, 1994; Black, 1994; Williams et al., 1994). There also exist parallels between the immune and central nervous systems in human immunodeficiency virus type 1 (HIV-1)-mediated secondary effects possibly triggered among others by cytokines leading to pathological abnormalities. In the immune system, although the T-helper cells are affected by the direct infection with HIV-1, other subsets of cells or their function are also altered by the virus, despite the lack of infection of these other cell subsets by the virus. These include the CD8-positive suppressor T cells whose numbers are diminished and/or their cytotoxic function reduced indirectly by the virus (Ho et al., 1993; Watret et al., 1993). Similarly, the ability of B lymphoid cells to provide help to T cells is also affected, although HIV-1 does not productively infect B lymphoid cells (Maggi et al., 1994). Analogously, in the central nervous system (CNS), the blood-borne macrophages infected with the virus carry the virus across the blood-brain barrier to fuse with and infect microglial cells in the brain, and spread the infection to parenchymal microglial cells in vivo. While this gives rise to white matter pathology, the neurons in the gray matter of the CNS are not infected by the virus. But the damage done to oligodendrocytes and neurons is again indirect as for non-CD4 cells in the immune system and can occur even in the absence of viral infection of these cells and by interactions between viral envelope proteins and glial cells in the CNS.