MODELING HIV-1 INFECTION OF BONE MARROW PROGENITOR CELLS: IMPLICATIONS FOR HIV-1 CNS DISEASE

Open Access
- Author:
- Quiterio, Shane Joseph
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 28, 2003
- Committee Members:
- Brian Wigdahl, Committee Chair/Co-Chair
Shao Cong Sun, Committee Member
Robert Harold Bonneau, Committee Member
Robert G Levenson, Committee Member
Andrew Thomas Henderson, Committee Member - Keywords:
- HIV-1
bone marrow
CNS
CD34
progenitor
differentiation
coreceptor
LTR
dementia
HAD - Abstract:
- HIV-1-associated dementia (HAD), which arises as a consequence of HIV-1 infection of the central nervous system (CNS), may be initiated by events within the bone marrow and peripheral immune system that lead to HIV-1 infection and activation of monocytic cells or their progenitors. Cells of the monocyte/macrophage lineage, which play important roles in HIV-1-associated neuropathogenesis, arise from CD34+ hematopoietic progenitor cells (HPCs) within the bone marrow and peripheral blood. CD34+ HPCs in the bone marrow are generally resistant to HIV-1 infection, likely due to deficiencies in HIV-1 coreceptor expression. However, cells of the monocytic lineage that arise from these precursor cells are able to support a productive HIV-1 infection, indicating that the process of differentiation from CD34+ HPCs to monocytes or macrophages may induce changes in cell surface receptors and transcription factors that favor viral entry and productive replication. In studies of HIV-1 genomic sequence compartmentalization, gp160 sequences isolated from deep white matter of a HAD patient’s brain were more closely related to sequences isolated from bone marrow and monocytes within the blood, suggesting either similar paths of viral evolution or migration of HIV-1-infected cells from the bone marrow to the brain. The differentiation of HIV-1-infected bone marrow cells and their migration to the brain could represent an important step in the development of HIV-1-associated neuropathogenesis and HAD. The central hypothesis of this thesis is that differentiation of bone marrow CD34+ progenitor cells leads to increased HIV-1 susceptability, LTR activation, and virus production that promotes the alteration of signaling pathways associated with increased cellular trafficking. The TF-1 erythromyeloid CD34+ progenitor cell line has been used to model bone marrow progenitor cell differentiation and to examine HIV-1 coreceptor expression levels during phorbol myristate acetate (PMA)-induced differentiation. Reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry analyses were used to measure the expression of TF-1 cell RNA and surface proteins that are either crucial for HIV-1 binding and entry or indicative of cellular differentiation. TF-1 cells express the HIV-1 receptor CD4 and very low or undetectable levels of the HIV-1 coreceptors CXCR4 and CCR5. However, TF-1 cells had increased expression of CXCR4 and CCR5 coreceptors after PMA treatment, with high expression levels of both coreceptors 48-72 hr post-treatment. Expression of the activation marker CD69 also increased dramatically within 24 hr after PMA treatment. These results indicate that during progenitor cell differentiation the cells may become more susceptible to HIV-1 infection as a consequence of increased levels of the chemokine receptors involved in HIV-1 binding and entry. Cellular differentiation of bone marrow-derived progenitor monocytic cells can produce a distinct array of transcription factors that can modulate the activity of the HIV-1 long terminal repeat (LTR) activity and subsequently regulate HIV-1 replication. TF-1 cells that are induced to differentiate by PMA are being utilized as a model system for examining LTR regulation and viral replication during the differentiation of bone marrow-derived monocytic cells. Studies of nuclear transcription factor availability using electrophoretic mobility shift (EMS) analyses demonstrated that PMA treatment of TF-1 progenitor cells altered the availability of factors important to HIV-1 LTR regulation. In transient expression experiments, PMA-induced differentiation resulted in large increases of about 100-fold in HIV-1 LTR activity compared to untreated cells. Finally, viral infection studies using HIV-1 strains IIIB (X4) and BaL (R5) indicated increased viral production (as measured by p24) in PMA-treated TF-1 cells compared to untreated, undifferentiated cells. Cumulatively, these results suggest that bone marrow progenitor cell differentiation results in cellular changes that promote HIV-1 LTR activation and viral replication. The ability of differentiated and/or activated TF-1 cells to secrete factors that can induce the differentiation of undifferentiated TF-1 cells and other cell lines was examined. Conditioned media taken from TF-1 cells that have undergone differentiation in response to PMA can induce differentiation and morphologic changes in undifferentiated TF-1 and U-937 cells. The TF-1 and U-937 cells treated with the conditioned media also had phenotypic changes in cell surface marker expression and had cell surface marker expression profiles similar to cells differentiated directly by PMA. Furthermore, cells exposed to conditioned media did not exhibit the decrease in the CD4 receptor expression that was normally associated with PMA treatment. However, increases in the expression of the HIV-1 coreceptors CXCR4 and CCR5 and the activation marker CD69 were observed. Experiments using CD69 expression as a measure of cellular activation also indicated that media conditioned for longer periods of time induced greater levels of activation, which is consistent with time-dependent accumulation of soluble factors secreted from PMA-treated TF-1 cells. In transient transfection experiments using LTR-luciferase reporter constructs, basal LTR activity was increased considerably in TF-1 and U-937 cells by the presence of conditioned media. Factors such as IL-1?, TNF-?, or IL-6, secreted by the cells and acting by an autocrine or paracrine mechanism, may be responsible for inducing cellular differentiation, as well as coreceptor upregulation and LTR activation. These studies provide potential insights into mechanisms that underlie the development of HAD. As CD34+ cells undergo differentiation (and activation) in vivo in response to inflammatory cytokines such as TNF-? or secreted viral proteins such as gp120 or Tat, they become increasingly susceptible to HIV-1 infection. The increases in infection are due to increases in HIV-1 receptor and coreceptor expression, as well as changes in nuclear transcription factor availability that promote HIV-1 expression. Progenitor cells, infected by HIV-1 in the BM or in the blood as they differentiate down the monocytic lineage, can then travel through the blood and migrate into the brain, delivering virus into the CNS. These activated cells are primed to migrate into tissues such as the brain because of increases in cell adhesion molecules that permit adhesion to the blood vessel endothelium and enable chemotaxis to sites of inflammation. HIV-1-infected cells and damaged bystander cells may also release inflammatory cytokines and chemokines that can, in turn, recruit additional monocyte/macrophage cells into the brain. ?-chemokines, such as macrophage inflammatory protein-1? (MIP-1?) and MIP-1?, can be secreted by monocyte/macrophages, promoting chemotaxis, leukocyte trafficking, and inflammation. Upregulation of the chemokine receptors CXCR4 and CCR5 (the receptor for MIP-1? and MIP-1?) in differentiating cells can thus serve a dual role of enabling HIV-1 binding and entry and mediating chemotaxis of the cells into the CNS. Increased susceptibility to infection combined with enhanced recruitment of both uninfected and HIV-1-infected monocytic cells to the CNS may form a positive feedback loop of cell migration and viral infection that results in the production of even greater levels of HIV-1 and inflammatory mediators. In vitro studies suggest that in vivo infection of CD34+ progenitor cells by HIV-1 as they differentiate into cells of monocye/macrophage lineage may lead to the promotion or acceleration of the development or progression of HIV-1-associated neuropathogenesis and HAD. These investigations, which contribute toward a basic understanding of HIV-1-associated nervous system disease, imply that antiretroviral treatments that target HIV-1-infected cells in the peripheral immune system may also lessen the risk for CNS disease. This hypothesis is supported by clinical studies that demonstrate correlations between a decreased incidence of HIV-1-associated CNS disease and treatment regimens involving single or multiple antiretroviral drugs.