Kathleen Borgmann

Regulation of tissue inhibitor of metalloproteinase-1 by astrocytes: Links to HIV-1 dementia

The neuropathogenesis of HIV‐1‐associated dementia (HAD) revolves around the secretion of toxic molecules from infected and immune‐competent mononuclear phagocytes. Astrocyte activation occurs in parallel but limited insights are available for its role in neurotoxicity and cognitive dysfunction. One means in which astrocytes may affect disease is through their production of tissue inhibitors of metalloproteinases (TIMPs). TIMPs are regulators of matrix metalloproteinases, enzymes that affect blood‐brain barrier integrity through altering the extracellular matrix. We hypothesized that in response to injury and inflammation in HAD, astrocytes regulate the production of TIMP‐1, the inducible type of TIMP that is important in inflammation. To address astrocyte‐mediated TIMP‐1 regulation in HAD, we evaluated the responses of primary human to IL‐1β and HIV‐1. TIMP‐1 levels in plasma, CSF, and brain tissue of control, HIV‐1 infected patients without cognitive impairment, and HAD patients were also studied. Our data show that an upregulation of TIMP‐1 results from astrocytes acutely activated with IL‐1β. In contrast, CSF and brain tissue samples from HAD patients showed reduced TIMP‐1 levels compared to seronegative controls. MMP‐2 levels in brains showed the opposite. Consistent with this, prolonged activation of astrocytes led to a reduction in TIMP‐1 and MMP‐2, but a sustained elevation in MMP‐1. Our data suggest that in diseased brain tissue, the ability of astrocytes to counteract the destructive effects of MMP through expression of TIMP‐1 is diminished by chronic activation. Our studies reveal new opportunities for repair‐based therapeutic strategies in HAD.

Role of activated astrocytes in neuronal damage: Potential links to HIV-1-associated dementia

HIV-1-associated dementia (HAD) is an important complication of HIV-1 infection. Reactive astrogliosis is a key pathological feature in HAD brains and in other central nervous system (CNS) diseases. Activated astroglia may play a critical role in CNS inflammatory diseases such as HAD. In order to test the hypothesis that activated astrocytes cause neuronal injury, we stimulated primary human fetal astrocytes with HAD-relevant pro-inflammatory cytokine IL-1β. IL-1β-activated astrocytes induced apoptosis and significant changes in metabolic activity in primary human neurons. An FITC-conjugated pan-caspase inhibitor peptide FITC-VAD-FMK was used for confirming caspase activation in neurons. IL-1β activation enhanced the expression of death protein FasL in astrocytes, suggesting that FasL is one of the potential factors responsible for neurotoxicity observed in HAD and other CNS diseases involving glial inflammation. Our data presented here add to the developing picture of role of activated glia in HAD pathogenesis.

HIV-1 and IL-1β regulate Fas ligand expression in human astrocytes through the NF-κB pathway

Reactive astrogliosis is a prominent pathological feature of HIV-1-associated dementia (HAD). We hypothesized that in HAD, astrocytes activated with proinflammatory stimuli such as IL-1beta express Fas ligand (FasL), a death protein. IL-1beta and HIV-1-activated astrocytes expressed FasL mRNA and protein. Luciferase reporter constructs showed that IL-1beta and HIV-1 upregulated FasL promoter activity (p<0.001). The NF-kappaB pathway was involved as shown by inhibition with SN50 and dominant negative IkappaBalpha mutants. Brain extracts from HAD patients had significantly elevated FasL levels compared to HIV-seropositive (p<0.001) and seronegative individuals (p<0.01). We propose that astrocyte expression of FasL may participate in neuronal injury in HAD.

Potential mechanisms for astrocyte-TIMP-1 downregulation in chronic inflammatory diseases.

The pathogenesis of many neurodegenerative disorders, including human immunodeficiency virus (HIV)‐1 associated dementia, is exacerbated by an imbalance between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). In the context of disease, TIMP‐1 has emerged as an important multifunctional protein capable of regulating inflammation. We previously reported differential TIMP‐1 expression in acute versus chronic activation of astrocytes. This study investigates possible mechanisms underlying TIMP‐1 downregulation in chronic neuroinflammation. We used interleukin (IL)‐1β as a model pro‐inflammatory stimulus and measured TIMP‐1 binding to extracellular matrix, cell death, receptor downregulation, TIMP‐1 mRNA stability and transcriptional regulation in activated astrocytes. TIMP‐1 remained localized to the cell body or was secreted into the cell supernatant. DNA fragmentation ELISA and MTT assay showed that prolonged IL‐1β activation of astrocytes induced significant astrocyte death. In acute and chronic IL‐1β‐activated astrocytes, IL‐1 receptor levels were not significantly different. TIMP‐1 mRNA stability was measured in astrocytes and U87 astroglioma cells by real‐time PCR, and TIMP‐1 promoter activation was studied using TIMP‐1‐luciferase reporter constructs in transfected astrocytes. Our results indicated that TIMP‐1 expression is regulated through multiple mechanisms. Transcriptional control and loss of mRNA stabilization are, however, the most likely primary contributors to chronic downregulation of TIMP‐1. These data are important for unraveling the mechanisms underlying astrocyte responses during chronic neuroinflammation and have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance.

Novel role of TGF‐β in differential astrocyte‐TIMP‐1 regulation: Implications for HIV‐1‐dementia and neuroinflammation

Astrocyte production of tissue inhibitor of metalloproteinase (TIMP)‐1 is important in central nervous system (CNS) homeostasis and inflammatory diseases such as HIV‐1‐associated dementia (HAD). TIMPs and matrix metalloproteinases (MMPs) regulate the remodeling of the extracellular matrix. An imbalance between TIMPs and MMPs is associated with many pathologic conditions. Our recently published studies uniquely demonstrate that HAD patients have reduced levels of TIMP‐1 in the brain. Astrocyte‐TIMP‐1 expression is differentially regulated in acute and chronic inflammatory conditions. In this and the adjoining report (Gardner et al., 2006 ), we investigate the mechanisms that may be involved in differential TIMP‐1 regulation. One mechanism for TIMP‐1 downregulation is the production of anti‐inflammatory molecules, which can activate signaling pathways during chronic inflammation. We investigated the contribution of transforming growth factor (TGF)‐signaling in astrocyte‐MMP/TIMP‐1‐astrocyte regulation. TGF‐β1 and β2 levels were upregulated in HAD brain tissues. Co‐stimulation of astrocytes with IL‐1β and TGF‐β mimicked the TIMP‐1 downregulation observed with IL‐1β chronic activation. Measurement of astrocyte‐MMP protein levels showed that TGF‐β combined with IL‐1β increased MMP‐2 and decreased proMMP‐1 expression compared to IL‐1β alone. We propose that one of the mechanisms involved in TIMP‐1 downregulation may be through TGF‐signaling in chronic immune activation. These studies show a novel extracellular regulatory loop in astrocyte‐TIMP‐1 regulation.

Isolation and HIV-1 infection of primary human microglia from fetal and adult tissue.

Glial inflammation, principally involving astrocytes and microglia, underlies the pathogenesis of a broad range of neurodegenerative disorders, including, most notably, human immunodeficiency virus (HIV-1)-associated dementia. Indeed, for the latter, disease mechanisms are attributed to viral infection and activation of microglia and perivascular macrophages and their resultant neurotoxic activities. Although monocyte-derived macrophages have served as models for microglia, they are limited both qualitatively and quantitatively in their immune responses and susceptibility to viral infection. Thus, the acquisition of primary human microglial cells is critical for laboratory studies of human neurological disease. In this chapter, we provide detailed methods of isolation, cultivation, characterization, HIV-1 infection, and experimental applications of primary human fetal and adult microglial cells, with particular emphasis on studies of HIV-1 neuropathogenesis.

A novel ligand on astrocytes interacts with natural cytotoxicity receptor NKp44 regulating immune response mediated by NK cells

NK cells play important role in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. NK cells were shown to be recruited to the CNS following several pathological conditions. NK cells could impact CNS physiology by killing glial cells and by secreting IFN-γ. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders. There is little data on NK-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. Natural cytotoxicity receptors (NCRs) play a critical role in the cytolytic function of NK cells. Among the NCRs, NKp44 is unique in expression and signal transduction. NKp44 is expressed only upon activation of NK cells and it can mediate both activating and inhibitory signals to NK cells. Here, we have studied the expression and function of natural cytotoxicity receptor NKp44 upon NK-astrocytes interactions in the presence or absence of an HIV peptide (HIV-3S peptide) shown to induce NK cell killing of CD4+ T cells during HIV–infection. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with HIV-3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study showed that NKp44 have a protective effect on astrocytes from NK cell mediated killing during HIV infection and impact astrocyte role in HAND.