Basic Science Research
The HIV/TB group is dedicated to studying the molecular mechanisms which regulate pathogenesis in both of these debilitating infections. The group is focused on the effector cells of innate immunity which are critical targets for both HIV and TB. We seek to identify molecular mechanisms regulating virulence and pathogenesis which may serve as potential therapeutic targets and translate to effective medical treatments for TB and HIV infection.
Despite effective therapies for treatment of mycobacterial infection and stemming the advancement of HIV infection, the combined prevalence of these diseases is staggering and exceeds 50 million people in the world. In HIV disease novel approaches for inhibiting viral replication, especially in the more difficult to treat host reservoirs, are in great need. TB infects one-third of the world’s population and causes disease in ten percent of these cases. The morbidity and mortality resulting from these active infections are significant and further promote the spread of infection. Thus, for HIV and TB, new therapeutic targets need to be identified to fight both active infection and prevent continued spread.
The effector arm of the innate immune system includes alveolar macrophages (AM) and polymorphonuclear cells (PMN). Best known for their eradicating function, these cells are increasingly being recognized as possessing a more complex immunologic role beyond antigen presentation and killing. In the lung alveolar macrophages predominate in numbers and are the first line of defense. In the setting of infection PMN are rapidly recruited to eradicate offending pathogens utilizing a number of mechanisms. In HIV and TB infection the normal function of both of these cell-types is altered to the advantage of the infecting organism. These changes support the continued survival of the microbe, as well as result in impaired lung function and defense.
The HIV research group focuses its investigations on the alveolar macrophage which is a unique environment for the virus. HIV-1 infection of macrophages, unlike T lymphocytes, is not cytotoxic and chronic infection is well tolerated. In the majority of asymptomatic HIV-1 infected people alveolar macrophages, which have a phenotype distinct from other macrophage lines, harbor proviral DNA and are an important reservoir of HIV-1 that can be reactivated under specific conditions. When HIV replicates in the lung its replication dynamics appear to be distinct from that in the serum.
Progression of HIV-infection is associated with a worsening pro-oxidative state and increased oxidative stress synergizes with inflammatory cytokines to induce NF-kB translocation leading to HIV-LTR activation. Preliminary studies show that increasing the intracellular superoxide dismutase and catalase activity of in vitro HIV-1 infected alveolar macrophages inhibits HIV-1 replication. Others have shown that increased nitric oxide levels are associated with increased HIV-1 replication in monocyte-derived macrophages. The lung is an exceedingly oxidative environment, especially in settings of infection and progressive respiratory failure. We hypothesize that reactive oxygen and nitrogen intermediates play an integral role in the activation of HIV-1 in alveolar macrophages and propose that this may be an important reservoir of latent HIV-1 which is induced to replicate during acute respiratory infections and exposures to hyperoxia.
Another project in the HIV group is examining the role of IL-16 in HIV-infection of alveolar macrophages. IL-16 is a cytokine that binds to CD4 and has been shown to inhibit HIV replication in T-cells and monocyte-derived macrophages. Serum levels have been reported to inversely correlate with worsening HIV disease. We are currently characterizing the effect of IL-16 on in vitro HIV infection of alveolar macrophages. We aim to determine if IL-16 inhibits HIV replication, as well as to determine if HIV-infection alters the expression of IL-16 in alveolar macrophages.
The Flow Cytometry laboratory has been investigating the roles of the polymorphonuclear and the mononuclear phagocyte in human lung disease. Recent research interests have focused on studies of the development of the activation response and in the expression of specific activation mechanisms in human neutrophil, monocyte, and macrophage subpopulations. Current efforts are directed at elucidating normal mechanisms of control of phagocyte function, such as early pH responses within the cell and its phagovacuoles, in order to learn how these mechanisms are controlled in “normal” circumstances, and how they may be subverted by pathogens such as Mycobacteria tuberculosis. Much of this work utilizes fluorescent ligands and probes, and specific blocking molecules and inhibitors, to examine the very early kinetic responses, within seconds of stimulation, of defined cell populations and subpopulations, using a fluorescence activated cell sorter (FACS) with multi-parameter analyses to study events in real-time. Using these methods, we recently discovered a Ca2+/H+ membrane channel in human neutrophils that plays a role in the maintenance of cellular pH — a property that appears to be important in controlling the cellular response to inflammatory stimuli. A new 3-laser MoFLO FACS permits study of up to 7 parameters, at stationary points or kinetically, in real time, simultaneously in single cells.
- Redox regulation of HIV replication in alveolar macrophages
- Role of IL-16 in alveolar macrophages and HIV disease
- HIV-induced impairment of alveolar macrophage fungicidal activity
- Control of the phagosomal environment in human phagocytic cells, and subversion of normal processes by specific mycobacterial pathogens
- Human neutrophil responses to mediators of inflammation
The HIV group has extensive experience in working with primary human cells obtained by bronchoscopy of volunteers under a institutionally approved IRB protocol. We utilize a model of in vitro HIV-1 infection of human alveolar macrophages as starting point for many of our experiments. We employ a number of cell and molecular biology techniques to study this system. Methods include flow cytometry, ELISA, immunobloting, electromobility shift assays, fluorescent reporters of reactive oxygen species, and QRTPCR. We are currently collaborating with the Kotton Lab to study the use of lentiviral driven protein expression in alveolar macrophages.
The Flow Cytometry group has more than 20 years’ experience developing and utilizing novel methods of flow cytometric and fluorescence image analysis. Dr Simons has extensive experience in designing and fluorescence probes, and our instrumentation is “open platform” in design, permitting user-developed adaptations of hardware to be utilized to answer research questions.
Principal Investigators and collaborators:
- John Bernardo, M.D.
- Elizabeth Simons, Ph.D.
- Michael Ieong, M.D.
- Marlynne Quig-Nicol, Ph.D.
- Paul Skolnik, M.D.
- Gregory Viglianti, Ph.D.
- Jens Herrmann, D.M.D
- Jeremy Richards, M.D.
- Heidi Long, B.S.
- Lehka Mikkilineni, B.S.
- Ieong MH, Farber HW. Non-infectious Pulmonary Complications of HIV-infection. (In press)
- Herrmann, J.M., Kantarci, A., Long, H., Bernardo, J., Hasturk, H., Wray, L.V., Jr., Simons, E.R., Van Dyke, T.E. Simultaneous measurements of cytoplasmic Ca2+ responses and intracellular pH in neutrophils of localized aggressive periodontitis (LAP) patients. J Leukoc Biol 2005 78: 612-619.
- Bernardo, J., Hartlaub, H., Yu, X., Long, H., Simons, E.R. Immune complex stimulation of human neutrophils involves a novel Ca2+/H+ exchanger that participates in the regulation of cytoplasmic pH: flow cytometric analysis of Ca2+/pH responses by subpopulations. J Leukoc Biol 2002 72: 1172-1179.
- Ieong MH, Reardon CC, Levitz SM, Kornfeld H. HIV-1 Infection of Alveolar Macrophages Impairs their Innate Fungicidal Activity. Am J Resp Crit Care Med 2000; 162 (3 Pt 1): 966-70.