Pathogenesis of HIV-1 and Its Copathogens in Human Tissues
- Leonid Margolis, PhD, Head, Section on Intercellular Interactions
- Christophe Vanpouille, PhD, Staff Scientist
- Anush Arakelyan, PhD, Staff Scientist
- Rogers Nahui Palomino, BA, Visiting Fellow
- Victor Silva, PhD, Visiting Fellow
- Sonia Zicary, PhD, Visiting Fellow
- Wendy Fitzgerald, BS, Technician
The general goal of the Section of Intercellular Interactions is to understand the mechanisms of pathogenesis of various pathogens in human tissues, including the human immunodeficiency virus (HIV). Given that the critical events of this pathogenesis occur in tissues and are thus difficult to study in vivo, we used for our study a system of human tissues ex vivo, developed in our Section and now adopted by many investigators, to study viral infections and to test antivirals.
During the past year, we focused on three aims, to: (1) investigate pathogens' interactions mediated by cytokine network; (2) study HIV-1 infectivity in human tissue by analyzing individual virions' individual spikes, that mediate virus-cell fusion; (3) extend our flow-virometry technology developed for HIV-1 analysis to other viruses, in particular to Dengue virus; and (4) investigate whether the immuno-activation that we demonstrated to be the consequence of HIV-1 infection plays a role in other human diseases. The research conducted during the last year provided new insights into general mechanisms of the interaction of human pathogens with the infected host, leading to new concepts in therapeutic strategies. We investigated different modes of HIV transmission in human tissue ex vivo. In particular, we compared the transmission of cell-free HIV with that of HIV adsorbed at the surface of seminal cells such as monocytes and lymphocytes in a continuing project.
This year, we also continued our studies on viral pathogenesis using the nanotechnology we developed in 2015 for the analysis of individual viral particles. In particular, it became clear that together with viruses, infected cells release extracellular vesicles (EVs) that are similar in size and physical properties to viruses. We investigated the role of extracellular vesicles (EVs) in the pathogenesis of HIV and one of its major copathogens, cytomegalovirus (CMV). In particular, we identified the possible role of CMV (or EVs that carry CMV surface markers) in atherosclerosis, in particular in the destabilization of atherosclerotic plaques. We analyzed EVs in normal and in different pathologic conditions including HIV infection, acute coronary syndrome (ACS), and uveitis. We concluded that EVs may not only be a disease marker but may also mediate pathologies. Moreover, viruses and EVs have common features, including the transfer of cellular components from one cell to another.
Transmission of HIV from human leucocytes to cervico-vaginal tissue
The vast majority of new HIV infections occurs through sexual transmission, whereby HIV is transferred from the semen of an infected male to an uninfected partner. In semen, HIV-1 particles may exist as free-floating virions, inside infected cells, or attached to the surface of cells, whether they are infected or not. The majority of seminal cells in HIV–infected individuals carry HIV proviral DNA. We investigated whether cells carrying attached HIV on their surface can transmit infection. In particular, we investigated whether HIV attached at the surface of monocytes or lymphocytes could transmit infection to human tissue ex vivo. This latter system, developed in our laboratory, is close to in vivo and does not require artificial activation or stimulation. We irradiated monocytes and lymphocytes to prevent them from becoming infected and producing HIV, which then would act as a cell-free source of virus in our system. After washing off the unbound viruses, we quantified the amount of attached virus (by measuring p24) and incubated the cells carrying HIV on their surface with target T cells or with explants of human lymphoid tissue.
Even though comparable amounts of HIV were attached to monocytes and lymphocytes, the results of incubation of these cells with human lymphoid tissues was dramatically different. While monocytes readily transferred infection to human tissue, lymphocytes did not. Using chemotaxis chambers to physically separate irradiated monocytes carrying HIV on their surface from target cells, we found that cell-to-cell contacts were needed for effective HIV transfer.
In summary, our results show that infected and non-infected monocytes/macrophages (some of which would inevitably adsorb virus in HIV–containing semen) but not lymphocytes present in semen of HIV–infected men may adsorb virus and, together with productively infected cells, are able to transfer infection to mucosal tissues. Our results highlight the important role that seminal monocytes/macrophages may play in HIV transmission in vivo, especially given that monocytes/macrophages far outnumber lymphocytes in semen of HIV–infected individuals. Understanding the mechanisms of HIV-1 sexual transmission may help in designing new preventive strategies.
Cytomegalovirus and destabilization of atherosclerotic plaques
Atherosclerosis of coronary arteries, the major cause of acute coronary syndrome (ACS), may often progress for years and even decades and then suddenly result in the rupture of an atherosclerotic plaque, leading to myocardial infarction. Despite the long history of investigation, the triggers and mechanisms of such sudden ruptures remain largely unknown. However, it is well established that immuno-activation is associated with the destabilization of atherosclerotic plaques.
We previously found that both the spectrum of lymphocytes and their activation status in plaques are different from those in blood. In particular, plaques are enriched in CD8 and CD4 T lymphocytes expressing CD25 and HLA-DR, and in effector memory lymphocytes rather than naive lymphocytes. The lymphocyte phenotypes may indicate the presence of a specific antigen, possibly of the infectious agent, toward which the lymphocytes are reactive. Herpesviruses (HHV) have been thought to be associated with atherosclerosis. However, the reports supporting such an association are controversial and, in most cases, are based on serological evidence or on the presence of cell-associated HHV DNA, but they do not provide information about actual viral replication.
To evaluate productive infection, rather than focusing on HHV replication in particular cells, we investigated, by quantitative real-time PCR, the presence of HHV DNA of all eight types of HHV in blood samples from 71 patients with ACS, from 26 patients with stable coronary artery disease (SCAD), and from 53 healthy volunteers, as well as in atherosclerotic plaques of 22 patients with peripheral artery disease who underwent endarterectomy. The analysis revealed the presence of all eight HHV types in plasma of healthy controls and patients with ACS. HHV-5 (cytomegalovirus, CMV) was the only HHV the amount of which was higher in ACS patients than in the control group. We found that, for CMV, the DNA copy numbers in plasma positively correlated with ACS. There were no such correlations for other HHVs. With the development of our protocol of flow analysis of individual T cells in plaques, we were able to link the status of these T cells with CMV load. We demonstrated enrichment of intermediate-differentiation (CD27+/–CD28+), but not late-differentiated (CD27–CD28–) T effector memory (Tem) cells. Furthermore, there were fewer naive CD4 and CD8 T cells and fewer CD4 T central memory (Tcm) cells in plaques than in blood. Our results thus indicate that only plaques with a high amount of CMV (which we found to be associated with ACS) are characterized by high levels of T cell activation. It is therefore conceivable that CMV contributes to T cell activation and triggers the rupture of these plaques.
In summary, we found that, of all HHV levels, only CMV was higher in patients with stable coronary artery disease and acute coronary syndrome than in the healthy group and that its load correlated with the level of high-sensitivity C-reactive protein. The level of CMV in atherosclerotic plaques correlated with the state of immuno-activation of lymphocytes in plaques, suggesting that the reactivation of CMV may contribute to the immune activation associated with the progression of atherosclerosis. CMV is strongly associated with the atherosclerotic process and, in particular, with ACS. In general, our results support the hypothesis that acute coronary events are synchronized with acute viral replication of CMV, possibly because of its reactivation, and that this reactivation, which may occur both at the systemic level and in plaques, may contribute to plaque destabilization.
Extracellular vesicles in human pathologies
Membrane vesicles (extracellular vesicles, EVs) are released by almost all cells in vivo and ex vivo. The most abundant type of EV, often called "exosomes," are lipid bilayer–enclosed entities of 50 to 100 nm containing proteins and RNA. Initially, EVs were considered to be ‘cellular dust’ or garbage and did not attract much scientific attention. However, it was recently found that EVs have an important biological function and that, in both structural and functional aspects, they resemble viruses. Published data indicate that EVs share with viruses an important function, namely delivering bioactive material from one cell to another. Therefore, the composition of EVs reflects properties of the cells that release them, can be an indicator of pathological alteration, and may play a role in the disease progression. We tested this hypothesis in two models. (1) We compared EVs in blood of healthy volunteers with those in blood of patients with acute coronary syndrome (ACS); and (2) we investigated the effect on lymphocytes and monocytes of EVs released by normal retinal cells and retinal cells treated with pro-inflammatory cytokines, which simulate the conditions typical for uveitis.
EVs in patients with ACS. Multiple bulk analysis studies report that the total amounts of EVs are associated with cardiovascular diseases. These bulk analyses do not reflect the individual characteristics of EVs that are released by cells of different types and activation status. Theoretically, analysis of individual EVs could reflect the pathological states of the cells of their origin, provided that they carry cellular antigens by which they can be traced to these particular cells. Using our new nanotechnology-based flow analysis, we analyzed the antigenic composition of individual EVs isolated from the blood of healthy volunteers and from the blood of patients with acute forms of coronary artery disease, in particular myocardial infarction. Plasma EVs were captured with 15-nm magnetic nanoparticles coupled to antibodies against EVs. We chose a set of antibodies specific for antigens that, in different combinations, characterize platelet- and endothelium-derived EVs, namely CD31, CD41a, CD62E, CD63, CD105, and CD146, and we evaluated their presence on EVs isolated from blood. The total amounts of EVs were higher in the ACS patients than in the controls, predominantly owing to patients with acute myocardial infarction. For all captured fractions, the differences in the EV amounts were restricted to EVs expressing CD41a+, a platelet marker. The increase in the numbers of these EVs in the ACS patients probably reflects platelet activation and may indicate disease progression. Future studies should reveal whether the increase in the number of platelet-derived EVs precedes other symptoms of ACS; if so, this increase may be one of the early indicators of the disease and may lead to the development of new diagnostic tools.
EVs released by human retinal cells. Retinal pigment epithelium (RPE) cells are critical to the normal function of overlying photoreceptors and hence for normal vision. In the steady state, RPE cells possess immunosuppressive properties and presumably contribute to the inhibition of inflammatory damage to the retina. RPE cells may become damaged during degenerative or inflammatory eye diseases. Recently, RPE cells were shown to release EVs, considered to be important for intercellular communication. Using the ARPE-19 cell line, we investigated whether EVs released from RPE cells, either in homeostatic or inflammatory environments, alter T cell responses and monocyte phenotype in vitro. In particular, we compared the size, concentration, and immunomodulatory function of EVs released by RPE, stimulated with the inflammatory cytokines IL-1beta, IFN-gamma, and TNF-alpha, with those released by nonstimulated RPE. Analysis by flow cytometry using human-specific antibodies revealed an approximately 3-fold higher concentration of EVs released from cytokine-stimulated RPE than from nonstimulated controls. EVs from both resting and cytokine-stimulated RPE inhibited the proliferation of T cells in peripheral blood mononuclear cell (PBMC) cultures. Importantly, RPE–derived EVs did not induce T cell death.
We next tested the ability of RPE–derived EVs to alter the phenotype and activation status of normal human monocytes. Human monocytes exposed to EVs released by nonstimulated RPE cells resulted in an enrichment of the CD14++CD16+ intermediate monocyte population without affecting monocyte viability. The findings suggest that resting ARPE-19 cells, which are known to possess immunosuppressive properties, may use EVs to mediate these functions. This may represent one mechanism by which RPE cells maintain a homeostatic immunosuppressive environment to protect neighboring RPE as well as adjacent photoreceptors from inflammatory damage. However, incubation of human monocytes with EV released by cytokine-stimulated ARPE-19 cells led to monocyte cell death and upregulation of several proinflammatory cytokines. We hypothesize that the EVs released by RPE cells exposed to inflammatory conditions are modified to express proapoptotic molecules that may reduce the number of infiltrating immune cells in an attempt to abrogate immune-mediated damage to surrounding RPE cells and photoreceptors. Taken together, our results indicate that RPE cells release EVs that suppress immune responses under both resting and inflammatory conditions. Under resting conditions, RPE cells constitutively release EVs that inhibit T cell proliferation and render monocytes immunosuppressive without affecting their survival. Under inflammatory conditions, RPE release EVs that inhibit T cell proliferation without affecting T cell viability but that induce monocyte cell death, perhaps to dampen an actively destructive immune response. The results suggest that EVs secretion may be an important mechanism used by RPE cells to exert their known immunomodulatory effects.
Additional Funding
- Office of AIDS Research (OAR), NIH, Intramural Award
Publications
- Barreto-de-Souza V, Arakelyan A, Zicari S, Margolis L, Vanpouille C. Monocytes but not lymphocytes carrying HIV-1 on their surface transmit infection to human tissue ex vivo. J Virology 2016;21:9833-9840.
- Vanpouille C, Introini A, Morris SR, Margolis L, Daar ES, Dube MP, Little SJ, Smith DM, Lisco A, Gianella S. Distinct cytokine/chemokine network in semen and blood characterize different stages of HIV infection. AIDS 2016;30:193-201.
- Zicari S, Arakelyan A, Fitzgerald W, Zaitseva E, Chernomordik LV, Margolis L, Grivel JC. Evaluation of the maturation of individual Dengue virions with flow virometry. Virology 2016;488:20-27.
- Vagida M, Arakelyan A, Lebedeva A, Grivel J, Shpektor A, Vasilieva E, Margolis L. Flow analysis of individual blood extracellular vesicles in acute coronary syndrome. Platelets 2016;4:382-391.
- Nikitskaya E, Lebedeva A, Ivanova O, Maryukhnich E, Shpektor A, Grivel JC, Margolis L, Vasilieva E. Cytomegalovirus-productive infection is associated with acute coronary syndrome. J Am Heart Assoc 2016;5(8):e003759.
- Knickelbein JE, Liu B, Arakelyan A, Zicari S, Hannes S, Chen P, Li Z, Grivel JC, Chaigne-Delalande B, Sen HN, Margolis L, Nussenblatt RB. Modulation of immune responses by extracellular vesicles from retinal pigment epithelium. Invest Ophthalmol Vis Sci 2016;57:4101-4107.
- Nolte-'t Hoen E, Cremer T, Gallo RC, Margolis LB. Extracellular vesicles and viruses: are they close relatives? Proc Natl Acad Sci USA 2016;113:9155-9161.
Collaborators
- Robert Gallo, MD, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD
- Jean-Charles Grivel, PhD, Sidra Medical and Research Center, Doha, Qatar
- Michael Lederman, MD, Case Western University, Cleveland, OH
- Esther Nolte-'t Hoen, PhD, Utrecht University, Utrecht, The Netherlands
- Robert B. Nussenblatt, MD, MPH, Laboratory of Immunology, NEI, Bethesda, MD
- Robin Shattock, PhD, St. George's Hospital Medical School, University of London, UK
- Alexandr Shpektor, MD, Moscow Medical University, Moscow, Russia
- Elena Vasilieva, MD, Moscow Medical University, Moscow, Russia
Contact
For more information, email margolis@helix.nih.gov or visit irp.nih.gov/pi/leonid-margolis.
