Extracellular Vesicles in Pathogenesis of Human Tissue
- Leonid Margolis, PhD, Head, Section on Intercellular Interactions
- Christophe Vanpouille, PhD, Staff Scientist
- Anush Arakelyan, PhD, Staff Scientist
- Rogers Nahui Palomino, BA, Visiting Fellow
- Wendy Fitzgerald, BS, Technician
- Vincenzo Mercurio, MS, Predoctoral Visiting Fellow
Our 2019 activity focused on two interrelated projects regarding pathologic immune activation in tissues. Viruses, in particular HIV-1 and cytomegalovirus (CMV) are involved in the immune activation, leading to various diseases, in particular cardiovascular diseases.
Immune activation in tissues infected with HIV-1 and treated with antivirals
Immune activation is now considered to be a driving force of various human pathologies, including HIV-1 disease. Currently, antiretroviral therapy (ART) has proven to be efficient in suppressing HIV-1 replication. However, lengthy suppression of HIV-1 replication by ART is associated with an increased risk of complications, including neurological and cardiovascular diseases, which appear to be related to the residual immune activation in patients undergoing ART. Cytokines may play an important role in the residual immune activation. Earlier, we found that cytokines, which are generally considered to be classical soluble immune-regulating molecules, can be associated with extracellular vesicles (EVs). In this form, they can be delivered to (target) cells and elicit cellular responses. We found that the spectra of EV–associated cytokines in HIV-1–infected tissues is different from that of soluble cytokines. We focused on both soluble and EV–associated cytokines that become up-regulated in HIV infection in human tissues ex vivo. After HIV-1 is fully suppressed by antiviral drugs, cytokines remain upregulated, with EV–associated cytokines more likely to be elevated than soluble ones. Similarly, cytokines are upregulated in myocardial infarction, and EV–associated cytokines form a distinct group regulated differently from soluble cytokines. The findings indicate a physiological role of the EV–associated cytokines distinct from that of soluble molecules.
While ART efficiently suppresses viral replication in infected individuals, various diseases develop years earlier than in the control population. Moreover, some infected individuals with total suppression of HIV replication fail to fully restore their immune system (immune non-responder [INR]); in particular, they have an inability to reconstitute the CD4+ T cell pool after antiretroviral therapy. General residual immune activation seems to be involved in these pathologies. Understanding the mechanisms of these phenomena requires the development of ex vivo models in which these mechanisms can be studied under controlled laboratory conditions. We developed a model of human tissues infected ex vivo with HIV-1 and investigated immune activation after HIV-1 was suppressed by antivirals. Also, we identified mitochondrial defects in the INRs that may contribute to their failure to reconstitute CD4+ T cells.
Immune activation in cardiovascular disease: the role of CMV (cytomegalovirus)
Various human diseases have immune activation as a common denominator; myocardial infarction is one such diseases. In blood, immune activation is associated with activation of various cells, in particular, platelets that release vesicles upon activation. We studied this phenomenon and found that these EVs lead to aggregation of monocytes with platelets or with vesicles released by platelets. Aggregation of monocytes with platelets and platelet-derived EVs may represent an early marker of disease progression. It remains to be determined what triggers such persistent immune activation. Earlier, we found that the development of unstable angina is associated with the presence of CMV RNA in blood. We further studied the role of this virus in immune activation and the development of atherosclerosis. We found that productive CMV infection negatively correlates with endothelial function in myocardial infarction. The results further implicate CMV in cardiovascular disease.
HIV-1 pathogenesis and immune activation in tissue treated with antiretroviral therapy
To investigate the role that cytokines play in residual immune activation, it is necessary to develop an ex vivo laboratory-controlled system reflecting what happens in vivo. As an experimental model, we used ex vivo human lymphoid tissues, where critical events in HIV-1 infection occur in vivo. We evaluated concentrations of 33 cytokines released by donor-matched human lymphoid tissues ex vivo productively infected with HIV-1 over 16 days of infection and treated or not with the antivirals. We evaluated concentrations of soluble and EV–associated cytokines separately.
Soluble and EV–associated cytokines in HIV infection of human tissues ex vivo.
Specifically, we found that two strains of HIV-1 (R5 and X4) efficiently replicated in tissues and triggered an upregulation of numerous soluble cytokines as early as day three post infection. Some of the cytokines are typical of the acute TH1 (T helper cell 1) response, which protects against intracellular pathogens. Evaluation of EV–associated cytokines demonstrated that some of the same cytokines upregulated in soluble form were also upregulated in EV–associated form. The beta-chemokines (MIP-1a, MIP-1b, and RANTES), in particular, were consistently upregulated throughout infection with both virus strains in soluble and EV–associated forms. However, several cytokines were uniquely upregulated in the EV form, particularly upon X4 infection. Also, in early HIV-1 infection, there was a significant increase in the percentage of the soluble chemokines RANTES and TNF-alpha compared with EV–associated cytokines. Additionally, RANTES significantly increased in the percentage of surface-associated EVs compared with internal EVs.
Persistent immune activation in ex vivo HIV–infected tissues under ART
We investigated whether the HIV-1–triggered immune activation is reduced when viral replication is suppressed. In particular, we demonstrated that ritonavir and AZT-3TC treatment of tissues efficiently suppressed viral replication (over 99% suppression for both treatments). Despite control of viral replication, cytokines remained upregulated after 13 days of ART treatment, and EV–associated cytokines were less likely to decrease than soluble ones. Also, X4 elicited stronger immune responses, as measured by increased soluble and EV–associated cytokines, particularly pro-inflammatory cytokines and the beta-chemokines compared with the R5 strain.
We identified mitochondrial defects in the INRs that may contribute to their failure to reconstitute CD4+ T cells. Thus, the phenomenon of residual immune activation after successful ART can be reproduced in a laboratory experimental system with human lymphoid tissue ex vivo, opening a way to study this phenomenon under controlled laboratory conditions. We showed that cells with the phenotype and transcriptional profile of Tregs (regulatory T cells) were enriched among cycling cells in health and in HIV infection. However, there were diminished frequencies and numbers of Tregs among cycling CD4+ T cells in INRs, and cycling CD4+ T cells from INR subjects displayed transcriptional profiles associated with the impaired development and maintenance of functional Tregs. Flow-cytometric assessment of TGF-beta activity confirmed the dysfunction of Tregs in INR subjects. Transcriptional profiling and flow cytometry revealed diminished mitochondrial fitness in Tregs among INRs, and cycling Tregs from INRs had low expression of the mitochondrial biogenesis regulators peroxisome proliferator activated receptor coactivator 1- (PGC1) and transcription factor A for mitochondria. In vitro exposure to the cytokine IL-15 (interleukin-15) allowed cells to complete division, restored the expression of PGC1, and regenerated mitochondrial fitness in the cycling Tregs of INRs. Our data suggest that rescuing mitochondrial function could correct the immune dysfunction characteristic of Tregs in INRs and enhance immune restoration in these subjects.
In conclusion, our analysis showed that HIV-1–infected lymphoid tissues ex vivo upregulated production of many cytokines, both free and EV–associated, and that the majority of the cytokines remained upregulated despite suppression of viral replication by ART. Also, in spite of ART, many patients do not reconstitute their immune system, in part owing to mitochondrial dysfunction. The mechanisms of both these phenomena can now be investigated under controlled laboratory conditions, which should result in the development of new therapeutic strategies.
Cytomegalovirus and EVs in atherosclerosis: immune activation
Acute cardiovascular syndrome (ACS) is associated with a general activation of the immune system, which includes activation of many cells, in particular monocytes and platelets, leading to destabilization and rupture of coronary atherosclerotic plaques and to acute myocardial infarction (AMI). Recently, it was found that immune activation is associated with the release of EVs by activated cells; the EVs mediate cell-cell communication and play an important role in immune activation. One of the manifestations of the activation of monocytes and platelets is the formation of monocyte-platelet complexes (MPCs).
We analyzed MPCs in vivo and in vitro and investigated the abilities of various monocyte subclasses to form MPCs, the characteristics of the cells and EVs involved in MPC formation, and MPC changes in AMI. We identified MPCs by co-staining for the platelet antigen CD41a and for the monocyte antigens CD14 and CD16. Platelet activation was evaluated by expression of phosphatidylserine (PS). Monocytes of some classes disproportionately formed MPCs: although classical monocytes (CD14++CD16–) constituted the majority, MPCs were preferentially formed by intermediate monocytes (CD14++CD16+). CD41a–positive events in MPCs exposed more PS than in the circulating monocytes. AMI was associated with a 50% increase in circulating monocytes and with a threefold increase in MPCs, in particular in those formed by classical monocytes. In AMI patients, MPCs formed by intermediate monocytes contained more CD41a–positive events than MPCs with other monocyte subsets, whereas in controls, MPCs formed by classical monocytes carried more platelets than other MPCs. The sizes of some of the CD41a+ events in MPCs were smaller than those of regular platelets and may represent platelet-derived EVs. Some of the aggregates seem to consist of monocytes and platelet-derived EVs. Binding of EVs to monocytes was confirmed in in vitro experiments when monocytes were co-incubated with platelet-generated EVs. There was association between complications of AMI and the increase in MPCs and their composition. Aggregation of EVs and platelets with monocytes in AMI patients is another manifestation of immune activation associated with atherosclerosis that plays an important role in this pathology and can be used as an AMI correlate.
However, it is not yet understood what causes the immune activation, given that, in general, the destabilization of atherosclerotic plaques and development of recurrent cardiovascular events are strongly associated with activation of the immune system. Perturbation of the T cell repertoire, with higher expression of effector memory and activation markers, was found not only in blood of patients with cardiovascular diseases but also within their atherosclerotic plaques. Herpes viruses are one of the main candidates causing persistent immunoactivation within plaques owing to their ubiquity and ability to cycle between dormancy and replication. Specifically, it has been shown that accumulation of intermediate and late-differentiated T effector memory cells in blood is associated with CMV. In our previous work, we showed that productive CMV infection is more common in patients with acute coronary syndrome than in patients with chronic coronary artery disease or in healthy volunteers. Moreover, we also found a positive correlation between the cytomegaloviral DNA load and T lymphocyte differentiation within the atherosclerotic plaques of patients with cardiovascular diseases.
We analyzed the presence of CMV DNA in plasma and endothelial function in 33 patients with ST-elevation myocardial infarction (STEMI) and 33 volunteers without cardiovascular diseases, using real-time polymerase chain reaction and a noninvasive test of flow-mediated dilation (FMD). We found that the presence of CMV DNA in plasma of STEMI patients was significantly higher than in volunteers without cardiovascular diseases. Also, we found a significant prevalence in the number of copies of CMV DNA in older hypertensive patients, indicating that one of the possible mechanisms of the development of hypertension in patients with CMV infection could be the development of endothelial dysfunction triggered by CMV; endothelial function plays an important role in patients with atherosclerosis. We addressed this issue by using FMD test given that CMV was shown earlier, and confirmed here, to be an independent risk factor of STEMI. Using this test, we demonstrated for the first time the negative correlation between productive CMV infection and endothelial function in patients with STEMI. In a multivariate analysis, we confirmed that the identified correlation persisted, regardless of sex, hypertension, CRP (C-reactive protein) level, or patient age, factors that were significantly associated with CMV production and endothelial function. In summary, we found that CMV infection is strongly associated with endothelial dysfunction in STEMI patients.
Although we found an association between CMV in plasma and endothelial dysfunction, it is possible that other pathogens, not studied in the present work, may also be associated with this pathological state. In general, our results support a hypothesis that acute coronary events are synchronized with CMV replication, and that this viral reactivation contributes to endothelial dysfunction. Therefore, it is conceivable that preventing the reactivation of CMV would enhance endothelial function and affect the outcome of coronary artery disease.
Additional Funding
- Office of AIDS Research (OAR), NIH, Intramural Award
Publications
- Lebedeva A, Maryukhnich E, Grivel JC, Vasilieva E, Margolis L, Shpektor A. Productive cytomegalovirus infection is associated with impaired endothelial function in ST-elevation myocardial infarction. Am J Med 2019;133(1):133-142.
- Margolis L, Sadovsky Y. The biology of extracellular vesicles: The known unknowns. PLoS Biol 2019;17(7):e3000363.
- Fitzgerald W, Freeman ML, Lederman MM, Vasilieva E, Romero R, Margolis L. A system of cytokines encapsulated in extracellular vesicles. Sci Rep 2018;8:8973.
- Fitzgerald W, Gomez-Lopez N, Erez O, Romero R, Margolis L. Extracellular vesicles generated by placental tissues ex vivo: a transport system for immune mediators and growth factors. Am J Reprod Immunol 2018;80:e12860.
- Zicari S, Arakelyan A, Palomino R, Fitzgerald W, Vanpouille C, Lebedeva A, Schmitt A, Bomsel M, Britt W, Margolis L. Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins. Virology 2018;524:97-105.
- Younes S-A, Talla A, Ribeiro SP, Saidakova EV, Korolevskaya LB, Shmagel K, Shive CL, Freeman ML, Panigrahi S, Zweig S, Balderas R, Margolis L, Douek DC, Anthony DD, Pandiyan P, Cameron M, Sieg SF, Calabrese LH, Rodriguez B, Lederman MM. Cycling CD4+ T cells in HIV-infected immune nonresponders have mitochondrial dysfunction. J Clin Invest 2018;128(11):5083-5094.
Collaborators
- Leonid Chernomordik, PhD, Section on Membrane Biology, NICHD, Bethesda, MD
- Sara Gianella Weibel, MD, University of California San Diego, La Jolla, CA
- Sergey Kochetkov, PhD, Engelhard Institute of Molecular Biology, Moscow, Russia
- Michael Lederman, MD, Case Western University, Cleveland, OH
- David D. Roberts, PhD, Laboratory of Pathology, Center for Cancer Research, NCI, Bethesda, MD
- Roberto Romero-Galue, MD, DMedSci, Perinatology Research Branch, NICHD, Detroit, MI
- Alexandr Shpektor, MD, Moscow Medical University, Moscow, Russia
- Elena Vasilieva, MD, Moscow Medical University, Moscow, Russia
- Beatrice Vitali, PhD, Università di Bologna, Bologna, Italy
Contact
For more information, email margolis@helix.nih.gov or visit http://irp.nih.gov/pi/leonid-margolis.
