Immune Activation and Viral Pathogenesis
- Leonid Margolis, PhD, Head, Section on Intercellular Interactions
- Christophe Vanpouille, PhD, Staff Scientist
- Wendy Fitzgerald, BS, Biologist
The general goal of the Section is to understand the mechanisms of pathogenesis of human pathogens in the context of complex tissue microenvironments. In particular, we focused on the role of cytokines in viral pathogenesis. Our studies include extracellular vesicles (EVs) that mediate cell-cell communications and can carry cytokines. We found that EVs, including ones that carry cytokines, are altered in women experiencing pregnancy complications and can be used as biomarkers of such pathologies. Also, EVs in people with psycho-neurological symptom clusters are different from control ones. Both free cytokines and EV–associated cytokines are associated with the pattern of HIV infection and may determine the distinct course of HIV disease in women living with HIV undergoing anti-retroviral therapy. Our current studies are designed according to NICHD Strategic goals on reproductive health, healthy pregnancies, life-long wellness, and gender-based health disparities.
Cytokines in HIV disease
Among the determinants of an effective pathogen immune responses, to viruses in particular, the complex network of cytokines plays an important role. Cytokines are protein messengers of intercellular communication between multiple immune and nonimmune cells. They are part of the language used by the innate and adaptive immune system to orchestrate an effective immune response to infectious pathogens. Although antiretroviral therapy (ART) efficiently suppresses HIV replication to undetectable levels, it does not fully restore immune functions. We investigated whether ART differentially affects the cytokine network in different body compartments. In addition to cytokines in blood plasma, our study focused on cytokines in semen, given that HIV transmission mostly occurs through sexual intercourse. We characterized the cytokine network in blood and semen in individuals longitudinally sampled before they began ART and after achieving suppression of HIV RNA, by measuring the concentrations of 34 cytokine/chemokines, using a multiplex bead-based assay. We used Partial Least Squares Discriminant Analysis (PLS-DA) to visualize the differences in cytokine patterns between the time points. Cytokines with VIP (variable importance in projection) scores (which reflect the difference in cytokines before and after antiretroviral therapy) exceeding 1 were deemed important for predicting suppression status and were subsequently tested using Wilcoxon Signed Rank Tests. While PLS-DA projections in blood were fairly similar before and after viral suppression, they were very different in semen before and after ART. When tested individually, four cytokines were significantly different across time points in semen (MIG, IL-15, IL-7, I-TAC), and two in blood (MIG and IP-10). Our results identified specific changes in the cytokine networks in semen and blood as the immune system acclimates to chronic, suppressed HIV infection.
Distinct biological manifestations of sex-based differences have been described in people living with HIV. Women with HIV have lower plasma viral loads, especially during the early phases of infection, and a 1.6‑fold higher risk of developing AIDS when accounting for viral load levels in chronic infection. Substantial differences in immune activation between men and women with HIV have been described, but the reasons for these differences have not been fully elucidated. One important factor may be the cytokine response evoked during infection. We investigated the sex differences in blood plasma of men and women with chronic HIV on suppressive ART, using Luminex, and investigated whether cytokines contribute to maintaining higher immune activation in women despite suppressive therapy with ART. We identified five cytokines that were significantly higher in women than in men, namely the pro-inflammatory chemokines CXCL1 (Gro-α), CCL5 (RANTES), CCL3 (MIP-1α), CCL4 (MIP-1β), as well as the T cell homeostatic factor IL-7. Thus, inflammatory cytokines remained higher in virologically suppressed women than in men living with HIV, suggesting that cytokines contribute to maintaining higher immune activation in women despite suppressive therapy with ART. Moreover, CCL3, CCL4, and CCL5, which are β chemokines that share the same receptor, CCR5, have been identified as strong HIV–suppressive factors produced by CD8 T cells. This observation could provide a possible explanation for the differences of viral loads previously reported between women and men. Overall, our results are consistent with the higher cellular activation observed in women. More studies are needed to better understand the underlying mechanisms that contribute to sex-based immune-cell regulation in people with HIV. Our study, together with others, reveals the importance of sex/gender–specific studies, which too often remain a neglected area of viral pathogenesis.
Although ART dramatically reduces HIV viral load and improves the life span and quality of life for most patients, people living with HIV are more likely to develop serious non–AIDS comorbidities, in part because of recurrent immune activation. The drivers of recurrent immune activation in both men and women living with HIV under ART remain to be fully understood. Cytomegalovirus (CMV) is considered to be one of the driving forces of persistent immune activation. Although the associations between CMV replication and systemic inflammation in people living with HIV during suppressive ART have been well documented, it is not clear whether CMV replication is associated with systemic immune activation already during the earliest phase of untreated HIV infection, and thus a potential target for early CMV intervention. We investigated whether genital CMV shedding contributed to systemic immune activation, as evaluated by the concentration of 34 blood cytokines in people living with HIV in the acute/early phase of HIV infection. Independent of CMV, we found that the concentrations of the chemokines IP-10, MIG, MCP-1, I-TAC 10, IL-16, and MIP-1β were modulated in the earliest phase of HIV infection compared with control individuals without HIV. In people with HIV, there was no difference in blood cytokines among CMV shedders vs non-shedders. Our results suggest that CMV shedding in the male genital tract is not the main driver of systemic immune activation in the early phase of HIV infection, in contrast with the later phase of HIV infection. Early ART initiation should remain the priority. Similar studies in women should reveal whether there is a sex-related difference in the role of CMV in early infection. Whatever the role of CMV is in HIV acquisition, our results on cytokine distribution may already explain why women progress faster to AIDS than men at a given viral load.
Extracellular vesicles as markers of complicated pregnancies
Extracellular vesicles (EVs) are released by all cells of the human body and report on the physiology of their cells of origin. Therefore, EVs can, in principle, report on the pathologic development in the organism. In particular, EVs can serve as biomarkers in complicated pregnancies. We found that the composition of EVs in maternal blood is significantly changed in the case of fetal death. Preliminary results found that 16 soluble cytokines, growth factors, and angiogenic factors are significantly different between maternal blood plasma of mothers experiencing fetal death and age-matched mothers without complications. Eight proteins were significantly different in the EV fractions, many of which were the same as the significant soluble proteins, but one protein, CD163, was significantly downregulated only in EV–associated form. Ongoing analysis will determine whether the EV–associated cytokines improve the prediction accuracy of poor outcomes, and future functional studies will focus on determining whether these EV–associated cytokines play distinct roles in pathology.
The cause of fetal death is not always known, but viral infection is a major cause of fetal mortality, in particular CMV infection. Congenital CMV (cCMV) infection is the most common congenital infection and is often associated with severe neurological disabilities or perinatal death. The prediction of neonatal status in the case of CMV infection is limited and therefore the identification of new prognostic markers in amniotic fluid that is sampled by amniocentesis for the diagnosis of fetal infection could improve timely prenatal assessment of infected fetuses. Among biological processes involved in the innate immunity, many cytokines are involved in the immune control of cCMV infection in fetuses. Earlier, we reported that EVs carry cytokines and that these cytokine-carrying EVs constitute a system of cytokine delivery to particular cells. EVs are present in many biological fluids, including amniotic fluid.
We studied whether cytokines measured in the amniotic fluid, in particular cytokines associated with EVs, can report on CMV infection in pregnancy. The study enrolled 80 pairs of women and fetuses/newborns, including 40 infected fetuses and 40 negative controls. Our data suggest that cCMV infection and related symptoms at birth are associated with changes in the immunological signature of the amniotic fluid. Four soluble pro-inflammatory mediators (IP-10, IL-18, ITAC, and TRAIL) and one mediated by EV (IP-10) were elevated in the case of cCMV infection. Among these proteins, five were related to symptoms at birth (IP-10 internal, IP-10 surface, IP-10 soluble, IL-18 soluble, and TRAIL soluble). Seven other cytokines, not related to cCMV infection, were significantly associated with symptomatic status at birth; therefore, a pattern for severe infection can be related with a specific increase in the presence and concentration of six mediators (IL-18 soluble, TRAIL soluble, CRP soluble, TRAIL surface, MIG internal, and RANTES internal). Thus, our data suggest that cCMV infection and its severity are associated with differential expression of cytokines, in particular EV–associated ones, in amniotic fluid at mid-gestation, and may thus serve as candidate biomarkers of severity in case of fetal infection diagnosed by CMV–PCR. Considering trafficking of EVs, EV–associated cytokines may also prove to be promising biomarkers in maternal blood, allowing less invasive tests than amniocentesis.
Extracellular vesicles as markers of psychoneurological symptom clusters
We investigated the associations between EV–associated and soluble cytokines with immune markers and symptom clusters in men with non-metastatic prostate cancer and women with breast cancer. Because extracellular vesicle EV–associated cytokines, both encapsulated and surface bound, have been associated with symptom severity, and may vary over the lifespan, they may be potential biomarkers to uncover underlying mechanisms of various conditions.
Psycho-neurological symptom clusters are co-occurring and interrelated physiological symptoms that may include cancer-related fatigue, pain, depressive symptoms, cognitive disturbances, and sleep disturbances. It is hypothesized that these symptoms share a common systemic pro-inflammatory etiology. We investigated the associations between extracellular vesicle EV–associated and soluble cytokines with immune markers and symptom clusters in men with non-metastatic prostate cancer. Our observational study included 40 men with non-metastatic prostate cancer at the start (T1) of external beam radiation therapy (EBRT) and three months post treatment (T2), as well as 20 men with non-metastatic prostate cancer on active surveillance (AS) seen at one time point.
Both EV–associated and soluble forms of the chemokine RANTES significantly correlated with the symptom cluster for EBRT at T1, whereas, at T2, soluble IFNα2, IL-9, and IL-17 correlated with the corresponding symptom cluster. For the AS group, soluble survivin (an inhibitor of apoptosis highly expressed in most cancers) correlated with psycho-neurological symptoms. Linking specific inflammatory cytokines with psycho-neurological symptom clusters in men receiving prostate cancer treatment can enhance our understanding of the underlying mechanisms of this phenomenon and aid in developing targeted interventions.
We also investigated the associations of soluble and EV–associated cytokine concentrations with distinct symptom profiles reported by 290 women with breast cancer. Patients were classified into older (60 years or more, n = 93) and younger (less than 60 years, n = 197) cohorts within two previously identified distinct symptom-severity profiles, which included pain, depressive symptoms, sleep disturbance, and fatigue (i.e., High Fatigue Low Pain and All Low). Results of this study suggest that levels of cytokine concentrations differ between EV and soluble fractions. Several EV and soluble pro-inflammatory cytokines had positive associations with depressive symptoms and fatigue within both age cohorts and symptom profiles. In addition, in the older cohort with the High Fatigue Low Pain symptom profile, EV GM-CSF (a white cell growth factor) concentrations were significantly higher than for the All Low symptom profile. These exploratory analyses provide new information on the association between cytokines and symptom profiles of older and younger cohorts. We found unique EV–associated cytokines in older patients and in specific symptom classes. These results suggest that EVs are potential biomarker discovery tools, as well as underlying distinct symptom class profiles, and may thus inform intervention trials and offer precision-medicine approaches.
Additional Funding
- NICHD Director Award
Publications
- Ñahui-Palomino RA, Vanpouille C, Costantini PE, Margolis L. Microbiota–host communications: bacterial extracellular vesicles as a common language. PLoS Pathog 2021 17(5):e1009508.
- Mercurio V, Fitzgerald W, Vanpouille C, Molodtsov I, Margolis L. Mechanisms of residual immune activation in HIV-1-infected human lymphoid tissue ex vivo. AIDS 2021 35:1179.
- Sass D, Saligan L, Fitzgerald W, Berger AM, Torres I, Barb JJ, Kupzyk K, Margolis L. Extracellular vesicle associated and soluble immune marker profiles of psychoneurological symptom clusters in men with prostate cancer: an exploratory study. Translat Psychiatry 2021 11:440.
- Vanpouille C, Wells A, Wilkin T, Mathad JS, Morris S, Margolis L, Gianella S. Sex differences in cytokine profiles during suppressive antiretroviral therapy. AIDS 2022 36:1215.
- Rawlings SA, Torres F, Wells A, Lisco A, Fitzgerald W, Margolis L, Gianella S, Vanpouille C. Effect of HIV suppression on the cytokine network in blood and seminal plasma. AIDS 2022 36:621.
Collaborators
- Michael Bukrinsky, MD, PhD, George Washington University, Washington, DC
- Leonid Chernomordik, PhD, Section on Membrane Biology, NICHD, Bethesda, MD
- Sara Gianella Weibel, MD, University of California San Diego, La Jolla, CA
- Michael Lederman, MD, Case Western University, Cleveland, OH
- Eva Povedra, PhD, Galicia Sur Health Research Institute, Vigo, Spain
- Roberto Romero, MD, DMedSci, Perinatology Research Branch, NICHD, Detroit, MI
- Leorey Saligan, PhD, RN, CRNP, FAAN, Symptom Biology Unit, NINR, Bethesda, MD
- Yves Ville, MD, Paris Descartes University, Paris, France
- Beatrice Vitali, PhD, Università di Bologna, Bologna, Italy
Contact
For more information, email margolis@helix.nih.gov.
