- Pathogenesis of sexual human immunodeficiency virus (HIV) transmission
- Immunoregulatory functions of extracellular microvesicles and their RNA cargo in human semen
- Development and toxicity evaluation of microbicides (antiviral drugs that are applied topically to the mucosa to prevent sexual HIV transmission)
- Application of systems biology tools to understand mucosal physiology and pathology
- Impact of nucleoside reverse transcriptase inhibitor drugs on HIV latency
- Curing HIV infection by curbing the proliferation of cells latently infected with HIV
- Optimization and standardization of mucosal sampling techniques and cryopreservation
- Role of herpes simplex virus (HSV) infection in HIV latency
- Effects of progestin-containing contraceptives on HIV risk
- Discovering biomarkers of placental disease and premature birth
- Impact of opioid use on regulatory RNA molecules in human semen
Pathogenesis of sexual human immunodeficiency virus (HIV) transmission
For over a decade, a major focus of our work has been to understand how HIV is transmitted across the human mucosal linings and how the mucosal immune system reacts to the virus. We use human mucosal tissues from surgeries to model HIV infection ex vivo. We were the first to isolate viable dendritic cells from the female genital tract and test their role in HIV transmission. We discovered the importance of specialized dendritic cells in the genital mucosa, intraepithelial Langerhans cells [ILC 1] [ILC 2] , which hide the virus like a Trojan horse and sneakily pass it on to T lymphocytes when the two cell types interact. More recently, we found that HIV target cells in the large intestine enrich toward the rectum.
Infectious synapse formation between a vaginal Langerhans cell and a T cell. A Langerhans cell (large red cell) was imaged in situ within the outer epithelium of the human vagina by confocal microscopy. A T lymphocyte (small red cell) binds to it. The left and the right pictures show different sections of the same confocal image. HIV-1 (green) is seen engulfed within the Langerhans cell (left image, cell is visually sliced through its center) and concentrated near the contact zone between the two cells (right image). Enrichment of HIV in the contact zone is called an “infectious synapse”.
CCR5 expression of Langerhans cells (CD1a+) and T cells (CD3+) infiltrating the squamous epithelium of the vagina. The CD3 label is shown in green, CCR5 in red and CD1a in blue. Yellow signifies co-expression of CD3 and CCR5. Pink signifies co-expression of CD1a and CCR5. Reflection-enhanced backscatter was used to visualize overall tissue structure (grey).
M. Juliana McElrath, Seattle
Immunoregulatory functions of extracellular microvesicles and their RNA cargo in human semen
While using electron microscopy to study mucosal fluids and HIV, we observed an intriguing phenomenon: several HIV virions in a rosette around a microvesicle. This prompted our interest in extracellular microvesicles, which are produced by many cell types, and are abundant in semen, with trillions in each ejaculation. We have since shown that these vesicles are rapidly taken up by antigen-presenting cells and carry a distinct repertoire of small regulatory RNA molecules. We hypothesize that the microvesicles in semen interact with HIV, changing the transmissibility of the virus, and depress immune responses to HIV in the recipient partner.
HIV “rosetting” around a mucosal microvesicle. This electron microscopic discovery initiated our attention on microvesicles in semen.
Entry of seminal microvesicles into vaginal Langerhans cells (LCs). Vaginal LCs (large green cell with large blue nucleus) efficiently internalize microvesicles transported in human semen (red particles). An adjacent T cell (small blue nucleus) does not internalize the microvesicles.
Distribution of small regulatory exRNA species in seminal microvesicles
For more detailed information see our recent paper in Nucleic Acids Research.
Our work on extracellular microvesicles in semen was funded by R21 AI095023, and just received NIH/NIDA R01 DA040386-01 (2015 – 2020). We are also participating in the NIH extracellular RNA consortium as collaborators on U01 OD019746 (2014 – 2019).
Development and toxicity evaluation of microbicides (antiviral drugs that are applied topically to the mucosa to prevent sexual HIV transmission)
Collaborating with the Microbicide Trials Network (MTN) since its inception in 2006, we have helped develop mucosal explant models to assess candidate drugs for their antiviral efficacy and potential side effects on the mucosa. Our recent publication in eLife reports the first in vivo, systems biology evaluation of topical tenofovir gel, which is currently the most clinically advanced microbicide. We uncovered tenofovir’s broad and largely unexpected effects on the mucosa, raising potential safety concerns for its longer-term use. This work led to R01 AI116292, where we are using systems biology tools to broadly assess the side effects of topical microbicides on the human mucosa and developing new transgenic mouse models to expediently predict the potential of microbicide carcinogenicity.
Interleukin 10 (IL-10) protein (brown) in columnar epithelial cells of the human rectum in vivo before (left image) and after 7 daily topical tenofovir 1% gel applications (right image). Strong inhibition of IL-10 production by tenofovir 1% gel was confirmed by gene expression (PCR) and soluble protein (ELISA) assays.
Application of systems biology tools to understand mucosal physiology and pathology
We use broad systems biology approaches to understand physiological and pathological processes in the mucosa, at both the RNA and protein levels.
Effects of tenofovir 1% gel treatment on gene expression in the human rectum. Genes depicted in red were induced by the treatment, genes depicted in green were inhibited. Cellular compartments are indicated (CP, cytoplasm; PM, plasma membrane). The majority of genes encoding for proteins that locate to the cell nucleus were inhibited (lower two thirds of the figure). This included many transcription factors. The reverse was seen for genes encoding for extracellular proteins – these were mostly induced (upper third of the figure). These included a number of chemokines. A notable exception in the extracellular space was the anti-inflammatory cytokine interleukin 10, which was strongly inhibited by tenofovir. Direct (solid lines) and indirect (dashed) interactions between gene products are indicated. Line colors are arbitrary and indicate relationships between groups of genes. For more detailed information see our recent publication in eLife.
Impact of nucleoside reverse transcriptase inhibitor drugs on HIV latency
Our finding that tenofovir 1% gel has broad effects on the human mucosa could have ramifications reaching beyond the microbicide field. Tenofovir and other NRTIs have strong antiviral activity and are mainstays of combination antiretroviral treatment (ART) in HIV-infected patients. However, if oral administration causes similar changes as observed in our microbicide studies (see figure), these drugs could exhibit a paradoxical, HIV latency-promoting activity, which may hypothetically interfere with HIV cure.
Optimization and standardization of mucosal sampling techniques and cryopreservation
Mucosal HIV prevention studies require proper sampling, handling and evaluation of mucosal specimens. As a founding partner of the NIH- and Gates Foundation-funded HVTN Mucosal Immunology Group, we have been centrally involved in the effort to optimize and standardize mucosal cell and antibody sampling and assay techniques. These important studies are still ongoing, for example focusing on improving the functional cryopreservation of mucosal cells and tissues, so that precious mucosal specimens from international trial sites can be shipped, banked and, if necessary, pooled for specialized analyses in central laboratories. These studies are currently funded by the Bill and Melinda Gates Foundation.
When collecting mucosal specimens, it is equally important to record relevant demographic and clinical data from the study subjects. We contributed to an investigator’s guide for capturing participant information for mucosal sampling.
Comparison of immune cell yield and distribution in endocervical cytobrush (CB) and ectocervical biopsy samples. Percentage contribution of each cell subset, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ macrophages, and CD19neg/HLA-DQ+ dendritic cells (DC), as well as of cells that do not fit one of the described populations (‘‘unknown’’), to the total CD45+ population from cytobrush and biopsy samples. Data are averaged across three trial sites (Seattle, Chicago, Nairobi).
Role of herpes simplex virus (HSV) infection in sexual HIV transmission
Working with Drs. Jia Zhu, Anna Wald and Larry Corey, we have shown that genital HSV-2 infection increases the pool of mucosal CD4+ T cells available for HIV to infect [See also] . These infiltrates persist despite resolution of clinical lesions during acyclovir treatment, potentially explaining why acyclovir prophylaxis failed to lower HIV-1 incidence in clinical trials. This work has led to an ongoing collaboration in P01 AI030731, where we are evaluating how becoming infected with HSV-2 changes the genital immune milieu of adolescent women in Kenya. We also hope to provide insight into why women in sub-Saharan Africa tend to have more inflammation in the genital tract than their American counterparts, as has previously been reported.
Induction of interleukin 1 alpha (IL-1α) gene expression in vaginal epithelial cells by herpes simplex virus type 2 (HSV-2). Primary vaginal epithelial cells cultured from a healthy woman were exposed to either HSV-2 strain 186 or SD90, at a multiplicity of infection (MOI) of 0.1 and for the indicated time periods. IL-1α copy numbers were measured by reverse transcription digital droplet PCR assay. Unpublished data.
Effects of progestin-containing contraceptives on HIV risk
Injectable hormonal contraceptives have been reported to make HIV acquisition more likely. We have evaluated several possible biological explanations for this increase in risk. We are currently active collaborators on further projects that investigate possible connections between hormonal contraceptives and HIV risk, which are funded by the National Institutes of Health and the Centers for Disease Control and Prevention.
Discovering mucosal biomarkers of premature birth
We are using mass cytometry (CyToF) to analyze the phenotypes and functions of neutrophil granulocytes in endocervical cytobrushes from pregnant women. The goal of these studies is to discover novel biomarkers of preterm birth that could be used to predict early in pregnancy which women are at risk of prematurity. This project is a collaboration supported through the Global Alliance to Prevent Prematurity and Stillbirth (GAPPS), which is funded by the Bill and Melinda Gates Foundation.
Impact of opioid use on regulatory RNA molecules in human semen
Rodent studies suggest transgenerational inheritance of epigenetic traits triggered by substance abuse. Inheritance that is not manifested in the genetic code could be delivered by regulatory RNA molecules in the germ line. As part of a grant from the National Institute on Drug Abuse (NIDA), we will survey exRNA molecules in human semen from heroin users and normal controls.