ex-vivo-infection-human-lymphoid-tissue-female-genital-mucosa-with

<em>Ex Vivo</em> Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Karolinska Institutet

National Institutes of Health

Sex hormones can influence the immune defenses of the female genital tract (FGT) and its susceptibility to infections. Here we investigated the effect of different hormonal contraceptives on the production of antimicrobial peptides (AMPs) in different compartments of the female genital mucosa (FGM), secretions and tissue.

Expression profiles of antimicrobial peptides in the genital tract of women using progesterone intrauterine devices versus combined oral contraceptives.

Currently, whether hormonal contraceptives affect male to female human immunodeficiency virus (HIV) transmission is being debated. In this study, we investigated whether the use of progesterone-based intrauterine devices (pIUDs) is associated with a thinning effect on the ectocervical squamous epithelium, down-regulation of epithelial junction proteins, and/or alteration of HIV target cell distribution in the human ectocervix. Ectocervical tissue biopsies from healthy premenopausal volunteers using pIUDs were collected and compared to biopsies obtained from two control groups, namely women using combined oral contraceptives (COCs) or who do not use hormonal contraceptives. In situ staining and image analysis were used to measure epithelial thickness and the presence of HIV receptors in tissue biopsies. Messenger RNA levels of epithelial junction markers were measured by quantitative PCR. The epithelial thickness displayed by women in the pIUD group was similar to those in the COC group, but significantly thinner as compared to women in the no hormonal contraceptive group. The thinner epithelial layer of the pIUD group was specific to the apical layer of the ectocervix. Furthermore, the pIUD group expressed significantly lower levels of the tight junction marker ZO-1 within the epithelium as compared to the COC group. Similar expression levels of HIV receptors and coreceptors CD4, CCR5, DC-SIGN, and Langerin were observed in the three study groups. Thus, women using pIUD displayed a thinner apical layer of the ectocervical epithelium and reduced ZO-1 expression as compared to control groups. These data suggest that pIUD use may weaken the ectocervical epithelial barrier against invading pathogens, including HIV. 

Progesterone-based intrauterine device use is associated with a thinner apical layer of the human ectocervical epithelium and a lower ZO-1 mRNA expression.

The effects of sex hormones on the immune defenses of the female genital mucosa and its susceptibility to infections are poorly understood. The injectable hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may increase the risk for HIV-1 acquisition. We assessed the local concentration in the female genital mucosa of cationic polypeptides with reported antiviral activity in relation to DMPA use.

Depot Medroxyprogesterone Acetate Use Is Associated With Elevated Innate Immune Effector Molecules in Cervicovaginal Secretions of HIV-1-Uninfected Women.

Human tissue explants are a valuable tool to study the interactions between host and infectious agents. They reliably mimic many important aspects of tissue cytoarchitecture and functions and allow us the investigation of the mechanisms of microbial pathogenesis under controlled laboratory conditions. One of the advantages of this system is that, unlike isolated cells, infection of tissue blocks with HIV-1 does not require exogenous stimulation with mitogens or activating factors. Here we describe a protocol to infect with HIV-1 human lymphoid tissue from tonsils and cervico-vaginal tissue and maintain them in culture in a non-polarized setting. These ex vivo infected tissues can be used as fruitful models to study HIV-1 pathogenesis and HIV-1 vaginal transmission, respectively, as well as an efficient platform for testing anti-HIV therapeutic and preventative strategies.

An ex vivo Model of HIV-1 Infection in Human Lymphoid Tissue and Cervico-vaginal Tissue.

The female genital tract (FGT) mucosa is a critically important site for immune defense against microbes. Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population that recognizes microbial riboflavin metabolite antigens in an MR1-dependent manner. The role of MAIT cells in the FGT mucosa is unknown. Here, we found that MAIT cells and MR1 antigen-presenting cells were present in the upper and lower FGT, with distinct tissue localization of MAIT cells in endometrium vs. cervix. The MAIT cells from the FGT and blood displayed a distinct phenotype with expression of interleukin (IL)-18Rα, CD127, α4β7, PD-1, as well as the transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, Eomes, and T-bet. Their expression levels of PLZF and Eomes were lower in the FGT compared with blood. When stimulated with Escherichia coli, MAIT cells from the FGT displayed a bias towards IL-17 and IL-22 expression, whereas blood MAIT cells produced primarily IFN-γ, TNF, and Granzyme B. Furthermore, both FGT- and blood-derived MAIT cells were polyfunctional and contributed to the T-cell-mediated response to E. coli. Thus, MAIT cells in the genital mucosa have a distinct IL-17/IL-22 profile and may have an important role in the immunological homeostasis and control of microbes at this site.

MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell-mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

Innate Invariant NKT Cell Recognition of HIV-1-Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion.

Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been determined. We previously showed that CD4 T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals. Here we show that these CD4CD44CD62L T helper cells by gene expression are a distinct T-cell population defined by ChAT (CD4 T). Mice lacking ChAT expression in CD4 cells have elevated arterial blood pressure, compared to littermate controls. Jurkat T cells overexpressing ChAT (JT) decreased blood pressure when infused into mice. Co-incubation of JT and endothelial cells increased endothelial cell levels of phosphorylated endothelial nitric oxide synthase, and of nitrates and nitrites in conditioned media, indicating increased release of the potent vasorelaxant nitric oxide. The isolation and characterization of CD4 T cells will enable analysis of the role of these cells in hypotension and hypertension, and may suggest novel therapeutic strategies by targeting cell-mediated vasorelaxation.

Blood pressure regulation by CD4 lymphocytes expressing choline acetyltransferase.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8 Trm cells on a compartmental and functional basis. In human skin epithelia, CD8CD49a Trm cells produced interferon-γ, whereas CD8CD49a Trm cells produced interleukin-17 (IL-17). In addition, CD8CD49a Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8CD49a Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8CD49a Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8 Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

CD49a Expression Defines Tissue-Resident CD8 T Cells Poised for Cytotoxic Function in Human Skin.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.

Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.

Genital mucosa is the main portal of entry for various incoming pathogens, including human immunodeficiency virus (HIV), hence it is an important site for host immune defenses. Tissue-resident memory T (TRM) cells defend tissue barriers against infections and are characterized by expression of CD103 and CD69. In this study, we describe the composition of CD8+ TRM cells in the ectocervix of healthy and HIV-infected women.

Human Immunodeficiency Virus-Infected Women Have High Numbers of CD103-CD8+ T Cells Residing Close to the Basal Membrane of the Ectocervical Epithelium.

Susceptibility to HIV is associated with the menstrual cycle and vaginal microbiome, but their collective impact on vaginal inflammation remains unclear. Here, we characterized the cervicovaginal proteome, inflammation, and microbiome community structure and function during the menstrual cycle.

Article	Year
Expression profiles of antimicrobial peptides in the genital tract of women using progesterone intrauterine devices versus combined oral contraceptives. American journal of reproductive immunology (New York, N.Y. : 1989)\| PubMed ID: 25087845	2014
Progesterone-based intrauterine device use is associated with a thinner apical layer of the human ectocervical epithelium and a lower ZO-1 mRNA expression. Biology of reproduction\| PubMed ID: 25588510	2015
Depot Medroxyprogesterone Acetate Use Is Associated With Elevated Innate Immune Effector Molecules in Cervicovaginal Secretions of HIV-1-Uninfected Women. Journal of acquired immune deficiency syndromes (1999)\| PubMed ID: 25622059	2015
An ex vivo Model of HIV-1 Infection in Human Lymphoid Tissue and Cervico-vaginal Tissue. Bio-protocol\| PubMed ID: 25774380	2014
MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation. Mucosal immunology\| PubMed ID: 27049062	2017
Innate Invariant NKT Cell Recognition of HIV-1-Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 27481843	2016
Blood pressure regulation by CD4 lymphocytes expressing choline acetyltransferase. Nature biotechnology\| PubMed ID: 27617738	2016
CD49a Expression Defines Tissue-Resident CD8 T Cells Poised for Cytotoxic Function in Human Skin. Immunity\| PubMed ID: 28214226	2017
Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants. PLoS pathogens\| PubMed ID: 28542587	2017
Human Immunodeficiency Virus-Infected Women Have High Numbers of CD103-CD8+ T Cells Residing Close to the Basal Membrane of the Ectocervical Epithelium. The Journal of infectious diseases\| PubMed ID: 29272532	2018
The vaginal microbiome amplifies sex hormone-associated cyclic changes in cervicovaginal inflammation and epithelial barrier disruption. American journal of reproductive immunology (New York, N.Y. : 1989)\| PubMed ID: 29709092	2018

Andrea Introini

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