Juan Sabatté

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells.

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells

Results Flow cytometry showed that heparan sulfate is expressed in spermatozoa. Heparan sulfate plays an important role in the capture of HIV-1, as demonstrated by the inhibitory effect induced by heparine (50 U/ml) (>70% capture inhibition, n = 15) and heparinase II pre-treatment of the spermatozoa (>50% capture inhibition, n = 6). By contrast, treatment with the inhibitor of mannose receptor mannan (5 mg/ml) slightly inhibited virus attachment (> 20% capture inhibition, n = 10). Spermatozoa-attached viruses were efficiently transmitted to DCs through a cellto-cell contact-dependent mechanism. Fluorescence, confocal and electronic microscopy showed that this process was associated to the internalization of a fraction of the spermatozoa. This interaction also resulted in the phenotypic maturation of DCs (up-regulation of CD80, CD86, CD40, CD83 and CCR7), and the production of IL-10 but not IL-12p70. Finally, we found that acidic extracellular pH levels, similar to those found in the vaginal mucosa after sexual intercourse, increased more than four times (n = 12) the binding of HIV-1 to the spermatozoa and the subsequent transmission of HIV-1 to DCs.

Human Seminal Plasma Abrogates the Capture and Transmission of Human Immunodeficiency Virus Type 1 to CD4+ T Cells Mediated by DC-SIGN

ABSTRACT Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is expressed by dendritic cells (DCs) at mucosal surfaces and appears to play an important role in the dissemination of human immunodeficiency virus type 1 (HIV-1) infection. DC-SIGN binds HIV-1 gp120 and efficiently transmits the virus to T CD4+ cells, which become the center of viral replication. Semen represents the main vector for HIV-1 dissemination worldwide. In the present study we show that human seminal plasma (SP), even when used at very high dilutions (1:104 to 1:105), markedly inhibits the capture and transmission of HIV-1 to T CD4+ cells mediated by both DCs and B-THP-1-DC-SIGN cells. In contrast, SP does not inhibit the capture of HIV-1 by DC-SIGN-negative target cells, such as the T-cell line SupT-1, monocytes, and activated peripheral blood mononuclear cells. The SP inhibitor has a high molecular mass (>100 kDa) and directly interacts with DC-SIGN-positive target cells but not with HIV-1. Moreover, the inhibitor binds to concanavalin A, suggesting that it contains high-mannose N-linked carbohydrates. Of note, using biotin-labeled SP we found that the binding of SP components to DCs was abrogated by mannan, while their interaction with B-THP-1 cells was almost completely dependent on the expression of DC-SIGN. Since epithelium integrity is often compromised after vaginal or anal intercourse, as well as in the presence of ulcerative-sexually transmitted diseases, our results support the notion that components of the SP might be able to access to the subepithelium, inhibiting the recognition of HIV-1 gp120 by DC-SIGN-positive DCs.

Semen Clusterin Is a Novel DC-SIGN Ligand

The C-type lectin receptor dendritic cell-specific ICAM-3–grabbing nonintegrin (DC-SIGN) is an important player in the recognition of pathogens by dendritic cells. A plethora of pathogens including viruses, bacteria, parasites, and fungi are recognized by DC-SIGN through both mannose and fucose-containing glycans expressed on the pathogen surface. In this study, we identified semen clusterin as a novel DC-SIGN ligand. Semen clusterin, but not serum clusterin, expresses an extreme abundance of fucose-containing blood-type Ags such as Lex and Ley, which are both excellent DC-SIGN ligands. These motifs enable semen clusterin to bind DC-SIGN with very high affinity (Kd 76 nM) and abrogate the binding of HIV-1 to DC-SIGN. Depletion of clusterin from semen samples, however, did not completely prevent the ability of semen to inhibit the capture of HIV-1 by DC-SIGN, supporting that besides clusterin, semen contains other DC-SIGN ligands. Further studies are needed to characterize these ligands and define their contribution to the DC-SIGN–blocking activity mediated by semen. Clusterin is an enigmatic protein involved in a variety of physiologic and pathologic processes including inflammation, atherosclerosis, and cancer. Our results uncover an unexpected heterogeneity in the glycosylation pattern of clusterin and suggest that the expression of high concentrations of fucose-containing glycans enables semen clusterin to display a unique set of biological functions that might affect the early course of sexually transmitted infectious diseases.

The role of semen in sexual transmission of HIV: beyond a carrier for virus particles.

Unprotected sexual intercourse between discordant couples is by far the most frequent mode of HIV-1 (human immunodeficiency virus type 1) transmission being semen the main vector for HIV-1 dissemination worldwide. Semen is usually considered merely as a vehicle for HIV-1 transmission. In this review we discuss recent observations suggesting that beyond being a carrier for virus particles semen markedly influences the early events involved in sexual transmission of HIV through the mucosal barriers.

Mechanisms regulating neutrophil survival and cell death

Neutrophils not only play a critical role as a first line of defense against bacteria and fungi infections but also contribute to tissue injury associated with autoimmune and inflammatory diseases. Neutrophils are rapidly and massively recruited from the circulation into injured tissues displaying an impressive arsenal of toxic weapons. Although effective in their ability to kill pathogens, these weapons were equally effective to induce tissue damage. Therefore, the inflammatory activity of neutrophils must be regulated with exquisite precision and timing, a task mainly achieved through a complex network of mechanisms, which regulate neutrophil survival. Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis although new forms of cell death have been characterized over the last few years. The lifespan of neutrophils can be dramatically modulated by a large variety of agents such as cytokines, pathogens, danger-associated molecular patterns as well as by pharmacological manipulation. Recent findings shed light about the complex mechanisms responsible for the regulation of neutrophil survival in different physiological, pathological, and pharmacological scenarios. Here, we provide an updated review on the current knowledge and new findings in this field and discuss novel strategies that could be used to drive the resolution of neutrophil-mediated inflammatory diseases.

Semen Promotes the Differentiation of Tolerogenic Dendritic Cells

Seminal plasma is not just a carrier for spermatozoa. It contains high concentrations of cytokines, chemokines, and other biological compounds that are able to exert potent effects on the immune system of the receptive partner. Previous studies have shown that semen induces an acute inflammatory response at the female genital mucosa after coitus. Moreover, it induces regulatory mechanisms that allow the fetus (a semiallograft) to grow and develop in the uterus. The mechanisms underlying these regulatory mechanisms, however, are poorly understood. In this study, we show that seminal plasma redirects the differentiation of human dendritic cells (DCs) toward a regulatory profile. DCs differentiated from human monocytes in the presence of high dilutions of seminal plasma did not express CD1a but showed high levels of CD14. They were unable to develop a fully mature phenotype in response to LPS, TNF-α, CD40L, Pam2CSK4 (TLR2/6 agonist), or Pam3CSK4 (TLR1/2 agonist). Upon activation, they produced low amounts of the inflammatory cytokines IL-12p70, IL-1β, TNF-α, and IL-6, but expressed a high ability to produce IL-10 and TGF-β. Inhibition of the PG receptors E-prostanoid receptors 2 and 4 prevented the tolerogenic effect induced by seminal plasma on the phenotype and function of DCs, suggesting that E-series PGs play a major role. By promoting a tolerogenic profile in DCs, seminal plasma might favor fertility, but might also compromise the capacity of the receptive partner to mount an effective immune response against sexually transmitted pathogens.

Sphingosylphosphorylcholine activates dendritic cells, stimulating the production of interleukin‐12

Compared with other lysophospholipid mediators such as sphingosine‐1‐phosphate and lysophosphatidic acid, little is known about the physiological significance of the related bioactive lysosphingolipid sphingosylphosphorylcholine (SPC), which is present in high‐density lipoprotein particles. The present study was undertaken to evaluate the effect of SPC on human immature dendritic cells (DCs). Reverse transcription–polymerase chain reaction and flow cytometry assays revealed that DCs express two putative receptors for SPC, ovarian cancer G‐protein‐coupled receptor 1 and G‐protein‐coupled receptor 4. Exposure to SPC induced a rapid and transient increase in intracellular free calcium concentrations but did not stimulate endocytosis or chemotaxis of DCs. SPC increased the expression of HLA‐DR, CD86 and CD83 and improved the T‐cell priming ability of DCs, as well as the ability of DCs to stimulate the production of interferon‐γ by allogeneic peripheral blood mononuclear cells during the mixed lymphocyte reaction. Consistent with these results, we also observed that SPC stimulated the production of interleukin (IL)‐12 and IL‐18 by DCs. Taken together, our results support the notion that the accumulation of SPC in peripheral tissues during the course of inflammatory processes may favour the development of T helper type 1 immunity.

Analysis of Suppressor and Non-Suppressor FOXP3+ T Cells in HIV-1-Infected Patients

Recently, it was shown that peripheral blood FOXP3+CD4+ T cells are composed of three phenotypic and functionally distinct subpopulations. Two of them having in vitro suppressive effects were characterized as resting Treg cells (rTregs) and activated Treg cells (aTregs). A third subset, identified as FOXP3+ non-Tregs, does not display any suppressor activity and produce high levels of Th1 and Th17 cytokines upon stimulation. In the present study we focus on the characteristics of these three subsets of FOXP3+CD4+ T cells in untreated HIV-1-infected patients. We found that the absolute counts of rTregs, aTregs and FOXP3+ non-Tregs were reduced in HIV-1 patients compared with healthy donors. The relative frequency of rTregs and aTregs was similar in HIV-1 patients and healthy donors, while the frequency of FOXP3+ non-Tregs was significantly higher in HIV-1 patients, reaching a maximum in those patients with the lower values of CD4 counts. Contrasting with the observations made in FOXP3- CD4+ T cells, we did not find a negative correlation between the number of rTregs, aTregs or FOXP3+ non-Tregs and virus load. Studies performed with either whole PBMCs or sorted aTregs and FOXP3+ non-Tregs cells showed that these two populations of FOXP3+ T cells were highly permissive to HIV-1 infection. Upon infection, FOXP3+ non-Tregs markedly down-regulates its capacity to produce Th1 and Th17 cytokines, however, they retain the ability to produce substantial amounts of Th2 cytokines. This suggests that FOXP3+ non-Tregs might contribute to the polarization of CD4+ T cells into a Th2 profile, predictive of a poor outcome of HIV-1-infected patients.

Fucosylated clusterin in semen promotes the uptake of stress-damaged proteins by dendritic cells via DC-SIGN

STUDY QUESTION Could seminal plasma clusterin play a role in the uptake of stress-damaged proteins by dendritic cells? SUMMARY ANSWER Seminal plasma clusterin, but not serum clusterin, promotes the uptake of stress-damaged proteins by dendritic cells via DC-SIGN. WHAT IS KNOWN ALREADY Clusterin is one of the major extracellular chaperones. It interacts with a variety of stressed proteins to prevent their aggregation, guiding them for receptor-mediated endocytosis and intracellular degradation. The concentration of clusterin in semen is almost 20-fold higher than that found in serum, raising the question about the role of seminal plasma clusterin in reproduction. No previous studies have analyzed whether seminal plasma clusterin has chaperone activity. We have previously shown that seminal plasma clusterin, but not serum clusterin, expresses an extreme abundance of fucosylated glycans. These motifs enable seminal plasma clusterin to bind DC-SIGN with very high affinity. STUDY DESIGN, SIZE, DURATION In vitro experiments were performed to evaluate the ability of seminal plasma clusterin to inhibit the precipitation of stressed proteins, promoting their uptake by dendritic cells via DC-SIGN (a C-type lectin receptor selectively expressed on dendritic cells (DC)). Moreover, the ability of seminal plasma clusterin to modulate the phenotype and function of DCs was also assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS Clusterin was purified from human semen and human serum. Catalase, bovine serum albumin, glutathione S-transferase, and normal human serum were stressed and the ability of seminal plasma clusterin to prevent the precipitation of these proteins, guiding them to DC-SIGN expressed by DCs, was evaluated using a fluorescence-activated cell sorter (FACS). Endocytosis of stressed proteins was analyzed by confocal microscopy and the ability of seminal plasma clusterin-treated DCs to stimulate the proliferation of CD25+FOXP3+CD4+ T cells was also evaluated by FACS. MAIN RESULTS AND THE ROLE OF CHANCE Seminal plasma clusterin interacts with stressed proteins, inhibits their aggregation (P < 0.01) and efficiently targets them to dendritic cells via DC-SIGN (P < 0.01). DCs efficiently endocytosed clusterin-client complexes and sorted them to degradative compartments involved in antigen processing and presentation. Moreover, we also found that the interaction of seminal plasma clusterin with DC-SIGN did not change the phenotype of DCs, but stimulates their ability to induce the expansion of CD25+FOXP3+CD4+ T lymphocytes (P < 0.05 versus control). LIMITATIONS, REASONS FOR CAUTION All the experiments were performed in vitro; hence the relevance of our observations should be validated in vivo. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that by inducing the endocytosis of stress-damaged proteins by DCs via DC-SIGN, seminal plasma clusterin might promote a tolerogenic response to male antigens, thereby contributing to female tolerance to seminal antigens. STUDY FUNDING/COMPETING INTERESTS The present research was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas, the Buenos Aires University School of Medicine, and the Agencia Nacional de Promoción Científica y Tecnológica (Argentina). The authors have no conflicts of interest to declare.