Breast milk contains several immunological components known to be important for protection and development of the child's immune system . However, vertical transmission of HIV-1 from mother to child can occur through breastfeeding . The risk of transmission through breastfeeding is, however, less than 30%  and studies have shown a lower risk of postnatal HIV-1 infection in exclusively breastfed infants than in mixed breastfed children during the first months of life [4,5]. These observations point towards a protective effect of breast milk components against HIV-1 transmission to the breastfed child. In accordance, studies have identified components in milk, such as bile-salt stimulated lipase (BSSL)  and soluble mucin 1 (MUC1) , which protect against HIV-1 infection of dendritic cells.
Dendritic cells are abundant in skin and mucosa and act as a first line of defense against invading pathogens. Dendritic cells are efficient in antigen processing and presentation and in initiating potent primary immune responses . Pathogens are recognized by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors and C-type lectin receptors (CLRs). In contrast to other PRRs, CLRs can mediate internalization of bound ligands as well as modulate signalling by other PRRs. The CLR intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is important for recognition of pathogens by dendritic cells. A broad range of pathogens including viruses, bacteria, parasites and fungi are recognized by DC-SIGN via mannose and fucose-containing glycans expressed on the pathogen surface. The HIV-1 envelope glycoprotein, gp120, has multiple N-glycans that mediate binding to several receptors on dendritic cells, including DC-SIGN  and Langerin . Although binding to Langerin results in degradation of the virus, HIV-1 capture by DC-SIGN results in internalization of the virus to endosomes or multivesicular bodies wherein it remains protected from degradation [9,11,12]. This can lead to subsequent transfer of intact virions to T cells in the lymph node .
Exosomes are approximately 100 nm membrane vesicles released from most cells in the body and have been found to serve functions in cell–cell communication and immune regulation . Exosomes are formed as vesicles budding off the membrane within multivesicular bodies or late endosomes . When the endosomal membrane fuses with the plasma membrane, the vesicles are released into the extracellular space and are then termed exosomes. Exosomes are found in all body fluids so far analysed and can be produced by cells in vitro. Exosomes are capable of immune stimulation, explored in tumour therapy using dendritic cell derived exosomes [16,17], and of immune inhibition, exemplified by cancer cell derived exosomes [18,19]. We have previously reported the presence of exosomes in human breast milk  with the ability to induce Foxp3 expression and inhibit anti-CD3 induced interleukin (IL)-2 expression in peripheral blood mononuclear cells (PBMCs) in vitro, suggesting a possible immune regulatory role in the child. Some viruses use the same intracellular pathways as exosomes and it has been suggested that viruses exploit the exosome pathway to increase viral spread [21,22]. Accordingly, HIV-1 has been detected to be associated with exosomes  and has been proposed to be a way for HIV-1 to avoid immune detection and accelerate infection.
Exosomes derived from human breast milk carry MUC1 on their surface , a known DC-SIGN ligand . Therefore, in the present study, we addressed whether breast milk derived exosomes could block HIV-1 infection and transmission in monocyte-derived dendritic cells (MDDCs) by binding DC-SIGN. We show that milk exosomes inhibit productive HIV-1 infection of MDDCs as well as subsequent viral transfer to CD4+ T cells. Furthermore, we show that milk exosomes bind to MDDCs, and that their binding can be partially blocked by anti-DC-SIGN antibodies. This suggests that milk-derived exosomes may play a role in lowering the risk of HIV-1 transmission by breastfeeding.
Materials and methods
Generation of monocyte-derived dendritic cells
PBMCs were prepared from buffy coats from healthy blood donors (Karolinska University Hospital, Stockholm, Sweden) using Ficoll-Pacque PLUS (GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradient centrifugation. Monocytes were positively selected for CD14 expression using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14+ monocytes were cultured at a density of 4 × 105 cells/ml at 37°C, 6% CO2 in RPMI 1640 medium (Invitrogen, Carlsbad, California, USA) supplemented with 2 mmol/l L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 50 μmol/l β-mercaptoethanol (Sigma, St. Louis, Missouri, USA), 10% heat-inactivated FCS (HyClone, Thermo Scientific), IL-4 (800 IU/ml) and GM-CSF (550 IU/ml) (Biosource International, Camarillo, California, USA) (complete media). On day three, half of the culture medium was exchanged with fresh complete media. On day six, the cells were phenotyped by flow cytometry (FACSCalibur; BD Biosciences, San Jose, California, USA) with antibodies against CD1a (Coulter Corporation, Hialeah, Florida, USA), CD11c, CD14 and CD83 (BioLegend, San Diego, California, USA), and corresponding isotype controls, labelled with fluorescein isothiocyanate (FITC) or phycoerythrin. The immature phenotype of the MDDCs was confirmed with CD83 and CD14 expression lower than 10% and CD1a and CD11c expression higher than 80%. This work was approved by the local ethics committee.
Isolation of exosomes from human breast milk and plasma
Fresh breast milk was collected from healthy volunteers using a manual breast pump in sterile tubes. All mothers were nonsmokers, had vaginal deliveries at full term of healthy, normal birth weight infants. The mature milk was collected at home and kept at 4°C until delivered to the laboratory, and processed within 24 h. Exosomes from human breast milk were isolated as previously described . For preparations of exosomes from plasma, fresh plasma was collected from healthy donors (Karolinska University Hospital, Stockholm, Sweden) and centrifuged at 11 000g for 45 min (Beckman Coulter, Fullerton, California, USA), passed through a 100 μm cell strainer and centrifuged again at 11 000g for 45 min. The plasma was thereafter passed through a 100 μm cell strainer and subjected to ultracentrifugation (Beckman Coulter) at 140 000g for 2 h. The exosome pellet was dissolved in PBS, filtered through a 0.2 μm filter and washed twice with PBS by ultracentrifugation at 142 000g for 90 min (Beckman Coulter). The exosome pellet was thereafter dissolved in a small volume of PBS. Exosomal protein concentration was determined by DC protein assay according to the manufacturer's instructions (Bio-Rad, Hercules, California, USA). This work was approved by the local ethics committee.
Sucrose gradient and phenotypic analysis of exosomes by flow cytometry
300 μg milk or plasma-derived exosomes were added on top of a linear sucrose gradient (0.25 mol/l sucrose/20 mmol/l Hepes (pH 7) and 2 mol/l sucrose/20 mmol/l Hepes (pH 7) (Sigma-Aldrich) and centrifuged for 20 h at 80 000g (Beckman Coulter) at 4°C. One millilitre fractions were collected and the fraction density was determined by refraction index measurements. Two hundred microlitres of each fraction was added to sulfate-aldehyde latex microspheres (4 μm, Invitrogen) for 30 min at room temperature (RT) before addition of 1 ml PBS and rotation overnight at RT. The following day, the beads were pelleted by centrifugation at 10 000g for 10 min at RT and re-suspended in 1 ml of bead blocking buffer [PBS, 0.1% BSA (Sigma), 0.01% sodium azide] for 30 min. Thereafter, the beads were pelleted by centrifugation and washed twice with PBS before being labelled with FITC-conjugated antibody against MUC1 and corresponding isotype-matched antibody (BD Biosciences). The presence of MUC1 was analysed by flow cytometry (FACS Calibur) and FlowJo software (Tree Star Inc., Ashland, Oregon, USA) analysis.
PKH67 labelling of exosomes
Exosomes were pelleted by ultracentrifugation (Beckman Coulter) at 100 000g for 70 min and re-suspended in Diluent C (Sigma-Aldrich). The green fluorescent membrane dye PKH67 (4 μmol/l) (Sigma-Aldrich) was filtered through a 0.2 μm syringe filter before mixing with the exosomes. To control for unspecific PKH67 staining, same amount of membrane dye was solubilized in Diluent C and treated similarly. The reaction was stopped by adding 1% BSA before ultracentrifugation (Beckman Coulter) at 100 000g for 70 min before re-suspension of the exosomes in PBS.
Binding of exosomes to monocyte-derived dendritic cell
Ten or 20 μg of PKH67-labelled exosomes were added per well to MDDCs and incubated at 37°C, 6% CO2 in complete media. After 30 min, 1, 2 or 4 h, the cells were centrifuged at 300g for 10 min and fixated with 2% paraformaldehyde before subjected to flow cytometry or stained with anti-HLA-DR antibody (BD Biosciences) and Alexa fluor 546 (Invitrogen) for confocal microscopy analyses. Twenty thousand live cells were collected using flow cytometry (FACSCalibur, BD Biosciences) and the number of PHK67-positive cells was analysed using FlowJo software (Tree Star Inc.). For confocal analyses, 1 × 105 MDDCs were mounted on Superfrost Plus glass slides (Thermo Scientific, Bremen, Germany) by cytospin centrifugation at 500 rpm for 3 min (Shandon Cytospin 3, Minnesota, USA). A drop of mounting media (Dako, Stockholm, Sweden) and a glass coverslip was added on top of the cells and sealed with nail polish.
Blocking of DC-SIGN or intercellular adhesion molecule-1 on monocyte-derived dendritic cell
To block Fc-receptors, heat-inactivated serum (1 : 100) from healthy AB-positive donors (Karolinska University Hospital, Stockholm, Sweden) was added to the MDDCs in PBS. The serum was washed away after 15 min incubation at RT and anti-DC-SIGN (Beckman Coulter), anti-ICAM-1 (Thermo Scientific, UK) or isotype control (BD Biosciences) (20 μg/ml) was added to the MDDCs for 30 min at RT. The cells were thereafter washed and re-suspended in complete media. Ten or 20 μg of PKH67-labelled exosomes were thereafter added to 0.25 × 106 MDDCs. After 30 min, 1 or 4 h incubation at 37°C, 6% CO2, the MDDCs were fixated with 2% paraformaldehyde and subjected to flow cytometry or stained with anti-HLA-DR antibody (BD Biosciences) and Alexa flour 546 (Invitrogen) for confocal microscopy analyses.
HIV-1 preparation, infection of monocyte-derived dendritic cell and transfer to CD4+ T cells
HIV-1BaL was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, Maryland, USA) and grown in PBMC cultures stimulated with PHA (2.5 mg/ml; Sigma-Aldrich) and IL-2 (20 ng/ml; PeproTech, Rocky Hill, New Jersey, USA). The supernatant was centrifuged at 12 000g for 5 min, filtered (0.22 μm), aliquoted and stored at –80°C. MDDCs were preincubated with the exosome preparations (10 and 20 μg/well) for 1 h at 37oC before incubation with HIV-1BaL for 4 h. Cells were then extensively washed, re-suspended with medium and incubated at 37oC for 5 days. For viral transfer experiments, CD4+ T cells were prepared using RosetteSep enrichment kits (Stem Cell Technology, Vancouver, British Columbia, Canada) and added at a 1 : 2 ratio, together with IL-2 (10 ng/ml), to washed MDDCs preexposed to exosomes and HIV-1BaL. Productive infection was determined by staining for CD11c or CD3 (BD Biosciences) and p24 (clone KC57; Beckman Coulter). Each condition was performed in duplicates. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analysed using FlowJo software (Tree Star Inc.).
Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, California, USA). To test for statistical significance, nonparametric two-tailed Mann–Whitney analysis was performed.
Milk exosomes, but not plasma exosomes, inhibit HIV-1 infection of monocyte-derived dendritic cells
Milk and plasma exosomes were generated from healthy donors using sequential centrifugation and MUC1 expression was analysed with sucrose gradient fractionation and flow cytometry. MUC1 expression was only detected in fractions corresponding to typical exosome densities (1.10–1.20 g/ml)  on milk-derived exosomes, but not on plasma-derived exosomes (Fig. 1a). To investigate whether exosomes had an impact on HIV-1 infection of MDDCs, milk or plasma-derived exosomes (10 or 20 μg/well) were added to human immature MDDCs generated from healthy donors for 1 h before addition of HIV-1BaL. Five days postinfection, the number of productively infected (p24+) MDDCs was analysed by flow cytometry. Exosomes derived from milk (Fig. 1b, c) but not plasma (Fig. 1d) significantly (P < 0.05) reduced the productive infection of MDDCs by approximately 50% as compared with MDDCs not treated with exosomes. Thus, milk-derived exosomes expressed MUC1 and had the capacity to inhibit productive HIV-1 infection of MDDCs, an effect that was not detected for plasma-derived exosomes.
Milk exosomes block HIV-1 transfer from monocyte-derived dendritic cells to CD4+ T cells
We next investigated whether exosomes had an impact on HIV-1 transfer from MDDCs to CD4+ T cells. To test this, MDDCs were incubated with 10 or 20 μg milk or plasma-derived exosomes before addition of HIV-1BaL. The MDDCs were thereafter cocultured with allogeneic CD4+ T cells. The presence of productive infection (p24+) in CD4+ T cells was then analysed by flow cytometry (Fig. 2). Milk-derived exosomes significantly (P < 0.05, median = 51.75% of full infection) reduced the ability of MDDCs to transfer HIV-1Bal to CD4+ T cells (Fig. 2a, b). In contrast, plasma-derived exosomes did not inhibit viral transfer in parallel experiments (P = 0.13, median = 64.49% of full infection) (Fig. 2c). These data indicate that milk-derived exosomes have the capacity to inhibit viral transfer from HIV-1 exposed MDDCs to CD4+ T cells, whereas plasma-derived exosomes do not.
Increased binding of milk exosomes to monocyte-derived dendritic cells over time
As milk-derived exosomes, but not plasma-derived exosomes, were potent inhibitors of HIV-1 infection of MDDCs, we next investigated the binding and uptake of exosomes by MDDCs. Milk and plasma-derived exosomes were labelled with a fluorescent membrane dye and incubated with MDDCs for 30 min, 1, 2 or 4 h. By confocal microscopy, milk exosome uptake was detected after 1 h incubation, with more pronounced uptake in MDDCs incubated with the highest dose of milk-derived exosomes (Fig. 3a). At later time points, the majority of cells had taken up milk-derived exosomes. MDDCs treated with the membrane dye alone did not stain positive for the dye, indicating exosome uptake specificity (data not shown). With the more sensitive flow cytometry based method, we detected exosome-positive cells already after 30 min incubation, with more positive cells in cultures incubated with 20 μg of exosomes than with 10 μg (Fig. 3b). The most prominent milk exosome binding occurred within the first hour of incubation. In MDDCs incubated with plasma-derived exosomes, no exosome binding could be detected up to 4 h, either with confocal microscopy or flow cytometry (data not shown). These data indicate that milk-derived exosomes, but not plasma-derived exosomes, are taken up by MDDCs within 4 h and that most of the binding occurs within the first hour of coincubation.
Binding of milk exosomes to monocyte-derived dendritic cells occurs partly via DC-SIGN
To investigate what receptor is engaged on MDDCs by milk-derived exosomes, we blocked DC-SIGN or intercellular adhesion molecule 1 (ICAM-1) on MDDCs before addition of fluorescent membrane dye-labelled exosomes. Milk-derived exosomes express MUC1, a known DC-SIGN ligand, as well as lymphocyte function associated antigen 1 (LFA-1), a ligand for ICAM-1 that is important for uptake of dendritic cell derived exosomes . Moreover, HIV-1 has been shown to bind both DC-SIGN and ICAM-1  and are therefore potential receptors for binding by both milk-derived exosomes and HIV-1. By confocal microscopy, milk exosome uptake was detected despite treatment with ICAM-1 blocking or isotype control antibody at all time points tested. In contrast, cells treated with DC-SIGN blocking antibodies displayed no milk exosome uptake (Fig. 4a). With flow cytometry, milk exosome binding was detected in DC-SIGN treated MDDCs, but to a lower extent than ICAM-1 and isotype control antibody treated cells (Fig. 4b). However, the difference between DC-SIGN and ICAM-1 antibody treated cells was reduced over time, possibly due to internalization of receptor/antibody complexes and recirculation of ICAM-1 and DC-SIGN to the cell surface. Taken together, these data indicate that milk-derived exosomes bind to and are taken up by MDDCs partly via DC-SIGN and not via ICAM-1/LFA-1 interactions.
In this study, we have shown that preexposure to human breast milk derived exosomes reduces productive HIV-1 infection of MDDCs and subsequent viral transfer to CD4+ T cells. Interestingly, plasma-derived exosomes did not reduce HIV-1 infection of MDDCs or the viral transfer to CD4+ T cells, suggesting that exosomes from different body fluids have different capacity in this regard. Furthermore, we have shown that milk-derived exosomes adhere to MDDCs via DC-SIGN. Our results suggest that exosomes from milk might be involved in reducing the risk of transmission of HIV-1 from mother to child through breastfeeding.
Transmission of HIV-1 from mother to child through breastfeeding is relatively inefficient , suggesting that breast milk contains components that might interfere with HIV-1 infection. It was reported that antibodies to DC-SIGN as well as soluble MUC1 are present in breast milk, which can reduce dendritic cell mediated HIV-1 transfer to CD4+ T cells . In the present study, we show that human breast milk derived exosomes can also mediate this effect. The notion that exosomes directly interact with cells is well established in vitro, but whether they fuse with target cells, are engulfed or bind through specific receptor–ligand interactions is much less understood and probably varies with regard to exosome-type as well as the recipient cell. We have previously shown that milk-derived exosomes preferentially bind to monocytes within PBMCs  relative to B-cells, CD4+ and CD8+ T cells. In the present study, we show that exosomes found in human breast milk also bind to MDDCs, and that the binding is, at least partly, mediated through DC-SIGN. Interestingly, plasma-derived exosomes did not bind to MDDCs, suggesting a specific interaction between milk-derived exosomes and MDDCs. This observation suggests that exosomes from different body fluids target specific cell types, possibly influenced by variability in exosome phenotype. We propose, based on the finding that binding of milk-derived exosomes to MDDC is partly DC-SIGN mediated, together with the previously reported involvement of DC-SIGN in HIV-1 infection, that the protective effect against HIV-1 infection carried by milk-derived exosomes involves competition for DC-SIGN binding. Putative ligands on milk-derived exosomes responsible for the binding to DC-SIGN could be MUC1, not present on plasma-derived exosomes, or possibly other glycoproteins containing DC-SIGN binding domains. However, as a complete block of MUC1 on exosomes is difficult to obtain and control, due to extensive glycosylation with many different isoforms, we could not definitely conclude in this study that MUC1 is the DC-SIGN binding ligand on the milk-derived exosomes. Further investigation is also needed to conclude whether MUC1 levels or the degree of glycosylation of MUC1 on milk-derived exosomes impact viral infection.
It has been shown that several of the molecules found on milk-derived exosomes, such as MUC1 and CD36, are more resistant to gastrointestinal digestion when associated with the membrane of milk fat globulins . In addition, the higher gastric pH of the newborn relative to the adult , as well the finding that exosomes are long-lasting in vivo , suggest that exosomes might be protected from degradation in the stomach and could potentially impact the immune system of the breastfed child. Milk-derived exosomes might also be efficient in reaching distant sites, due to binding to specific target cells.
Milk exosome preparations from different donors were not equally efficient in their ability to reduce productive HIV-1 infection of MDDCs in our in-vitro system. This is consistent with results suggesting that the DC-SIGN binding capacity of BSSL in breast milk varies highly among mothers , which could be due to differences in number of glycans as well as the structure of the protein. Perhaps this could partially explain why HIV-1 transmission through breastfeeding occurs only in some cases. Interestingly, semen contain the DC-SIGN ligand Semen Clusterin, which has also been shown to limit HIV-1 infection of dendritic cells and subsequent viral transfer to CD4+ T cells . Moreover, semen-derived clusterin, but not clusterin from serum, bind DC-SIGN supporting our finding that different body fluids can interact with certain cells that could lead to diverse protection against pathogens. HIV-1 can be transferred from dendritic cells to CD4+ T cells by different ways. The virus can be trapped inside the dendritic cells and then released or new viruses can be released after productive infection. An alternative mechanism for the effect of milk exosomes on T-cell infection could be that they would change the activation state of the T cells. However, as free exosomes were washed away from the dendritic cells before the addition of T cells, this explanation seems unlikely. Moreover, exosomes did not activate the dendritic cells, as measured by a nonaltered expression of CD86 (data not shown), suggesting that more likely explanation for the findings is that exosomes inhibit the HIV binding and infection of dendritic cells, which in turn leads to lower ability to transfer to CD4+ T cells. Further studies are needed to conclude on what mode of viral transmission is inhibited by milk-derived exosomes.
Thus, we suggest that exosomes from human breast milk might be one of the components that confer protection from HIV-1 transmission from mother to child through breastfeeding. However, we need to consider the possible alternative that exosomes may also be the point of entry for HIV-1 to infect recipient cells in the child. Indeed, it has been shown that HIV-1 can associate with exosomes from HIV-1 infected dendritic cells and initiate infection of CD4+ T cells [23,35]. Hence, our finding that milk-derived exosomes efficiently adhere to MDDCs could be a potential mechanism exploited by HIV-1 for allowing effective transmission of HIV-1, perhaps also infecting cell types not targeted by the virion itself. However, as many of the infants breastfeed by HIV-1 infected mothers do not get infected, the net effect of the breast milk derived exosomes may be protective for the infant. Possibly, the ratio between HIV-associated and HIV-nonassociated exosomes might determine the infection risk of the infant. Further studies will have to investigate whether breast milk derived exosomes from HIV-1 positive mothers carry HIV-1 and what effect this would have on the transmission rate.
The authors want to thank all the breast milk donors. This study was supported by the Swedish Research Council, Swedish Medical Society, the Stockholm County Council, the IMTAC consortium at Karolinska Institutet, The Milk drop, Torsten Söderberg's, the Swedish Cancer Society, King Gustaf V's 80-years’, Magnus Bergvall's, Swedish Heart-Lung, Hesselman's and David and Astrid Hageléns’ Foundations. D.P.-P. is the recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research.
T.I.N., D.P.P., P.T.P., J.K.S., S.G. designed the study. T.I.N., D.P.P. designed and performed experiments and analysed the results. H.V. performed experiments and analysed the results. T.I.N. wrote the manuscript and D.P.P., H.V., P.T.P., J.K.S., S.G. revised.
Conflicts of interest
There are no conflicts of interest.
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