MUC1 in human milk blocks transmission of human immunodeficiency virus from dendritic cells to T cells

Molecular Immunology 46 (2009) 2309–2316

Contents lists available at ScienceDirect

Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

MUC1 in human milk blocks transmission of human immunodeficiency virus from dendritic cells to T cells夽 Eirikur Saeland ∗ , Marein A.W.P. de Jong, Alexey A. Nabatov, Hakan Kalay, Teunis B.H. Geijtenbeek, Yvette van Kooyk Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 23 March 2009 Accepted 28 March 2009 Available online 29 April 2009 Keywords: Human milk Human immunodeficiency virus Dendritic cells MUC1 Glycosylation

a b s t r a c t Mother-to-child transmission of human immunodeficiency virus-1 (HIV-1) occurs frequently via breastfeeding. HIV-1 targets DC-SIGN+ dendritic cells (DCs) in mucosal areas that allow efficient transmission of the virus to T cells. Here, we demonstrate that the epithelial mucin MUC1, abundant in milk, efficiently bound to DC-SIGN on DC. The O-linked glycans within the mucin domain contained Lewis X structures, that were specifically recognized by the receptor. Interestingly, MUC1 prevented DC-SIGNmediated transmission of HIV-1 from DCs to CD4+ T cells. We hypothesize that repetitive units of Lewis X, within the mucin domain, play an important role in inhibiting transmission of HIV-1 from mother to child. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Dendritic cells (DCs) express a variety of receptors for capturing pathogens, and initiating immune responses. One of these receptors is the DC-specific ICAM-3-grabbing non-integrin (DC-SIGN) that binds a variety of pathogens, via a carbohydrate recognition domain that recognizes specific carbohydrate structures (Geijtenbeek et al., 2004). These pathogens include the hepatitis C virus (Ludwig et al., 2004), human immunodeficiency virus (HIV) (Geijtenbeek et al., 2000a), Mycobacterium tuberculosis (Geijtenbeek et al., 2003; Tailleux et al., 2003), Helicobacter pylori (Bergman et al., 2004), measles virus (de Witte et al., 2008) and herpes simplex virus (de Jong et al., 2008a). Recognition of pathogens by DC generally results in the initiation of immune responses to clear the infection, but pathogens may also target immune cells, such as DC, to evade immune responses. The gp120 envelope glycoprotein of HIV (types 1 and 2) is known to interact with DC-SIGN and this interaction results in enhanced transmission of the virus to CD4+ T lymphocytes in vitro (Geijtenbeek et al., 2000a). This can be an important mechanism of how the virus efficiently travels from the site of infec-

夽 ES was supported by the Nutricia Research Foundation; MAWPdJ was supported by Dutch Scientific Organization (NWO) grant 91204025; AN was supported by the Dutch Aids Foundation (grant no. 20005033). ∗ Corresponding author at: Department of Molecular Cell Biology and Immunology, VU University Medical Center, Postbox 7057, 1007 MB Amsterdam, The Netherlands. Tel.: +31 20 444 8259; fax: +31 20 444 8081. E-mail address: [email protected] (E. Saeland). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.03.025

tion (at mucosal surfaces) to T cell areas in the draining lymph nodes. Mother-to-child transmission (MTCT) of HIV-1 occurs frequently via breast-feeding. The mechanism of MTCT by breast-feeding has been extensively studied, mainly because replacement feeding is unavailable or unacceptable in many parts of the world and because breast-feeding provides protection against common causes of mortality in infants (Kourtis et al., 2007; Hartmann et al., 2006). A large clinical trial in Nairobi estimated HIV-1 transmission attributable to milk to be 44% MTCT (Nduati et al., 2001). Macrophages, present in milk, can be stimulated with IL-4 to express DC-SIGN and have been shown to efficiently transmit virus to T cells in vitro (Satomi et al., 2005). This may be one mechanism how the virus is transmitted to the infant’s T cells. However, recent studies indicate that exclusive breast-feeding during the early months of life protects against HIV infection (Coovadia and Kindra, 2008; Fowler, 2008; Kuhn et al., 2007) which suggests that certain components in human milk may protect against transmission of the virus to the child. Along with anti-viral factors present in milk, factors that block interaction of HIV with DC-SIGN have been identified. These include the bile salt-stimulated lipase (BSSL), a Lewis X-containing component in milk that has been demonstrated to block DC-SIGN-mediated HIV transmission to T cells (Naarding et al., 2005, 2006) and natural antibodies to the carbohydrate recognition domain (CRD) of DC-SIGN present in breast milk (Requena et al., 2008). Here, we demonstrate that the epithelial mucin MUC1, another Lewis X-containing factor abundantly present in human milk, specifically bound to DC-SIGN on DC and effectively blocked DC-SIGN-mediated HIV-1 transmission to T cells.

2310

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

2. Materials and methods 2.1. Human milk Milk was collected from healthy Dutch mothers. Aqueous phase was obtained by centrifuging the milk samples in two sequential steps (collecting the aqueous phase from underneath the fat layer and centrifuging again): 10 at 680 × g followed by 30 at 10,000 × g at 4 ◦ C. The samples were stored at −80 ◦ C. The aqueous layer of milk (50 ml) was freeze-dried overnight and dissolved in 15 ml water, mixed with 15 ml N-butanol and 30 ml di-isopropyl ether and incubated at 4 ◦ C for 2 h, rolling. After centrifugation, the upper (organic) layer was removed and aqueous layer mixed again with 30 ml di-isopropyl ether. The solution was incubated for 2 h at 4 ◦ C, centrifuged and the organic layer removed. The aqueous layer was freeze-dried overnight. Twenty milliliters PBS was added and proteins solubilised for 1 h by sonication and filtered through a 0.45 ␮m filter. Milk proteins were purified by two methods. Proteins were either separated on a gel filtration column (Sepharose 6 (10 × 300), GE Healthcare Europe, Diegem, Belgium) or by an anti-MUC1 affinity column. The affinity column was generated as follows: antiMUC1 antibodies (clone 214D4; 1 mg/ml) were coupled to 1 ml N-hydroxysuccinimidyl sepharose 4 (Sigma). Milk proteins were applied, the column was washed with PBS and MUC1 eluted with 100 mM glycine pH 2.7, directly into 1 M Tris buffer pH 9.0. The eluate was desalted against 20 mM ammonium formate through a G25 desalting column (GE Healthcare). Finally, the protein was brought to dryness by speedvac overnight and dissolved in PBS. Protein concentration was measured by a BCA assay (Pierce, Rockford, IL, USA). 2.2. Antibodies and Fc-chimeric proteins DC-SIGN-Fc contains the extracellular portion of DC-SIGN (amino acid residues 64–404) and the Fc domain of human IgG1 fused at the COOH terminus of DC-SIGN (Geijtenbeek et al., 2002). Similarly, gp120–Fc was made by fusing the whole length gp120 construct to the Fc domain of human IgG1. The following antibodies were used: MUC1 antibodies clone 214D4 (IgG1 isotype, provided by John Hilkens, Netherlands Cancer Institute, Amsterdam); clone 214D4 was biotinylated by using a biotinylation kit (Pierce, Rockford, USA) following the manufacturer’s instructions; antibodies to DC-SIGN clone AZN-D1 (Geijtenbeek et al., 2000b); antibodies to Lewis X, Y, A, and B antigens (Calbiochem, Gibbstown, NJ, USA); PE-labeled antibodies to the DC maturation markers CD80, CD86 (BD Biosciences Pharmingen, San Jose, CA, USA) and CD83 (Beckman Coulter, Mijdrecht, The Netherlands). The following secondary antibodies were used: peroxidase-labeled anti-human IgG (Jackson ImmunoResearch Europe, Suffolk, UK); FITC-labeled goat anti-mouse IgG (Jackson ImmunoResearch); biotinylated F(ab’)2 fragments of goat anti-mouse IgG (Jackson Immunoresearch); peroxidase-labeled streptavidin (Vector Laboratories, Burlingame, CA, USA). 2.3. Cells Chinese hamster ovary cells (CHO), transfected with DC-SIGN, were cultured in the presence of G418 (1 mg/ml) in RPMI containing 10% fetal bovine serum (FBS) and streptomycin/penicillin. DC-SIGNtransduced Raji cells were cultured in RPMI containing 10% FBS and streptomycin/penicillin. Monocyte-derived dendritic cells (MoDCs) were cultured from buffycoats. Buffycoats were mixed with PBS containing 0.45% citrate (PBS-citrate) and peripheral blood mononuclear cells (PBMC) were isolated by a Ficoll gradient step. The PBMC were washed

in PBS-citrate and monocytes isolated by a Percoll (GE Healthcare Europe, Diegem, Belgium) gradient step, extensively washed in PBScitrate to remove the remaining platelets and finally washed in RPMI. The monocytes were cultured in RPMI containing 10% FBS and streptomycin/penicillin in the presence of recombinant IL-4 and GM-CSF (500 and 800 U/ml, respectively; Biosource, Nivelles, Belgium) for 6 days. The phenotype of immature DC was confirmed by flow cytometry (DC-SIGNhigh, CD80low, CD83low, CD86low). PHA-activated-enriched CD4+ T lymphocytes (CCR5+/+) were obtained using MACS beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultured as described earlier (Nabatov et al., 2006) and Jurkat T cells expressing CCR5 were generated by retroviral transduction as previously described (Arrighi et al., 2004a,b). 2.4. Fluorescent beads adhesion assay Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0 ␮m; Molecular Probes, Eugene, OR) were coated with streptavidin as previously described (Geijtenbeek et al., 1999). Gp120 beads were made as follows: streptavidin-coated beads were incubated with biotinylated F(ab’)2 fragments of goat anti-mouse IgG (6 ␮g/ml, Jackson Immunoresearch) in 0.5 ml PBS 0.5% BSA for 2 h at 37 ◦ C, washed and further incubated with gp120–Fc fusion proteins at 4 ◦ C overnight. MUC1-beads were similarly generated by incubating streptavidin-coated beads with biotinylated F(ab’)2 fragments of goat anti-mouse IgG (6 ␮g/ml) in 0.5 mL PBS 0.5% BSA for 2 h at 37 ◦ C. The beads were washed and incubated with anti-MUC1 antibodies at 4 ◦ C overnight (rotating), followed by washing and incubation in human skim milk at 4 ◦ C overnight (rotating). All beads were stored in 100 ␮l PBS 0.5% BSA, and used within 1 week. The beads adhesion assay was performed as previously described (Geijtenbeek et al., 1999). DCs were incubated with coated beads in Tris–sodium buffer (20 mM Tris–HCl, pH 7, 150 mM NaCl, 2 mM CaCl2 , 2 mM MgCl2 ) with 0.5% BSA and adhesion was determined in the absence or presence of blocking agents (10 mM EGTA, 20 ␮g/ml AZN-D1 or 500 ␮g/ml Mannan (Sigma–Aldrich, Zwijndrecht, The Netherlands)) at 37 ◦ C for 45 min. Cells were washed and analysed by flow cytometry (Beckton Dickinson, NJ, USA) and binding determined as percentage of fluorescent positive DC. 2.5. Binding assay The solid phase adhesion assay was performed by coating ELISA plates (Maxisorp, Nunc, Roskilde, Denmark) with anti-MUC1 antibodies (10 ␮g/ml), diluted in 0.2 M Na2 CO3 and incubated overnight at 4 ◦ C. The plates were washed and incubated with Tris–sodium buffer containing 1% BSA for 30 min at 37 ◦ C. The plates were emptied and further incubated with milk (1:100) for 1 h at room temperature (RT). After washing, biotinylated anti-MUC1 antibodies (2 ␮g/ml), DC-SIGN-Fc supernatant (0.5 ␮g/ml) or anti-glycan antibodies (1 ␮g/ml) were added and incubated for 2 h at RT. Unbound lectins/antibodies were washed away with Tris–sodium–0.02% Tween and binding was detected by peroxidase-labeled anti-human Fc or peroxidase-labeled streptavidin. The reaction was developed by 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate (Sigma–Aldrich) and optical density measured by a spectrophotometer. 2.6. Viruses Two different type of HIV-1 viruses were used. Replicationcompetent HIV-1 stocks (subtype B molecular cloned viruses JR-CSF (R5)) were generated by the passage of viruses through CD4+ lymphocytes, with tissue culture infectious dose (TCID50/ml)

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

Fig. 1. Blocking of HIV-gp120 interaction with DC by milk. DCs were incubated in with HIV-gp120-coated fluorescent beads in the presence or absence of skimmed milk or inhibitors (20 ␮g/ml anti-DC-SIGN or 10 mM EGTA). Binding was measured by flow cytometry and represented as %DC binding fluorescent beads. Experiment was performed in triplicates (error bars show standard deviation) and one experiment out of two is shown.

determined by limiting dilution on CD4+-enriched T lymphocytes. HIV-1 NL4.3-BaL-eGFP (20 ng/well) were produced in 293T cells as described before. Virus stocks were quantified by p24 ELISA (PerkinElmer Life Sciences) and titrated using the indicator cells TZM-blue (de Jong et al., 2008b). 2.7. DC-mediated HIV-1 transmission assay Immature DCs or DC-SIGN-transfected Raji cells were plated at 4 × 104 cells/well in a 96-well format and pulsed with the appropriate virus. After incubation, cells were washed three times with culture medium before addition of CD4+-enriched T lymphocytes (2 × 105 cells/well) or CCR5+ Jurkat T cells (3 × 104 cells/well). Either the JR-CSF (R5) strain or NL4.3-BaL-eGFP (20 ng/well) were

2311

Fig. 2. MUC1 in milk blocks interaction of HIV-gp120 with DC. DCs were incubated in with HIV-gp120-coated fluorescent beads in the presence or absence of milk fractions (“Fraction” refers to fraction number represented in Fig. 3) or inhibitors (20 ␮g/ml anti-DC-SIGN, mannan (500 ␮g/ml) or 10 mM EGTA). Binding was measured by flow cytometry and represented as percentage of DC binding beads. One representative experiment of two is shown.

used for the experiments. The inhibitory effects of MUC1 and other compounds on the DC-mediated HIV-1 transfer to T cells cultures was determined by preincubation of DCs with the compounds for 2 h at 37 ◦ C and subsequent washing. The infection was followed with HIV-1 CA-p24 ELISA when the JR-CSF (R5) strain was used or by flow cytometry when the NL4.3-BaL-eGFP strain was used.

2.8. Statistical analysis Statistical differences between groups were analysed by Dunnet multiple comparisons test using Graphpad Instat from Graphpad Software, Inc. Significance was accepted at the p < 0.05 level.

Fig. 3. MUC1 concentration in different fractions. ELISA plate was coated with anti-MUC1 antibodies (214D4) and the different fractions of milk added (diluted to the same protein concentration; fraction 1 contains proteins of the highest molecular weight and fraction 36 proteins of the lowest molecular weight) and incubated overnight at 4 ◦ C. MUC1 was detected with a biotinylated anti-MUC1 antibody (214D4).

2312

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

3. Results 3.1. Human milk blocks HIV-gp120 interaction with dendritic cells To study the HIV-1 blocking capacity of human milk we investigated the interaction of DC with HIV-1 gp120 using the fluorescent beads adhesion assay. Fluorescent beads, coated with gp120–Fc were incubated with DC in the presence of human milk samples in different dilutions. Milk (1:10) efficiently blocked the interaction of gp120 to DC (Fig. 1). To obtain an indication whether and which protein(s) were responsible for the inhibition, milk was defatted and proteins were separated by gel filtration. The different purified fractions were used as blocking agents in the beads adhesion assay, using identical protein concentrations of each fraction. Proteins of high molecular weight efficiently blocked interaction of gp120 with DC, whereas proteins of less than 100 kDa did not inhibit the binding (Fig. 2). 3.2. MUC1 in milk binds to DC-SIGN and blocks gp120 interaction with DC Bile salt-stimulated lipase (BSSL, 112 kDa), a Lewis X-positivefactor in human milk, has been identified as a potential mediator of blocking HIV interaction with DC (Naarding et al., 2006). Other high molecular weight proteins have not been studied, in particular not glycoproteins that may contain high amounts of Lewis X, the structure responsible for blocking and high affinity binding to DC-SIGN (van Liempt et al., 2006). The C-terminus of BSSL is composed of a mucin-like domain, rich in proline, serine, and threonine, bearing repeating units of O-linked Lewis X structures (Baba et al., 1991; Wang et al., 1995). An obvious candidate of high molecular weight glycoproteins in milk, containing such a domain, is the epithelial mucin MUC1. MUC1 is one of the main proteins in the human milk fat globule membrane (estimated around 420 ␮g/ml, measured in 44 samples, Peterson et al., 1998) and is also shed into the aqueous phase of the milk. MUC1 in human milk contains diverse O-linked glycan structures that have been demonstrated to contain fucose (Hanisch et al., 1989). These glycans, in particular Lewis antigens, may interact with DC-SIGN (van Liempt et al., 2006) and inhibit interaction with the HIV-1 gp120 glycoprotein. As expected, MUC1 was detected in milk fractions containing high molecular weight proteins (Fig. 3). To determine glycosylation of MUC1 (in fraction 3) ELISA plates were coated with anti-MUC1 antibodies before incubation with MUC1. Lewis carbohydrates were determined with specific antibodies: Lewis A, Lewis B, Lewis X, and Lewis Y. Strikingly only Lewis X was detected to be present on MUC1 (Fig. 4). Furthermore, DC-SIGN-Fc bound strongly to MUC1 in ELISA and this interaction was blocked by removing the essential calcium ions from the buffer (using EGTA) (Fig. 4C). Calcium ions are required for ligand binding of DC-SIGN. To demonstrate that MUC1, isolated from human milk, can be involved in blocking gp120 interaction with DC-SIGN, MUC1 binding to DC-SIGN on DC and DC-SIGN-transfected CHO cells was studied (Fig. 5A). MUC1 (from milk) was captured on fluorescent beads by specific anti-MUC1 antibodies, prior to incubation with DC. The MUC1-coated beads bound efficiently to DC and the interaction was blocked anti-DC-SIGN antibodies, mannan (a ligand for DC-SIGN) and EGTA (Fig. 5B). Blocking antibodies to the mannose receptor did not inhibit this binding (data not shown). These data were confirmed using DC-SIGN-transfected CHO cells, that specifically bound to the MUC1-coated beads in contrast to mocktransfected CHO cells (Fig. 5C). Taken together, these data suggest that MUC1 interacts with DC-SIGN, probably via Lewis X moieties within the mucin domain, and can specifically block HIV-1 gp120 interaction with DC-SIGN on DC.

Fig. 4. DC-SIGN-Fc binds to Lewis X on MUC1. ELISA plate was coated with anti-MUC1 antibodies (214D4), followed by MUC1-containing milk fractions and incubated overnight at 4 ◦ C. (A) Lewis B, Lewis X, and Lewis Y were determined with specific antibodies of the IgM isotype and peroxidase-labeled anti-mouse IgM. (B) Lewis A was detected with specific IgG antibody followed by peroxidase-labeled anti-mouse IgG. Black bars represent negative controls: wells containing only coating antibodies. (C) DC-SIGN binding was detected by DC-SIGN-Fc (1 ␮g/ml) and specificity determined by co-incubation in the presence of 10 nM EGTA. Milk fractions 3, 4, and 6 were used (see Fig. 3). The experiment was done in triplicates and error bars represent standard deviations.

3.3. MUC1 in milk blocks HIV transmission to T cells HIV-1 is efficiently transmitted via DC-SIGN to CD4+ T cells. To determine the possible role of MUC1 in blocking HIV-1 transmission, Raji and DC-SIGN-transfected Raji cells were pre-incubated with a MUC1-containing fraction and an irrelevant milk fraction before incubation with live HIV-1 virus. Activated CD4+ T cells were added and the cells were co-incubated for 10 days. Strikingly, the MUC1-containing fraction efficiently blocked DC-SIGN-mediated transmission of the virus to T cells (Fig. 6A), to a similar extent as anti-DC-SIGN blocking antibodies and mannan. As expected, the

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

2313

Fig. 6. MUC1 blocks HIV transmission. Dendritic cells and DC-SIGN-transfected Raji cells were pre-incubated with blocking agents (40 ␮g/ml AZN-D1; 1 mg/ml mannan; milk proteins) for 1 h at 37 ◦ C before incubation with HIV-1JR-CSF. T cells were added and the cells co-cultured for 10 days. Transmission experiments using (A) Raji cells and DC-SIGN transfected Raji or (B) dendritic cells from two donors. Numbers on the X-axis represent concentration of proteins used (␮g/ml) (B). Experiments were performed in triplicates two times in independent experiments. The results of the p24 ELISA were plotted as average of three samples with bars reflecting standard deviation (SD). The groups were compared using Dunnett multiple comparisons test and statistically significant difference from “No block” is represented by *p < 0.05; **p < 0.01.

Fig. 5. MUC1 binding to DC-SIGN. DC and DC-SIGN transfected CHO cells highly express DC-SIGN (A). DC (B), CHO, and CHO-DC-SIGN (C) were incubated in with MUC1-coated fluorescent beads in the presence or absence of inhibitors (20 ␮g/ml anti-DC-SIGN or anti-mannose receptor (MR), mannan (500 ␮g/ml) or 10 mM EGTA). Binding was measured by flow cytometry and represented as percentage of cells binding beads. One representative experiment of three is shown.

MUC1-negative milk fraction did not block the transmission. Similar observations were made using monocyte-derived DC where the MUC1+ milk fraction blocked transmission of HIV-1 to T cells (Fig. 6B). To exclude the possibility that contaminating proteins in the MUC1+ fraction were responsible for the inhibiting effect, we made an anti-MUC1 affinity column to purify MUC1 from milk. MUC1 was purified from milk of two donors and these were used in the transmission assay. MUC1 sample from Donor 1 blocked transmission of the virus from DC to targets cells, and Donor 2 showed either no or reduced block (Fig. 7A). The effect correlated with the amount of Lewis X present on MUC1 and with DC-SIGN binding. MUC1 from Donor 1 contained significantly higher Lewis X and bound much more efficiently to DC-SIGN compared to Donor 2. 4. Discussion In this study we demonstrate that the highly glycosylated mucin MUC1 in human milk interacts with DC-SIGN on moDC. More importantly, MUC1 efficiently blocked interaction of the HIV-1 gp120 glycoprotein with DC and DC-SIGN-mediated transmission of HIV-1 to CD4+ T cells. It has previously been demonstrated that

2314

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

Fig. 7. Lewis X on MUC1 blocks transmission of HIV-1. Dendritic cells from two different donors (A and B) were pre-incubated with purified MUC1 for 2 h at 37 ◦ C before incubation with HIV-1 NL4.3-BaL-eGFP. Target cells were added and the cells co-cultured for 5 days and infection measured by flow cytometry (%infected T cells). MUC1 concentration in samples was measured by a sandwich ELISA (C). Lewis X was detected on MUC1 in the ELISA (D) and DC-SIGN-Fc binding was measured (E). Experiment was performed in triplicates. The results of infection were plotted as average of three samples with bars reflecting standard deviation (SD).

a high molecular weight, Lewis X-positive-factor in human milk can inhibit DC-SIGN-mediated transmission of HIV-1 (Naarding et al., 2005). Later, the same authors identified a milk protein, BSSL (112 kDa), that could be responsible for this inhibiting effect (Naarding et al., 2006). BSSL (between 100 and 200 ␮g/ml in milk) is an enzyme responsible for fat digestion in the newborn and the C-terminus is composed of a mucin-like domain, rich in proline, serine and threonine, containing the carbohydrates responsible for DC-SIGN binding (Lewis X) (Baba et al., 1991). These amino acids are found within ∼16 tandem repeats of 11 amino acids each (Wang et al., 1995). Other high molecular weight proteins (>100 kDa) were not excluded as potential inhibitors in this particular study which prompted us to study the role of the mucin MUC1, also an abundant

glycoprotein in human milk (420 ␮g/ml, primarily within milk fat globules, Peterson et al., 1998). We demonstrated that milk fractions of high molecular weight proteins (∼150–700 kDa), efficiently blocked HIV-1 binding to DC-SIGN. MUC1 was identified in these fractions, containing repetitive O-linked glycans and carrying the DC-SIGN-ligand Lewis X. Purified MUC1 (through anti-MUC1 affinity column) was also shown to block transmission of the virus and this was related to the Lewis X carbohydrates. The mucin domain of MUC1 is extensively glycosylated (with carbohydrates representing as much as 50–80% of the molecular weight), underlining the potential of this molecule to inhibit HIV-1 transmission to T cells similar to BSSL, by competing with DC-SIGN on the DC surface.

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

Mucin-like domains are composed of several tandem repeats (TR). MUC1 contains 20–120 TR of 20 amino acids, each containing 5 potential O-glycosylation sites (Gendler et al., 1990). Similarly, although much shorter, BSSL contains ∼16 TR of 11 amino acids (Baba et al., 1991). The repetitive units of O-glycans in mucin-like domains exhibit high avidity binding to DC-SIGN, which may be responsible for efficient competition with HIV-1. MUC1 may be a much stronger player than BSSL due to the extensive amount of glycans present. Interestingly, both MUC1 and BSSL exhibit a significant polymorphism in the number of TR (Gendler et al., 1990; Ligtenberg et al., 1990; Lindquist et al., 2002). Variable number of TR within the mucin-like domain significantly attributes to the total amount of O-glycans on the protein. It remains to be determined how polymorphism influences the protective effects of the molecules and whether this may have clinical consequences. Another variable is that glycans may change in the course of lactation. Lewis X structures on BSSL have been demonstrated to increase with time (Landberg et al., 2000), and whether this occurs on MUC1 remains to be determined. MUC1 in human milk has been considered to play a role in innate immune defence of the infant, functioning as a decoy receptor for pathogens. MUC1 prevents adhesion of enteropathogenic strains of Escherichia coli to buccal epithelial cells, by interacting directly with the bacteria and preventing colonisation (Schroten et al., 1992). Furthermore, purified MUC1 incubated with HIV1 has been demonstrated to inhibit direct infection of CEM-SS cells expressing CD4, CXCR4, ICAM-3 and MHC class II (Habte et al., 2008). Interestingly, both MUC1 and BSSL have been shown to bind Norwalk virus, an important cause of viral gastroenteritis, and inhibit adhesion to epithelial cells (Ruvoen-Clouet et al., 2006). This occurred via carbohydrate structures that are recognized by the virus, again preventing attachment of the virus to the epithelium. Collectively these data demonstrate the role for MUC1 as a decoy receptor, preventing the first stage of pathogen infection (attachment). Our data further implicate the role of MUC1 in innate immune defence of the infant preventing vertical transmission of HIV from mother to child, by competing with HIV for the receptor on DC. Thus, although the virus is transmitted via milk, milk also contains factors (MUC1, BSSL, antibodies) that protect against HIV transmission to T cells. The importance of these protective factors is highlighted in studies demonstrating that exclusive breast-feeding during the early months of life protect against HIV infection (3.5–4-fold more chance of transmission to infants not exclusively breast-fed) (Coovadia and Kindra, 2008; Fowler, 2008; Kuhn et al., 2007). More frequent breast-feeding provides the infant with quantitatively more protective factors that are also beneficial against other infections. References Arrighi, J.F., Pion, M., Garcia, E., Escola, J.M., van Kooyk, Y., Geijtenbeek, T.B., Piguet, V., 2004a. DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells. J. Exp. Med. 200, 1279– 1288. Arrighi, J.F., Pion, M., Wiznerowicz, M., Geijtenbeek, T.B., Garcia, E., Abraham, S., Leuba, F., Dutoit, V., Ducrey-Rundquist, O., van Kooyk, Y., Trono, D., Piguet, V., 2004b. Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells. J. Virol. 78, 10848–10855. Baba, T., Downs, D., Jackson, K.W., Tang, J., Wang, C.S., 1991. Structure of human milk bile salt activated lipase. Biochemistry 30, 500–510. Bergman, M.P., Engering, A., Smits, H.H., van Vliet, S.J., van Bodegraven, A.A., Wirth, H.P., Kapsenberg, M.L., Vandenbroucke-Grauls, C.M., van Kooyk, Y., Appelmelk, B.J., 2004. Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DCSIGN. J. Exp. Med. 200, 979–990. Coovadia, H., Kindra, G., 2008. Breastfeeding to prevent HIV transmission in infants: balancing pros and cons. Curr. Opin. Infect. Dis. 21, 11–15. de Jong, M.A., de Witte, L., Bolmstedt, A., van Kooyk, Y., Geijtenbeek, T.B., 2008a. Dendritic cells mediate herpes simplex virus infection and transmission through the C-type lectin DC-SIGN. J. Gen. Virol. 89, 2398–2409.

2315

de Jong, M.A., de Witte, L., Oudhoff, M.J., Gringhuis, S.I., Gallay, P., Geijtenbeek, T.B., 2008b. TNF-alpha and TLR agonists increase susceptibility to HIV-1 transmission by human langerhans cells ex vivo. J. Clin. Invest. 118, 3440–3452. de Witte, L., de Vries, R.D., van der Vlist, M.V., Yuksel, S., Litjens, M., de Swart, R.L., Geijtenbeek, T.B., 2008. DC-SIGN and CD150 have distinct roles in transmission of measles virus from dendritic cells to T-lymphocytes. PLoS. Pathog. 4, e1000049. Fowler, M.G., 2008. Further evidence that exclusive breast-feeding reduces motherto-child HIV transmission compared with mixed feeding. PLoS. Med. 5, e63. Geijtenbeek, T.B., van Duijnhoven, G.C., van Vliet, S.J., Krieger, E., Vriend, G., Figdor, C.G., van Kooyk, Y., 2002. Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1. J. Biol. Chem. 277, 11314–11320. Geijtenbeek, T.B., van Vliet, S.J., Engering, A., ‘t Hart, B.A., van Kooyk, Y., 2004. Self- and nonself-recognition by C-type lectins on dendritic cells. Annu. Rev. Immunol. 22, 33–54. Geijtenbeek, T.B., van Vliet, S.J., Koppel, E.A., Sanchez-Hernandez, M., Vandenbroucke-Grauls, C.M., Appelmelk, B., van Kooyk, Y., 2003. Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp. Med. 197, 7–17. Geijtenbeek, T.B.H., Kwon, D.S., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C.F., Middel, J., Cornelissen, I.L., Nottet, H.S., KewalRamani, V.N., Littman, D.R., Figdor, C.G., van Kooyk, Y., 2000a. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, 587–597. Geijtenbeek, T.B.H., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C.F., Adema, G.J., van Kooyk, Y., Figdor, C.G., 2000b. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100, 575–585. Geijtenbeek, T.B.H., van Kooyk, Y., van Vliet, S.J., Renes, M.H., Raymakers, R.A., Figdor, C.G., 1999. High frequency of adhesion defects in B-lineage acute lymphoblastic leukemia. Blood 94, 754–764. Gendler, S.J., Lancaster, C.A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E.N., Wilson, D., 1990. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J. Biol. Chem. 265, 15286–15293. Habte, H.H., de, B.C., Lotz, Z.E., Tyler, M.G., Kahn, D., Mall, A.S., 2008. Inhibition of human immunodeficiency virus type 1 activity by purified human breast milk mucin (MUC1) in an inhibition assay. Neonatology 93, 162–170. Hanisch, F.G., Uhlenbruck, G., Peter-Katalinic, J., Egge, H., Dabrowski, J., Dabrowski, U., 1989. Structures of neutral O-linked polylactosaminoglycans on human skim milk mucins. A novel type of linearly extended poly-N-acetyllactosamine backbones with Gal beta(1–4)GlcNAc beta(1–6) repeating units. J. Biol. Chem. 264, 872–883. Hartmann, S.U., Berlin, C.M., Howett, M.K., 2006. Alternative modified infant-feeding practices to prevent postnatal transmission of human immunodeficiency virus type 1 through breast milk: past, present, and future. J. Hum. Lact. 22, 75–88. Kourtis, A.P., Jamieson, D.J., de Vries, I., Taylor, A., Thigpen, M.C., Dao, H., Farley, T., Fowler, M.G., 2007. Prevention of human immunodeficiency virus-1 transmission to the infant through breastfeeding: new developments. Am. J. Obstet. Gynecol. 197, S113–S122. Kuhn, L., Sinkala, M., Kankasa, C., Semrau, K., Kasonde, P., Scott, N., Mwiya, M., Vwalika, C., Walter, J., Tsai, W.Y., Aldrovandi, G.M., Thea, D.M., 2007. High uptake of exclusive breastfeeding and reduced early post-natal HIV transmission. PLoS ONE 2, e1363. Landberg, E., Huang, Y., Stromqvist, M., Mechref, Y., Hansson, L., Lundblad, A., Novotny, M.V., Pahlsson, P., 2000. Changes in glycosylation of human bile-saltstimulated lipase during lactation. Arch. Biochem. Biophys. 377, 246–254. Ligtenberg, M.J., Vos, H.L., Gennissen, A.M., Hilkens, J., 1990. Episialin, a carcinomaassociated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J. Biol. Chem. 265, 5573–5578. Lindquist, S., Blackberg, L., Hernell, O., 2002. Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11. Eur. J. Biochem. 269, 759–767. Ludwig, I.S., Lekkerkerker, A.N., Depla, E., Bosman, F., Musters, R.J., Depraetere, S., van Kooyk, Y., Geijtenbeek, T.B., 2004. Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation. J. Virol. 78, 8322–8332. Naarding, M.A., Dirac, A.M., Ludwig, I.S., Speijer, D., Lindquist, S., Vestman, E.L., Stax, M.J., Geijtenbeek, T.B., Pollakis, G., Hernell, O., Paxton, W.A., 2006. Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells. Antimicrob. Agents Chemother. 50, 3367–3374. Naarding, M.A., Ludwig, I.S., Groot, F., Berkhout, B., Geijtenbeek, T.B., Pollakis, G., Paxton, W.A., 2005. Lewis X component in human milk binds DC-SIGN and inhibits HIV-1 transfer to CD4+ T lymphocytes. J. Clin. Invest 115, 3256–3264. Nabatov, A.A., van Montfort, T., Geijtenbeek, T.B., Pollakis, G., Paxton, W.A., 2006. Interaction of HIV-1 with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-expressing cells is influenced by gp120 envelope modifications associated with disease progression. FEBS J. 273, 4944– 4958. Nduati, R., Richardson, B.A., John, G., Mbori-Ngacha, D., Mwatha, A., Ndinya-Achola, J., Bwayo, J., Onyango, F.E., Kreiss, J., 2001. Effect of breastfeeding on mortality among HIV-1 infected women: a randomised trial. Lancet 357, 1651–1655. Peterson, J.A., Patton, S., Hamosh, M., 1998. Glycoproteins of the human milk fat globule in the protection of the breast-fed infant against infections. Biol. Neonate 74, 143–162. Requena, M., Bouhlal, H., Nasreddine, N., Saidi, H., Gody, J.C., Aubry, S., Gresenguet, G., Kazatchkine, M.D., Sekaly, R.P., Belec, L., Hocini, H., 2008. Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural anti-

2316

E. Saeland et al. / Molecular Immunology 46 (2009) 2309–2316

bodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins. Immunology 123, 508–518. Ruvoen-Clouet, N., Mas, E., Marionneau, S., Guillon, P., Lombardo, D., Le, P.J., 2006. Bile-salt-stimulated lipase and mucins from milk of ‘secretor’ mothers inhibit the binding of Norwalk virus capsids to their carbohydrate ligands. Biochem. J. 393, 627–634. Satomi, M., Shimizu, M., Shinya, E., Watari, E., Owaki, A., Hidaka, C., Ichikawa, M., Takeshita, T., Takahashi, H., 2005. Transmission of macrophage-tropic HIV-1 by breast-milk macrophages via DC-SIGN. J. Infect. Dis. 191, 174–181. Schroten, H., Hanisch, F.G., Plogmann, R., Hacker, J., Uhlenbruck, G., Nobis-Bosch, R., Wahn, V., 1992. Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect

of the protective function of mucins in the nonimmunoglobulin fraction. Infect. Immun. 60, 2893–2899. Tailleux, L., Schwartz, O., Herrmann, J.-L., Pivert, E., Jackson, M., Amara, A., Legres, L., Dreher, D., Nicod, L.P., Gluckman, J.C., Lagrange, P.H., Gicquel, B., Neyrolles, O., 2003. DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells 197 (1). van Liempt, E., Bank, C.M., Mehta, P., Garcia-Vallejo, J.J., Kawar, Z.S., Geyer, R., Alvarez, R.A., Cummings, R.D., van Kooyk, Y., van Die, I., 2006. Specificity of DC-SIGN for mannose- and fucose-containing glycans. FEBS Lett. 580, 6123–6131. Wang, C.S., Dashti, A., Jackson, K.W., Yeh, J.C., Cummings, R.D., Tang, J., 1995. Isolation and characterization of human milk bile salt-activated lipase C-tail fragment. Biochemistry 34, 10639–10644.