Soluble Toll-like receptor 2 is significantly elevated in HIV-1 infected breast milk and inhibits HIV-1 induced cellular activation, inflammation and infection
Henrick, Bethany M.a; Yao, Xiao-Dana; Drannik, Anna G.a; Abimiku, Alash’leb; Rosenthal, Kenneth L.a; the INFANT Study Team
aMcMaster Immunology Research Centre, Michael G. DeGroote Institute for Infectious Disease Research, Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
bInstitute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
*The INFANT Study Team: Innate Factors Associated with Nursing Transmission.
Correspondence to Kenneth L. Rosenthal, Department of Pathology & Molecular Medicine, McMaster University, MDCL 4019, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada. Tel: +1 905 525 9140 x22375; fax: +1 905 522 6750; e-mail: firstname.lastname@example.org
Received 24 January, 2014
Revised 11 June, 2014
Accepted 11 June, 2014
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).
Objectives: We previously demonstrated that immunodepletion of soluble Toll-like receptor 2 (sTLR2) from human breast milk significantly increased HIV infection in vitro. The aims of this study were to characterize sTLR2 levels in breast milk from HIV-infected and uninfected women, and identify a mechanism by which sTLR2 inhibits HIV-induced cellular activation and infection.
Design: Blinded studies of breast milk from HIV-infected and uninfected Nigerian and Canadian women were evaluated for levels of sTLR2, proinflammatory cytokines and viral antigenemia. In-vitro experiments were conducted using cell lines to assess sTLR2 function in innate responses and effect on HIV infection.
Results: Breast milk from HIV-infected women showed significantly higher levels of sTLR2 than uninfected breast milk. Further, sTLR2 levels correlated with HIV-1 p24 and interleukin (IL)-15, thus suggesting a local innate compensatory response in the HIV-infected breast. Given the significantly higher levels of sTLR2 in breast milk from HIV-infected women, we next demonstrated that mammary epithelial cells and macrophages, which are prevalent in milk, produced significantly increased levels of sTLR2 following exposure to HIV-1 proteins p17, p24 and gp41 or the TLR2 ligand, Pam3CSK4. Our results also demonstrated that sTLR2 physically interacts with p17, p24 and gp41 and inhibits HIV-induced nuclear factor kappa-light-chain-enhancer of activated B cells activation, and inflammation. Importantly, binding of sTLR2 to HIV-1 proteins inhibited a TLR2-dependent increase in chemokine receptor 5 expression, thus resulting in significantly reduced HIV-1 infection.
Conclusion: These results indicate novel mechanisms by which sTLR2 plays a critical role in inhibiting mother-to-child HIV transmission.
Breast milk is an extraordinary mucosal fluid that provides an optimal source of nutrition for the developing infant while conferring protection against environmental pathogens through a milieu of innate and adaptive immune factors. An intervention that promotes exclusive breastfeeding, regardless of the mother's HIV status, has led to significantly decreased mother-to-child transmission (MTCT) compared with mixed feeding [1–4]. Although it is poorly understood how infants breastfeeding from HIV-infected mothers remain uninfected despite repeated and prolonged exposure to HIV, the apparent protection is attributable to innate factors in breast milk that possess potent antiviral activities [5,6]. Indeed, lactoferrin, mucin, secretory leukocyte protease inhibitor (SLPI) and soluble Toll-like receptor 2 (sTLR2) all are found at high levels in breast milk and have documented HIV-1 inhibitory properties by interfering with virus/host cell attachment [5,7–9]. Our recent data, using neutralization and immunodepletion of sTLR2 from breast milk, strongly suggested that sTLR2 directly interacted with the virus, thus inhibiting cell-free HIV-1 infection in vitro. Moreover, our results indicated that mammary epithelial cells (MECs) as well as breast milk cells produced sTLR2 , thus motivating us to hypothesize that sTLR2 in breast milk likely plays an important role in inhibiting MTCT of HIV.
sTLRs provide the most direct attenuation of innate immune responses to pathogens by binding to pathogen-associated molecular patterns (PAMPs) before they engage membrane-bound TLRs, thus effectively inhibiting PAMP-pattern recognition receptor (PRR) engagement . Numerous publications, originating with LeBouder et al., have highlighted sTLR2's role in significantly inhibiting bacterially induced pro-inflammatory cytokine production [12,13], and together indicate that sTLR2 is critically important in regulating bacteria-induced cellular activation.
sTLR2-dependent regulation of immune activation during virus infection remains poorly understood. However, accruing evidence indicates that the immune system uses a range of soluble molecules, including defensins, antiproteases, interferons and chemokines to control viral infections . Indeed, our laboratory has recently reported that elafin/trappin-2, an antiprotease, directly interferes with viral PAMPs/host engagement, thus modulating immune responses . Furthermore, the sTLR2 precursor, TLR2, has been shown to recognize multiple viral proteins that culminate in increased cellular activation and infection in a TLR2-dependent manner. Specifically, cytomegalovirus glycoprotein gB and gH, hepatitis C core and structural proteins, as well as measles haemagglutinin protein, trigger TLR2-dependent pro-inflammatory responses in permissive cells [16–18]. The outcome of virus-specific cellular activation can mediate a wide range of responses, including acceleration of antiviral clearance, inflammation and recruitment of additional target cells, as well as establishing a favourable microenvironment that facilitates viral replication . Indeed, we and others have identified higher levels of TLR2 expression in peripheral blood mononuclear cells (PBMCs) from untreated HIV-infected than from uninfected individuals [20,21]. Therefore, one aim of this study was to characterize TLR2 expression levels and sTLR2 concentrations in breast milk from HIV-uninfected and HIV-infected women and their associations with correlates of disease progression. Further, we sought to identify a mechanism by which sTLR2 inhibits viral-induced cellular activation and HIV-1 infection in vitro.
Materials and methods
Study cohorts and breast milk
This study was approved by the McMaster Research Ethics Board and the University of Maryland Baltimore, and Plateau State Specialist Hospital Nigeria Institutional Review Boards. Historic Nigerian breast milk HIV-infected (n = 40) and uninfected (n = 15) samples, and HIV-uninfected breast milk samples from our Hamilton, Ontario cohort (n = 13) were blindly tested. All participants provided voluntary written informed consent. Women were excluded if they had caesarean sections, their pregnancies were not full-term or they were diagnosed with mastitis postpartum. The average age of HIV-infected women was 27 years, while the HIV-uninfected Nigerian and Canadian women were 30 and 31 years old, respectively. Average parity was 2. Milk samples were self-collected within the first 6 months postpartum into sterile containers and processed in our laboratory where they were separated into lipid, supernatant and cellular fractions, and stored at −80°C and/or liquid nitrogen.
Quantitative reverse-transcriptase real-time PCR
Total RNA was extracted from cells with TRIzol as per manufacturer's instructions (Life Technologies, Burlington, Canada). Reverse transcription reactions and quantitative reverse-transcriptase real-time PCR (qRT-PCR) of selected genes and internal control, RPL13A, were completed as previously described . PCR primers for TLR2, interleukin (IL)-8, chemokine receptor 5 (CCR5) and HIV Pol were designed using the program, Primer 3.0 (http://frodo.wi.mit.edu), and supplied by Mobix (McMaster University).
OptiEIA was used to measure IL-8 and IL-15 levels in cell and breast milk supernatants according to manufacturer's instructions (BD Biosciences, Oakville, Canada). TLR2 ELISA (R&D Biosciences, Burlington, Canada) and p24 ELISA (Advanced Bioscience Laboratories, Burlington, Canada) were used to determine sTLR2 and p24 levels, respectively, in supernatants of cells and breast milk. Endotoxin detection assay (kindly provided by Dr Bowdish, McMaster University) was completed according to the manufacturer's instructions (Lonza, Burlington, Canada).
Cell lines and reagents
The cell line, TZMbl cell line stably expressing TLR2 (TZMbl-2), was generated from TZMbl cells using pIRES2-ZsGreen1 vector (Clontech, Burlington, Canada) cloned with human TLR2 cDNA from PBMC. Three rounds of selection were conducted using Dulbecco's modified eagle's medium (DMEM) medium containing 0.8 mg/ml G418. Long-term expression of TLR2 was confirmed using RT-PCR and western blot analyses. Michigan Cancer Foundation-10A (MCF-10A) cells were cultured with DMEM medium that was supplemented with 20 ng/ml epidermal growth factor (Sigma, Burlington, Canada), 10 μg/ml insulin, 0.5 μg/ml hydrocortisone (Sigma) and 100 ng/ml cholera toxin. Human monocytic cell line (THP-1) cells were differentiated into macrophages using 50 ng/ml phorbol 12-myristate 13-acetate (Sigma) supplemented medium and cultured for 48 h before use. HIV-1 components included p17 (Virogen, Mississauga, Canada), p24, nef, gp41 (Genway), gp120 (NIH AIDS Reference & Reagent Program) and ssRNA40 (Mobix, McMaster University). Pam3CSK4 (InvivoGen, Burlington, Canada) was reconstituted in PBS. Anti-TLR2 antibodies used for western, dot blot and neutralization included goat polyclonal IgG, N-17 (Santa Cruz Biotechnology, Santa Cruz, California, USA); goat IgG (R&D); mouse monoclonal IgG1, T2.5 (Santa Cruz).
Viral stocks and reporter assay
HIV-1 R5-tropic BaL was prepared and tissue culture infectious doses (TCIDs) of pooled supernatants as well as in-vitro functional assays were determined using TZMbl and TZMbl-2 cells, as previously described . Luciferase activity was determined using Bright-Glo reagents (Promega, Madison, Wisconsin, USA), analysed using a Veritas luminometer (Promega) and reported as relative light units (RLUs).
The source of sTLR2 was cell lysates from HEK293 cells transfected with sTLR2 plasmid, pDR2sTLR2Myc, kindly provided by Dr Mario O. Labéta, Cardiff University. The co-immunoprecipitation (co-IP) reaction was completed by mixing 50 μg sTLR2 lysate with 2–50 μg HIV-1 proteins in PBS overnight at 4°C. The negative controls were empty-transfected HEK293 lysates and a reaction without specific HIV-1 proteins. Either anti-TLR2 or anti-HIV-1 protein antibodies were added to the mixture, and rotated at 4°C for 1 h. The complexes were captured with 20 μl protein G-nanobeads (Invitrogen, Burlington, Canada) at room temperature for 15 min, isolated and washed with PBS in a 96-well magnet ring stand. Beads were boiled, centrifuged and the supernatant was subjected to western blot analyses.
Nuclei extraction and western blotting
TZMbl-2, THP-1 and MCF-10A cells were grown in 12-well plates and treated with HIV-1 proteins and Pam3CSK4 in the presence of sTLR2 or sTLR2-free medium for 1 h at 37°C. Nuclei were isolated as described previously . Briefly, cells were washed with dilute PBS, immersed in 160 μl of a hypotonic buffer followed by 200 μl of 1% Triton X-100 buffer to rupture their membranes. After centrifugation at 12 000g for 3 min, the nuclear pellets were dissolved in a high salt buffer containing protease inhibitor and phosphotase inhibitor cocktails (PhosStop Mini Complete; Roche Applied Science, Burlington, Canada). The supernatants were collected and protein concentrations were determined using protein DC assay (Bio-Rad). The nucleic extracts were separated using SDS-PAGE gels. Primary antibodies included anti-p65 and anti-histone 3 (Cell Sciences, Burlington, Canada) antibodies. Secondary antibodies included horseradish peroxidase (HRP)-labelled donkey antigoat IgG, HRP-labelled chicken antimouse IgG (Santa Cruz) and HRP-labelled mouse antirabbit IgG (Pierce Biotechnology, Mississauga, Canada). The blots were detected with Femto chemiluminescent substrates (Thermo, Burlington, Canada). Optical densitometry was determined using Un-Scan-It image digitizing software (Silk Scientific Inc., Orem, Utah, USA).
All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad, San Diego, California, USA). Spearman's correlation was used to test for independence between sTLR2 concentration and viral antigenemia or immune responses in breast milk. The Mann–Whitney U-test was used to directly compare two groups for sTLR2 concentration, TLR2 expression, immune responses, cellular activation, HIV integration and CCR5 expression. All P-values are two-tailed and considered statistically significant if P value was less than 0.05.
Significantly elevated Toll-like receptor 2 expression in breast milk cells and soluble Toll-like receptor 2 in HIV-infected women's breast milk
We initially determined the effect that HIV-1 infection had on TLR2 expression levels in breast milk cells and sTLR2 levels in milk. These studies made use of historic HIV-infected (n = 40) and uninfected (n = 15) breast milk samples from Nigeria, as well as milk obtained from HIV-uninfected women in Canada (n = 13). Our results showed significantly higher TLR2 expression in breast milk cells from HIV-infected Nigerian women than from HIV-uninfected breast milk cells from Nigerian and Canadian women (Fig. 1a; P = 0.001, P = 0.002, respectively). Furthermore, sTLR2 levels in breast milk from HIV-infected women were significantly elevated compared with milk from HIV-uninfected women from Nigeria and Canada (Fig. 1b; P = 0.001, P = 0.0002, respectively).
Soluble Toll-like receptor 2 levels in HIV-1 infected breast milk correlates with p24 and interleukin-15 concentration
We evaluated the association between p24 and sTLR2 concentrations in breast milk and revealed that p24 antigenemia positively correlated with sTLR2 levels in breast milk from HIV-infected women (Fig. 1c; P = 0.016, r = 0.379). Furthermore, we sought to determine whether IL-15 and sTLR2 levels correlate in breast milk, and clearly demonstrate a highly significant correlation between sTLR2 and IL-15 concentrations in breast milk from HIV-infected women (Fig. 1d; P < 0.0001, r = 0.570).
Taken together, these data reveal significantly higher TLR2 expression levels in breast milk cells and sTLR2 concentrations in HIV-infected than in uninfected breast milk. These results suggest that there might be a local innate compensatory mechanism in the breast by which sTLR2 levels significantly increase in HIV-infected milk as the p24 concentration increases. In addition, IL-15, which previously has been associated with protection against MTCT , positively correlated with sTLR2 levels in infected breast milk.
Soluble Toll-like receptor 2 significantly inhibits cell-free R5 HIV-1 inflammation and infection
In order to determine the function of significantly higher levels of sTLR2 in HIV-infected milk, we performed in-vitro studies. Cell-free R5 HIV-1 was incubated with sTLR2-containing (sTLR2) or sTLR2-free supernatant (-sTLR2) for 1 h before addition to TZMbl cells, and after a 48-h infection. Data indicated that IL-8 production was significantly decreased in cell supernatants containing sTLR2 compared with sTLR2-free supernatants (Fig. 2a; P = 0.001), whereas it was significantly increased when sTLR2 was neutralized using TLR2-specific mAbs after exposure to HIV-1 (Fig. 2a; P = 0.015).
HIV-1 at 1, 10 and 100 TCID50 infectious doses incubated with sTLR2-containing supernatant before addition to TZMbl cells significantly reduced HIV-1 infection/integration rates 2.3, 4.2 and 2.6-fold, respectively, compared with sTLR2-free supernatants (Fig. 2b; P = 0.014, P = 0.0004, P = 0.0003, respectively). Importantly, neutralization of sTLR2 using TLR2-specific antibodies significantly increased HIV infection 1.3, 1.8 and 2.8-fold compared with sTLR2 supernatant, respectively (Fig. 2b; P = 0.05; P = 0.0018), indicating that sTLR2 was responsible for suppressing HIV replication.
HIV-1 proteins significantly elevate soluble Toll-like receptor 2 concentration in vitro
Given the significantly higher levels of sTLR2 in breast milk from HIV-infected women, we determined whether MECs and/or macrophages exposed to HIV-1 proteins (p17, p24 and gp41) significantly increased sTLR2 levels. Supernatants from MECs and macrophages exposed to recombinant HIV-1 proteins (p17, p24 and gp41) or TLR2 ligand (Pam3CSK4) were collected and western blot analyses revealed substantially elevated sTLR2 protein levels in supernatants of MECs exposed to recombinant proteins compared to medium alone (Fig. 3a). Quantification of sTLR2 in these supernatants showed significantly augmented sTLR2 concentrations in mammary epithelial and macrophage cell lines compared with medium alone [Fig. 3a; (MCF-10A) P = 0.014, 0.012 and 0.039; (THP-1) P = 0.0008, 0.006, 0.013 and 0.026, respectively].
These data support the notion that HIV and its proteins promote innate immune activation in the nursing mammary gland environment, and suggest that elevated sTLR2 levels in breast milk might be the result of local viral-induced release and/or production of sTLR2 from resident macrophages and MECs.
Soluble Toll-like receptor 2 inhibits HIV-1 protein-induced inflammation
TZMbl-2 cells, which stably express TLR2 and endogenously express TLR1, allowed us to determine whether sTLR2 could inhibit TLR2-dependent activation. IL-8 mRNA analysis of TZMbl-2 cells exposed to Pam3CSK4 in the presence of varying concentrations of sTLR2 supernatant significantly inhibited IL-8 production 4, 9 and 10.5-fold, respectively, compared with sTLR2-free supernatants (Fig. 3b; P = 0.015, 0.005, 0.02). Likewise, gp41-induced IL-8 expression was significantly inhibited in the presence of varying concentrations of sTLR2 supernatant 1.9, 2.5 and 4.3-fold compared with sTLR2-free supernatant (Fig. 3d; P = 0.009, 0.002 and 0.0007, respectively). p17-induced cellular activation was reduced 3.5-fold at all concentrations of sTLR2 supernatant (Fig. 3c; P = 0.003, 0.004 and 0.004) Collectively, these data indicate that sTLR2 is an important innate factor inhibiting HIV-1 protein-induced cellular activation by competitively suppressing ligand interaction with TLR2.
Soluble Toll-like receptor 2 physically interacts with p17, p24 and gp41
Viral protein–sTLR2 interactions were identified by dot blotting as previously described . The detection indicated that HIV-1 p17, p24 and gp41 directly interacted with sTLR2, whereas no interaction was detected between sTLR2 and viral components, gp120, nef or synthetic viral RNA (ssRNA) (Fig. 4a). Interactions were also detected between sTLR2 and the positive control, Pam3CSK4 and CD14. As well, the membrane control indicated that the anti-TLR2 antibodies were at a sufficient concentration to detect sTLR2, when present (Fig. 4a). Importantly, when sTLR2 was immunodepleted from breast milk, less or no interaction was observed between sTLR2 and p17, p24, and gp41 (Fig. 4b).
Co-IP assays validated our above findings to show that sTLR2 was coimmunoprecipitated with p24 and p17 (Fig. 4c), and with gp41 (Fig. 4d). Taken together, these results demonstrate, for the first time, that sTLR2 directly interacts with HIV-1 structural proteins p17, p24 and gp41.
Potential mechanism of action of soluble Toll-like receptor 2 in inhibiting HIV-1 infection in vitro
As sTLR2 binds to HIV proteins, we determined whether this may trigger innate signalling, therefore HIV-1 proteins (p17, p24 and gp41) and Pam3CSK4, as a control, were incubated with sTLR2-containing or sTLR2-free supernatant for 1 h prior to addition to TZMbl-2 cells. Results showed substantially increased nuclear translocation of p65, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), following treatment with Pam3CSK4 p17 and gp41 in the absence of sTLR2 (Fig. 5a). In contrast, substantially less p65 was detected in the nucleus of cells exposed to Pam3CSK4, p17 and gp41 in the presence of sTLR2 supernatant (Fig. 5a). A similar pattern was demonstrated under the same conditions in the two most prominent cell types in breast milk: mammary epithelial cells (MCF-10A) and macrophages (THP-1) (data not shown).
It was critically important to clarify whether the activation of the NFκB signalling pathway observed was due to possible endotoxin contamination during the production of viral proteins. Therefore, proteinase K digestion followed by heat denaturation was performed to test whether cells expressing TLR2 responded to specific ligands or possible endotoxin contamination. Proteinase K did not reduce the ability of synthetic lipopeptide, Pam3CSK4, to induce IL-8 production in TZMbl-2 cells. In contrast, proteinase K treatment of viral proteins (p17, p24 and gp41) significantly abrogated IL-8 production in TZMbl-2 cells (Fig. 5b). Furthermore, all reagents were tested for the presence of endotoxin using a highly sensitive modified limulus amebocyte lysate assay, which indicated that all components used in our tests had undetectable to very low levels of endotoxin that were well below levels found in the foetal bovine serum used in cell medium (Table S1, http://links.lww.com/QAD/A545), thus excluding the possibility that these effects are due to lipopolysaccharide contamination.
CCR5 mRNA analysis of macrophages exposed to HIV-1 proteins (p17, p24 and gp41) with supernatants that contained sTLR2 significantly decreased CCR5 expression compared with sTLR2-free supernatants (Fig. 5c; P = 0.009). Together, these data indicate that sTLR2 may inhibit HIV infection by binding to HIV-1 structural proteins, inhibiting NFκB-dependent cellular activation and HIV-1 coreceptor CCR5 expression.
TLR2 is classically considered in the context of Gram-positive bacteria recognition and signalling; however, an expanding body of evidence indicates that TLR2 is intimately involved in viral sensing [16–18,24]. Indeed, we recently reported significantly higher TLR2 expression in genital mucosal cells from HIV-infected than from uninfected or highly HIV-exposed seronegative commercial sex workers . Moreover, TLR2 activation has been shown to enhance HIV infection in T cells  and viral replication in macrophages of HIV-1 infected patients . Conversely, sTLR2 has recognized antimicrobial properties in which it significantly reduces production of proinflammatory cytokine [11–13]. Furthermore, we previously revealed that sTLR2 significantly inhibited cell-free HIV-1 infection in vitro; however, the mechanism remained undetermined. In the present study, we evaluated TLR2 expression in breast milk cells; sTLR2 concentration in HIV-1 uninfected and infected breast milk, and assessed its correlation to antigenemia and proinflammatory cytokine levels. Our collective results highlighted a mechanism through which sTLR2 directly inhibited HIV-1 induced cellular activation and infection in vitro, which supports the notion that sTLR2 plays a critical role in the prevention of MTCT. Furthermore, these data may possibly support a role of sTLR2 for prophylactic and therapeutic strategies in the prevention and care of HIV-1 infection and warrant further investigation.
Our analyses of TLR2 in cells from HIV-1 infected breast milk revealed a significant elevation of TLR2 expression compared with uninfected breast milk cells. Although these data are novel, we and others have documented a similar trend in PBMCs, cervical mononuclear cells and cervical epithelial cells in HIV-infected compared with uninfected individuals [21,25,27]. The predominant cell type involved in cell-associated HIV transmission is not yet established; however given that macrophages are a major cell population in breast milk [28,29] and previous speculation that this cell type is involved in vertical HIV transmission , our current findings indicating higher TLR2 expression in breast milk cells highlights a novel HIV infection induced alteration in innate immune PRR expression in breast milk.
Conversely, and possibly as a means to regulate aberrant cellular activation through increased expression of PRRs, we revealed that sTLR2 concentration was significantly elevated in HIV-1 infected compared with uninfected breast milk. Furthermore, evaluation of sTLR2 concentration in breast milk from HIV-infected women showed a positive correlation with HIV p24 and IL-15, and further extended these findings in vitro to show that HIV-1 specific cellular activation promoted sTLR2 release from MECs and macrophages, one of the predominant cell types found in breast milk [28,29]. These data provide a possible mechanism explaining why sTLR2 concentration positively correlated with p24 levels. Indeed, similar findings have been reported for α-defensin level, which correlated with viral burden in breast milk , as well as monocyte/macrophage cell lines and placental explants exposed to Pam3CSK4 that increased sTLR2 secretion in cell supernatants [12,13]. However, these data are in contrast to Heggelund et al. whose results did not indicate significant differences in sera sTLR2 levels between HIV-uninfected and HIV-infected people. Furthermore, sTLR2 levels strongly correlated with IL-15 concentration in HIV-1 infected breast milk. This result is particularly intriguing, as IL-15 is associated with protection against HIV transmission through breastfeeding  and is similar to a TLR2-dependent mechanism of proinflammatory production and release of IL-15 from macrophages exposed to HSV envelope glycoproteins [24,34]. Taken together, we suggest that breast milk macrophages provide crucial local compensatory protection that likely plays an important role in inhibiting mother-to-child HIV transmission. Moreover, IL-15 increases function and proliferation of natural killer (NK) cells, which have previously been shown to control HIV replication . Given the strong correlation between sTLR2 and IL-15 in breast milk, we speculate that these innate factors act in concert to help control HIV infection.
Our findings are particularly intriguing for three reasons: first, sTLR2 has known anti-inflammatory properties effected by bacterial ligands, and, we show here, significantly inhibited HIV-1 induced NFκB activation and IL-8 production in a dose-dependent manner; second, sTLR2 bound directly to HIV-1 structural proteins; and third, sTLR2 inhibited TLR2-dependent, HIV-1-induced increases in CCR5 coreceptor expression. Together, these likely all contribute to significant decreases in HIV-1 infection.
To our knowledge, this is the first study to demonstrate that sTLR2 inhibited viral protein induced NFκB activation, which is similar to other viral-induced TLR2-dependent NFκB activation [17,24], and is particularly important given the critical role NFκB plays in inflammation and HIV pathogenesis . Importantly, we confirmed that the cellular activation mediated by specific HIV antigens used in these studies was not due to endotoxin contamination. These findings are similar to previous reports indicating that SLPI can affect NFκB nuclear translocation and gene expression . However, contrary to SLPI's mechanism, we showed direct protein-to-protein interaction between sTLR2 and HIV-1 structural proteins p17, p24 and gp41. Furthermore, our data demonstrated a sTLR2-dependent decrease in HIV-1 protein-induced CCR5 expression that led to lower HIV-1 infection. Thus, sTLR2 might play a pivotal role in controlling virulence levels in breast milk. Moreover, it is increasingly apparent that neonatal lymphocyte activation is required for HIV-1 reverse transcriptase to complete the transmission event . Taken together with the fact that viral entry itself might not equal transmission until integration is established , our data indicating that sTLR2 binds directly to HIV-1 proteins could inhibit infection in at least two possible ways: first, sTLR2 inhibits HIV-1 proteins from inducing cellular activation, thus retaining low CCR5 expression levels, typical of unstimulated cells for low inflammation  and/or second, sTLR2-gp41 binding might directly impede virus-host membrane fusion, which is critical to HIV entry and infection.
In conclusion, we demonstrate that sTLR2 inhibits HIV-1 cellular activation, increased CCR5 expression and viral infection through direct interaction with structural proteins. Moreover, our data demonstrate that sTLR2 levels are significantly higher in breast milk from HIV-infected women and positively correlate with p24 and IL-15 concentrations. Importantly, MECs and macrophages exposed to HIV-1 proteins led to significantly increased sTLR2 levels in vitro. Therefore, these data revealed an innate compensatory mechanism controlling HIV in breast milk in which sTLR2, along with other innate factors, is likely pivotal in the prevention of HIV-1 MTCT, and underscore the importance of innate factors in infant health and development.
We would like to especially thank the Nigerian and Canadian women who participated in our study. We also thank Drs Mark McDermott and Clive Gray for proof reading and review of this manuscript. As well, we thank Dr Chris Verschoor, McMaster University, for his technical assistance in endotoxin testing of reagents used in the experiments included here.
B.M.H. and K.L.R. conceived the project; B.M.H., X-D.Y., A.G.D., A.A. and K.L.R. provided ideas and designed experiments; B.M.H. and A.A. recruited patients; B.M.H. and A.A. collected and processed breast milk specimens; B.M.H., X-D.Y., A.G.D. performed experiments; B.M.H., X-D.Y., A.G.D., A.A. and K.L.R. analysed and interpreted the data; B.M.H. wrote the manuscript with editing provided by X-D.Y., A.A. and K.L.R.
This research was supported by a large team grant from the Canadian Institutes of Health Research (CIHR) as part of the Canadian HIV Vaccine Initiative (CHVI).
Conflicts of interest
The authors declared no conflict of interest.
1. Coovadia HM, Rollins NC, Bland RM, Little K, Coutsoudis A, Bennish ML, et al. Mother-to-child transmission of HIV-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort study
2. Kuhn L, Sinkala M, Kankasa C, Semrau K, Kasonde P, Scott N, et al. High uptake of exclusive breastfeeding and reduced early postnatal HIV transmission
. PLoS One
3. Iliff PJ, Piwoz EG, Tavengwa NV, Zunguza CD, Marinda ET, Nathoo KJ, et al. Early exclusive breastfeeding reduces the risk of postnatal HIV-1 transmission and increases HIV-free survival
4. Coutsoudis A, Kuhn L, Pillay K, Coovadia HM. Exclusive breast-feeding and HIV transmission
5. Kazmi SH, Naglik JR, Sweet SP, Evans RW, O'Shea S, Banatvala JE, et al. Comparison of human immunodeficiency virus type 1-specific inhibitory activities in saliva and other human mucosal fluids
. Clin Vaccine Immunol
6. Lyimo MA, Howell AL, Balandya E, Eszterhas SK, Connor RI. Innate factors in human breast milk inhibit cell-free HIV-1 but not cell-associated HIV-1 infection of CD4+ cells
. J Acquir Immune Defic Syndr
7. Saeland E, de Jong MAWP, Nabatov AA, Kalay H, Geijtenbeek TBH, van Kooyk Y. MUC1 in human milk blocks transmission of human immunodeficiency virus from dendritic cells to T cells
. Mol Immunol
8. Groot F, Geijtenbeek TBH, Sanders RW, Baldwin CE, Sanchez-Hernandez M, Floris R, et al. Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN--gp120 interaction
. J Virol
9. Henrick BM, Nag K, Yao X-D, Drannik AG, Aldrovandi GM, Rosenthal KL. Milk matters: soluble Toll-like receptor 2 (sTLR2) in breast milk significantly inhibits HIV-1 infection and inflammation
. PLoS One
10. Liew FY, Xu D, Brint EK, O’Neill LAJ. Negative regulation of toll-like receptor-mediated immune responses
. Nat Rev Immunol
11. LeBouder E, Rey-Nores JE, Rushmere NK, Grigorov M, Lawn SD, Affolter M, et al. Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk
. J Immunol
12. Kuroishi T, Tanaka Y, Sakai A, Sugawara Y, Komine K-I, Sugawara S. Human parotid saliva contains soluble toll-like receptor (TLR) 2 and modulates TLR2-mediated interleukin-8 production by monocytic cells
. Mol Immunol
13. Dulay AT, Buhimschi CS, Zhao G, Oliver EA, Mbele A, Jing S, et al. Soluble TLR2 is present in human amniotic fluid and modulates the intraamniotic inflammatory response to infection
. J Immunol
14. DeVico AL, Gallo RC. Control of HIV-1 infection by soluble factors of the immune response
. Nat Rev Microbiol
15. Drannik AG, Nag K, Yao X-D, Henrick BM, Sallenave J-M, Rosenthal KL. Trappin-2/elafin modulate innate immune responses of human endometrial epithelial cells to PolyI:C
. PLoS One
16. Boehme KW, Guerrero M, Compton T. Human cytomegalovirus envelope glycoproteins B and H are necessary for TLR2 activation in permissive cells
. J Immunol
17. Dolganiuc A, Oak S, Kodys K, Golenbock DT, Finberg RW, Kurt-Jones E, et al. Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation
18. Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, Duprex WP, et al. Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling
. J Virol
19. Mogensen TH, Melchjorsen J, Larsen CS, Paludan SR. Innate immune recognition and activation during HIV infection
20. Heggelund L, Müller F, Lien E, Yndestad A, Ueland T, Kristiansen KI, et al. Increased expression of toll-like receptor 2 on monocytes in HIV infection: possible roles in inflammation and viral replication
. Clin Infect Dis
21. Lester RT, Yao X-D, Ball TB, McKinnon LR, Kaul R, Wachihi C, et al. Toll-like receptor expression and responsiveness are increased in viraemic HIV-1 infection
22. Mian MF, Ahmed AN, Rad M, Babaian A, Bowdish D, Ashkar AA. Length of dsRNA (poly I:C) drives distinct innate immune responses, depending on the cell type
. J Leukoc Biol
23. Matucci A, Rossolillo P, Baroni M, Siccardi AG, Beretta A, Zipeto D. HLA-C increases HIV-1 infectivity and is associated with gp120
24. Leoni V, Gianni T, Salvioli S, Campadelli-Fiume G. Herpes simplex virus glycoproteins gH/gL and gB bind Toll-like receptor 2, and soluble gH/gL is sufficient to activate NF-kB
. J Virol
25. Yao XD, Omange RW, Henrick BM, Lester RT, Kimani J, Ball TB, et al. Acting locally: innate mucosal immunity in resistance to HIV-1 infection in Kenyan commercial sex workers
. Mucosal Immunol
26. Ding J, Rapista A, Teleshova N, Mosoyan G, Jarvis GA, Klotman ME, et al. Neisseria gonorrhoeae enhances HIV-1 infection of primary resting CD4+ T cells through TLR2 activation
. J Immunol
27. Hernandez JC, Stevenson M, Latz E, Urcuqui Inchima S. HIV-1 infection up-regulates TLR2 and TLR4 expression and function in vivo and in vitro
. AIDS Res Hum Retroviruses
28. Goldman AS. Human milk, leukocytes, and immunity
. J Pediatr
29. Jain N, Mathur NB, Sharma VK, Dwarkadas AM. Cellular composition including lymphocyte subsets in preterm and full term human colostrum and milk
. Acta Paediatr Scand
30. Satomi M, Shimizu M, Shinya E, Watari E, Owaki A, Hidaka C, et al. Transmission of macrophage-tropic HIV-1 by breast-milk macrophages via DC-SIGN
. J Infect Dis
31. Bosire R, John-Stewart GC, Mabuka JM, Wariua G, Gichuhi C, Wamalwa D, et al. Breast milk defensins are associated with HIV type 1 RNA and CC chemokines in breast milk but not vertical HIV type 1 transmission
. AIDS Res Hum Retroviruses
32. Heggelund L, Flo T, Berg K, Lien E, Mollnes TE, Ueland T, et al. Soluble toll-like receptor 2 in HIV infection: association with disease progression
33. Walter J, Ghosh MK, Kuhn L, Semrau K, Sinkala M, Kankasa C, et al. High concentrations of interleukin 15 in breast milk are associated with protection against postnatal HIV transmission
. J Infect Dis
34. Ahmad A, Ahmad R, Toma E, Morisset R, Menezes J. Impaired induction of IL-15 in response to herpes simplex virus type 1 infection in peripheral blood mononuclear cells of HIV-infected patients
35. Oliva A, Kinter AL, Vaccarezza M, Rubbert A, Catanzaro A, Moir S, et al. Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro
. J Clin Invest
36. Pereira LA, Bentley K, Peeters A, Churchill MJ, Deacon NJ. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter
. Nucleic Acids Res
37. Taggart CC, Cryan S-A, Weldon S, Gibbons A, Greene CM, Kelly E, et al. Secretory leucoprotease inhibitor binds to NF-kappaB binding sites in monocytes and inhibits p65 binding
. J Exp Med
38. Legrand FA, Nixon DF, Loo CP, Ono E, Chapman JM, Miyamoto M, et al. Strong HIV-1-specific T cell responses in HIV-1-exposed uninfected infants and neonates revealed after regulatory T cell removal
. PLoS One
39. Kourtis AP, Lee FK, Abrams EJ, Jamieson DJ, Bulterys M. Mother-to-child transmission of HIV-1: timing and implications for prevention
. Lancet Infect Dis
40. Viard M, Parolini I, Sargiacomo M, Fecchi K, Ramoni C, Ablan S, et al. Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells
. J Virol
breast milk; cellular activation; gp120; gp41; HIV; inflammation; innate immunity; p17; p24; soluble Toll-like receptor 2
Supplemental Digital Content
© 2014 Lippincott Williams & Wilkins, Inc.
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.
Readers Of this Article Also Read