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PLoS One. 2015 Mar 20;10(3):e0122020. doi: 10.1371/journal.pone.0122020. eCollection 2015.
Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.
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- Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
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- Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, United States of America.
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- Department of Dermatology, Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands; STI outpatient clinic, Cluster Infectious Diseases, Public Health Service Amsterdam and Centre for Infections and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
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- Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands.
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- Division of Infectious Diseases, Faculty of Medicine, Imperial College, London, United Kingdom.
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- Department of Medical Biochemistry, Medical Centre of the University of Amsterdam, Amsterdam, the Netherlands.
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- Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
Abstract
Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.
Fig 1CM binds DC-SIGN thereby preventing gp140 binding.
(A) DC-SIGN binding ELISA with 300 fold diluted CM coated on an ELISA plate and DC-SIGN-Fc as detection antibody, demonstrates DC-SIGN-Fc binds CM compared to EGTA treated product (negative control) (p<0.0001). (B) DC-SIGN-Fc was pre-incubated with mannan (positive control) and CM dilutions before being added to a gp140 coated plate. Depicted is the percentage by which DC-SIGN-Fc binding to gp140 is blocked, pre-incubation with mannan was set to 100% blocking and pre-incubation with medium to 0%. Pre-incubating DC-SIGN-Fc with up to a 1000 fold diluted CM inhibits HIV-1 envelope gp140 trimer binding. Data points were performed in triplicate.
PLoS One. 2015;10(3):e0122020.
Fig 2CM does not inhibit HIV-1 direct infection but does trans-infection.
(A) NSI-18 (R5) or LAI (X4) (5ng/ml p24) was pre-incubated with medium (control) or 100 fold and 1000 fold diluted CM after which the mixture was added to TZM-bl cells. Two days post infection the cells were lysed and the luciferase activity was measured demonstrating that the level of infection was similar whether CM was present or not. (B) CD4+ T-lymphocytes were incubated with medium (control) or 100 fold and 1000 fold diluted CM prior to addition of LAI. Viral outgrowth, supernatant p24, was measured over several days, with no difference observed. (C) Raji DC-SIGN cells were incubated with medium (negative control) or 100 fold diluted CM or 300 fold diluted CM prior to addition of either NSI-18 or LAI virus, washed and added to CD4+ T-lymphocytes. Viral outgrowth, determined by capsid p24 ELISA, is depicted in Raji DC-SIGN cell—CD4+ T lymphocyte co-cultures. For both NSI-18 and LAI inhibition is observed with 100 fold diluted CM and less with 300 fold diluted CM. Data points were performed in triplicate.
PLoS One. 2015;10(3):e0122020.
Fig 3CM inhibits HIV-1 trans-infection by iDCs.
(A) iDCs were pre-incubated with 100 fold diluted CM, 20μg/ml AZN-D1 (DC-SIGN blocking antibody), 50μg/ml mannan or medium (control) before addition of LAI. Using flow cytometry viral outgrowth in iDC – CD4 T-lymphocyte co-cultures was measured by intracellular staining for p24. Depicted is the number of p24+ cells per 1x105 CD3+ T cells. (B) Shown are representative dot plots of the data depict in (A). Data points were performed in triplicate.
PLoS One. 2015;10(3):e0122020.
Fig 4Biochemical analysis of the DC-SIGN binding component in CM.
(A) Unfractionated CM (input) or <30, 30–100 or >100kDa CM fractions were coated to an ELISA plate and DC-SIGN-Fc binding was determined. Binding of unfractionated CM is set to 100% and binding of the fractions is expressed as a relative percentage. The <30kDa fraction shows no DC-SIGN binding, between 30–100kDa shows limited binding whilst >100kDa shows stronger binding. (B) DC-SIGN-Fc was incubated with untreated, heated (10 min at 95°C), proteinase K treated CM or medium (negative control) prior to addition to a gp140 coated plate. Compared to the media alone control incubating DC-SIGN-Fc with treated or untreated CM led to similar reductions in gp140 binding. (C) DC-SIGN-Fc was incubated with CM, BSA or mannan prior to addition to a gp140 coated plate. Untreated, both CM and mannan inhibit DC-SIGN-Fc from binding gp140 compared to BSA (negative control). Depletion of mannose structures from CM, BSA and mannan by a pull-down with Galanthus Nivalis lectin does not alter the DC-SIGN-Fc binding capacity of CM while mannan loses its ability to prevent gp140 binding by DC-SIGN-Fc. Data points were performed in triplicate.
PLoS One. 2015;10(3):e0122020.
Fig 5DC-SIGN binding capacity of CM varies greatly between individuals.
(A) Serial dilutions of the 21 CM samples were coated onto an ELISA plate and the end-point dilution showing binding of DC-SIGN-Fc is depicted. For all samples maximal binding was achieved when 11.7μg/ml CM was coated. A large variation between donors was observed ranging from high to no DC-SIGN binding and where the samples can be divided into three groups as indicated with dotted lines (B). The ability of samples to prevent DC-SIGN-Fc binding to trimeric gp140 was determined with a blocking ELISA. 11.7μg/ml CM was pre-incubated with DC-SIGN-Fc before addition to a gp140 coated plate. Next, DC-SIGN binding to gp140 was correlated with the OD found in the DC-SIGN binding ELISA where 11.7μg/ml CM was coated (P<0.01). As a control 5μg/ml mannan was included, depicted as an open square.
PLoS One. 2015;10(3):e0122020.
Fig 6Mass spectrometry indicates that human CM lactoferrin binds DC-SIGN.
(A) 4–12% SDS PAGE gel loaded (from left to right) with a 250kD protein marker, agarose beads, DC-SIGN-Fc, the supernatant from the first wash, supernatant from the second wash, and the DC-SIGN-Fc coated agarose beads loaded with the DC-SIGN binding component from CM. Band #2, 3 and 4 potentially contain the DC-SIGN binding component of CM. Ion trap mass spectrometry of in gel digests identified human lactoferrin fragments (highly abundant in band #3) and immunoglobulins in all three bands and intelectin-1 in band #4 (with trace amounts in the other bands). (B) CM representing a high DC-SIGN binder, an intermediate DC-SIGN binder and a low/no DC-SIGN binder were coated on an ELISA plate and were tested for DC-SIGN and lactoferrin binding. The first graph (left) confirms the DC-SIGN binding status while the second graph (right) shows the binding capacity of polyclonal anti-lactoferrin, which is high for CM from a DC-SIGN high binder, intermediate for an intermediate DC-SIGN binder and not present in CM from a low/no DC-SIGN binder.
PLoS One. 2015;10(3):e0122020.
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