Stax Martijn J, Mouser Emily E I M, van Montfort Thijs, Sanders Rogier W, de Vries Henry J C, Dekker Henk L, Herrera Carolina, Speijer Dave, Pollakis Georgios, Paxton William A
Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
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.
PLoS One. 2015 Mar 20;10(3):e0122020. doi: 10.1371/journal.pone.0122020. eCollection 2015.
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.
包括母乳和精液在内的身体分泌物含有调节HIV-1感染的因子。由于肛交是HIV-1传播的最高风险途径之一，我们的目的是确定结肠直肠黏液（CM）是否也含有干扰HIV-1感染和复制的因子。收集了许多个体的CM，并检测其结合DC-SIGN以及抑制HIV-1对CD4+ T淋巴细胞进行顺式或反式感染的能力。为此，使用了DC-SIGN结合ELISA、gp140三聚体竞争ELISA以及HIV-1捕获/转移试验。随后，我们旨在通过生化特性分析和质谱分析来鉴定DC-SIGN结合成分。结果表明，CM能结合DC-SIGN并与HIV-1 gp140三聚体竞争结合。用CM对Raji-DC-SIGN细胞或未成熟树突状细胞（iDC）进行预孵育，能有效抑制DC-SIGN介导的HIV-1毒株对表达CCR5和CXCR4的CD4+ T淋巴细胞的反式感染，而对直接感染没有影响。初步生化特性分析表明，该成分似乎很大（>100kDa），耐热且耐蛋白酶K，以不依赖α1-3甘露糖的方式结合，并且个体之间差异很大。使用DC-SIGN-Fc包被的琼脂糖珠进行免疫沉淀，随后进行质谱分析，结果表明乳铁蛋白（片段）及其受体（intlectin-1）是候选成分。我们通过ELISA表明，CM中的乳铁蛋白水平与DC-SIGN结合能力相关。总之，CM可以结合C型凝集素DC-SIGN，并阻断HIV-1毒株对表达CCR5和CXCR4的细胞的反式感染。此外，我们的数据表明乳铁蛋白是CM的DC-SIGN结合成分。这些结果表明，CM有可能干扰病原体传播并调节结肠直肠黏膜处的免疫反应。
