The protease allergen Der p 1 cleaves cell surface DC-SIGN and DC-SIGNR: experimental analysis of in silico substrate identification and implications in allergic responses

Background The cysteine protease Der p 1 from the house dust mite Dermatophagoides pteronyssinus is one of the most potent allergens known. An attractive mechanism for a component of Der p 1 allergenicity lies in its ability to cleave key regulatory molecules from leucocyte surfaces, subverting cellular function and driving abnormal immunoglobulin E (IgE) responses. Objective Although CD23, CD25 and CD40 have already been identified as major Der p 1 targets, other significant substrates may also exist. Methods To investigate this, knowledge of the proteolytic properties of Der p 1 was used to perform in silico digestion of human dendritic cell surface proteins, using the prediction of protease specificity (PoPS) bioinformatics tool, in conjunction with cellular in vitro analysis and cleavage site determination. Results Targets identified included DC‐SIGN and DC‐SIGNR, two C‐type lectins implicated mostly in pathogen trafficking. Treatment of positively expressing cells with Der p 1 led to loss of detectable surface DC‐SIGN and DC‐SIGNR. Digestion of purified soluble recombinant DC‐SIGN and DC‐SIGNR, followed by N‐terminal sequencing and MALDI mass spectrometry, indicated in each case one major cleavage site and several minor sites, the former correlating well with Der p 1 enzymology and the folded state of the substrate proteins. Loss of DC‐SIGN from the cell surface led to reduced binding of intracellular adhesion molecule‐3, an endogenous DC‐SIGN ligand expressed on naïve T cells which is thought to be involved in T‐helper type 1 cytokine signalling. Conclusion These data provide evidence of lectin involvement in the initiation of the allergic response and the value of using genome‐wide in silico digestion tools.     

DC-SIGN activation mediates the differential effects of SAP and CRP on the innate immune system and inhibits fibrosis in mice

Fibrosis is caused by scar tissue formation in internal organs and is associated with 45% of deaths in the United States. Two closely related human serum proteins, serum amyloid P (SAP) and C-reactive protein (CRP), strongly affect fibrosis. In multiple animal models, and in Phase 1 and Phase 2 clinical trials, SAP affects several aspects of the innate immune system to reduce fibrosis, whereas CRP appears to potentiate fibrosis. However, SAP and CRP bind the same Fcγ receptors (FcγR) with similar affinities, and why SAP and CRP have opposing effects is unknown. Here, we report that SAP but not CRP binds the receptor DC-SIGN (SIGN-R1) to affect the innate immune system, and that FcγR are not necessary for SAP function. A polycyclic aminothiazole DC-SIGN ligand and anti–DC-SIGN antibodies mimic SAP effects in vitro. In mice, the aminothiazole reduces neutrophil accumulation in a model of acute lung inflammation and, at 0.001 mg/kg, alleviates pulmonary fibrosis by increasing levels of the immunosuppressant IL-10. DC-SIGN (SIGN-R1) is present on mouse lung epithelial cells, and SAP and the aminothiazole potentiate IL-10 production from these cells. Our data suggest that SAP activates DC-SIGN to regulate the innate immune system differently from CRP, and that DC-SIGN is a target for antifibrotics.Fibrosing diseases such scleroderma, pulmonary fibrosis, and renal fibrosis are caused by aberrant scar tissue formation in internal organs and are associated with 45% of deaths in the United States (1). At a fibrotic lesion, monocytes leave the blood, enter the tissue, and differentiate into cells such as macrophages and fibrocytes (2). Fibrocytes and macrophages then secrete extracellular matrix (ECM) proteins, ECM modifying enzymes, and/or cytokines such as IL-4 to promote scar tissue formation and fibrosis (3, 4).Pentraxins are a family of highly conserved secreted proteins that have a profound effect on the development of fibrosis and the regulation of the innate immune system (5–7). The pentraxin serum amyloid P (SAP) reduces neutrophil activation and recruitment (8, 9), inhibits the differentiation of monocytes into fibroblast-like cells called fibrocytes (8, 10), and promotes IL-10–secreting macrophages (11–13). In animal models and two human trials (6, 14, 15), injections of SAP decrease fibrosis, indicating that SAP has a dominant effect on a disease that is mediated in part by the innate immune system. Conversely, the closely related pentraxin C-reactive protein (CRP) is proinflammatory and promotes fibrosis (5, 16). However, under some conditions, CRP decreases inflammation, indicating that much remains to be understood about this molecule (5, 17). Despite the strong effects of pentraxins on the innate immune system and fibrosis (5, 6), little is known about their mechanism of action. For instance, pentraxins such as SAP and CRP appear to bind the same Fcγ receptors (FcγR) with similar affinities (7, 8, 18), but they generally have opposite effects. What causes this functional difference is not known.Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN/CD209) is a C-type lectin found on dendritic cells, macrophages, and monocytes (19, 20). DC-SIGN mainly binds to mannosylated and fucosylated proteins (19, 20). Humans have DC-SIGN and L-SIGN, whereas mice have eight DC-SIGN orthologs called SIGN-R1–8 (21). SIGN-R1 most closely resembles DC-SIGN (21). DC-SIGN and SIGN-R1 also bind sialylated IgG (sIgG) (20). This interaction appears to be a protein:potein interaction and not a sialic acid:DC-SIGN interaction (22). Both sIgGs and SAP have α(2,6)-linked terminal sialic acids on the protein surface, and both sIgGs and SAP alleviate inflammation in mice (6, 20, 23).In this report, we show that in absence of all of the FcγR, neutrophils, monocytes, and macrophages still respond to SAP, indicating that SAP uses other receptors. For SAP, we show that one of the other receptors is DC-SIGN. We also found that anti–DC-SIGN antibodies and a small-molecule DC-SIGN ligand mimic the effects of SAP. The synthetic DC-SIGN ligand shows efficacy in murine models of acute lung inflammation and pulmonary fibrosis. In contrast to SAP, we find that CRP requires the FcγR to regulate neutrophils and IL-10 secretion from macrophages, but not to increase ICAM-I⁺ macrophages. This finding suggests that there are additional CRP receptors that regulate macrophage polarization. Our findings suggest the presence of a previously unidentified pentraxin target that accounts for the functional difference between SAP and CRP, and which might be useful as a therapeutic target to regulate the innate immune system and fibrosis.     

DC-SIGNR基因绞链区重复序列多态性检测方法的建立与应用

目的建立树突状细胞表面的蛋白质DC-SIGNR基因绞链区重复序列多态性的检测方法，了解宿主因素在抗艾滋病病毒1型（HIV-1）所起的作用。方法设计特定引物，用PCR方法对DC-SIGNR基因绞链区重复序列进行扩增，用凝胶电泳法对其产物进行分析。结果根据DC-SIGNR绞链区重复序列的数量，DC-SIGNR绞链区重复序列具有高度基因多态性，而DC-SIGN则罕见基因多态性。结论所建立的检测DC-SIGNR绞链区重复序列的方法简单，易于掌握。由于DC-SIGNR重复序列数量的改变可能会影响其正常功能，从而影响宿主与HIV-1的结合能力，因此该法为研究宿主因素在抗HIV-1感染中所起作用的一种好方法。     

Expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin on dendritic cells generated from human peripheral blood monocytes

AIM:To generate dendritic cells(DCs)from human pe-ripheral blood and to detect the expression of dendriticcell-specific intercellular adhesion molecule 3 grabbingnonintegrin(DC-SIGN;CD209)for the further study ofDC-SIGN in hepatitis C virus(HCV)transmission.METHODS:Peripheral blood monocytes were isolatedfrom blood of healthy individuals by Ficoll—Hypaquesedimentation and cultured in complete medium contain-ing rhGM-CSF and rhIL-4.Cells were cultured for sevendays,with cytokine addition every two days to obtainimmature DCs.Characteristics of the cultured cells wereobserved under light and scanning microscope,and theexpression of DC-SIGN was detected by immunofluores-cence staining.RESULTS:After seven-day culture,a large number ofcells with typical characteristics of DCs appeared.Theircharacteristics were observed under light and scanningelectron microscope.These cells had a variety of cellshapes such as those of bipolar elongate cells,elaboratestellate cells and DCs.DC-SIGN was detected by immu-nofluorescence staining and its expression level on culti-vated dendritic cells was high.CONCLUSION:DCs with a high expression of DC-SIGNcan be generated from human peripheral blood mono-cytes in complete medium containing rhGM-CSF andrhIL-4.     

DC-SIGN and immunoregulation

Dendritic cells （DCs） are known to be the most powerful professional antigen-presenting cells so far. It could activate primary immune response, and also downregulate immune response. DCs have a unique character of immunoregulation. DC-SIGN, a molecule designated as CD209, is one member of the C-type lectin superfamily. It is not only a pattern recognition receptor but implicated in immunoregulation of DCs. DC-SIGN has become hotspot of recent studies because of its important role in mediating DC adhesion, migration, inflammation, activating primary T cell, triggering immune response and participating in immune escape of pathogens and tumors. These studies on DC-SIGN involved in primary and secondary immune response and relevant mechanism will certainly provide us with a new method in treating and preventing certain diseases.     

Impact of polymorphisms in the DC-SIGNR neck domain on the interaction with pathogens

The lectins DC-SIGN and DC-SIGNR augment infection by human immunodeficiency virus (HIV), Ebolavirus (EBOV) and other pathogens. The neck domain of these proteins drives multimerization, which is believed to be required for efficient recognition of multivalent ligands. The neck domain of DC-SIGN consists of seven sequence repeats with rare variations. In contrast, the DC-SIGNR neck domain is polymorphic and, in addition to the wild type (wt) allele with seven repeat units, allelic forms with five and six sequence repeats are frequently found. A potential association of the DC-SIGNR genotype and risk of HIV-1 infection is currently under debate. Therefore, we investigated if DC-SIGNR alleles with five and six repeat units exhibit defects in pathogen capture. Here, we show that wt DC-SIGNR and patient derived alleles with five and six repeats bind viral glycoproteins, augment viral infection and tetramerize with comparable efficiency. Moreover, coexpression of wt DC-SIGNR and alleles with five repeats did not decrease the interaction with pathogens compared to expression of each allele alone, suggesting that potential formation of hetero-oligomers does not appreciably reduce pathogen binding, at least under conditions of high expression. Thus, our results do not provide evidence for diminished pathogen capture by DC-SIGNR alleles with five and six repeat units. Albeit, we cannot exclude that subtle, but in vivo relevant differences remained undetected, our analysis suggests that indirect mechanisms could account for the association of polymorphisms in the DC-SIGNR neck region with reduced risk of HIV-1 infection.     

Evidence that multiple defects in murine DC-SIGN inhibit a functional interaction with pathogens

Certain viruses, bacteria, fungi and parasites target dendritic cells through the interaction with the cellular attachment factor DC-SIGN, making this C-type lectin an attractive target for therapeutic intervention. Studies on DC-SIGN function would be greatly aided by the establishment of a mouse model, however, it is unclear if the murine (m) homologue of human (h) DC-SIGN also binds to pathogens. Here, we investigated the interaction of mDC-SIGN, also termed CIRE, with the Ebolavirus glycoprotein (EBOV-GP), a ligand of hDC-SIGN. We found that mDC-SIGN neither binds EBOV-GP nor enhances infection by reporterviruses pseudotyped with EBOV-GP. Analysis of chimeras between mDC-SIGN and hDC-SIGN provided evidence that determinants in the carbohydrate recognition domain and in the neck domain of mDC-SIGN inhibit a functional interaction with EBOV-GP. Moreover, mDC-SIGN was found be monomeric, suggesting that lack of multimerization, which is believed to be required for efficient pathogen recognition by hDC-SIGN, might be one factor that prevents binding of mDC-SIGN to EBOV-GP. Our results suggest that mDC-SIGN on murine dendritic cells is not an adequate model for pathogen interactions with hDC-SIGN.     

CD16+ monocytes exposed to HIV promote highly efficient viral replication upon differentiation into macrophages and interaction with T cells

The CD16+ subset of monocytes is dramatically expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unknown. Here, we demonstrate that CD16+ but not CD16- monocytes promote high levels of HIV replication upon differentiation into macrophages and interaction with T cells. Conjugates formed between CD16+ monocyte-derived macrophages and T cells are major sites of viral replication. Furthermore, similar monocyte-T cell conjugates detected in peripheral blood of HIV-infected patients harbor HIV DNA. Thus, expansion of CD16+ monocytes during HIV infection and their subsequent recruitment into tissues such as lymph nodes, brain, and intestine may contribute to HIV dissemination and establishment of productive infection in T cells.     

Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection

Studies were undertaken to determine whether previously described reductions in splenic DC-SIGN expression in simian acquired immune deficiency syndrome (AIDS) are limited to pathogenic simian immunodeficiency virus (SIV) infection. DC-SIGN expression was evaluated by immunohistochemistry in lymphoid tissues from AIDS-susceptible Asian macaque monkeys as compared with AIDS-resistant sooty mangabey monkeys in the presence and absence of SIV infection. The phenotype of DC-SIGN+ cells in susceptible and resistant species was identical and most consistent with macrophage identity. Significantly lower levels of DC-SIGN expression were identified in spleen, mesenteric lymph node, and bone marrow of macaques with AIDS (P<0.05). Reduced levels of splenic DC-SIGN correlated significantly with CD4T cell depletion in long-term pathogenic infection of macaques (P<0.01), whereas SIV-infected mangabeys retained high levels of DC-SIGN expression in spleen despite persistent infection. Reduced expression of DC-SIGN in spleen specifically characterizes pathogenic forms of SIV infection, correlates with disease progression, and may contribute to SIV pathogenesis.