Glycopeptides as Targets for Dendritic Cells: Exploring MUC1 Glycopeptides Binding Profile toward Macrophage Galactose-Type Lectin (MGL) Orthologs

The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL

Tailored Presentation of Carbohydrates on a Coiled Coil-Based Scaffold for Asialoglycoprotein Receptor Targeting

The coiled-coil folding motif represents an ideal scaffold for the defined presentation of ligands due to the possibility of positioning them at specific distances along the axis. We created a coiled-coil glycopeptide library to characterize the distances between the carbohydrate-binding sites of the asialoglycoprotein receptors (ASGPR) on hepatocytes. The components of the glycopeptide library vary for the number of displayed ligands (galactose), their position on the peptide sequence, and the space between peptide backbone and carbohydrate. We determined the binding of the glycopeptides to the hepatocytes, and we established the optimal distance and orientation of the galactose moieties for interaction with the ASGPR using flow cytometry. We confirmed that the binding occurs through endocytosis mediated by ASGPR <i>via</i> inhibition studies with cytochalasin D; fluorescence microscopy studies display the uptake of the carrier peptides inside the cell. Thus, this study demonstrates that the coiled-coil motif can be used as reliable scaffold for the rational presentation of ligands

Scoring for assessment of the disease activity index.

<p>Scoring for assessment of the disease activity index.</p

Local cytokine concentrations in the colon of wild-type, MCL^−/−, and DCIR^−/− mice.

<p>Colons from untreated wild-type mice or from wild-type and MCL^−/− mice (n = 6) (<b>A</b>), or wild-type and DCIR^−/− mice (n = 7 for wild-type and n = 8 for DCIR^−/− mice) (<b>B</b>) treated with 3% DSS for seven consecutive days were homogenized and used for cytokine determination by cytometric bead array. Data are expressed as mean + SEM. Significance is indicated by asterisks (*), ns = no significance.</p

The C-Type Lectin Receptor Mincle Binds to <i>Streptococcus pneumoniae</i> but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response

<div><p>The innate immune system employs C-type lectin receptors (CLRs) to recognize carbohydrate structures on pathogens and self-antigens. The Macrophage-inducible C-type lectin (Mincle) is a FcRγ-coupled CLR that was shown to bind to mycobacterial cord factor as well as certain fungal species. However, since CLR functions during bacterial infections have not yet been investigated thoroughly, we aimed to examine their function in <i>Streptococcus pneumonia</i> infection. Binding studies using a library of recombinantly expressed CLR-Fc fusion proteins indicated a specific, Ca²⁺-dependent, and serotype-specific binding of Mincle to <i>S. pneumonia</i>. Subsequent experiments with different Mincle-expressing cells as well as Mincle-deficient mice, however, revealed a limited role of this receptor in bacterial phagocytosis, neutrophil-mediated killing, cytokine production, and antibacterial immune response during pneumonia. Collectively, our results indicate that Mincle is able to recognize <i>S. pneumonia</i> but is not required for the anti-pneumococcal innate immune response.</p></div

Histological analysis of colon sections from wild-type and DCIR^−/− mice.

<p>Paraffin sections of the colon from untreated or 3% DSS-treated wild-type and DCIR^−/− mice were prepared at day seven and were stained with hematoxylin and eosin (H&E) for histological evaluation in a blinded manner. (<b>A</b>) Representative images of paraffin-embedded sections of the rectal part of the colon are shown (40x magnification). Arrows indicate a severe ulcer in the colon from DCIR^−/− mice. Each colon was divided into three segments of identical length (oral, middle, rectal) which were separately analyzed. The degree of leukocyte infiltration (<b>B</b>) and mucosal erosion/ulceration (<b>C</b>) was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4). The scores for both, cell infiltration as well as mucosal ulceration in the rectal part of the colon from DCIR^−/− mice were significantly increased compared to wild-type mice. Data are expressed as mean + SEM (n = 5). The <i>p</i>-values were determined using Mann-Whitney’s U test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p

Scoring for the histological evaluation of intestinal lesions.

<p>Scoring for the histological evaluation of intestinal lesions.</p

MCL and DCIR recognize commensal intestinal microbiota and modulate APC and T cell cytokine production.

<p>Binding of MCL- and DCIR-hFc fusion proteins to stained gut microbes was analyzed by flow cytometry. (<b>A</b>) Representative dot plots of one binding experiment with MCL- and DCIR-hFc, with hFc as negative control, and with MGL1-hFc as positive control. Gating and frequencies indicate binding events of CLR-hFc fusion proteins to commensal microbiota. For analysis, it was first gated on Syto 61 positive events ( = stained microbiota) followed by gating on PE positive events ( = CLR-Fc fusion proteins). Data are representative of three independent experiments (triplicates each). (<b>B</b>) MCL^−/− and wild-type BMMs or (<b>C</b>) DCIR^−/− and wild-type BMDCs were stimulated with various concentrations of heat-killed gut microbiota, LPS or coated zymosan for 18 h (triplicates each). TNF-α levels in the culture supernatants were determined by ELISA. TNF-α production was significantly increased for MCL^−/− BMMs and DCIR^−/− BMDCs compared to wild-type APCs. Data are representative of three independent experiments. For analysis of T cell activation, purified OT-II transgenic T cells were co-cultivated with BMMs or BMDCs in the presence of heat-killed gut microbiota and 30 µg/mL OVA for 72 h. (<b>D</b>) IL-2 and (<b>E</b>) IFN-γ levels were determined in the culture supernatants of stimulated MCL^−/− and wild-type BMMs. Similarly, (<b>F</b>) IL-2 and (<b>G</b>) IFN-γ levels were analyzed in the culture supernatants of stimulated DCIR^−/− and wild-type BMDCs. Data are representative of three independent experiments (triplicates each) and are expressed as mean + SEM. The <i>p</i>-values were determined with unpaired Student’s t-test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p

Lack of Mincle or FcRγ does not influence the antibacterial host response during pneumococcal pneumonia.

<p>(A) C57Bl/6 mice were intranasally infected with 5×10⁶ CFU/mouse <i>S. pneumoniae</i> ST3 or control treated with PBS and Mincle expression levels in the lungs at the indicated time points were determined by quantitative RT-PCR. (B) WT, <i>Fcerg1^-/-</i> and <i>Mincle^-/-</i> mice were intranasally infected with 7.5×10⁴ CFU/mouse <i>S. pneumoniae</i> ST3 and survival was monitored every 12 h over 10 days. (C-K) WT, <i>Fcer1g^-/-</i> and <i>Mincle^-/-</i> mice were intranasally infected with 5×10⁶ CFU/mouse <i>S. pneumoniae</i> ST3 or treated with PBS. Bacterial loads were determined in the (C) bronchoalveolar lavage fluid (BALF) and (D) blood. (E) Neutrophils and (F) macrophages in the BALF were quantified by flow cytometry. (G) IL-6 and (H) TNFα (H) levels in the BALF were analyzed by ELISA. Relative expression of (I) <i>Cxcl1</i>, (J) <i>Ccl2</i> and (K) <i>Gcsf</i> in the lung was determined by quantitative RT-PCR. Data are shown as mean + SEM; (A) n = 3, (B) n = 8–10, (C-F) n = 6–22, (G-H) n = 6–8, (I-K) n = 4–5 mice each group. n.s. not significant.</p

Lack of FcRγ or Mincle does not affect the innate immune response to <i>S. pneumoniae</i> in different cell types.

<p>(A) AECs, MVECs and AMΦs were isolated from the lung, and PMNs and BMMs from the bone marrow of C57Bl/6 mice. Cells were left untreated or infected with <i>S. pneumoniae</i> ST2 (D39). (B-G) AMΦs, BMMs and PMNs were isolated from WT, <i>Fcer1g^-/-</i> and <i>Mincle^-/-</i> mice. (B, C) AMΦs or (D) BMMs were infected with <i>S. pneumoniae</i> ST2 or (E) BMMs were stimulated with TDM as a positive control for 16 h and cytokine release was quantified by ELISA. (F) Untreated or LPS-treated (100 ng/ml, 4 h) BMMs were infected with <i>S. pneumoniae</i> ST2Δ<i>cps</i> (MOI = 2.5) and were treated with gentamicin (50 mg/ml) after 30 min. Intracellular, viable bacteria were determined 60 min or 120 min post infection. (G) Rabbit serum-opsonized <i>S. pneumoniae</i> ST2Δ<i>cps</i> were incubated with PMNs and neutrophil-mediated killing was assessed after 45 min incubation. Data are shown as mean + SEM of (A) two (A: PMN, E, F), three (C, G), four (A: AEC, MVEC, B, D) or five (A: AMΦ, BMM) independent experiments carried out in duplicates (A-F) or quadruplicates (G); * = p<0.05; ** = p<0.01; *** = p<0.001; n.s. not significant.</p