Specific Intercellular Adhesion Research Articles

Engineered Nanovesicles Expressing Bispecific Single Chain Variable Fragments to Protect against SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in high morbidity and mortality rates worldwide. Although the epidemic has been controlled in many areas and numerous patients have been successfully treated, the risk of reinfection persists due to the low neutralizing antibody titers and weak immune response. To provide long-term immune protection for infected patients, novel bispecific CB6/dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (SIGN) nanovesicles (NVs) were constructed to target both the SARS-CoV-2 spike protein (S) and the DC receptors for virus neutralization and immune activation. Herein, we designed NVs expressing both CB6 and DC-SIGN single chain variable fragments (scFvs) on the surface to block SARS-CoV-2 invasion and activate DC function. Monophosphoryl lipid A (MPLA) was loaded into the CB6/DC-SIGN NVs as an adjuvant to promote this process. The CB6/DC-SIGN NVs prevented a pseudovirus expressing the S protein from infecting the target cells expressing high levels of angiotensin-converting enzyme 2 in vitro. Additionally, CB6/DC-SIGN NVs admixed with S-expressing pseudoviruses activated the DCs, which was promoted by the adjuvant MPLA loaded in the NVs. Using a mouse model, we also confirmed that the CB6/DC-SIGN NVs effectively improved the neutralizing antibody titer and inhibited the growth of tumors expressing the S protein after 3 weeks of treatment. This potential NV-based treatment not only exerts a blocking effect by binding the S protein in the short term but may also provide patients with long-term protection against secondary infections.

DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes.

Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

DC-SIGN Binding to the Oligomannose-Type Glycans on the Surface Immunoglobulin Variable Region of Follicular Lymphoma Promotes Selective Adhesion to Microenvironmental VCAM-1

The selective acquisition of oligomannose-type glycans at the antigen-binding site of the surface immunoglobulin (sIg-Mann) is a tumor-specific post-translational modification of all follicular lymphomas (FL). Our immunogenetic and structural analyses have revealed that these oligomannoses at N-glycosylation sites are universally acquired and persist during the entire natural history of FL from the early stages throughout transformation into EZB lymphoma (Chiodin G, Blood 2021). If a FL cell loses these sites, which is rare, the progeny disappears unless a new glycosylation site is acquired (Odabashian M, Blood 2020). These data indicate that sIg-Mann is a fundamental functional requirement for the entire clonal population of all FLs. The low-affinity interaction of sIg-Mann with its specific ligand dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), expressed on interfollicular M2-macrophages and follicular dendritic cells (FDC) of FL (RadtkeA, BioRxiv, 2022), distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via AKT and MYC and not endocytosis (Linley A, Blood 2015). However, the function of DC-SIGN:Ig-Mann remains to be further elucidated. In this study, we investigated the hypothesis that the specific engagement of DC-SIGN:Ig-Mann interaction functions to promote homing, while maintaining selective growth and survival signals. We found that DC-SIGN induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1 and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN with an anti-DC-SIGN antibody specific to the carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion, confirming that adhesion was mediated by the selective interaction with the oligomannose of sIg-Mann. Although intracellular signals were significantly lower than those by soluble anti-IgM F(ab)2 (here used to mimic the binding of cross-linking polyvalent antigen to Ig protein), the level of adhesion induced by DC-SIGN was similar to that induced by anti-IgM F(ab)2. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable by immunoblotting. However, either proximal inhibition of the PI3K/AKT pathway or distal inhibition of ARP2/3, formin, Rac and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. This suggested that Ig-Mann-mediated adhesion required both low-level intracellular signals and membrane adaptation (Valle-Argos, Sci Rep 2021). Remarkably, unlike anti-IgM F(ab)2, adhesion was specific to VCAM-1 but not to other stromal elements including fibronectin (FN). Instead, using the sIg-Mann+ve (IGHV4-34+ve) WSU-FSCCL cells, DC-SIGN appeared to mimic the interaction of monovalent ligands, including Fab fragments to either the Ig constant (anti-IgM Fab) or Ig variable region (9G4 Fab, which recognizes an epitope in the framework region 1 of IGHV4-34). Like DC-SIGN, Fab fragments induced selective adhesion to VCAM-1 and low-level signals via AKT, but neither endocytosis nor adhesion to FN. These results identify an important functional role of the DC-SIGN:sIg-Mann in mediating a signal that is tuned at the right threshold to promote selective and, in the absence of Ig endocytosis, persistent adhesion to environmental VCAM-1, determining homing of the FL tumor cells in the lymph node. This tumor-specific antigen-independent low-affinity DC:SIGN-Ig-Mann interaction contrasts to conventional high-affinity antigen:Ig protein interaction, which is prevented in FL (Schneider D, Blood 2015) and would instead lead to endocytosis and death. Therapeutic interruption of the interaction of DC-SIGN+ve cells with sIg-Mann+ve FL cells would provide a new way to displace the tumor cells from their prosurvival microenvironment.

Interaction of Host Pattern Recognition Receptors (PRRs) with Mycobacterium Tuberculosis and Ayurvedic Management of Tuberculosis: A Systemic Approach.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), infects the lungs' alveolar surfaces through aerosol droplets. At this stage, the disease progression may have many consequences, determined primarily by the reactions of the human immune system. However, one approach will be to more actively integrate the immune system, especially the pattern recognition receptor (PRR) systems of the host, which notices pathogen-associated molecular patterns (PAMPs) of Mtb. Several types of PRRs are involved in the detection of Mtb, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Dendritic cell (DC) -specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), Mannose receptor (MR), and NOD-like receptors (NLRs) related to inflammasome activation. In this study, we focus on reviewing the Mtb pathophysiology and interaction of host PPRs with Mtb as well as adverse drug effects of anti-tuberculosis drugs (ATDs) and systematic TB treatment via Ayurvedic medicine.

Equipping Cancer Cell Membrane Vesicles with Functional DNA as a Targeted Vaccine for Cancer Immunotherapy.

By inducing tumor-specific immune responses, tumor vaccines have recently aroused great research interest. Herein, we design a targeted nanovaccine by equipping cell membrane vesicles (CMVs) harvested from tumor cells with functional DNA including CpG oligonucleotide, an agonist for toll-like receptor 9, as well as an aptamer targeting the dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3 grabbing nonintegrin (DC-SIGN) receptor overexpressed on DCs. Such DNA-modified CMVs could target DCs and further stimulate their maturation. Notably, our nanovaccines could trigger robust antitumor immune responses to effective delay the tumor growth. Moreover, the combination of CMV-based nanovaccines with an immune checkpoint blockade could result in improved therapeutic responses by eliminating the majority of the tumors as well as long-term immune memory to prevent tumor recurrence. Therefore, by simply assembling functional DNA on CMVs harvested from tumor cells, we propose a general platform of DC-targeted personalized cancer vaccines for effective and specific cancer immunotherapy.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior

AbstractGlycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

Functional role of surface layer proteins of Lactobacillus acidophilus L-92 in stress tolerance and binding to host cell proteins.

Lactobacillus acidophilus surface layer proteins (SLPs) self-assemble into a monolayer that is non-covalently bound to the outer surface of the cells. There they are in direct contact with the environment, environmental stressors and gut components of the host in which the organism resides. The role of L. acidophilus SLPs is not entirely understood, although SLPs seem to be essential for bacterial growth. We constructed three L. acidophilus L-92 strains, each expressing a mutant of the most abundant SLP, SlpA. Each carried a 12-amino acid c-myc epitope substitution at a different position in the protein. A strain was also obtained that expressed the SlpA paralog SlpB from an originally silent slpB gene. All four strains behaved differently with respect to growth under various stress conditions, such as the presence of salt, ox gall or ethanol, suggesting that SlpA affects stress tolerance in L. acidophilus L-92. Also, the four mutants showed differential in vitro binding ability to human host cell proteins such as uromodulin or dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). Furthermore, co-culture of murine immature DCs with a mutant strain expressing one of the recombinant SlpA proteins changed the concentrations of the cytokines IL-10 and IL-12. Our data suggest that SlpA and SlpB of L. acidophilus participate in bacterial stress tolerance and binding to uromodulin or DC-SIGN, possibly leading to effective immune-modification.

Association of Genetic Polymorphisms in DC-SIGN, Toll-Like Receptor 3, and Tumor Necrosis Factor α Genes and the Lewis-Negative Phenotype With Chikungunya Infection and Disease in Nicaragua.

Chikungunya infections range from subclinical infection to debilitating arthralgia and to chronic inflammatory rheumatism. Tumor necrosis factor (TNF) α, DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), Toll-like receptor (TLR) 3, and blood groups have been directly or indirectly implicated in the susceptibility and pathogenesis of chikungunya. To test the hypothesis that polymorphisms in genes coding for these molecules determine clinical outcomes of chikungunya infection, a retrospective case-control study was performed in León, Nicaragua. The study included 132 case patients and 132 controls, matched for age, sex and neighborhood. Case patients had clinical symptoms of chikungunya, which was diagnosed by means of polymerase chain reaction. Controls were individuals not reporting abrupt presentation of clinical chikungunya-like symptoms. Polymorphisms were identified by TaqMan single-nucleotide polymorphism genotyping assays. After adjustment for sociodemographic risk factors, chikungunya disease was associated with polymorphism in DC-SIGN and TLR3 genes (odds ratios, 5.2 and 3.3, respectively), and TNF-α with reduced persistent joint pain (0.24). Persistent joint pain was also associated with age, female sex and other comorbid conditions. Most interestingly, the Lewis-negative phenotype was strongly associated with both symptomatic chikungunya and immunoglobulin G seropositivity (odds ratios, 2.7, and 3.3, respectively). This study identified polymorphisms in DC-SIGN, TLR3, and TNF-α genes as well as Lewis-negative phenotype as risk factors for chikungunya infection and disease progression.

Poor clinical outcomes of intratumoral dendritic cell–specific intercellular adhesion molecule 3–grabbing non-integrin–positive macrophages associated with immune evasion in gastric cancer

AimTumour-associated macrophages (TAMs) are prominent immune cells infiltrating in solid tumours with phenotypic and functional heterogeneity. However, the clinical significance of heterogeneous subtypes of TAMs in gastric cancer still remains obscure. Here, we aimed to explore the clinical significance of TAMs expressing dendritic cell–specific intercellular adhesion molecule 3–grabbing non-integrin (DC-SIGN) and its relevance with immune contexture in gastric cancer. MethodsWe selected 453 formalin-fixed and paraffin-embedded samples and 51 fresh tissue specimens of patients with gastric cancer from Zhongshan Hospital. The association of DC-SIGN+ macrophages with clinicopathological parameters, overall survival (OS) and responsiveness to fluorouracil-based adjuvant chemotherapy (ACT) was inspected. Immunohistochemistry (IHC) and flow cytometry (FCM) were applied to characterize immune cells in gastric cancer. ResultsWe demonstrated that high intratumoral DC-SIGN+ macrophages infiltration predicted poor OS and inferior therapeutic responsiveness to fluorouracil-based ACT in patients with gastric cancer. Furthermore, higher infiltration of DC-SIGN+ macrophages indicated an increased number of Foxp3+ regulatory T cells (Tregs), CD8+ T cells and a higher ratio of Foxp3+/CD8+ within the tumour microenvironment (TME). In addition, CD8+ T cells in DC-SIGN+ macrophages high subgroup were functionally impaired, showing decreased interferon-γ (IFN-γ), granzyme B (GZMB) and perforin production yet elevated programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression. ConclusionsDC-SIGN+ macrophages were associated with immunoinvasive TME and indicated poor prognosis and inferior therapeutic responsiveness to fluorouracil-based ACT. DC-SIGN+ macrophages might be an independent prognosticator and a potential immunotherapeutic target for gastric cancer.

Serum Amyloid P and a Dendritic Cell–Specific Intercellular Adhesion Molecule-3–Grabbing Nonintegrin Ligand Inhibit High-Fat Diet–Induced Adipose Tissue and Liver Inflammation and Steatosis in Mice

High-fat diet (HFD)-induced inflammation is associated with a variety of health risks. The systemic pentraxin serum amyloid P (SAP) inhibits inflammation. SAP activates the high-affinity IgG receptor Fcγ receptor I (FcγRI; CD64) and the lectin receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209). Herein, we show that for mice on an HFD, injections of SAP and a synthetic CD209 ligand (1866) reduced HFD-increased adipose and liver tissue inflammation, adipocyte differentiation, and lipid accumulation in adipose tissue. HFD worsened glucose tolerance test results and caused increased adipocyte size; for mice on an HFD, SAP improved glucose tolerance test results and reduced adipocyte size. Mice on an HFD had elevated serum levels of IL-1β, IL-23, interferon (IFN)-β, IFN-γ, monocyte chemoattractant protein 1 [MCP-1; chemokine (C-C motif) ligand 2 (CCL2)], and tumor necrosis factor-α. SAP reduced serum levels of IL-23, IFN-β, MCP-1, and tumor necrosis factor-α, whereas 1866 reduced IFN-γ. Invitro, SAP, but not 1866, treated cells isolated from white fat tissue (stromal vesicular fraction) produced the anti-inflammatory cytokine IL-10. HFD causes steatosis, and both SAP and 1866 reduced it. Conversely, compared with control mice, SAP knockout mice fed on a normal diet had increased white adipocyte cell sizes, increased numbers of inflammatory cells in adipose and liver tissue, and steatosis; and these effects were exacerbated on an HFD. SAPand1866 may inhibit some, but not all, of the effects of a high-fat diet.

Preparation and identification of anti-human ICAM-1 scFv

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein–carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly-N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule–grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.—Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Protein-Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer.

Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein–protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.

SIgA–Shigella Immune Complexes Interact with Dectin-1 and SIGNR3 to Differentially Regulate Mouse Peyer's Patch and Mesenteric Lymph Node Dendritic Cell's Responsiveness

In addition to contributing to immune exclusion at mucosal surfaces, secretory IgA (SIgA) made of polymeric IgA and secretory component is able to selectively reenter via microfold cells into Peyer's patches (PPs) present along the intestine and to associate with dendritic cells (DCs) of the CD11c+CD11b+MHCII+F4/80−CD8−phenotype in the subepithelial dome region and the draining mesenteric lymph nodes (MLNs). However, the nature of the receptor(s) for SIgA on murine PP and MLN DCs is unknown. We find that glycosylated secretory component moiety and polymeric IgA are both involved in the specific interaction with these cells. Using blocking antibodies and competition experiments, we identify Dectin-1 and specific intercellular adhesion molecule-3 grabbing non-integrin receptor 3 (SIGNR3) as receptors for SIgA. While SIgA-commensal immune complexes (ICs) contribute to local homeostasis upon interaction with mucosal DCs, the picture is less clear for pathogenic agents. We find that in comparison with incubation of Shigella flexneri alone, association of the enteropathogen with SIgA prompts freshly isolated DCs from PPs and MLNs to invert the production of pro- versus non-inflammatory cytokines/chemokines. The sum of the data suggests that in contrast to IgG-based ICs boosting immune reactivity of antigen-presenting cells, SIgA produced during an ongoing immune response can, in addition to its known function of immune exclusion, modulate mucosal DC conditioning via specific interaction with Dectin-1 and SIGNR3.

Prion pathogenesis is unaltered following down-regulation of SIGN-R1

Prion diseases are infectious neurodegenerative disorders characterised by accumulations of abnormal prion glycoprotein in affected tissues. Following peripheral exposure, many prion strains replicate upon follicular dendritic cells (FDC) in lymphoid tissues before infecting the brain. An intact splenic marginal zone is important for the efficient delivery of prions to FDC. The marginal zone contains a ring of specific intercellular adhesion molecule-3-grabbing non-integrin related 1 (SIGN-R1)-expressing macrophages. This lectin binds dextran and capsular pneumococcal polysaccharides, and also enhances the clearance of apoptotic cells via interactions with complement components. Since prions are acquired as complement-opsonized complexes we determined the role of SIGN-R1 in disease pathogenesis. We show that transient down-regulation of SIGN-R1 prior to intravenous prion exposure had no effect on the early accumulation of prions upon splenic FDC or their subsequent spread to the brain. Thus, SIGN-R1 expression by marginal zone macrophages is not rate-limiting for peripheral prion disease pathogenesis.

N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

Vitamin C attenuates hemorrhagic shock induced dendritic cell accumulation and lung injury

Objective To investigate the effect of hemorrhagic shock on dendritic cell accumulation in rat lung and lung injury, and to observe the protective effect of vitamin C. Methods Forty-two Sprague-Dawley rats (6 to 7 weeks old) were randomly divided into 7 groups (n=61 group): control group, hemorrhagic shock groups (HS 2 hours, HS 6 hours and HS 24 hours), and hemorrhagic shock+ vitamin C groups (HS+ VitC 2 hours, HS+ VitC 6 hours and HS+ VitC 24 hours). The rat hemorrhagic shock model was established by withdrawing blood through femoral arteriy. We used immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) technology to detect the expression level of dendritic cell specific intercellular adhesion molecule -3 (DC-SIGN) in rat lung tissues. The mRNA expression levels of TNF-α and IL-6, the myeloperioxidase activity in the lung tissues were tested, the lung injury scours were assessed simultaneously. Results There were few DC-SIGN proteins in rat lungs in control group. The expression levels of DC-SIGN protein increased markedly in hemorrhagic shock groups. The DC-SIGN mRNA concentration in rat lungs increased significantly 2 hours after hemorrhagic shock (P<0.05), and kept in high levels in 24 hours. The TNF-α and IL-6 mRNA concentration, the myeloperoxidase activity in lung tissues, and the histological damage scores of lungs were all increased in hemorrhagic shock groups compared with control group (P<0.05). All these phenomena induced by hemorrhagic shock were relieved when vitamin C was treated before resuscitation (P<0.05). Conclusions Perhaps hemorrhagic shock induced acute lung injury by promoting dendritic cell accumulation in rat lungs.Vitamin C curbed this phenomenon. Key words: Shock, Hemorrhagic; Dendritic cells; Acute lung injury

Lectin binding to surface Ig variable regions provides a universal persistent activating signal for follicular lymphoma cells

AbstractThe vast majority of cases of follicular lymphoma (FL), but not normal B cells, acquire N-glycosylation sites in the immunoglobulin variable regions during somatic hypermutation. Glycans added to sites are unusual in terminating at high mannoses. We showed previously that the C-type lectins, dendritic cell–specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor, bound to FL surface immunoglobulin (sIg), generating an intracellular Ca2+ flux. We have now mapped further intracellular pathways activated by DC-SIGN in a range of primary FL cells with detection of phosphorylated ERK1/2, AKT, and PLCγ2. The SYK inhibitor (tamatinib) or the BTK inhibitor (ibrutinib) each blocked phosphorylation. Activation by DC-SIGN occurred in both IgM+ and IgG+ cases and led to upregulation of MYC expression, with detection in vivo observed in lymph nodes. Unlike cells of chronic lymphocytic leukemia, FL cells expressed relatively high levels of sIg, unchanged by long-term incubation in vitro, indicating no antigen-mediated downregulation in vivo. In contrast, expression of CXCR4 increased in vitro. Engagement of sIg in FL cells or normal B cells by anti-Ig led to endocytosis in vitro as expected, but DC-SIGN, even when cross-linked, did not lead to significant endocytosis of sIg. These findings indicate that lectin binding generates signals via sIg but does not mediate endocytosis, potentially maintaining a supportive antigen-independent signal in vivo. Location of DC-SIGN in FL tissue revealed high levels in sinusoidlike structures and in some colocalized mononuclear cells, suggesting a role for lectin-expressing cells at this site.

DC-SIGN–expressing macrophages trigger activation of mannosylated IgM B-cell receptor in follicular lymphoma

AbstractFollicular lymphoma (FL) results from the accumulation of malignant germinal center (GC) B cells leading to the development of an indolent and largely incurable disease. FL cells remain highly dependent on B-cell receptor (BCR) signaling and on a specific cell microenvironment, including T cells, macrophages, and stromal cells. Importantly, FL BCR is characterized by a selective pressure to retain surface immunoglobulin M (IgM) BCR despite an active class-switch recombination process, and by the introduction, in BCR variable regions, of N-glycosylation acceptor sites harboring unusual high-mannose oligosaccharides. However, the relevance of these 2 FL BCR features for lymphomagenesis remains unclear. In this study, we demonstrated that IgM+ FL B cells activated a stronger BCR signaling network than IgG+ FL B cells and normal GC B cells. BCR expression level and phosphatase activity could both contribute to such heterogeneity. Moreover, we underlined that a subset of IgM+ FL samples, displaying highly mannosylated BCR, efficiently bound dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), which could in turn trigger delayed but long-lasting BCR aggregation and activation. Interestingly, DC-SIGN was found within the FL cell niche in situ. Finally, M2 macrophages induced a DC-SIGN–dependent adhesion of highly mannosylated IgM+ FL B cells and triggered BCR-associated kinase activation. Interestingly, pharmacologic BCR inhibitors abolished such crosstalk between macrophages and FL B cells. Altogether, our data support an important role for DC-SIGN–expressing infiltrating cells in the biology of FL and suggest that they could represent interesting therapeutic targets.