Human Cytomegalovirus Fcγ Binding Proteins gp34 and gp68 Antagonize Fcγ Receptors I, II and III

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.DFG grant He 2526/6-2.European Commission grants QLRT-2001-01112 and MRTN-CT-2005-019248.Helmholtz Association through VISTRIE VH-VI-242.UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Characterization and functional analysis of mouse Ficolin B

The innate immune system contributes day by day to the elimination of invading pathogens. The lectin pathway of the complement system is initiated when mannose-binding-lectin (MBL) or ficolins interact with MASP-2. The aim of the present work was to characterize the mouse FcnB and define its function in the innate immune system and especially its role in the complement activation. Results from this thesis revealed that immature macrophages and dendritic cells as well as PMN express mouse FcnB (immature myeloid cells). While inflammatory stimuli can clearly enhance the FcnB expression in PMN no further enhancement was observed in mature macrophages or dendritic cells from bone marrow cultures of wild type mice, but was also seen in dendritic cells derived from mice that lack TNFR2. Previous results from our group (Runza et al., 2006) were supported by demonstrating the presence of FcnB on protein level in lysates of PMN, bone marrow-derived macrophages and dendritic cells, using newly generated monoclonal antibodies specific for mouse FcnB. In native PMN from Balb/c mice FcnB was localized in lysosomal granules similarly as previously found for peritoneal macrophages (Runza et al., 2006). In addition, in serum from Balb/c mice FcnB was detected. Further, binding properties of FcnB to various ligands were investiagted in this thesis. Our results indicate that FcnB might act as a potent pattern recognition molecule by recognizing sialic acid (present in the glycoprotein fetuin), DNA (potentially a target in dying cells) and chitin (potentially a target in fungi). The fact that FcnB binds to DNA shown in this thesis might be of importance concerning the finding that PMN have stored FcnB and produce NET structures upon stimulation, which contain DNA. Furthermore, recombinant FcnB bound to various strains of GBS and to serotype T-5 of S. aureus. One major function of ficolins is their interaction with MASP-2 what subsequently leads to the activation of the lectin pathway. In this thesis it was demonstrated for the first time that also mouse FcnB is able to activate the lectin pathway. Contradictory results regarding the role of mouse FcnB within the complement system are probably due to different oligomerization forms of the used FcnB. All results obtained during this work lead to the conclusion that mouse FcnB plays a role in the innate immune system, serving as a pattern recognition molecule for the detection of microorganisms and activating the complement system by binding to MASP-2, like the other ficolins or MBL

Immature mouse granulocytic myeloid cells are characterized by production of ficolin-B

Ficolins activate the lectin pathway of the complement system upon binding to carbohydrate patterns on pathogens. To characterize the producer cells of ficolin-B the expression of mouse ficolin-B, the orthologue of human M-ficolin, was studied in macrophages and dendritic cells during differentiation from bone marrow cells, in primary granulocytes, and during differentiation of granulocytes derived from ER-Hoxb8 cells. Expression of ficolin-B mRNA declined in all myeloid cell types to low levels during terminal differentiation. However, in contrast to macrophages and dendritic cells, ficolin-B expression was enhanced upon activation in granulocytes. High expression of ficolin-B was observed in primary immature neutrophilic CD11b+ Ly-6Cint Ly-6Ghigh granulocytes when isolated from the bone marrow, in particular during sepsis. Ficolin-B was demonstrated in lysates of primary granulocytes, ER-Hoxb8-derived granulocytes, bone marrow-derived macrophages, and dendritic cells. Native ficolin-B from cell lysates and supernatants of granulocytes activated the lectin pathway as measured by binding to MASP-2 and inducing C4 deposition. Specific staining demonstrated intra-cellular or cell associated ficolin-B protein in activated immature granulocytes deposited in a granular fashion. This study shows that ficolin-B is stored in and set free from immature granulocytic myeloid cells indicating a role in the early infection-induced cellular response of these inflammatory cells.Fil: Weber Steffens, Dorothea. Universitat Regensburg; AlemaniaFil: Hunold, Katja. Universitat Regensburg; AlemaniaFil: Kürschner, Johanna. Universitat Regensburg; AlemaniaFil: Giraldez Martinez, Sonia. Universitat Regensburg; AlemaniaFil: Elumalai, Preetham. Universitat Regensburg; AlemaniaFil: Schmidt, Dominic. Universitat Regensburg; AlemaniaFil: Trevani, Analía Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Runza, Valeria L.. Universitat Regensburg; AlemaniaFil: Männel, Daniela N.. Universitat Regensburg; Alemani

Right Ventricular Cardiomyopathy Meeting the Arrhythmogenic Right Ventricular Dysplasia Revised Criteria? Don't Forget Sarcoidosis!

A 53-year-old woman was referred for ventricular fibrillation with resuscitation. A CT-angiography showed signs of a right ventricular enlargement without obvious cause. A cardiac MRI demonstrated a dilated and hypokinetic right ventricle with extensive late gadolinium enhancement. Arrhythmogenic right ventricular dysplasia (ARVD) was suspected according to the "revised ARVD task force criteria". An endomyocardial biopsy was inconclusive. The patient developed purulent pericarditis after epicardial ablation therapy and died of toxic shock syndrome. The post-mortem pathologic examination demonstrated sarcoidosis involving the heart, lungs, and thyroid gland

Soluble ectodomains of HCMV vFcγR interfere with antibody dependent NK cell degranulation.

<p>(<b>A</b>) Cytotect was coated to a plate in binding buffer (0.1 M Na₂HPO₄ pH 9.0) at a concentration of 0.5 mg/ml and incubated for 2.5 hours at 37°C. After blocking for 30 minutes and washing unbound antibodies, soluble proteins, rIL-2 pre-activated primary NK cells and α-CD107a-PECy5 antibody were added and incubated for 4 hours at 37°C. Duplicates were measured for CD107a surface expression after dead cell exclusion with DAPI staining in a FACS Canto II. Means are shown with standard deviations (error bars). Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>B</b>) To compare the amounts of soluble proteins used in (A), SDS-PAGE and anti-V5 immunobotting was performed.</p

Ectopic expression of HSV-1 gE, HCMV gp68 and HCMV gp34 inhibit IgG1 (trastuzumab) mediated activation of FcγRs.

<p>(<b>A</b>) SKOV-3 cells were infected for 24 h with 2 PFU/cell VACV wt and rVACV expressing gE or (<b>B</b>) rVACV expressing gp68 or gp34 before opsonized with trastuzumab at different concentrations for 30 min. After removing of unbound antibodies by repeated washing with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR-ζ activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured, means with standard deviations (error bars) are shown for 3 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

The presence of the vFcγRs on the surface of infected cells inhibits antibody dependent NK cell degranulation.

<p>(<b>A</b>) MRC-5 fibroblasts were infected with HSV-1F wt, ΔgE and ΔgE revertant for 24 h before cells were opsonized with HSV IgG positive and negative human sera. After 30 min of incubation, unbound antibodies were washed away and NK cells at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells was measured in FACS. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a-positive cells with the immune antibody opsonization - % of CD107a-positive cells with non-immune antibody). (<b>B</b>) MRC-5 fibroblasts were infected with HCMV HB5 wt, HB5Δgp68, HB5ΔIRLΔgp34 and HB5ΔIRLΔgp68/Δgp34 for 72 h before cells were opsonized with HCMV IgG positive and negative human sera. After 30 min of incubation, unbound antibodies were washed away and NK cells at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells was measured in FACS. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a-positive cells with the immune antibody opsonization - % of CD107a-positive cells with non-immune antibody) (<b>C</b>) MRC-5 fibroblasts were infected with HCMV HB5 wt and HB5ΔIRLΔgp68/Δgp34 for 72 h before opsonized with Cytotect or human HCMV-IgG negative sera at 1∶10 and 1∶100 dilutions. After 30 min of incubation, unbound antibodies were washed away and NK cells from six different donors at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells against HCMV-infected cells was measured. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a positive cells with the immune antibody opsonization - % of CD107a positive cells with non-immune antibody). Single measurements of CD107a cells are shown. One of three (A, B) or two (C) representative experiments is shown.</p

Soluble ectodomains of HCMV vFcγRs interfere with FcγR activation.

<p>(<b>A</b>) SKOV-3 cells were opsonized with 100 ng/ml trastuzumab for 30 minutes and washed three times with D-MEM 10% FCS (vol/vol) before soluble proteins were added in graded concentrations concomitantly with BW:FcγRIIIA-ζ transfectants. mIL-2 was determined in supernatants (which were harvested after 16 h of co-cultivation of responder cells with target cells) by ELISA. (<b>B</b>) As in (A) but SKOV-3 cells were opsonized with 500 µg/ml trastuzumab for 30 minutes and washed three times with D-MEM 10% FCS (vol/vol) before soluble proteins were added in graded concentrations concomitantly with BW:FcγRI-ζ cells. (<b>C</b>) MRC-5 cells were infected with HCMV HB5ΔIRLΔgp68/Δgp34 (2 PFU/cell) for 72 h before soluble proteins were added in graded concentrations concomitantly with BW:FcγRIIIA-ζ responder cells. MRC-5 fibroblasts were opsonized with 1∶50 diluted Cytotect for 30 min. After removing of unbound antibodies by washing, soluble proteins and BW:FcγR-ζ transfectants were added and co-cultivated overnight. (<b>D</b>) as in (C), but HCMV HB5ΔIRLΔgp68/Δgp34 infected target cells were opsonized with 1∶10 diluted Cytotect and BW:FcγRIIA-ζ cells were used as responders. n = 3 replicates, means with standard deviations (error bars) are shown for 2 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

HSV-1 gE, HCMV gp68 and HCMV gp34 interfere with host FcγR activation upon opsonization of cells with polyclonal immune IgG.

<p>(<b>A</b>) The HSV vFcγR gE inhibits FcγRIIIA and FcγRIIA activation but fails to inhibit FcγRI. Human MRC-5 fibroblasts were infected with 2 PFU/cell of HSV-1 strain F wt and ΔgE for 24 h. Cells were opsonized with Cytotect at different concentrations for 30 min. After removing of unbound antibodies with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured; means with standard deviations (error bars) are shown for 4 independent experiments. (<b>B</b>) HCMV vFcγR gp68 interferes with FcγRIIIA, FcγRIIA and FcγRI activation. MRC-5 cells were infected with HCMV HB5 wt virus or HB5Δgp68 (2 PFU/cell) for 72 h. Fibroblasts were opsonized with Cytotect at different concentrations for 30 min. After removing of unbound antibodies by washing, 1×10⁵ BW:FcγR-ζ transfectants were added per well. Measurement of mIL-2 in supernatants after 16 h of co-cultivation of reporter cells with targets was performed by ELISA. Values are presented in the graphic as OD 450 nm. Three independent wells were measured; means with standard deviations (error bars) are shown for 4 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>C</b>) HCMV vFcγR gp34 interferes with FcγRIIIA, FcγRIIA and FcγRI activation. As in (B) but MRC-5 cells were infected with HCMV HB5ΔIRL, HB5ΔIRLΔgp34 or HB5ΔIRLΔgp68/Δgp34 (2 PFU/cell) for 72 h. (<b>D</b>) gp34 and gp68 interfere with FcγR activation in AD169varL infected cells. As in (B) but MRC-5 cells were infected with AD169varL wt, AD169varLΔgp68, AD169varLΔgp34 or AD169varLΔgp68/Δgp34.</p