Hanne Van Gorp

Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy

Scavenger receptor CD163 contains nine scavenger receptor cysteine-rich (SRCR) domains and because of the presence of this ancient and highly conserved protein motif, CD163 belongs to the SRCR superfamily. Expression of CD163 is restricted to cells of the monocyte/macrophage lineage and is tightly regulated, with a general tendency of anti-inflammatory signals to induce CD163 synthesis, while pro-inflammatory signals rather seem to downregulate CD163 expression. The first-identified and most-studied function of CD163 is related to its capacity to bind and internalize haemoglobin-haptoglobin (HbHp) complexes. Later on, its functional repertoire was expanded, with the identification of CD163 as an erythroblast adhesion receptor, a receptor for tumour necrosis factor-like weak inducer of apoptosis (TWEAK), as well as a receptor for distinct pathogens encompassing bacteria and viruses. Interaction of one of these ligands with CD163 might result in receptor-mediated endocytosis, but might as well trigger a signalling cascade leading to the secretion of signalling molecules, which implicates that CD163 also acts as an immunomodulator. Not only the membrane-bound form of CD163 has an immunomodulating capacity, but also soluble CD163, which is generated via ectodomain shedding, is able to exert anti-inflammatory effects. Furthermore, the concentration of this soluble protein is significantly increased under specific pathological conditions, making it a useful marker protein for certain diseases. Finally, its restricted expression pattern and potential to internalize make CD163 an attractive candidate as gateway for cell-directed (immuno)therapy. This review aims to summarize current knowledge on CD163s biology and its different biological functions beyond HbHp scavenging, thereby mainly focussing on the more recently discovered ones. Furthermore, current data supporting the capacity of CD163 to serve as a diagnostic marker in certain diseases and its potential as a target molecule for cell-directed therapy are surveyed.

Sialoadhesin and CD163 join forces during entry of the porcine reproductive and respiratory syndrome virus

The porcine reproductive and respiratory syndrome virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in virus internalization but not in virus uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 degrees C with either sialoadhesin- or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 degrees C, only sialoadhesin- and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased virus production 10-100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection.

Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the late 1980s and rapidly became one of the most significant viral pathogens in the swine industry. In vivo, the virus shows a very narrow cell tropism and targets specific subsets of porcine macrophages. The entry of PRRSV into its host cell is the first crucial step in infection and has been the focus of many fundamental studies. This review provides a comprehensive overview of the current knowledge on PRRSV entry into the porcine macrophage, covering virus binding, internalization and genome release, and integrates these findings into a general model of the entry process.

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.

Identification of the CD163 Protein Domains Involved in Infection of the Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Scavenger receptor CD163 is a key entry mediator for porcine reproductive and respiratory syndrome virus (PRRSV). To identify the CD163 protein domains involved in PRRSV infection, deletion mutants and chimeric mutants were created. Infection experiments revealed that scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR 5) is essential for PRRSV infection, while the four N-terminal SRCR domains and the cytoplasmic tail are not required. The remaining CD163 protein domains need to be present but can be replaced by corresponding SRCR domains from CD163-L1, resulting in reduced (SRCR 6 and interdomain regions) or unchanged (SRCR 7 to SRCR 9) infection efficiency. In addition, CD163-specific antibodies recognizing SRCR 5 are able to reduce PRRSV infection.

The porcine reproductive and respiratory syndrome virus requires trafficking through CD163-positive early endosomes, but not late endosomes, for productive infection

The porcine reproductive and respiratory syndrome virus (PRRSV) enters its target cell via clathrin-mediated endocytosis. Using dominant-negative Rab5 and Rab7 mutants, we show that upon internalization, PRRSV enters early endosomes but does not continue through the endocytic pathway to late endosomes. This was confirmed via colocalization experiments visualizing PRRSV and markers for different compartments of the endocytic pathway. Furthermore, it was shown that PRRSV colocalizes with its internalization receptor, sialoadhesin, on the cell surface and beneath the plasma membrane, while CD163 and PRRSV only meet in early endosomes.

Susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and CD163

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV.ResultsDetailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHOSn-CD163 and PK15Sn-CD163 cells. Initially, this occurred less efficiently for macrophage grown virus than for Marc-145 grown virus. Upon internalization, disassembly of the virus particles was observed. The two cell lines could be infected with PRRSV strains LV and VR-2332. However, it was observed that Marc-145 grown virus infected the cells more efficiently than macrophage grown virus. If the cells were treated with neuraminidase to remove cis-acting sialic acids that hinder the interaction of the virus with Sn, the amount of infected cells with macrophage grown virus increased. Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line. Only 2 out of 5 PRRSV strains replicated well in CHOSn-CD163 cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15Sn-CD163 cells was similar to the virus titer of those strains produced in Marc-145 cells. Analysis of the sequence of the structural proteins of original virus and virus grown for 5 passages on PK15Sn-CD163 cells showed either no amino acid (aa) changes (VR-2332 and 07V063), one aa (LV), two aa (08V194) or three aa (08V204) changes. None of these changes are situated in known neutralizing epitopes.ConclusionsA PRRSV susceptible cell line was constructed that can grow virus to similar levels compared to currently available cell lines. Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes. Together, the results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation.

Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation

Significance Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by more than 310 mutations in the gene MEFV, which encodes Pyrin. Pyrin recently was shown to trigger inflammasome activation in response to Rho GTPase-modifying bacterial toxins. Here we report that Clostridium difficile infection and intoxication with its enterotoxin TcdA engage the Pyrin inflammasome. Moreover, activation of the Pyrin inflammasome, but not other inflammasomes, was hampered by microtubule-depolymerizing drugs in mouse and humans. Unexpectedly, we found that FMF mutations render Pyrin activation independent of microtubules. Thus, our findings provide a conceptual framework for understanding Pyrin signaling and enable functional diagnosis of FMF. Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

NOD-like receptors interfacing the immune and reproductive systems

Nucleotide‐binding oligomerization domain receptors (NOD‐like receptors, NLRs) are intracellular proteins that are chiefly known for their critical functions in inflammatory responses and host defense against microbial pathogens. Several NLRs have been demonstrated to assemble inflammasomes or to engage transcriptional signaling cascades that result in the production of pro‐inflammatory cytokines and bactericidal factors. In recent years, NLRs have also emerged as key regulators of early mammalian embryogenesis and reproduction. A subset of phylogenetically related NLRs represents a new class of maternal effect genes that are highly expressed in maturing oocytes and pre‐implantation embryos. Mutations in several of these NLRs have been linked to hereditary reproductive defects and imprinting diseases. In this review, we discuss the expression profiles, the emerging functions and molecular mode of action of these NLRs with newly recognized roles at the interfaces of the immune and reproductive systems. In addition, we provide an overview of coding mutations in NLRs that have been associated with human reproductive diseases, and outline crucial outstanding questions in this emerging research field.

Interaction of the European genotype porcine reproductive and respiratory syndrome virus (PRRSV) with sialoadhesin (CD169/Siglec-1) inhibits alveolar macrophage phagocytosis

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that shows a restricted in vivo tropism for subsets of porcine macrophages, with alveolar macrophages being major target cells. The virus is associated with respiratory problems in pigs of all ages and is commonly isolated on farms with porcine respiratory disease complex (PRDC). Due to virus-induced macrophage death early in infection, PRRSV hampers the innate defence against pathogens in the lungs. In addition, the virus might also directly affect the antimicrobial functions of macrophages. This study examined whether interaction of European genotype PRRSV with primary alveolar macrophages (PAM) affects their phagocytic capacity. Inoculation of macrophages with both subtype I PRRSV (LV) and subtype III PRRSV (Lena) showed that the virus inhibits PAM phagocytosis. Similar results were obtained using inactivated PRRSV (LV), showing that initial interaction of the virion with the cell is sufficient to reduce phagocytosis, and that no productive infection is required. When macrophages were incubated with sialoadhesin- (Sn) or CD163-specific antibodies, two entry mediators of the virus, only Sn-specific antibodies downregulated the phagocytic capacity of PAM, indicating that interaction with Sn, but not CD163, mediates the inhibitory effect of PRRSV on phagocytosis. In conclusion, this study shows that European genotype PRRSV inhibits PAM phagocytosis in vitro, through the interaction with its internalization receptor Sn. If similar events occur in vivo, this interaction may be important in the development of PRDC, as often seen in the field.