Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent.

BACKGROUND: In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. METHODS: All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. RESULTS: The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. CONCLUSION: These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses.

Mycoplasma suis infection results endothelial cell damage and activation: new insight into the cell tropism and pathogenicity of hemotrophic mycoplasma.

Hemotrophic mycoplasmas (HM) are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs, Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.

The free radical scavenger S-PBN significantly prolongs DSG-mediated graft survival in experimental xenotransplantation.

BACKGROUND: Nitrones such as 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) are known to trap and stabilize free radicals and to reduce inflammation. Recently, S-PBN was shown to reduce infiltration of T lymphocytes and the expression of adhesion molecules on the endothelium in experimental traumatic brain injury. We hypothesized that S-PBN could reduce infiltration of T lymphocytes during cell-mediated xenograft rejection and thereby increase graft survival. The concordant mouse-to-rat heart transplantation model was used to test the hypothesis. In this model, grafts undergo acute humoral xenograft rejection (AHXR) almost invariably on day 3 and succumb to cell-mediated rejection on approximately day 8 if AHXR is inhibited by treatment with 15-deoxyspergualin (DSG). MATERIAL AND METHODS: Hearts from Naval Medical Research Institute (NMRI) mice were transplanted to the neck vessels of Lewis rats. Recipients were treated with S-PBN (n=9), DSG (n=9), S-PBN and DSG in combination (n=10) or left untreated (n=9) for survival studies. S-PBN was given daily intraperitoneally at a dose of 150 mg/kg body weight (BW) on day -1 to 30, and DSG was given daily intraperitoneally at a dose of 10 mg/kg BW on day -1 to 4 and 5 mg/kg BW on day 5 to 21. Nine additional recipients were given S-PBN only on days -1 and 0 in combination with continuous DSG treatment. Grafts were monitored until they stopped beating. Additional recipients were treated with S-PBN (n=5), DSG (n=5), S-PBN and DSG in combination (n=6) or left untreated (n=5) for morphological, immunohistochemical and flow cytometry analyses on days 2 and 6 after transplantation. RESULTS: S-PBN treatment in combination with DSG resulted in increased median graft survival compared to DSG treatment alone (14 vs. 7 days; P=0.019). Lower number of T lymphocytes on day 6 (P=0.019) was observed by ex vivo propagation and flow cytometry when combining S-PBN with DSG, whereas immunohistochemical analyses demonstrated a significant reduction in the number of infiltrated CD4+, but not TCR+, cells. S-PBN treatment alone had no impact on graft survival compared to untreated rats (3 vs. 3 days). No differences were seen in ICAM-1 and VCAM-1 expression or in morphology between the groups. CONCLUSION: The combination of S-PBN and DSG treatment increases xenograft survival. The main effect of S-PBN appears to be in direct connection with the transplantation. Because of its low toxicity, S-PBN could become useful in combination with other immunosuppressants to reduce cell-mediated xenograft rejection.

Efficiency of porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin.

BACKGROUND: Human cytomegalovirus (HCMV) infection or reactivation has been linked to allograft rejection resulting from endothelial injury and immune activation. In pig-to-human xenotransplantation, currently investigated to circumvent the shortage of human organs in transplantation medicine, the porcine endothelium will inevitably be exposed to human pathogens such as HCMV. We investigated the susceptibility of porcine endothelial cells (pEC) to HCMV infection. METHODS: Immortalized porcine aortic (PEDSV15) and porcine microvascular bone-marrow derived EC (2A2) as well as a panel of primary pEC originated from different vascular beds were inoculated with the endotheliotropic (TB40/E) and the fibroblast propagated (TB40/F) HCMV strains at multiplicity of infection (MOI) ranging from 0.1 to 5. Viral replication kinetics, development of cytopathology and release of viral progeny were analyzed. RESULTS: All viral strains infected pEC with differences in both infection efficiency and kinetics of cytopathology. Moreover, differences in susceptibility of pEC derived from distinct vascular beds were observed. HCMV underwent a complete replication cycle in about 5% of the infected pEC. Comparing the permissiveness of pEC to human aortic EC (HAEC) revealed differences in strain susceptibility and lower rates of late antigen expression in pEC. Finally, HCMV-infected pEC released viral particles but with a lower efficiency than infected HAEC. CONCLUSIONS: Our data demonstrate that HCMV productively infects pEC, therefore finding strategies to render pEC resistant to HCMV infection will be of interest to reduce the potential risk carried by HCMV reactivation in xenotransplantation.

HLA-Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells.

BACKGROUND: Human natural killer (NK) cell-mediated cytotoxicity represents a hurdle in pig-to-human xenotransplantation. It was previously reported that the expression of human major histocompatibility complex class I molecules, including HLA-B27, -Cw3, -E, and -G, partially protects porcine endothelial cells (pEC) from human NK-mediated cytotoxicity and that HLA-G inhibits NK adhesion to pEC. Here, we tested if HLA-Cw4 expression on pEC alone, or concurrently with HLA-Cw3, prevents human NK adhesion and cytotoxicity against pEC via recognition of the killer-cell immunoglobulin-like receptors (KIR) CD158a (KIR2DL1) and CD158b (KIR2DL2/3), respectively. METHODS: Two pEC lines (2A2 and PEDSV.15) were transfected with HLA-Cw3 and HLA-Cw4. HLA and KIR expression on porcine and human cells were analyzed by flow cytometry. The effect of HLA expression on pEC on human NK-mediated cytotoxicity and adhesion was tested by (51)Cr-release and dynamic adhesion assays, respectively. RESULTS: HLA-Cw4 expression on pEC reduced cytotoxicity mediated by CD158a(+) polyclonal human NK cells by an average of 58%, and by CD158a(bright) NK cell clones by 68%, but not by NK cells expressing low levels of CD158. Co-expression of HLA-Cw3 and HLA-Cw4 on pEC did not mediate further protection against NK cytotoxicity. The expression of HLA-Cw4 reduced the adhesion of human NK cells on pEC by a mean of 53%. CONCLUSIONS: While transgenic expression of HLA-Cw4 on pEC reduces NK cell adhesion and cytotoxicity, co-expression with HLA-Cw3 is not sufficient to completely overcome human NK-mediated cytotoxicity in vitro.

Immune responses to alpha1,3 galactosyltransferase knockout pigs.

PURPOSE OF REVIEW: To summarize the current knowledge of the immune response generated against xenografts stemming from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs. In particular, we will address the nature of potentially remaining Gal epitopes, the role of non-Gal xenoantigens, and the cellular response directed against GalT-KO tissues. RECENT FINDINGS: New findings support the view that porcine cells do not express isoglobotrihexosylceramide 3, and GalT-KO pigs, if at all, express negligible levels of Gal. The anti-non-Gal antibody response to GalT-KO cells allowed the identification of several potentially relevant porcine xenoantigens, mainly carbohydrates. Coculture of wildtype pig aortic endothelial cells but not of GalT-KO pig aortic endothelial cells with whole human blood induces the secretion of porcine and human cytokines and the upregulation of E-selectin; in contrast, the transmigration of human leukocytes across porcine endothelium is not regulated by Gal. SUMMARY: New immunological problems are arising after the elimination of Gal by the generation of GalT-KO pigs; these include non-Gal antibodies and the identification of their elusive antigens, as well as cellular components of the immune system, including neutrophils, macrophages, natural killer cells, and T cells.

Characterization of porcine UL16-binding protein 1 endothelial cell surface expression.

BACKGROUND: Natural killer (NK) cells participate in the immune response against solid organ allo- and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL-16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell-surface expression on primary porcine aortic endothelial cells (PAEC). METHODS: A monoclonal antibody (mAb; aE5-63) directed against pULBP1 was generated by immunizing C57BL/6 mice with the pEC line PEDSV.15, and used in cellular ELISA to determine pULBP1 cell surface expression. PAEC were either left untreated or stimulated with human or porcine cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha)], human serum, cultured under hypoxic conditions, or infected with human or porcine cytomegalovirus (CMV). RESULTS: Neither human nor porcine IFN-gamma stimulation changed pULBP1 expression, whereas both human and porcine TNF-alpha stimulation as well as human and porcine CMV infection significantly decreased pULBP1 expression on PAEC. Coculture of PAEC with human serum strongly increased pULBP1 expression depending on the binding of human anti-pig antibodies. Exposure of PAEC to hypoxia only slightly increased pULBP1 expression. CONCLUSIONS: In conclusion, (i) the novel anti-pULBP1 IgM mAb aE5-63 represents a useful tool to study pULBP1/hNKG2D-mediated responses in xenotransplantation, and (ii) the expression of pULBP1, a human-pig cross-species functional hNKG2D ligand, on the surface of PAEC is modulated by various stimuli associated with transplantation.

Current cellular innate immune hurdles in pig-to-primate xenotransplantation.

PURPOSE OF REVIEW: The generation of pigs lacking Galalpha1,3Gal (alphaGal) or overexpressing human regulators of complement has largely overcome the barrier of hyperacute xenograft rejection. Nevertheless, delayed xenograft rejection remains a major hurdle, including humoral responses against nonalphaGal epitopes, and cellular innate immune responses. This review addresses the early interactions between the porcine endothelium and human natural killer cells, neutrophils, and monocytes/macrophages. RECENT FINDINGS: Whether human leukocyte recruitment to, and lysis of, porcine endothelial cells includes direct recognition of alphaGal remains a matter of debate. Although the human natural killer receptor natural killerRP1A may directly recognize alphaGal, several studies did not reveal significant differences regarding adhesion, transmigration, and cytotoxicity using porcine endothelial cells expressing or lacking alphaGal. The strong human monocyte response against pig cells partly relies on the inability of porcine ligands to ligate inhibitory human receptors, such as SIRPalpha. Strategies to overcome cellular innate immune responses include transgenic expression in porcine cells of human ligands for inhibitory receptors, together with species-specific blocking of porcine ligands mediating human leukocyte interactions. SUMMARY: Cellular human innate immune responses are increasingly recognized as barriers to successful pig-to-human xenotransplantation, and only a detailed knowledge of the molecular mechanisms involved will allow us to overcome this barrier.