Dr Kerstin Luhn
| Research Area: | Immunology |
|---|---|
| Technology Exchange: | Cellular immunology, Flow cytometry, Protein interaction and Transcript profiling |
| Keywords: | Dengue, macrophages, immune regulation, regulatory T cells, immunopathology and transcriptional profiling |
Innate immune responses in Dengue and other viral infections
Dengue is with 100 million cases each year a significant public health problem in tropical and sub-tropical areas of the world. The disease is caused by the Dengue virus that gets transmitted by mosquitoes. Infection results in a broad range of disease severities ranging from a mild febrile illness to the potentially fatal Dengue Haemorrhagic Fever. Although the pathogenesis is not well understood immunopathology seems to play a major role. Excessive levels of cytokines secreted by immune cells can be detected in severe Dengue. As these can affect blood vessels, plasma leakage occurs as the most prominent symptom.
Immune responses are normally tightly regulated enabling an effective immune response while limiting damage to the surrounding tissue. The focus of our work is on the regulatory potential of innate immune cells. Macrophages and dendritic cells are the first line of cellular defence as they set the stage for the immune response by releasing cytokines and activating further immune cells. They are particularly important in Dengue as they are the major target cells for the virus and are the main source for viral reproduction in vivo.
We are investigating the effect of Dengue infection of innate immune cells to understand why Dengue in contrast to several other viral infections is associated with high levels of immunopathology. For this we are using an in vitro Dengue infection model. Obtained results are then confirmed with Dengue patient samples from our cohort in Ho Chi Minh City, Viet Nam.
| Name | Department | Institution | Country |
|---|---|---|---|
| Prof Jeremy Farrar | Tropical Medicine | Oxford University | UK |
| Dr Menno de Jong | Academic Medical Centre, University of Amsterdam | The Netherlands | |
| Prof Sarah L Rowland-Jones | Experimental Medicine Division | Oxford University | UK |
| Joanna Miller | Oxford Glycobiology Institute, University of Oxford | UK | |
| Siamon Gordon | Sir William Dunn School of Pathology, University of Oxford | UK | |
| Prof Cameron P Simmons | Tropical Medicine | Oxford University | UK |
2008. Preservation of a critical epitope core region is associated with the high degree of flaviviral cross-reactivity exhibited by a dengue-specific CD4+ T cell clone. Eur J Immunol, 38 (4), pp. 1050-1057. Read abstract | Read more
Dengue is a member of the Flaviviridae, a large group of related viruses some of which co-circulate in certain regions (e.g. dengue and Yellow fever in South America). Immune responses cross-reactive between different dengue serotypes are important in the pathogenesis of dengue disease but it is not known whether previous infection with one flavivirus might affect the clinical course of subsequent infections with other members of the family. CD4+ T cells have been shown to be important in the production of cytokines in response to dengue infection and can demonstrate significant epitope cross-reactivity. Here, we describe the generation and characterisation of CD4+ T cell clones from a patient experiencing acute dengue infection. These clones were DRB1*15+ and recognised epitope variants not only within other dengue viruses but certain other flaviviruses. This cross-reactivity was dependent upon the presence of a five-amino acid core region, consistent with structural observations of class II MHC binding to TCR demonstrating that only a subset of residues within an epitope bound to a class II molecule are "read out" by the TCR. This capacity of CD4+ T cell clones to recognise a given epitope despite considerable variation between viruses may be of pathological significance, particularly in regions where related viruses co-circulate. Hide abstract
2007. Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J Exp Med, 204 (5), pp. 979-985. Read abstract | Read more
Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease. Hide abstract
2007. High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection. PLoS One, 2 (12), pp. e1192. Read abstract | Read more
Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. Hide abstract
2004. Identification and molecular cloning of a functional GDP-fucose transporter in Drosophila melanogaster. Exp Cell Res, 301 (2), pp. 242-250. Read abstract | Read more
Nucleotide sugar transporters play a central role in the process of glycosylation. They are responsible for the translocation of nucleotide sugars from the cytosol, their site of synthesis, into the Golgi apparatus where the activated sugars serve as substrates for a variety of glycosyltransferases. We and others have recently identified and cloned the first GDP-fucose transporters of H. sapiens and C. elegans. Based on sequence similarity, we could identify a putative homolog in Drosophila melanogaster showing about 45% identity on protein level. The gene (CG9620) encodes a highly hydrophobic, multi-transmembrane spanning protein of 38.1 kDa that is localized in the Golgi apparatus. In order to test whether this protein serves as a GDP-fucose transporter, we performed complementation studies with fibroblasts from a patient with LADII (leukocyte adhesion deficiency II) which exhibit a strong reduction of fucosylation due to a point mutation in the human GDP-fucose transporter gene. We show that transient transfection of these cells with the Drosophila CG9620 cDNA corrects the GDP-fucose transport defect and reestablishes fucosylation. This study gives experimental proof that the product of the in silico identified Drosophila gene CG9620 serves as a functional GDP-fucose transporter. Hide abstract
2001. The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter. Nat Genet, 28 (1), pp. 69-72. Read abstract | Read more
Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II. Hide abstract
2001. Discontinuation of fucose therapy in LADII causes rapid loss of selectin ligands and rise of leukocyte counts. Blood, 97 (1), pp. 330-332. Read abstract | Read more
Leukocyte adhesion deficiency type II (LADII) is a rare inherited disorder of fucose metabolism. Patients with LADII lack fucosylated glycoconjugates, including the carbohydrate ligands of the selectins, leading to an immunodeficiency caused by the lack of selectin-mediated leukocyte-endothelial interactions. A simple and effective therapy has recently been described for LADII, based on the administration of oral fucose. Parallel to this treatment the lack of E- and P-selectin ligands on neutrophils was corrected, and high peripheral neutrophil counts were reduced to normal levels. This study reports that discontinuation of this therapy leads to the complete loss of E-selectin ligands within 3 days and of P-selectin ligands within 7 days. Peripheral neutrophil counts increased parallel to the decrease of selectin ligands. Selectin ligands reappeared promptly after resumption of the fucose therapy, demonstrating a causal relationship between fucose treatment and selectin ligand expression and peripheral neutrophil counts. Hide abstract
1999. Correction of leukocyte adhesion deficiency type II with oral fucose. Blood, 94 (12), pp. 3976-3985. Read abstract
We describe a simple, noninvasive, and effective therapy for leukocyte adhesion deficiency type II (LAD II), a rare inherited disorder of fucose metabolism. This disorder leads to an immunodeficiency caused by the absence of carbohydrate-based selectin ligands on the surface of neutrophils as well as to severe psychomotor and mental retardation. The fucosylation defect in LAD II fibroblasts can be corrected by addition of L-fucose to the culture medium. This prompted us to initiate dietary fucose therapy on a patient with LAD II. Oral supplementation of fucose in this patient induced the expression of fucosylated selectin ligands on neutrophils and core fucosylation of serum glycoproteins. During 9 months of treatment, infections and fever disappeared, elevated neutrophil counts returned to normal, and psychomotor capabilities improved. Hide abstract
1999. Leukocyte adhesion deficiency II syndrome, a generalized defect in fucose metabolism. J Pediatr, 134 (6), pp. 681-688. Read abstract | Read more
Leukocyte adhesion deficiency II has been described in only 2 patients; herein we report extensive investigation of another patient. The physical stigmata were detected during prenatal ultrasonographic investigation. Sialyl-Lewis X (sLex) was absent from the surface of polymorphonuclear neutrophils, and cell binding to E- and P-selectin was severely impaired, causing an immunodeficiency. The elevation of peripheral neutrophil counts occurred within several days after birth. A severe hypofucosylation of glycoconjugates bearing fucose in different glycosidic links was present in all cell types investigated, demonstrating that leukocyte adhesion deficiency II is not only a disorder of leukocytes but a generalized inherited metabolic disease affecting the metabolism of fucose. Hide abstract
Subcellular localisation of Dengue virus and its impact on disease severity
Epidemiological studies in Dengue demonstrated that severe disease is linked predominantly to secondary infection. It is believed that immunopathology mediated by antibody dependent enhancement (ADE) of the infection is leading to more severe outcome. This implies that antibodies from a previous infection recognise the new serotype but can’t neutralise it: The virus is still capable to bind to its receptors on target cells and infect them. In addition the virus-antibody complexes can now also ...
The impact of the inflammasome in Dengue infection
Dengue infection is with 100 million cases each year a major public health problem in the tropics. The disease is caused by the Dengue virus, a mosquito-borne virus that circulates as four different serotypes. The disease severity ranges from mild Dengue Fever to potentially fatal Dengue Haemorrhagic Fever. Epidemiological studies demonstrated that a secondary infection with a different serotype is a major risk factor for severe disease. Therefore the immune system rather than the virus itself ...
