Studies on pathogenesis of nephropathia epidemica, a Swedish viral hemorrhagic fever.
The aim of the present project is to investigate, in depth, innate and cellular immunity and changes in haemostasis during a Swedish hemorrhagic fever with renal syndrome caused by hantavirus, an infection also denoted nephropathia epidemica (NE, “sorkfeber”). We hypothesize that these factors play a significant role in the pathogenesis of the disease. Because the mechanisms of viral pathogenesis are likely similar for most hemorrhagic fever viruses, the proposed studies will provide valuable information regarding the pathogenesis of other severe forms of hemorrhagic fevers, including those caused by Dengue, Crim-Congo, Ebola, Marburg, and Lassa hemorrhagic fever viruses. The unique clinical materials collected in our projects provide the possibility to characterize in detail innate and cellular immunity, and alterations in hemostasis from the very first days of disease.
Hantaviruses are rodent-borne viruses which cause two febrile illnesses, Hemorrhagic Fever with Renal Syndrome (HFRS) in Europe and Asia and Hantavirus Cardiopulmonary Syndrome (HCPS) in the Americas, depending on the species of hantavirus. HFRS accounts for more than 200,000 cases and thousands of deaths annually, while HCPS is much less frequent although more severe with a mortality rate above 40%. Puumala virus (PUUV), Dobrava, Seoul and Hantaan viruses causing HFRS in humans, and Sin-Nombre and Andes viruses causing HCPS. Humans get an acute and overt infection, including pathology in functions of various organs. NE caused by PUUV is characterized by acute onset of high fever, head- and backache, myalgia, and abdominal pain. Renal failure is common. One third of the patients in Sweden have hemorrhagic symptoms and severe bleedings may occur. Thrombocytopenia is frequent and diagnostic for the disease. Five to ten percentage of the patients with the more severe forms of HFRS, die from hypotensive shock or bleeding. An unexpected and large outbreak of PUUV infection in Sweden occured 2007-2008 with the highest numbers of patients in Västerbotten County. The patients mostly showed classical HFRS symptoms and displayed the whole spectra from mild to severe disease requiring hospitalization and occasionally intensive care and/or dialysis. As many as 30% of the diagnosed patients were hospitalized and at least 0.5% case fatality were recorded. Currently, there is no specific treatment against human hantavirus infections. A good prognostic marker for severe infection is not available at present. There is increasing evidence for a correlation between previous PUUV infection and later chronic renal affection and hypertension. Very little is known about sex and age differences in the pathogenesis of NE. Preliminary evidence suggest that while men may be more likely to be exposed to PUUV, women are more likely to die following exposure to PUUV; this dimorphism continues even after ‘recovery’, with a 2.8 fold increase in death rates among women compared with men one year after hospitalization with NE. Using a multiplex cytokine platform, we recently reported differential plasma cytokine profiles in female compared to male patients during the acute phase of NE. Females showed in the acute phase significantly higher levels of IL-9, FGF-basic, and GM-CSF and lower levels of IL-8 and IL-10 compared with males.
Type I interferons (IFNs) include several IFN-α subtypes and a single IFN-β subtype and represent another critical aspect of innate immunity during virus infection. In PUUV-infected patients, we found that the levels of IFN-α and IFN-β were similar, whereas the level of IFN-λ (type-III IFN) was significantly decreased, during the acute compared with the convalescent phase of infection. This finding and results from in vitro studies performed by us suggest that hantaviruses interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of subtypes of IFNs. Our previous studies illustrate that levels of serum perforin, granzyme B, and the epithelial cell apoptosis marker, caspase-cleaved cytokeratin-18 are increased during the acute phase of PUUV infection. Serum perforin levels positively correlated with signs of capillary damage. The increased levels of granzyme B and perforin are signs of elevated CD8+ and NK-cell activity. Therefore, these cells are of particular interest for the analysis in hantavirus infection since they express high levels of cytokines and have potent perforin-dependent cytotoxic capacities. NK cells are immune effector cells important in antiviral responses, representing 10-15% of peripheral blood mononuclear cells. Absolute numbers and proliferative profiles as well as the balance between activating and inhibitory receptors on different NK cell subsets were therefore measured in a prospective study. NK cell numbers were markedly increased in comparison to controls, and accounted for a major part of the total lymphocyte increase observed. During the course of infection, numbers of CD56dim and atypical CD56neg NK cells increased the most, whereas CD56bright NK cells did not change. The CD56dim NK cells which increased vigorously during the second week following symptom debut, coinciding with high levels of Ki67 expression suggesting ongoing proliferation. CD56dim NK cell numbers, strikingly, remained at elevated levels for up to 60 days following infection, concurrent with high expression-levels of anti-apoptotic Bcl-2. This finding is an unexpected and new feature of the innate immune response which is in contrast to previous experimental animal models of viral infection, where a rapid decline in NK cell numbers is seen. The receptor expression profile of the CD56dim NK cells during the acute phase of infection was characterized by increased expression of multiple activation receptors and effector molecules. A specific expansion and persistence of NKG2C+ NK cells was noted. We believe this to be one of the first more detailed analyses of the early events occurring in the NK cell response to an acute viral infection in humans, and the first study assessing NK cells in patients with acute hantavirus infection. During experimental infection of primary human endothelial cells, HLA-E, which is a ligand for NKG2C, was up regulated on the surface, whereas the expression of cell surface ligands for other NK cell activation receptors did not change significantly. Therefore, we speculate that NK cell receptors recognizing HLA-E might be involved in regulation of the NK cell response to human hantavirus-infection. Based upon the altered balance between activating and inhibitory receptors in NK-cells present during the acute phase of infection, we propose that NK cells might exhibit a previously unappreciated role as mediators of capillary damage / leakage and symptomatic disease in HFRS patients. The lungs are affected also in Puumala hantavirus infected patients. Severe and fatal cases fulfilled the criteria for hantavirus pulmonary syndrome and showed focal pulmonary histopathological features consisted of interstitial mononuclear positive for CD8. Presence of granzyme B and TIA-1 indicated also that these cells are cytotoxic T-lymphocytes and/or NK-cells. In a recent accepted publication consecutive patients were studied for local inflammation in the airways using bronchoscopy and bronchoalveolar lavage. BAL fluid showed higher proportions of CD8+ T cells, NK cells together with an increased expression of activation markers HLA-DR and CD25 on T cells. The findings of increased numbers of CD8+ T cells in both epithelium and submucosa together with the expression of vascular cell adhesion molecule-1 (VCAM-1) implies the importance of cytotoxic lymphocytes in the pathogenesis of pulmonary involvement in HFRS.