| In human, Hantaan virus(HTNV) infection can cause severe disease named hemorrhagic fever with renal syndrome(HFRS), with a high-mortality rate from 2% to 10%. More than 100,000 cases occurred each year in the world, over 90% of which are in China. Shannxi province is a main endemic area of HFRS. The classic symptoms of HFRS include fever, hemorrhage, thrombocytopenia, and acute renal failure. However, since the HTNV infection doesn’t cause disease in the rodent hosts, we are still lack of proper animal model of HFRS. All the researches are confined to limited human samples from HFRS patients. So it is still not clear about the pathogenesis of HFRS. The dysfunction of platelet and dysregulation of endothelial cell barrier functions are reported to be the two major histopathological changes. It is suggested that exaggerated host immune response and subsequent induced “cytokine storm” contribute to the immunopathologic injury of HFRS. However, till now, it is still unclear how the HTNV infection triggers the “cytokine storm”.Previously, we screened a panel of cytokines expressions using Luminex and found that, in the serum from HFRS patients, CXCL10 was the most-highly-expressed cytokine. CXCL10/Interferon-γ-inducible protein-10(IP-10) is a member of CXC chemokines, which is mainly secreted by endothelial cells, epithelial cells, and keratinocytes. After binding to its receptor CXCR3, CXCL10 induces chemotaxis, apoptosis, cell growth inhibition, and angiostasis. Accumulating data have indicated that CXCL10 plays an important role in many virus infectious diseases infected with hepatitis virus B(HBV), hepatitis virus C(HCV), HIV, influenza virus, and some other viruses. The elevated level of CXCL10 in serum has been found to be correlated with the development and the severity of these infectious diseases. CXCL10-CXCR3 signaling axis recruits virusspecific T cells into inflammatory sites and promotes viral clearance during virus infection. Some in vivo studies showed that elevated levels of CXCL10 also contributed to the tissue damages. The contradictory findings of CXCL10’s role in either protecting or promoting infection may depend on the host immune status and genetic background. However, it is still unknown about the role of CXCL10 plays in HTNV infection model.In studies of the regulation mechanism of CXCL10 production, some signaling pathways have been documented to contribute to the regulation of CXCL10’s expression, including JAK-STAT pathway, PI3K/AKT pathway, and TRAF2/TAK1 pathway. Transcriptional factors like NF-κB, IRF1, and IRF3 have also been proved to bind to the CXCL10’s promoter. Although Geimonen et al. have reported that HTNV induce CXCL10 expression in HUVECs, little is known about the molecular regulation mechanism of HTNV-induced CXCL10 production.In the first part of our project, we will address the following two questions. First, what is the role of CXCL10 plays in HFRS disease? Second, what is the molecular regulation mechanism of CXCL10 in HTNV infection?At first, we collected the serum of HFRS patients and quantified the serum CXCL10 levels in HFRS patients of different severities and in different disease stages, analyzed the relationship between CXCL10 and the disease severity-indicating parameters in vivo. And then, after establishing HTNV-infected-endothelium-cells model in vitro, we explored the underlying regulation mechanisms of CXCL10’s expression during HTNV infection. In this study, we have found that the serum CXCL10 level in HFRS patients is substantially higher than its corresponding level in healthy donors and the content of CXCL10 in the convalescent phase of HFRS. Importantly, the elevation of CXCL10 level is correlated significantly with the HFRS severity-indicating clinical parameters. The higher CXCL10 level is also shown in the higher viral load group. Our analyses of the membrane expression level of CXCR3 on different subsets of lymphocytes revealed that the level of CXCR3 increases on CD14+ subset in PBMCs isolated from HFRS patients. These results suggest that the increasing level of CXCL10 in the acute phase of HFRS may recruit monocytes. In vitro study suggested that HTNV-infected HUVEC cells could produce excessive amount of CXCL10. In addition, it is the dsRNA structure rather than the protein of HTNV induces the production of CXCL10. The dsRNA sensors in cells, like TLR3, RIG-I, and MDA-5 pathways are activated. After that, the transcription factors NF-κB and IRF7 are phosphorated and translocated from cytoplasma to nucleus. Subsequently, p50, p65 and IRF7 bind to the promoter site of CXCL10 to regulate the expression of CXCL10 on transcription level. The understanding of the role of CXCL10 in HFRS and the regulations of cytokines/chemokines during HTNV infection might provide a novel therapeutic target in HFRS.Interleukin-33(IL-33), a new member of the IL-1 family, serves as a ligand for the ST2 receptor. Recent studies have suggested that IL-33 is specifically released during necrotic cell death but is intracellular during apoptosis. Because of these properties, IL-33 is identified as an “alarmin” and is defined as a member of danger-associated molecular pattern(DAMP) for alerting the immune system after infection or injury. As a potent inducer of the T-helper 2(Th2) immune response, IL-33 promotes the production of Th2-associated cytokines, such as IL-4, IL-5, and IL-13, mostly released from polarized Th2 cells. In addition to Th2-related effects, IL-33 also induces inflammatory responses in endothelium and epithelium.The ST2 gene, a member of the IL-1RL1 superfamily, is known to encode at least 3 isoforms of ST2 proteins by alternative splicing: a membrane-anchored long form(ST2L), a secreted soluble form(sST2), and a membrane-anchored variant form(ST2V). sST2, serving as a decoy receptor for IL-33, can neutralize the function of IL-33. ST2 L has been reported to be constitutively expressed by mast cells as well as Th2 cells. Upon binding with IL-33, ST2 L forms a complex with the IL-1R accessory protein(IL-1RAcP), recruits the adaptor protein MyD88, activates MAP kinases(MAPK) and NF-κB pathways, and promotes the production of inflammatory mediators.Numerous studies have reported the expression and function of IL-33/ST2 signalling in various diseases. IL-33/ST2 overstimulation has been implicated in airway inflammatory diseases, autoimmune diseases, viral infection diseases, and many other diseases, suggesting an important role for IL-33/ST2 in the development of inflammatory pathologies. However, the mechanism by which the IL-33/ST2 axis exerts its immunomodulatory effects in HFRS has not yet been elucidated.In the second part of our project, we will resolve the following two questions. First, how the IL-33/ST2 axis exerts its immunomodulatory effects in HFRS? Second, what is the molecular mechanism underlying the synergic effect of HTNV infection and IL-33/ST2 activation?We quantified the plasma levels of IL-33 and s ST2 in HFRS patients, analyzed the relationships between IL-33, sST2, and disease severity-indicating parameters in vivo, and explored the role of IL 33/ST2 in regulating immune response in vitro during HTNV infection. We found that elevated plasma IL-33 and sST2 levels were associated with the development of HFRS. Our in vitro examination indicated that IL-33 could enhance the production of pro-inflammatory cytokines in HTNV-infected endothelial cells and induce the “cytokine storm”. Furthermore, this process could be inhibited by the recombinant sST2. We also demonstrated that NF-κB pathway may contribute to the synergistic effect on cytokine production induced by both HTNV and IL-33 in HUVECs.Overall, our results indicated that IL-33 might be the initiator of the “cytokine storm” and the IL-33/ST2 axis, serving as an important regulator of the inflammatory response during HTNV infection, may be involved in the pathogenesis of HFRS. The utilisation of sST2 to selectively reduce IL-33/ST2, with a consequent decrease in the inflammatory response in endothelial cells, may be exploited as a therapeutic target for Hantavirus infections. |
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