Hantaan Virus Activates The NLRP3Inflammasome In THP-1Cells

Hantaan virus (HTNV) is the major pathogen responsible for hemorrhagic fever withrenal syndrome (HFRS) in China, which features fever, increased vascular permeability,hypotensive shock, renal dysfunction, and multi-organ dysfunction syndrome. Since it wasfirst reported in1955, the incidence and fatality rate of HFRS is very high compared toother infectious diseases, and it has already become a serious public health problem inChina. Although the immunopathological mechanism in HFRS is still unclear, the knownpathogenesis of HFRS includes the direct endothelial damage caused by virus and indirectand more severe impairment by many detrimental factors in immune system, such asimmune complexes, inflammatory cytokines, and cytotoxic T cells.Inflammasomes are a group of cytosolic protein complexes that activate pro-caspase-1to proteolytically process the pro-inflammatory cytokines interleukin-1(IL-1β) andinterleukin-18(IL-18) and induce inflammation reaction in the local infection areas. As animportant component of the innate immune system, inflammasomes can recognize a variety of ligands, such as bacterial toxins, virus nucleic acids, fatty acids, and crystallinestructures. Recently, inflammasomes have gained considerable attention due to their vitalrole in the detection of diverse microranisms and their rapidly response to cytosolic dangersignals. It has also been noticed that many viruses can activate inflammasomes in theprocess of infection and trigger inflammation and immune reactions. At the same time,viruses also develop many strategies to escape from the recognition by inflammasomes tofacilitate their replication and propagation.Previous researches demonstrated that HTNV could induce endothelial cells andmonocytes to secrete IL-1β. Therefore, we hypothesized that HTNV may also activateinflammasomes in the target cells. In the present study, we investigated the secretion ofIL-1β and the expression of the NLRP3inflammasome in THP-1cells infected withHTNV and also evaluated the role of calcium in the infection process. The results are asfollowing:1. We constructed the HTNV-cRNA standards and developed a SYBR Green Ⅰ-basedreal-time quantitative PCR method to measure the viral load of HTNV. The viral load ofHTNV in the supernatant of THP-1cells were also detected by real-time PCR.2. The protein expression of pro-IL-1β and the secretion of IL-1β was increased withthe concentration of HTNV propagated in the THP-1cells. The viral replication wasrequired for the production of IL-1β in the THP-1cells, and stimulation with viral nucleicacids and protein could not induce IL-1β secretion.3. The expression of inflammation-associated genes were up-regulated inHTNV-infected THP-1cells, especially for the NLRP3inflammasome genes. Thelocalization of the NLRP3inflammasome was detected with the laser confocal microscope.Futhermore, the role of the NLRP3inflammasome in HTNV infection was alsoinvestigated by specific NLRP3siRNA and caspase-1inhibitor.4. The concentration of cytosolic free calcium increased in the HTNV infected THP-1cells, and the secretion of IL-1β was decreased after the outside calcium was removed byEGTA. The results suggested that the calcium may be involved in the process of activationof the NLRP3inflammasome. In conclusion, we found that HTNV activated the NLRP3inflammasome in THP-1cells,increased the concentration of cytosolic calcium, activated the downstream caspase-1, andfinally induced the secretion of IL-1β. The activation of the NLRP3inflammasome byHTNV may reveal new insight into the pathogenesis of HTNV and additional targets fortherapeutic in HFRS.