Inflammasome activation leading to the induction of host inflammatory responses was considered a critical mechanism of innate immunity in cells infected with microbes. Activation of inflammasomes is a complex process with multiple signaling pathways involved, including RIG-I-like receptors (RLRs), Toll-like receptors, and NF-κB signaling, leading to elevated expression of pro-IL-1β and pro-IL-18, processing and secretion of their mature forms, IL-1β and IL-18, and eventually pyroptosis [12, 24]. IL-1β is a highly potent proinflammatory mediator that induces vasodilation and attracts granulocytes to inflamed tissues [12, 16]. IL-1β is also an endogenous pyrogen prominent during the febrile phases of some viral infections and induces the production of prostaglandin E2 in the hypothalamus, resetting the hypothalamic thermostat to fever. In addition, IL-1β is associated with different clinical manifestations, such as coagulopathy and thrombocytopenia [25, 26]. We have considered that IL-1β is also a key element in viral pathogenesis of SFTS which is an acute febrile disease with a tendency for hemorrhage. In this study, increased levels of IL-1β were confirmed in sera from acute phase SFTS cases. A transient increase of IL-1β was detected in a murine model infected with SFTSV. Monocytes were found to be susceptible to SFTSV infection and could be the key source of IL-1β secreted into blood, contributing to pathogenicity in infected hosts.
In addition to proinflammatory responses, the activation of inflammasomes and induction of pyroptosis could be part of the protective mechanisms directed at combatting viral infection [11, 12]. Some RNA viruses are able to regulate the activation of inflammasome in order to counteract its effect during viral replication in host cells [12, 27]. In this study, we found that in SFTS patients, higher IL-1β levels in sera of acute phase cases were correlated with relatively mild symptoms (Fig. 1B). Correlation between IL-1β levels and viral load in SFTS patients was also confirmed, which showed that IL-1β levels in acute phase sera were negatively correlated with serum viral load (R2 = 0.68) (Fig. 1C). It appeared when higher virus loads were in the sera of the acute stage, more severe the clinical symptoms ensued in SFTS cases (Fig. 1D). Reversal correlation of IL-1β levels and disease severity or viral loads in the acute phage of sera suggested that the process associated with increased IL-1β secretion could be protective against SFTSV, consistent with a previous report indicating that IL-1β could have dual roles in both the proinflammatory response and antimicrobial immunity in SFTSV infections . Studies have shown that IL-1β can activate monocytes, macrophages and neutrophils, and is capable of driving the development of helper CD4 + T cells by regulating Th17 and Th1 responses [28, 29]. Therefore, in hosts infected with SFTSV, activation of inflammasomes was not only an essential part of innate immunity in response to viral replication, but also a critical component of the adaptive immune responses for viral clearance.
NLRP3 and AIM2-mediated inflammasomes are the most commonly activated complexes when some RNA viruses infect host cells. Previous studies showed that the NLRP3 inflammasome can be activated by infections of ZIKA virus, Foot-and-mouth disease virus, and Rift Valley Fever virus [22, 30, 31], while the AIM2 inflammasome can be triggered by Chikungunya virus and West Nile Virus (WNV) . ZIKV facilitates the NLRP3 inflammasome assembly and activation through viral NS5 protein interacting with the NACHT and LRR domains, which leads to the secretion of IL-1β and induction of an aggressive host inflammatory response closely related to viral pathogenicity . Inflammasome activation has been identified in viral diseases which are common in febrile symptoms, indicating a significant advance in understanding their pathogenicity. In this study, SFTSV infection induced processing of pro-caspase-1 and subsequent maturation and secretion of IL-1β/IL-18 were significantly suppressed in human PBMCs when NLRP3 gene was knocked down by specific shRNA (Fig. 4) or inhibited by a specific inhibitor, glibenclamide (Fig. 5). Knockdown of several other types of NLRs did not have any inhibitory effect on the elevated IL-1β/IL-18 levels in SFTSV-infected PBMCs, demonstrating that SFTSV infection triggered IL-1β/IL-18 secretion by activating the NLRP3 inflammasome. We further examined the effect of the NLRP3 inflammasome activation on viral replication in SFTSV infected human PBMCs. Our data showed that viral RNA copies increased, or that viral replication was enhanced in PBMCs when there was an NLRP3 knockdown or inhibition by glibenclamide. We further demonstrated that effect of the NLRP3 inflammasome on viral replication in SFTSV-infected PBMCs and mice was caspase-1 dependent. When treated with caspase-1 inhibitor, the induction of IL-1β secretion in sera was suppressed and the viral replication was promoted (Fig. 3). Taken together, these data show that inflammasome activation was NLRP3 dependent and increased IL-1β secretion was essential for NLRP3 inflammasome function in SFSTV-infected human PBMCs and mice.
Activation of inflammasome can induce pyroptosis, a form of programmed cell death, through activation of pro-caspase-1 and subsequent cleavage of GSDMD [31, 33, 34]. Pyroptosis, as a form of inflammatory cell death dependent on caspase-1 [34, 35], can have two important functions in protecting host cells against microbial infection. First, death of infected cells can lead to eradication of an intracellular pathogen and premature abortion of replicative process; Second, release of intracellular contents can enhance inflammation in infected tissues, distinct from anti-inflammatory apoptosis . It is no surprise that pyroptosis has been reported to play an important role in inhibiting the replication of many viruses [23, 31, 35]. In this study, we observed that SFTSV infection induced pyroptosis with cleaved GSDMD in a proportion of PBMCs (Fig. 6) which could be attenuated by inhibition of NLRP3 or caspase-1 (Fig. 6C-D). Further, our data showed that, when inflammasome activation was suppressed by inhibiting caspase-1, viral replication was enhanced in infected PBMCs (Fig. 5), demonstrating that the induction of pyroptosis in monocytes might be contributing to protection against SFTSV by eliminating infected cells and/or enhancing local inflammatory responses in the host.
We are aware that our study has limitations in several aspects. IL-1β may be secreted from monocytes, macrophages and many other cell types in the various tissues and organs of a host. We were unable to determine whether the IL-1β, detected in the sera, was solely secreted from the monocytes in SFTS patients, although monocytes were part of the PBMC culture studied in vitro in this report. Activation of NLRP3 or other NLR inflammasomes leading to IL-1β/IL−18 secretion and pyroptosis may also have different impact on viral infection and host cell status in different tissues and organs. Platelets could well be a source of IL-1β and may play an important role in the host inflammatory responses and viral pathogenicity in SFTSV-infected patients. We have preliminary data at this stage to indicate that platelets may also secrete IL-1β with pro-caspase-1 processed upon infection with SFTSV.
In conclusion, our report provides the evidence that SFTSV infection induced activation of the NLRP3 inflammasome, which led to elevated IL-1β/IL-18 secretion and pyroptosis in PBMCs and mice. Increased IL-1β and pyroptosis could be important in both pathogenicity and host protection against SFTSV as shown in infected PBMCs where viral replication was enhanced with knocked down or suppressed NLRP3 and caspase-1. Overall, our data support the notion that the NLRP3 inflammasome-dependent IL-1β secretion and pyroptosis characterized in this study may contribute to the viral pathogenesis in SFTSV infection. This study provides valuable insights for deciphering the mechanism about how this emerging phlebovirus causes a severe hemorrhagic fever in humans.