Nonstructural Protein NSs Activates Inflammasome and Pyroptosis through Interaction with NLRP3 in Human Microglial Cells Infected with Severe Fever with Thrombocytopenia Syndrome Bandavirus

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne febrile disease caused by SFTS virus (SFTSV), or Dabie bandavirus, in the Phenuiviridae family. Clinically neurological disorders in SFTS have been commonly reported, but their neuropathogenesis has rarely been studied. Microglia are a type of neuroglia accounting for 10 to 12% of all cells in the brain. As resident immune cells, microglial cells are the first line of immune defense present in the central nervous system (CNS). Here, we report that SFTSV was able to infect microglial cells and stimulate interleukin 1β (IL-1β) secretion in the brains of infected neonatal BALB/c mice. We characterized the cell death induced in infected human microglial HMC3 cells, also susceptible to SFTSV, and found that the NOD-like receptor protein 3 (NLRP3) inflammasome was activated, leading to secretion of IL-1β and pyroptosis. Knockdown of NLRP3 or inhibition of the NLRP3 inflammasome activation suppressed the viral replication, suggesting that the activation of the NLRP3 inflammasome may support SFTSV replication in microglial cells. Viral nonstructural protein NSs, a known modulator of immune responses, interacted and colocalized with NLRP3 for the inflammasome activation. It appeared that the N-terminal fragment, amino acids 1 to 66, of NSs was critical to promote the assembly of the inflammasome complex by interacting with NLRP3 for its activation in microglial cells. Our findings provide evidence that SFTSV may cause neurological disorders through infecting microglia and activating the inflammasome through its nonstructural protein NSs for neural cell death and inflammation. This study may have revealed a novel mechanism of SFTSV NSs in dysregulating host response. IMPORTANCE Encephalitis or encephalopathy during severe fever with thrombocytopenia syndrome (SFTS) is considered a critical risk factor leading to high mortality, but there have been no studies to date on the pathogenesis of encephalitis or encephalopathy caused by SFTS virus. Here, we report that SFTSV infection can active the NLRP3 inflammasome and induce IL-1β secretion in the brains of infected newborn mice. In infected human HMC3 microglia, SFTSV activated the NLRP3 inflammasome via the viral nonstructural protein NSs through interaction with its N-terminal fragment. Notably, our findings suggest that the activation of the NLRP3 inflammasome may promote SFTSV replication in infected microglial cells. This study may reveal a novel mechanism by SFTSV to dysregulate host responses through its nonstructural protein, which could help us understand viral neuropathogenesis in SFTS patients.

Keywords: NLRP3; SFTSV; inflammasome; microglia; severe fever with thrombocytopenia syndrome virus.

FIG 1

Susceptibility of microglial cells to SFTSV infection in infected mice. Newborn BALB/c mice were intracerebrally inoculated with either JS-2010 (5 × 10³ PFU) (A) or PBS (B). Three days postinoculation, brain tissue cross sections from the neonatal mice were prepared for immunofluorescence staining with antibodies for SFTSV nucleocapsid protein (NP) and IBA1, a cell marker of microglial cells. Nuclei were stained with DAPI before being viewed with confocal microscopy. Scale bar is 50 μm. (C and D) Quantitative analysis of microglial cells infected with SFTSV. Cells with staining of IBA1⁺ NP⁺, IBA1⁺ NP⁻, IBA1⁻ NP⁺, or IBA1⁻ NP⁻ were counted for quantitative analysis. (C) Percentages of microglial and nonmicroglial cells infected or not infected with SFTSV. (D) Percentages of SFTSV-infected microglial cells. Data are presented as means ± standard deviation (SD) of triplicate experiments.

FIG 2

Susceptibility of human microglial cells to SFTSV. (A) Human microglial HMC3 cells were infected with JS-2010 or JS-2014 (MOI = 1) and CPE was observed at 24, 48, and 72 h p.i. Magnification, ×100. (B) NSs protein of SFTSV strain JS-2010 was detected by immunofluorescence assay with an antibody for NSs. Blue, nucleus; green, viral NSs protein. Scale bars, 10 μm. (C and D) HMC3 cells were infected with different MOIs (0.04, 0.2, and 1) of JS-2010 (C) or JS-2014 (D), and cell viability was measured with the CCK8 assay at 24, 48, and 72 h p.i. (E) Viral replication was demonstrated with detection of viral S genomic segments measured by qRT-PCR in infected cells at various time points p.i. (F) Increased viral NP was measured in cytosol by Western blot analysis in JS-2010- or JS-2014-infected cells. The differences between groups of SFTSV- and mock-infected cells were evaluated by a two-tailed Student t test. Data are presented as means ± SD of triplicate experiments.

FIG 3

Programmed death in human microglial cells caused by SFTSV infection. (A) Morphological changes in infected cells, altered by caspase inhibitors. Cells were treated or not with small-molecule inhibitors prior to infection with JS-2010 at an MOI of 1. Light microscopic images were observed at 48 h p.i. Magnification, ×40. (B) Costaining of the infected or mock-infected cells, prepared at 48 h p.i., with annexin V and PI for flow cytometry analysis. (C and D) Quantitative analyses of PI-positive (C) and annexin V-positive (D) cells demonstrated with flow cytometry. The differences between SFTSV-infected groups pretreated with either an inhibitor or DMSO were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 4

Activation of pyroptosis in SFTSV-infected microglial cells. (A, B, and D) Human microglial HMC3 cells were infected with SFTSV strain JS-2010 at an MOI of 1. Cell lysates were prepared at 12, 24, 48, and 72 h p.i. for Western blot analyses performed with specific antibodies to detect activated RIPK1, RIPK3, and MLKL in programmed necrosis (A), processed PARP, caspase-3, and caspase-7 in apoptosis (B), and processed GSDMD, IL-1β, and caspase-1 in pyroptosis (D). HMC3 cells were treated with apoptosis activator 2 (5 μM), TNF plus BV6 plus Z-VAD (TBZ) (TNF, 1,000 U/mL; BV6, 1 μM; and Z-VAD, 20 μM) or nigericin (5 μM) for 12 h or 36 h. Cell lysates were collected as positive controls for apoptosis, necrosis and pyroptosis, respectively. (C) HMC3 cells were infected with the JS-2010 strain (MOI = 1) and apoptotic cells were observed by TUNEL staining at different time points p.i. Blue, nucleus; green, apoptotic cells. Scale bars, 20 μm. (E) Supernatants were collected at different time points p.i. to detect IL-1β secretion via ELISA. The differences between SFTSV- and mock-infected cells were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 5

The NLRP3 Inflammasome activated in SFTSV-infected microglial cells. (A) HMC3 cells, stably expressing shRNA targeting NLRP1, AIM2, NLRP3, or NLRC4, were infected or not with SFTSV strain JS-2010 for 48 h. Cell supernatants were sampled for IL-1β secretion and tested by ELISA. (B) HMC3 cells, stably expressing shRNA targeting NLRP1, AIM2, NLRC4, NLRP3, ASC, or procaspase-1, were shown to be knocked down of the corresponding genes as verified by Western blot analyses with specific antibodies for respective proteins. (C and D) HMC3 cells, expressing shRNA targeting NLRP3, ASC, or procaspase-1, were infected with SFTSV strain JS-2010 (C) or JS-2014 (D) at an MOI of 1, and cell media were sampled at 24, 48, and 72 h p.i. for measurement of IL-1β secretion. (E) HMC3 cells, expressing shRNA targeting either NLRP3 (sh-NLRP3) or scrambled NLRP3 (sh-scr) as a control, were infected with SFTSV strain JS-2010 or JS-2014 at an MOI of 1. Cell media were sampled for measurement of IL-1β secretion. The differences between SFTSV-infected and control cell lines, expressing shRNA or scrambled RNA, were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 6

Role of the NLRP3 inflammasome in the regulation of SFTSV infection-induced pyroptosis. HMC3 cells, stably expressing shRNA targeting NLRP3 (A and D), ASC (B and E), or procaspase-1 (C and F), were infected or not with SFTSV strain JS-2010 (A to C) or JS-2014 (D to F) (MOI = 1). HMC3 cells expressing control shRNA were used as controls. Cell lysates were prepared at 24, 48, and 72 h p.i. and procaspase-1, GSDMD, cleaved GSDMD, cleaved caspase-1, and viral NP were detected by Western blot analyses using specific antibodies.

FIG 7

Effects of the NLRP3 inflammasome on cell death in microglial cells infected with SFTSV. (A and B) Morphological changes in infected cells expressing shRNA. HMC3 cells, stably expressing shRNA targeting NLRP3, ASC, or procaspase-1, were infected with JS-2010 (A) or JS-2014 (B) at an MOI of 1 and observed by light microscopy to evaluate CPE after 72 h p.i. Magnifications, ×100 for panel A and ×40 for panel B. (C and D) Cell viability was assessed in infected or noninfected cells expressing shRNA by the CCK-8 assay at 24, 48, and 72 h p.i. The differences between groups of the infected and mock-infected cells were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 8

Inhibition of inflammasome activation and pyroptosis by inhibitors in SFTSV-infected microglial cells. HMC3 cells, pretreated with different concentrations of VX765 or MCC950, were infected or not with JS-2010 or JS-2014 (MOI = 1) for 48 h. (A and B) IL-1β was detected in JS-2010-infected (A) or JS-2014-infected (B) cells, pretreated with inhibitors. (C to F) The levels of procaspase-1, GSDMD, cleaved GSDMD, cleaved caspase-1, and viral NP were detected in infected cells, pretreated with inhibitors, by Western blot analyses. VX765 (C and D) is a caspase-1 inhibitor; MCC950 (E and F) is an NLRP3 inhibitor. The differences between the two groups were evaluated with two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 9

SFTSV replication was promoted by activation of the inflammasome in infected microglial cells. (A and B) Suppression of SFTSV replication in infected cells pretreated with inhibitors. HMC3 cells, pretreated with VX765 or MCC950, were infected with either the JS-2010 (A) or JS-2014 (B) strain for 48 h, and the number of copies of the viral S genomic RNA was measured by qRT-PCR. (C and D) HMC3 cells, stably expressing shRNA targeting NLRP3, ASC, or caspase-1, were infected with either JS-2010 (C) or JS-2014 (D) for 24, 48, or 72 h. The number of copies of the viral S genomic RNA in infected and noninfected cells expressing shRNA was measured by qRT-PCR. (E and F) Decreased infectious viral titers in infected cells, pretreated with inhibitors or expressing shRNA. Supernatants from infected cells, pretreated or not with VX765 or MCC950 (E) or expressing shRNA targeting NLRP3, ASC, or caspase-1 (F), were sampled at 72 h p.i. for titration of TCID₅₀. The difference between the two groups was evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 10

Replicative SFTSV and viral protein production were required for the activation of pyroptosis. (A to D) The replicative SFTSV was required for the secretion of IL-1β and processing of caspase-1 and GSDMD. HMC3 cells were infected with strain JS-2010 (A and C) or JS-2014 (B and D) (MOI = 1) or inoculated with heat-inactivated or UV-inactivated viruses for 48 h. IL-1β levels in the supernatant from the cells were measured by ELISA (A and B). The levels of cleaved GSDMD, cleaved caspase-1, and viral NP were detected in cell lysates by Western blot analyses (C and D). (E to H) Viral proteins were required for the secretion of IL-1β and processing of caspase-1 and GSDMD. HMC3 cells were infected with JS-2010 (E and G) or JS-2014 (F and H) (MOI = 1), and the cells were treated with two concentrations of cycloheximide (CHX) to inhibit protein synthesis. IL-1β levels in the supernatants from the cells were measured 48 h p.i. by ELISA (E and F). The levels of cleaved GSDMD, cleaved caspase-1, and viral NP were detected in the cell lysates by Western blot analyses (G and H). (I and J) Viral replication was confirmed by measuring viral S genomic RNA numbers. HMC3 cells, pretreated with CHX, were infected with JS-2010 (MOI = 1) (I). HMC3 cells were infected with the live virus or inoculated with UV- or heat-inactivated virus (J). Total RNA was prepared from the cells 48 h p.i. for measuring viral S genomic RNA numbers by qRT-PCR. The differences between two groups were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 11

Activation of the NLRP3 inflammasome and pyroptosis by nonstructural protein NSs. HMC3 cells were transfected with 0, 1, 2, and 5 μg of plasmids expressing HA-tagged NSs for 48 h. (A) Induced IL-1β secretion in the culture medium of the cells overexpressing NSs as detected by ELISA. (B) Increased cleaved GSDMD, cleaved caspase-1, and HA-tagged NSs in the cell lysates as detected by Western blot analyses. (C) NLRP3 was critical to NSs-induced activation of inflammasome. HMC3 cells, expressing shRNA targeting NLRP3, were transfected with various amounts of plasmids expressing HA-tagged NSs. The levels of NLRP3, cleaved GSDMD, cleaved caspase-1, and HA-tagged NSs were detected in cell lysates by Western blot analyses. (D) Coimmunoprecipitation of NSs and NLRP3. HEK 293T cells were transfected with plasmids expressing HA-tagged NSs and/or FLAG-tagged NLRP3 for 48 h. Cell lysates were prepared for coimmunoprecipitation and Western blot analyses to show that NSs was associated with NLRP3 in cotransfected cells. The differences between two groups were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 12

NSs colocalized with NLRP3 in SFTSV-infected or NSs-expressing cells. (A) Colocalization of NSs and NLRP3 in infected microglial cells. HMC3 cells were infected with JS-2010 and JS-2014 for 48 h, and the localization of viral NSs (green) and NLRP3 (red) was visualized under a laser confocal microscope. (B) Colocalization of NSs and NLRP3 in transfected cells. HeLa cells were transfected with plasmids expressing HA-tagged NSs and/or FLAG-tagged NLRP3 for 48 h. Colocalization of NSs (green) and NLRP3 (red) was observed under a laser confocal microscope. The nuclei were stained with DAPI (blue). Scale bar, 10 μm.

FIG 13

N-terminal NSs1-66 was required for activation of the NLRP3 inflammasome in microglial cells. HMC3 cells were transfected with plasmids expressing HA-tagged NSs1-66, NSs66-160, NSs66-205, or NSs66-293 for 48 h. (A) Increased IL-1β secretion was detected in the culture media of cells transfected with plasmids expressing different fragments of NSs by ELISA. (B) Increased levels of cleaved GSDMD, cleaved caspase-1, and HA-tagged NSs were detected in the lysates prepared from the cells transfected with the plasmids by Western blot analyses. (C and D) NLRP3 was required for activation of the inflammasome in microglial cells expressing fragmented NSs. HMC3 cells, stably expressing shRNA targeting NLRP3, were transfected with plasmids expressing HA-tagged NSs1-66, NSs66-160, NSs66-205, or NSs66-293 for 48 h. Lack of IL-1β secretion was shown in the culture media of the transfected cells expressing shRNA targeting NLRP3 as detected by ELISA (C). Absence of cleaved GSDMD and caspase-1 in lysates of transfected cells expressing shRNA targeting NLRP3 was detected by Western blot analyses (D). The differences between plasmids encoding fragments of NSs and the control group were evaluated by two-tailed Student t test. Data are presented as means ± SD of at least triplicate experiments.

FIG 14

Inflammasome activation in brain microglial cells infected with SFTSV or expressing N-terminal NSs1-66. Newborn BALB/c mice were intracerebrally inoculated with either JS-2010 (5 × 10³ PFU) (A and B) or lentivirus expressing NSs1-66 (Lenti-NSs1-66) (C and D). Mice were also inoculated with PBS or a lentivirus control (Lenti-NC) as mock controls. Three days postinoculation, brain tissue cross sections were prepared from neonatal mice, immunohistochemically stained with antibodies for NLRP3 antibody (A and C) and IL-1β (B and D), and examined under light microscopy. Scale bar is 200 μm.

FIG 15

Inflammasome activation in brain microglial cells expressing the N-terminal NSs1-66. Newborn BALB/c mice were intracerebrally inoculated with lentivirus expressing GFP-NSs1-66 (Lenti-NSs1-66) (A) or a lentivirus GFP control (Lenti-NC) (B). Three days postinoculation, brain tissue cross sections were prepared from neonatal mice for immunofluorescence staining with antibodies for NLRP3 and IBA1. The nuclei were stained with DAPI before confocal microscopy. Scale bar is 50 μm.

FIG 16

Interaction of the N-terminal NSs1-66 and NLRP3. HEK 293T cells were transfected with plasmids expressing HA-tagged NSs1-66, NSs66-160, NSs66-205, or NSs66-293 and/or FLAG-tagged NLRP3 for 48 h. (A) Expression of HA-tagged NSs fragments and FLAG-tagged NLRP3 was detected in lysates of transfected or cotransfected cells. (B and C) Cell lysates were prepared for coimmunoprecipitation and Western blot analyses of the fragmented NSs and NLRP3. The cell lysates were immunoprecipitated with anti-HA tag antibodies (B) and by anti-FLAG tag antibodies (C), respectively. Anti-FLAG (B) and anti-HA (C) tag antibodies were used for Western blot analyses. (D) Colocalization of NSs1-66 and NLRP3. HeLa cells were transfected with plasmids expressing HA-tagged NSs1-66 or NSs66-293 and FLAG-tagged NLRP3. Colocalization of NSs1-66 or NSs66-293 (green) and NLRP3 (red) was visualized with a laser confocal microscope. Scale bar, 10 μm.