RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux

doi:10.1038/s41419-019-1579-0

. 2019 Apr 25;10(5):346.

doi: 10.1038/s41419-019-1579-0.

RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux

Leandro Silva da Costa^{1

2}, Ahmed Outlioua^{3

4

5}, Adrienne Anginot^{3

4}, Khadija Akarid⁵, Damien Arnoult^{6

7}

Affiliations

¹ INSERM, UMR_S 1197, Hôpital Paul Brousse, Villejuif, France. silvadacostta@gmail.com.
² Université Paris-Saclay, Paris, France. silvadacostta@gmail.com.
³ INSERM, UMR_S 1197, Hôpital Paul Brousse, Villejuif, France.
⁴ Université Paris-Saclay, Paris, France.
⁵ Molecular Genetics and Immunophysiopathology Research Team, Health and Environment Laboratory, Aïn Chock Faculty of Sciences, Hassan II University of Casablanca, Casablanca, Morocco.
⁶ INSERM, UMR_S 1197, Hôpital Paul Brousse, Villejuif, France. damien.arnoult@inserm.fr.
⁷ Université Paris-Saclay, Paris, France. damien.arnoult@inserm.fr.

PMID: 31024004
PMCID: PMC6483999
DOI: 10.1038/s41419-019-1579-0

RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux

Leandro Silva da Costa et al. Cell Death Dis. 2019.

. 2019 Apr 25;10(5):346.

doi: 10.1038/s41419-019-1579-0.

Authors

Leandro Silva da Costa^{1

2}, Ahmed Outlioua^{3

4

5}, Adrienne Anginot^{3

4}, Khadija Akarid⁵, Damien Arnoult^{6

7}

Affiliations

¹ INSERM, UMR_S 1197, Hôpital Paul Brousse, Villejuif, France. silvadacostta@gmail.com.
² Université Paris-Saclay, Paris, France. silvadacostta@gmail.com.
³ INSERM, UMR_S 1197, Hôpital Paul Brousse, Villejuif, France.
⁴ Université Paris-Saclay, Paris, France.
⁵ Molecular Genetics and Immunophysiopathology Research Team, Health and Environment Laboratory, Aïn Chock Faculty of Sciences, Hassan II University of Casablanca, Casablanca, Morocco.
⁶ INSERM, UMR_S 1197, Hôpital Paul Brousse, Villejuif, France. damien.arnoult@inserm.fr.
⁷ Université Paris-Saclay, Paris, France. damien.arnoult@inserm.fr.

PMID: 31024004
PMCID: PMC6483999
DOI: 10.1038/s41419-019-1579-0

Abstract

Early detection of viruses by the innate immune system is crucial for host defense. The NLRP3 inflammasome, through activation of caspase-1, promotes the maturation of IL-1β and IL-18, which are critical for antiviral immunity and inflammatory response. However, the mechanism by which viruses activate this inflammasome is still debated. Here, we report that the replication of cytopathogenic RNA viruses such as vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) induced a lytic cell death leading to potassium efflux, the common trigger of NLRP3 inflammasome activation. This lytic cell death was not prevented by a chemical or genetic inhibition of apoptosis, pyroptosis, or necroptosis but required the viral replication. Hence, the viruses that stimulated type I IFNs production after their sensing did not activate NLRP3 inflammasome due to an inhibition of their replication. In contrast, NLRP3 inflammasome activation induced by RNA virus infection was stimulated in IFNAR-deficient or MAVS-deficient cells consequently to an increased viral replication and ensuing lytic cell death. Therefore, in a context of inefficient IFN response, viral replication-induced lytic cell death activates of the NLRP3 inflammasome to fight against infection.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

**Fig. 1. Replicating and cytopathogenic RNA viruses trigger inflammasome activation.**
a Primed (LPS, 100 ng/ml for 3 h) BMDMs were infected with different RNA viruses for 15 h. Then, cell supernatants and cell lysates were analyzed by WB for the indicated proteins. **b, c** Primed BMDMs were infected with different RNA viruses. After15 h, IL-1β (b) or IL-18 release (c) in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with mock BMDMs in Student’s t-test). ****P < 0.0001, ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05. d Primed BMDMs were infected with different RNA viruses. After 15 h, cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with mock BMDMs in Student’s t-test). ****P < 0.0001, ***P < 0.001, *0.01 < P < 0.05. ns, not significant. e BMDMs were infected with different RNA viruses for 8 or 16 h. Then, cell lysates were analyzed by WB for the indicated proteins. f BMDMs were infected with different RNA viruses for 8 or 16 h. Then, IFNβ production was assessed by ELISA. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with mock BMDMs in Student’s t-test). ****P < 0.0001, ***P < 0.001, **0.001 < P < 0.01. g BMDMs were infected with different RNA viruses. After 15 h, cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with mock BMDMs in Student’s t-test). ****P < 0.0001, **0.001 < P < 0.01, *0.01 < P < 0.05. h Analysis of the viral replication in BMDMs

**Fig. 2. VSV and EMCV promote NLRP3 inflammasome activation and inflammasome-independent cell death.**
a Primed BMDMs were infected with increased MOI of VSV or EMCV for 15 h. Then, cell supernatants and cell lysates were analyzed by WB for the indicated proteins. b Primed BMDMs were infected with increased MOI of VSV or EMCV. After 15 h, IL-1β release in the cell supernatant was assessed by ELISA. c Primed BMDMs were infected with increased MOI of VSV or EMCV. After 15 h, cell death was assessed using the LDH release assay. d Primed WT or NLRP3^−/− BMDMs were infected with VSV or EMCV for 15 h. Then, cell supernatants and cell lysates were analyzed by WB for the indicated proteins. e Primed WT, NLRP3^−/−, ASC^−/−, Casp1^−/−, or GSDMD^-/− BMDMs were infected with VSV or EMCV. After 15 h, IL-1β release in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments. ***P < 0.001 versus WT BMDMs (Student’s t-test). ns, not significant. f Primed WT, NLRP3^−/−, ASC^−/−, Casp1^−/−, or GSDMD^−/− BMDMs were infected with VSV or EMCV. After 15 h, cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. ****P < 0.0001 versus WT BMDMs (Student’s t-test). ns, not significant. g Analysis of the viral replication in the different BMDMs

**Fig. 3. Replicating VSV and EMCV promote caspase-independent cell death and NLRP3 inflammasome activation through K⁺ efflux.**
a BMDMs were infected with VSV or EMCV. At different times after infection, cell lysates were analyzed by WB for the indicated proteins. Actinomycin D treatment (ActD) (20 μM for 8 h) was used as a positive control. b BMDMs were infected with VSV or EMCV in the presence or the absence of the pan caspase inhibitor zVAD-fmk (10 μM) for 15 h. Cell death was then assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. ns, not significant. c Analysis of VSV or EMCV replication, either untreated or UV inactivated. d Primed BMDMs were infected with untreated or UV-inactivated VSV or EMCV for 15 h. Next, IL-1β release in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.001 versus untreated virus (Student’s t-test). e Primed BMDMs were infected with untreated or UV-inactivated VSV or EMCV. After 15 h, cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. ****P < 0.0001, ***P < 0.001 versus untreated virus (Student’s t-test). f Primed BMDMs were infected with VSV or EMCV for 15 h in the presence of increased concentrations of KCl or glyburide (a proton pump inhibitor that prevents the K⁺ efflux, 25 μg/ml). IL-1β release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.0001, ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05 versus 5 mM KCl (Student’s t-test). g Primed BMDMs were infected with VSV or EMCV for 15 h in the presence of increased concentrations of KCl or glyburide (25 μg/ml). Then, cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. ns, not significant versus 5 mM KCl (Student’s t-test). h Analysis of the viral replication in the presence of KCl or glyburide

**Fig. 4. Virus-induced inflammasome activation is independent of RNA sensing by RIG-I, MDA-5, or DXH33.**
a Primed WT or RIG-I^−/− BMDMs were infected with VSV or EMCV. After 15 h, IL-1β release in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments. ns, not significant versus WT BMDMs (Student’s t-test). b Analysis of the viral replication in WT or RIG-I^−/− BMDMs. c Primed WT, MAVS^−/−, or MDA5^−/− BMDMs were infected with VSV or EMCV. After 15 h, IL-1β release in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments. ns, not significant versus WT BMDMs (Student’s t-test). d Analysis of the viral replication in WT, MAVS^−/−, or MDA5^−/− BMDMs. e WT or RIG-I^−/− BMDMs were infected with VSV, VSV M, or EMCV for 8 h. IFNβ release in the cell supernatant was next assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.0001, ***P < 0.001, ns, not significant versus WT BMDMs (Student’s t-test). f WT, MAVS^−/−, or MDA5^−/− BMDMs were infected with VSV, VSV M, or EMCV for 8 h. IFNβ release in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.0001, **0.001 < P < 0.01, ns, not significant versus WT BMDMs (Student’s t-test). g BMDMs were transfected with siRNAs raised against DHX33 or nonspecific siRNA (NS). After 3 days, BMDMs were infected with VSV or EMCV for 15 h. IL-1β release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ns, not significant versus NS siRNA-transfected BMDMs (Student’s t-test). The efficiency of the knockdown of DHX33 was confirmed by WB

**Fig. 5. Virus-induced inflammasome activation is independent of necroptosis machinery and Drp1.**
a BMDMs were infected in the presence or the absence of zVAD-fmk (20 μM) with VSV or EMCV for 8 or 16 h. Then, cell lysates were analyzed by WB for the indicated proteins. As a positive control, BMDMs were treated with TNFα (50 ng/ml), cycloheximide (1 μg/ml), and zVAD-fmk (20 μM) (TCZ) for 2 h. b Primed WT, RIPK3^−/−, or MLKL^−/− BMDMs were infected with VSV or EMCV. After 15 h, IL-1β release in the cell supernatant was assessed by ELISA. The data shown are means ± SD from three independent experiments. ns, not significant versus WT BMDMs (Student’s t-test). c Analysis of the viral replication in WT, RIPK3^−/−, or MLKL^−/− BMDMs. d BMDMs were transfected with siRNAs raised against RIPK1 or nonspecific siRNA (NS). After 3 days, BMDMs were infected with VSV or EMCV for 15 h. IL-1β release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ns, not significant versus NS siRNA-transfected BMDMs (Student’s t-test). The efficiency of the knockdown of RIPK1 was confirmed by WB. e BMDMs were infected with VSV or EMCV for the indicated times. Cell lysates were analyzed by WB for the indicated proteins. As a positive control, BMDMs were treated with nocodazole (100 ng/ml) for 16 h. f BMDMs were transfected with siRNAs raised against Drp1 or nonspecific siRNA (NS). After 3 days, BMDMs were infected with VSV or EMCV for 15 h. IL-1β release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ns, not significant versus NS siRNA-transfected BMDMs (Student’s t-test). The efficiency of the knockdown of Drp1 was confirmed by WB

**Fig. 6. Viral replication is required for NLRP3 inflammasome activation.**
aVSV M or Sindbis virus (SindV) replication was assessed in WT, IFNAR^−/−, or MAVS^−/− BMDMs. b WT or IFNAR^−/− BMDMs were infected with VSV M or SindV for 24 h, then cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. ***P < 0.001, **0.001 < P < 0.01, versus WT BMDMs (Student’s t-test). c Primed WT or IFNAR^−/− BMDMs were infected with VSV M or SindV for 24 h. IL-1β release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.0001, **0.001 < P < 0.01, versus WT BMDMs (Student’s t-test). d IFNAR^−/− BMDMs were transfected with siRNAs raised against NLRP3 or GSDMD or nonspecific siRNA (NS). After 3 days, BMDMs were infected with VSV M or SindV for 24 h, then cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. ns, not significant versus NS siRNA-transfected BMDMs (Student’s t-test). The efficiency of the knockdown of NLRP3 and GSDMD was confirmed by WB. e WT or MAVS^−/− BMDMs were infected with VSV M or SindV for 8 or 16 h. IFNβ release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.0001, ***P < 0.001, versus WT BMDMs (Student’s t-test). f WT or MAVS^−/− BMDMs were infected with VSV M or SindV for 24 h, then cell death was assessed using the LDH release assay. The data shown are means ± SD from three independent experiments. **0.001 < P < 0.01, versus WT BMDMs (Student’s t-test). g Primed WT or MAVS^−/− BMDMs were infected with VSV M or SindV for 24 h. IL-1β release in the cell supernatant was then assessed by ELISA. The data shown are means ± SD from three independent experiments. ****P < 0.0001, **0.001 < P < 0.01, versus WT BMDMs (Student’s t-test)

**Fig. 7. Proposed model.**
NF-κB activation following TLR3, TNFR, or IL-1R stimulation or following RLR stimulation (not depicted) promotes pro-IL-1β expression (signal I). In parallel, after infection, RNA virus replicates into the cell triggering a lytic form of cell death and plasma membrane rupture. This lytic cell death allows K⁺ efflux (signal II) that activates NLRP3 inflammasome. Within this inflammasome, caspase-1 is activated then cleaves pro-IL-1β into its mature form. IL-1β is released in the extracellular medium independently of the gasdermin D pores, likely as a consequence of the viral-induced lytic cell death

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References

1. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 2009;27:519–550. doi: 10.1146/annurev.immunol.021908.132612. - DOI - PubMed
1. Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 2016;16:407–420. doi: 10.1038/nri.2016.58. - DOI - PubMed
1. Man SM, Karki R, Kanneganti TD. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 2017;277:61–75. doi: 10.1111/imr.12534. - DOI - PMC - PubMed
1. Lawlor KE, Vince JE. Ambiguities in NLRP3 inflammasome regulation: is there a role for mitochondria? Biochim. Biophys. Acta. 2014;1840:1433–1440. doi: 10.1016/j.bbagen.2013.08.014. - DOI - PubMed
1. Munoz-Planillo R, et al. K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity. 2013;38:1142–1153. doi: 10.1016/j.immuni.2013.05.016. - DOI - PMC - PubMed

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[1] Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 2009;27:519–550. doi: 10.1146/annurev.immunol.021908.132612. - DOI - PubMed

[2] Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 2009;27:519–550. doi: 10.1146/annurev.immunol.021908.132612. - DOI - PubMed

[3] Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 2016;16:407–420. doi: 10.1038/nri.2016.58. - DOI - PubMed

[4] Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 2016;16:407–420. doi: 10.1038/nri.2016.58. - DOI - PubMed

[5] Man SM, Karki R, Kanneganti TD. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 2017;277:61–75. doi: 10.1111/imr.12534. - DOI - PMC - PubMed

[6] Man SM, Karki R, Kanneganti TD. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 2017;277:61–75. doi: 10.1111/imr.12534. - DOI - PMC - PubMed

[7] Lawlor KE, Vince JE. Ambiguities in NLRP3 inflammasome regulation: is there a role for mitochondria? Biochim. Biophys. Acta. 2014;1840:1433–1440. doi: 10.1016/j.bbagen.2013.08.014. - DOI - PubMed

[8] Lawlor KE, Vince JE. Ambiguities in NLRP3 inflammasome regulation: is there a role for mitochondria? Biochim. Biophys. Acta. 2014;1840:1433–1440. doi: 10.1016/j.bbagen.2013.08.014. - DOI - PubMed

[9] Munoz-Planillo R, et al. K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity. 2013;38:1142–1153. doi: 10.1016/j.immuni.2013.05.016. - DOI - PMC - PubMed

[10] Munoz-Planillo R, et al. K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity. 2013;38:1142–1153. doi: 10.1016/j.immuni.2013.05.016. - DOI - PMC - PubMed

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RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux

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RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux

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