RNase L activates the NLRP3 inflammasome during viral infections - PubMed (original) (raw)

RNase L activates the NLRP3 inflammasome during viral infections

Arindam Chakrabarti et al. Cell Host Microbe. 2015.

Abstract

The NLRP3 inflammasome assembles in response to danger signals, triggering self-cleavage of procaspase-1 and production of the proinflammatory cytokine IL-1β. Although virus infection activates the NLRP3 inflammasome, the underlying events remain incompletely understood. We report that virus activation of the NLRP3 inflammasome involves the 2',5'-oligoadenylate (2-5A) synthetase(OAS)/RNase L system, a component of the interferon-induced antiviral response that senses double-stranded RNA and activates endoribonuclease RNase L to cleave viral and cellular RNAs. The absence of RNase L reduces IL-1β production in influenza A virus-infected mice. RNA cleavage products generated by RNase L enhance IL-1β production but require the presence of 2',3'-cyclic phosphorylated termini characteristic of RNase L activity. Additionally, these cleavage products stimulate NLRP3 complex formation with the DExD/H-box helicase, DHX33, and mitochondrial adaptor protein, MAVS, which are each required for effective NLRP3 inflammasome activation. Thus, RNA cleavage events catalyzed by RNase L are required for optimal inflammasome activation during viral infections.

Copyright © 2015 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

Co-author Luigi Franchi is currently an employee of Lycera, a biotechnology company that develops drugs for inflammation. The other co-authors do not have a conflict of interest.

Figures

Figure 1

Figure 1. RNase L deficiency in mice enhances the lethality of IAV while decreasing the induction of IL-1β

(A) Survival of wt and _Rnasel_−/− mice infected i.n. with IAV. (B) Mean body weights corresponding to panel (A). (C,D) IL-1β and (E,F) TNF-α levels determined by ELISAs at 2 days post-infection (dpi) with IAV in (C,E) lung homogenates and (D,F) BALF. (G) Viral titers from lungs harvested at 7 dpi, determined by plaque assays. horizontal lines, mean ± standard deviation (SD). Significance was determined by (A,B) Kaplan-Meier analysis or (C–G) two-tailed Student’s t tests. *** p<0.001; **p< 0.01; *p<0.05; ns, not significant. Numbers of mice used: (A,B) n= 14; (C,E) n=8; (D) n=11; (F) n=9; and (G) n=8. See also Figure S1.

Figure 2

Figure 2. Inflammasome activation in response to viral infections is reduced in the absence of RNase L

IL-1β levels by ELISAs from (A) wt and Rnasel−/− BMDC primed with LPS for 6 hrs and infected with VSV for 12 hrs or IAV for 24 hrs, (B) wt and Rnasel−/− BMDC primed with TNFα for 6 hrs and infected with VSV for 12 hrs and (C) BMDC primed with LPS for 6 hrs and treated with extracellular ATP (0.5 mM) for 1 hr. (D) IL-1β mRNA levels by qRT-PCR and (E) TNFα levels by ELISAs from LPS primed wt and Rnasel−/− BMDCs without or with VSV infection. (F) Cleavage of caspase-1 and pro-IL-1β in LPS primed wt and Rnasel−/− BMDCs without or with VSV or IAV infection determined in immunoblots. Cell lysates (Lys); cell supernatants (Sup); error bars, SD; **, p<0.01; ***, p<0.001 by two-tailed Student’s t tests. See also Figure S2.

Figure 3

Figure 3. Involvement of NLRP3, ASC, and MAVS in IL-1β induction but not TNFα induction in response to viral infections

(A,B) LPS primed wt, Nlrp3−/− and Asc−/− BMDC were infected with VSV or IAV. (C,D) LPS primed wt, Rig I−/−, _Mda5_−/−, RigI_−/−/Mda5_−/−, and Mavs−/− BMDC were infected with VSV or IAV. IL-1β and TNFα levels were measured by ELISAs. Error bars, SD; ***, p<0.001 by two-tailed Student’s t tests. See also Figure S3.

Figure 4

Figure 4. IL-1β production by direct activation of RNase L is dependent on both NLRP3 and ASC

BMDC were primed with Pam3Csk4 (200 ng/ml) for 16 hrs and mock transfected or transfected with (2′-5′)p3A3 for additional 8 hrs as indicated. (A) rRNA cleavage in response to (2′-5′)p3A3 transfection was monitored in RNA chips. (B,C) IL-1β and cleaved caspase-1 was measured in cell supernatants (Sup), whereas proIL-1β, procaspase-1 and β-actin were measured in cell lysates (Lys). Upper panels, ELISAs; lower panels, immunoblots. Error bars, SD; ***; p<0.001 by two-tailed Student’s t tests.

Figure 5

Figure 5. RNase L generated RNA cleavage products bearing 2′,3′-cyclic phosphates induce IL-1β production

(A–C) wt and Rnasel−/− BMDC were primed with Pam3Csk4 (200 ng/ml) for 16 hrs and transfected with intact or RNase L-cleaved IAV RNA or cellular RNA for 8 hrs. (D–G) BMDC of different genotypes (as indicated) were primed with Pam3Csk4 and transfected with uncleaved or RNase L-cleaved cellular RNAs for 8 hrs. (H) Intact, cleaved, or cleaved and PNK-treated cellular RNA were transfected into Pam3Csk4 treated BMDC. (A,B,D,F,&H upper) ELISAs; (C,E,G, and H lower) immunoblots. Cell supernatants (Sup); cell lysates (Lys). Error bars, SD;. **p<0.01;***, p<0.001 by two-tailed Student’s t tests. See also Figure S4.

Figure 6

Figure 6. A functional nuclease domain in RNase L is required for inflammasome stimulation

(A) RNase L levels in THP-1 cells or expressing sh-Rnasel alone or with Flag-tagged wt or R667A mutant RNase L cDNAs. Immunoblots were probed with (upper panel) monoclonal antibody to human RNase L, (middle panel) antibody to Flag epitope, and (bottom panel) antibody to β-actin. (B) IL-1β and caspase-1 (p20) in cell supernatants (Sup) and procaspase-1 (p45) and β-actin in cell lysates (Lys) from THP-1 cells expressing sh-control or sh-Rnasel and infected with IAV, IAV/ΔNS1 or VSV. (C,D) THP-1 cells expressing Rnasel shRNA or reconstituted with wt or mutant R667A RNase L infected with (C) IAV/ΔNS1 or (D) VSV. (B–D), upper panels, ELISAs; lower panels, immunoblots. Error bars, SD; ***, p<0.001 by two-tailed Student’s t tests. See also Figure S5.

Figure 7

Figure 7. Involvement of DHX33 in RNase L-mediated inflammasome activation

(A) shRNA mediated depletion of DDX1 or DHX33 in THP-1 macrophages. (B–E) THP-1 macrophages expressing different shRNAs [sh-control, sh-Ddx1 or sh-_Dhx33_] were (B) transfected with pIC (2 μg/ml for 8 hrs), (C) infected with VSV, (D) infected with IAV/ΔNS1, and (E) mock transfected or transfected with cell RNA cleaved with RNase L or transfected with (2′-5′)p3A3. Upper panels, ELISAs; lower panels, immunoblots (IB). (F) THP-1 macrophages were transfected with intact, cleaved, cleaved and PNK-treated cellular RNA or pIC. Immunoprecipitations (IP) were with anti-NLRP3, anti-DHX33, or an isotype control IgG. Cell lysates were used as controls (bottom panel). (G) HEK293T cells were co-transfected with myc-MAVS and flag-NLRP3 cDNAs. After 72 hrs, cells were transfected with intact, cleaved or cleaved and PNK treated cellular RNA. IPs were with anti-myc followed by immunoblotting (IB) with anti-flag, anti-DHX33 or anti-myc antibodies. Cell lysates were used as controls (lower panel). (H) Purified flag-DHX33 was incubated with biotinylated IAV M gene RNA (intact, cleaved, or cleaved and PNK-treated). Biotinylated RNAs were precipitated with streptavidin beads followed by IB with anti-flag antibody to detect DHX33. cell supernatants (Sup); cell lysates (lys). Error bars, SD; **p<0.01;***, p<0.001 by two-tailed Student’s t tests. See also Figure S6.

Similar articles

Cited by

References

    1. Allen IC, Scull MA, Moore CB, Holl EK, McElvania-TeKippe E, Taxman DJ, Guthrie EH, Pickles RJ, Ting JP. The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity. 2009;30:556–565. - PMC - PubMed
    1. Banerjee S, Chakrabarti A, Jha BK, Weiss SR, Silverman RH. Cell-Type-Specific Effects of RNase L on Viral Induction of Beta Interferon. mBio. 2014:5. - PMC - PubMed
    1. Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and inflammation. Nature reviews Microbiology. 2009;7:99–109. - PMC - PubMed
    1. Cai X, Chen J, Xu H, Liu S, Jiang QX, Halfmann R, Chen ZJ. Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation. Cell. 2014;156:1207–1222. - PMC - PubMed
    1. Carroll SS, Chen E, Viscount T, Geib J, Sardana MK, Gehman J, Kuo LC. Cleavage of oligoribonucleotides by the 2′,5′-oligoadenylate- dependent ribonuclease L. The Journal of biological chemistry. 1996;271:4988–4992. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources