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. 2011 Sep;13(9):1371-84.
doi: 10.1111/j.1462-5822.2011.01625.x. Epub 2011 Jul 11.

Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis

Ka-Wing Wong  1 , William R Jacobs Jr
Affiliations
Free PMC article

Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis

Ka-Wing Wong et al. Cell Microbiol. 2011 Sep.
Free PMC article

Abstract

Induction of necrotic death in macrophages is a primary virulence determinant of Mycobacterium tuberculosis. The ESX-1 secretion system and its substrate ESAT-6 are required for M. tuberculosis to induce necrosis, but host factors that mediate the ESAT-6-promoted necrosis remain unknown. Here we report that ESAT-6-promoted necrotic death in THP-1 human macrophages is dependent on the NLRP3 inflammasome, as shown by RNA interference and pharmacological inhibitions. Phagosomes containing ESAT-6-expressing M. tuberculosis recruit markers previously associated with damaged phagosomal membrane, such as galectin-3 and ubiquitinated protein aggregates. In addition, ESAT-6 promoted lysosomal permeabilization by M. tuberculosis. ESAT-6 mutants defective for ubiquitination were unable to trigger NLRP3 activation and necrotic death. Furthermore, Syk tyrosine kinase, recently implicated in NLRP3 activation during fungal and malarial infections, was necessary for mediating the ESAT-6-promoted necrosis and NLRP3 activation. Our results thus link phagosomal damage and Syk activity to NLRP3-mediated necrotic death triggered by M. tuberculosis ESAT-6 during infection.

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Figures

Figure 1
Figure 1
The effect of ESAT-6 on NLRP3 functions during M. tuberculosis infections. Differentiated THP-1 macrophages (A–E) or primary human macrophages (E) were infected with the indicated strains of M. tuberculosis H37Rv for 2 days. A. The levels of active caspase-1 were determined by staining of active caspase-1 with zYVAD-FMK using fluorescence microscopy. ELISA measurements of IL-18 secretion (B) or IL-1beta (C) were performed on culture supernatants of THP-1 macrophages infected with the indicated strains. D. The effect of 50 µM of the caspase-1 inhibitor, z-YVAD, on IL-1beta secretion from and necrosis of THP-1 macrophages infected with H37Rv. E. The percentages of infected THP-1 macrophages (day 2) or primary human macrophages derived from PBMC (day 3) that became necrotic were determined by fluorescence microscopy. Results are summarized as means ± standard errors. **P < 0.005; *P < 0.05 (relative to DMSO control unless otherwise stated).
Figure 2
Figure 2
Induction of necrosis in response to M. tuberculosis infection is mediated by NLRP3. A. Glyburide treatment. 50 µM glyburide or DMSO as vehicle control was used to pretreat THP-1 macrophages and incubate infected macrophages. IL-1 β levels of culture supernatant and necrosis of infected THP-1 macrophages were analysed at 22 and 46 h after infection. 50 µM glipizide was used as negative control for glyburide. **P < 0.005 (relative to DMSO control unless otherwise stated). B. NLRP3 knockdown by siRNA. THP-1 macrophages were treated with 100 nM siRNA against NLRP3 or non-targeting siRNA as control, as described in Experimental procedures. Efficiency of NLRP3 siRNA was determined by real-time PCR as described in Experimental procedures. IL-1β levels of culture supernatant and necrosis of infected THP-1 macrophages were analysed at 21 and 41 h post infection. **P < 0.005; *P < 0.05; #P = 0.05 (relative to non-targeting siRNA control). C–D. THP-1 macrophages were treated with DMSO, 10 µM parthenolide (C) or 10 µM parthenolide (D). Necrosis levels were measured at 2 days after infection. All results were summarized as means ± standard errors. **P < 0.005; NS: not significant (relative to DMSO control).
Figure 3
Figure 3
The role of Zmp1 on NLRP3 functions during M. tuberculosis infections. Differentiated THP-1 macrophages were infected with the indicated strains of M. tuberculosis H37Rv for 2 days. A. The percentages of infected THP-1 macrophages that became necrotic were determined by fluorescence microscopy. B. The levels of active caspase-1 were determined by staining of active caspase-1 with zYVAD-FMK using fluorescence microscopy. C. ELISA measurement of IL-18 was performed from culture supernatants of THP-1 macrophages infected with the indicated strains. Results are summarized as means ± standard errors. **P < 0.005; *P < 0.05; NS: not significant (relative to H37Rv).
Figure 4
Figure 4
Phagosomal membrane damage by M. tuberculosis. A–B. ESAT-6-dependent accumulation of ubiquitinated proteins based on FK2 antibody staining (green) (A) or the phagosomal lysis marker Galectin-3 (green) (B) occurs on phagosomes containing M. tuberculosis (red) after 2 days post infection. Summarized results are shown in the right panel. M. tuberculosis is visualized by its autofluorescence upon excitation at blue wavelength and is pseudocoloured in red. Summarized results are shown in the right panel. **P < 0.001.
Figure 5
Figure 5
Recruitment of endolysosomes to M. tuberculosis-containing phagosomes and the subsequent permeabilization of endolysosomal membranes. Endolysosomal compartments of THP-1 macrophages were preloaded with 0.2 mg ml−1 of dextran-10 kDa (red) overnight, followed by a 2 h chase, as described in Experimental procedures, before infection with M. tuberculosis. A. Endolysosome (red) recruitment to M. tuberculosis (green) at 16 h post infection. The right panel shows the summarized results of the percentages of macrophages containing endolysosomal recruitment to M. tuberculosis, which were determined from 50 macrophages in triplicate. B. Endolysosomal permeabilization as evidenced by diffused localization of the dextran endolysosomal content marker (red) at 44 and 64 h post infection. M. tuberculosis was visualized by its autofluorescence and was pseudocoloured in green. At 64 h, H37Rv-infected macrophages were not scored because most of them were completely lysed, leaving no more detectable endolysosomal marker. Summarized results of the percentages of infected cells (n = 50 in triplicate) with diffused dextran localization are shown in the right panels. **P < 0.001; NS: not significant.
Figure 6
Figure 6
Triggering NLRP3 functions by ESAT-6 depends on the ability of ESAT-6 to cause phagosomal damage. PMA-differentiated THP-1 macrophages were infected with H37Rv, ΔesxA, ΔesxA::esxA (+ WT), ΔesxA::esxA (+ F8I), ΔesxA::esxA (+ I11R) or ΔesxA::esxA (+ I18R). A. Phagosomal damage of infected cells was assessed by accumulation of ubiquitinated proteins. IL-18 production (B) and necrosis (C) were examined as described in Experimental procedures. Results are summarized as means ± standard errors. **P < 0.005 [relative to ΔesxA::esxA (+ WT)].
Figure 7
Figure 7
Inhibition of Syk kinase blocks IL-18 production and necrosis. PMA-differentiated THP-1 macrophages were pretreated with DMSO or 10 µM syk inhibitor. After 4 h of infection, cells were incubated in the presence of DMSO or syk inhibitor overnight. A. IL-18 levels in supernatants of THP-1 cells infected with H37Rv were assayed by ELISA. B. The percentage of necrotic cells infected with H37Rv was determined from propidium iodide staining under fluorescence microscopy. **P < 0.001; *P < 0.01; NS: not significant (relative to DMSO control).
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References

    1. Abdul-Sater A, Sa d-Sadier N, Padilla E, Ojcius D. Chlamydial infection of monocytes stimulates IL-1 [beta] secretion through activation of the NLRP3 inflammasome. Microbes Infect. 2010;12:652–661. - PMC - PubMed
    1. Bardarov S, Bardarov S, Jr, Pavelka MS, Jr, Sambandamurthy V, Larsen M, Tufariello J, et al. Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology. 2002;148:3007–3017. - PubMed
    1. Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and inflammation. Nat Rev Microbiol. 2009;7:99–109. - PMC - PubMed
    1. Brodin P, de Jonge MI, Majlessi L, Leclerc C, Nilges M, Cole ST, Brosch R. Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity. J Biol Chem. 2005;280:33953–33959. - PubMed
    1. van Bruggen R, Koker M, Jansen M, van Houdt M, Roos D, Kuijpers T, van den Berg T. Human NLRP3 inflammasome activation is Nox1-4 independent. Blood. 2010;115:5398–5400. - PubMed

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