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. 2012 Apr 1;188(7):3469-77.
doi: 10.4049/jimmunol.1102272. Epub 2012 Feb 24.

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Mikhail A Gavrilin  1 , Dalia H A Abdelaziz, Mahmoud Mostafa, Basant A Abdulrahman, Jaykumar Grandhi, Anwari Akhter, Arwa Abu Khweek, Daniel F Aubert, Miguel A Valvano, Mark D Wewers, Amal O Amer
Affiliations

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Mikhail A Gavrilin et al. J Immunol. .

Abstract

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Human monocytes with knocked-down pyrin release less IL-1β and active caspase-1 in response to B. cenocepacia. (A) Human monocytes, either noninfected or infected with B. cenocepacia, show decreased pyrin expression at mRNA (MEFV relative copy numbers) and protein levels after nucleofection with siPyrin RNA compared with siControl. (B) B. cenocepacia and S. typhimurium induce similar IL-1β mRNA expression in human monocytes nucleofected with siControl and siPyrin RNA. Inflammasome-dependent release of mature IL-1β (C) and active caspase-1 (D) was significantly lower for siPyrin monocytes infected with B. cenocepacia but not with S. typhimurium. Inflammasome-independent IL-8 mRNA synthesis (E) and cytokine release (F) was similar between siControl and siPyrin monocytes infected with B. cenocepacia and S. typhimurium. Data are expressed as mean ± SD; n = 5 independent experiments. B.c., B. cenocepacia; NT, noninfected; siCtr, siControl; S.t., S. typhimurium.
FIGURE 2
FIGURE 2
Pyrin downregulation decreases IL-1β release by THP-1 cells infected with B. cenocepacia. THP-1 cells, stably expressing siControl or siPyrin, were infected with B. cenocepacia, and cell culture medium was collected 6 and 24 h later for cytokine detection by ELISA. (A) THP-1 stably suppressing pyrin (siPyrin) showed significantly lower release of inflammasome-dependent IL-1β at 24 h postinfection compared with the THP-1 expressing siControl. (B) Inflammasome-independent IL-8 release was equal between siControl- and siPyrin-expressing THP-1 cells. (C and D) The same cells as in (A) were infected with S. typhimurium for 4 h. There was no difference in IL-1β and IL-8 release between siControl- and siPyrin-expressing THP-1 cells. Data are expressed as mean ± SD; n = 4 independent experiments. B.c., B. cenocepacia; NT, noninfected; siCtr, siControl; S.t., S. typhimurium.
FIGURE 3
FIGURE 3
Pyrin overexpression enhances IL-1β release in response to B. cenocepacia. (A) THP-1 cells, stably expressing YFP–pyrin, showed significantly higher inflammasome-dependent IL-1β release in response to B. cenocepacia compared with the control THP-1 cells. (B) Inflammasome-independent IL-8 release was similar between control and YFP–pyrin–overexpressing THP-1 cells infected with B. cenocepacia. (C and D) The same cells were infected with S. typhimurium for 4 h. There was no difference in IL-1β and IL-8 release between THP-1 varying in pyrin levels. Data are expressed as mean ± SD; n = 4 independent experiments. B.c., B. cenocepacia; NT, noninfected; S.t., S. typhimurium.
FIGURE 4
FIGURE 4
THP-1 cells overexpressing pyrin show higher cell death in response to B. cenocepacia infection. THP-1 cells with different levels of pyrin expression were left untreated or infected with B. cenocepacia for 6 and 24 h and with S. typhimurium for 4 h. Cell culture medium was used to measure percentage of LDH release relative to the total LDH content in the cell. (A) Pyrin overexpression leads to a significant increase in LDH release 6 and 24 h postinfection with B. cenocepacia compared with the control THP-1 cells. In contrast, pyrin knockdown did not affect LDH release by THP-1 cells infected with B. cenocepacia. (B). LDH release in response to S. typhimurium infection was equally high between control THP-1 cells overexpressing pyrin (C) and knockdown of pyrin (D). Data are expressed as mean ± SD; n = 3 independent experiments. B.c., B. cenocepacia; NT, noninfected; siCtr, siControl; S.t., S. typhimurium.
FIGURE 5
FIGURE 5
ASC important to B. cenocepacia response. THP-1 cells, plain and overexpressing YFP–ASC, were infected with B. cenocepacia for 6 and 24 h; cell culture medium was collected and analyzed for the IL-1β and IL-8 release by ELISA. Data are expressed as mean ± SD; n = 4 independent experiments. (A) Inflammasome-dependent IL-1β release in response to B. cenocepacia was significantly higher in cells overexpressing ASC. (B) Inflammasome-independent IL-8 release in response to B. cenocepacia infection was similar between cells differing in ASC expression. (C) ASC is colocalized with pyrin after infection with B. cenocepacia. THP-1 cells were immunoprecipitated with anti-ASC Ab and immunoblotted for ASC and pyrin. (D) Pyrin is colocalized with ASC after infection with B. cenocepacia. YFP–pyrin from stably transfected THP-1 cells was captured on magnetic beads conjugated with anti-EGFP Ab, washed, and eluted. Cell extract (CE), flow through (F), and eluate from untreated (−) or B. cenocepacia treated (+) cells were immunoblotted. ASC was bound to YFP–pyrin only after infection with B. cenocepacia. B.c., B. cenocepacia; NT, noninfected.
FIGURE 6
FIGURE 6
ASC and pyrin are colocalized with intracellular B. cenocepacia. (A) Transmission electron microscope imaging of human monocytes infected with B. cenocepacia (B.c.) shows that this bacteria is taken up intracellularly (black arrow). (B) THP-1 cells stably expressing YFP–pyrin were infected with an RFP-expressing K56.2 clinical isolate of B. cenocepacia. Intracellular RFP-Burkholderia (B.c.) was colocalized with YFP–pyrin (white arrow) just prior to microscopic evidence of cell death. This is a representative image from Supplemental Video 1. (C) THP-1 cells stably expressing YFP–ASC were infected with RFP-B. cenocepacia. Intracellular B. cenocepacia (B.c.) induced rapid pyroptosis after colocalization with YFP–ASC (white arrow). This is a representative image from Supplemental Video 2.
FIGURE 7
FIGURE 7
T6SS is important in inflammasome activation in response to B. cenocepacia. Human monocytes (HM) (A) and THP-1 cells (B) were infected with B. cenocepacia WT and with mutants of type VI and type III secretion systems, respectively (T6SS and T3SS). Cells were lysed (CE) for detection of the levels of pro–IL-1β synthesis upon bacterial infection, and cell culture medium was used to determine whether mature IL-1β and active caspase-1 are released, as a signature of inflammasome activation. All bacteria types equally induce pro–IL-1β synthesis in human monocytes (A) and THP-1 cells (B). However, mutation of T6SS reduces inflammasome activation and caspase-1 and IL-1β release into cell culture medium. NT, noninfected.
FIGURE 8
FIGURE 8
Internalization of live B. cenocepacia is important for T6SS-dependent inflammasome activation. Human monocytes were infected with live or heat-killed B. cenocepacia for 6 h. To inhibit bacteria internalization, monocytes were pretreated for 30 min with 5 μg/ml cytochalasin D. NF-κB activation, measured by mRNA expression for IL1B (A), IL8 (D), and IL-8 release (E), shows no difference between WT and T6SS mutants of B. cenocepacia. Pro–IL-1β synthesis was also equal between experimental groups, based on immunoblot of cell lysates (C). Inflammasome-dependent IL-1β release was significantly decreased in monocytes infected with T6SS mutant, and also when bacteria internalization was inhibited by cytochalasin D or when bacteria were killed (B). Cell death, measured by LDH release, correlates well with IL-1β release (F). Data are expressed as mean ± SD; n = 3 independent experiments. *p < 0.05, **p < 0.005. cD, cytochalasin D; K, heat-killed B. cenocepacia; L, live B. cenocepacia; NT, noninfected.
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