NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis - PubMed (original) (raw)

. 2011 Jun;17(6):1359-72.

doi: 10.1002/ibd.21478. Epub 2010 Sep 24.

Jeffrey Ng, Alan Lueng, Maitham Khajah, Ken Parhar, Yan Li, Victor Lam, Mireille S Potentier, Kelvin Ng, Misha Bawa, Donna-Marie McCafferty, Kevin P Rioux, Subrata Ghosh, Ramnik J Xavier, Sean P Colgan, Jurg Tschopp, Daniel Muruve, Justin A MacDonald, Paul L Beck

Affiliations

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis

Simon A Hirota et al. Inflamm Bowel Dis. 2011 Jun.

Abstract

Background: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18.

Methods: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis.

Results: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota.

Conclusions: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations.

Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

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Figures

Figure 1

Figure 1

Nlrp3−/− mice exhibit enhanced susceptibility to DSS-induced colitis as exhibited by accelerated weight loss (A); reduced hematocrit (B); increased colonic MPO (C). Nlrp3−/− mice exposed to DSS exhibited significantly more inflammation, elevated damage score and overall magnitude of ulceration (D-E; Inflammation score ×10), which is evident in colonic sections stained with H&E. N=8-12; * denotes p<0.05 compared to WT mice; ** denotes p<0.005 compared to WT mice.

Figure 2

Figure 2

Nlrp3−/− mice exhibit enhanced susceptibility to TNBS-induced colitis as exhibited by accelerated weight loss (A) and increased colonic MPO (B). Nlrp3−/− mice exposed to TNBS exhibited significantly more inflammation and elevated damage which is evident in colonic sections stained with H&E (C) and quantified by blinded histological assessment (D). N=8/group; * denotes p<0.05 compared to WT mice.

Figure 3

Figure 3

Cytokine, chemokine and NOx levels are altered in Nlrp3−/− mice exposed to a 7-day course of DSS colitis when compared to WT mice. N=3-8/group; * denotes p<0.05 compared to levels observed in WT mice.

Figure 4

Figure 4

Peritoneal macrophages isolated from Nlrp3-/- mice fail to respond to bacterial muramyl dipeptide (A) Macrophages pre-treated with ultra-pure lipopolysaccharide (LPS, 10ng/mL for 1h) exhibit IL-1β processing and release into culture media when exposed (6h) to muramyl dipeptide (MDP, 10μg/mL), monosodium urate crystals (MSU, 50μg/mL), aluminum adjuvant (Alum, 500μg/mL) and adenosine triphospate (ATP, 5mM). IL-1β processing is inhibited by glyburide (100μM), a selective inhibitor of the NLRP3-inflammasome, and absent in NLRP3-deficient macrophages (B). Western blot are representative of 4 separate experiments.

Figure 5

Figure 5

Chemotaxis and spontaneous apoptosis are altered in BM-derived neutrophils isolated from Nlrp3−/− mice. (A) Overall neutrophil movement in response to PBS (vehicle control), KC (1.25 μM), leukotriene B4 (1 μM), WKTMVm (1 μM) and C5a (1 μM) as assessed with an under-agarose assay. (B) Neutrophil chemotaxis expressed as the number of cells that migrated towards the chemoattractant as a percentage of the total number of cells that moved in any direction. N=4; * denotes p<0.05 compared to WT cells. (C) Spontaneous apoptosis of BM-derived neutrophils after 12h in culture as assessed by flow cytometry. N=4; * denotes p<0.05 compared to WT cells. (D-E) Assessment of PMA(8 μM; D)- and fMLP(8 mM; E)-induced superoxide production in BM-derived neutrophils isolated from WT and Nlrp3−/− mice. N=4, * denotes p<0.05 compared to WT cells. Addition of superoxide dismutase (SOD, 1500 U) was employed as a control to quench superoxide accumulation (D).

Figure 6

Figure 6

Nlrp3−/− mice display altered expression of β-defensin transcripts and exhibit a unique intestinal microbiota. (A) qPCR assessment of β-defensin transcripts in naïve WT and Nlrp3−/− mice expressed as a fold-change. (B) qPCR assessment of β-defensin transcripts in DSS-treated WT and Nlrp3−/− mice expressed as a fold-change (7-day course of 2.5% DSS). _defb1_–mouse β-defensin 1; _defb2_–mouse β-defensin 2; _defb3_–mouse β-defensin 3; _defb4_–mouse β-defensin 4. * denotes p<0.05 compared to naïve WT mice; N=5/group. (C) The antimicrobial capacity of crypt secretions derived from WT and Nlrp3−/− mice. Overnight colony growth in secretion-treated E. coli cultures was compared to untreated cultures and expressed as percent reduction in colony numbers, termed percent killing. * denotes p<0.05 compared to WT mice; N=3. (D) Supervised OPL-S discriminant analysis of TRFLP binary data shows that naïve Nlrp3−/− mice display a unique microbiota when compared to WT littermates. (E) Several unique TRFs were found to significantly distinguish the two groups. N=7/group for TRFLP analysis.

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