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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Journal of Inflammation 1/2012

Expression analysis of inflammasomes in experimental models of inflammatory and fibrotic liver disease

Zeitschrift:
Journal of Inflammation > Ausgabe 1/2012
Autoren:
Sorina Georgiana Boaru, Erawan Borkham-Kamphorst, Lidia Tihaa, Ute Haas, Ralf Weiskirchen
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-9255-9-49) contains supplementary material, which is available to authorized users.
Sorina Georgiana Boaru, Erawan Borkham-Kamphorst contributed equally to this work.

Competing interests

The authors declare that they have no competing interest.

Authors’ contributions

SGB, EBK, LT, and UH performed the experiments and designed figures; RW designed the study and drafted the manuscript. All authors read and approved the final manuscript.

Abstract

During inflammation, the inflammasomes representing a group of multi-protein complexes trigger the biological maturation of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 by proteolytic activation of caspase-1 from its inactive proforms. The individual genes encoding components of the inflammasome machinery are regulated at transcriptional and post-transcriptional levels. Once activated, they drive a wide variety of cellular responses that are necessary to mediate host defense against microbial pathogens and to guarantee tissue homeostasis. In the present work, we have studied the expression of the different inflammasomes in various primary hepatic cell subpopulations, in models of acute inflammation and during experimental liver fibrogenesis. We demonstrate that NLRP-1, NLRP-3 and AIM2 are prominently expressed in Kupffer cells and liver sinusoidal endothelial cells, moderately expressed in periportal myofibroblasts and hepatic stellate cells, and virtually absent in primary cultured hepatocytes. We found that the challenge with the lipopolysaccharides results in a time- and concentration-dependent expression of the NOD-like receptor family members NLRP-1, NLRP-3 and NLRC4/NALP4 in cultured hepatic stellate cells and a strong transcriptional activation of NLRP-3 in hepatocytes. Moreover, we detect a diverse regulatory network of the different inflammasomes in the chosen experimental models of acute and chronic liver insult suggesting that the various inflammasomes might contribute simultaneously to the outcome of inflammatory and fibrotic liver insult, irrespectively of the underlying inflammatory stimulus.
Zusatzmaterial
Additional file 1:Statistics to Figure 2. Expression of inflammasome components in rat cirrhotic fat storing cell line CFSC-2G subjected to LPS stimulation. Statistics to Figure 3: Expression of inflammasome components in rat livers after bile duct ligation (BDL). Statistics to Figure 4: Expression of inflammasome components in rat livers after application of CCl4 . Statistics to Figure 5: Expression of inflammasomes in mice after Con A injection. Statistics to Figure 6: Expression of inflammasomes in primary murine hepatocytes after stimulation with LPS. Statistics to Suppl. Figure 3: Induction of acute phase response in mice after LPS injection. (DOC 120 KB)
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Additional file 4: Figure S3: Analysis of DNA fragmentation after treatment with LPS. (A) TUNEL assay in CFSC-2G cells that were stimulated in short (30 min) and long term (16 h) with 200 ng/ml LPS kept their cellular integrity. The nuclei are counterstained with DAPI. (TIFF 9 MB)
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Additional file 5: Figure S4: Analysis of cell cytotoxicity after treatment with LPS. (A) The standard curve for measurement of LDH activity was established with a preparation of hog LDH. (B) The chosen kit system allows to measure LDH activity in a wide range from 0.001 (Background), to 5,083 (high control). (C) Measurement of LDH in cellular supernatants of CFSC-2G cells that were incubated with indicated concentrations of LPS for indicated time intervals reveal only low cellular toxicity of LPS. (TIFF 946 KB)
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Additional file 7: Figure S6: Expression analysis of Kupffer cells after stimulation with LPS. Primary KC were stimulated with indicated concentration of LPS for 2 hrs and the expression of (A) NLRP-1, NLRC4/NALP4, AIM2, IL-18, ASC and (B) NLRP-3, IL-1β and TNF-α determined by quantitative RT-PCR. In this analysis, the target gene expression without stimulation with LPS was set to 1. (TIFF 222 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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Authors’ original file for figure 7
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Authors’ original file for figure 8
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Authors’ original file for figure 9
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