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.
The authors declare that they have no competing interest.
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.
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.
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)12950_2012_272_MOESM1_ESM.doc
Additional file 2: Figure S1: Establishment of TaqMan tests for expression analysis of individual inflammasome genes in rats. TaqMan assays for rat NLRP-3 (A), NLRC-4 (B), AIM2 (C), IL-1β (D), IL-18 (E), ASC (F), TNF-α (G), and rS6 (H) were established. Representative melting curves for each gene are depicted. Amplification of respective target gene sequences were performed under the same cycling conditions using a melting temperature of 95°C and amplification/extension temperatures of 60°C, respectively. The individual primer combinations used in each test are depicted in Table 1. (PDF 342 KB)
Additional file 3: Figure S2: Establishment of TaqMan tests for expression analysis of individual inflammasome genes in mouse. TaqMan assays for murine NLRP-1b (A), NLRP-1c (B), NLRP-3 (C), NLRC-4 (D), AIM2 (E), IL-1β (F), IL-18 (G), ASC (H), TNF-α (I), IL-6 (J), IL-10 (K), IFN-γ (L) and GAPDH (M) were established. Amplification of the different gene sequences were essentially performed under the same cycling conditions each using a melting temperature of 95°C and amplification/extension temperatures of 60°C, respectively. Representative melting curves for each gene are depicted. The individual primer combinations used in each test are depicted in Table 1. (PDF 261 KB)
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)12950_2012_272_MOESM4_ESM.tiff
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)12950_2012_272_MOESM5_ESM.tiff
Additional file 6: Figure S5: Induction of acute phase response in mice after LPS injection. (A) Mice were subjected to LPS stimulation and livers samples were taken after 2 and 6 hours. The expression of pro-inflammatory, anti-inflammatory cytokines as well as indicated inflammasome components was analyzed by qRT-PCR. (B) Expression analysis of the monocyte chemotactic protein (MCP-1, CCL2) in livers after LPS injection. For detailed statistical analysis of this set of experiments see Additional file 1. (TIFF 167 KB)
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)12950_2012_272_MOESM7_ESM.tiff
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Hsu LC, Ali SR, McGillivray S, Tseng PH, Mariathasan S, Humke EW, Eckmann L, Powell JJ, Nizet V, Dixit VM, Karin M: A NOD2-NALP1 complex mediates caspase-1-dependent IL-1β secretion in response to Bacillus anthracis infection and muramyl dipeptide. Proc Natl Acad Sci USA. 2008, 105: 7803-7808. 10.1073/pnas.0802726105. PubMedCentralCrossRefPubMed
Kanneganti TD, Ozören N, Body-Malapel M, Amer A, Park JH, Franchi L, Whitfield J, Barchet W, Colonna M, Vandenabeele P, Bertin J, Coyle A, Grant EP, Akira S, Núñez G: Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature. 2006, 440: 233-236. 10.1038/nature04517. CrossRefPubMed
Ting JP, Lovering RC, Alnemri ES, Bertin J, Boss JM, Davis BK, Flavell RA, Girardin SE, Godzik A, Harton JA, Hoffman HM, Hugot JP, Inohara N, Mackenzie A, Maltais LJ, Nunez G, Ogura Y, Otten LA, Philpott D, Reed JC, Reith W, Schreiber S, Steimle V, Ward PA: The NLR gene family: a standard nomenclature. Immunity. 2008, 28: 285-287. 10.1016/j.immuni.2008.02.005. PubMedCentralCrossRefPubMed
Stienstra R, van Diepen JA, Tack CJ, Zaki MH, van de Veerdonk FL, Perera D, Neale GA, Hooiveld GJ, Hijmans A, Vroegrijk I, van den Berg S, Romijn J, Rensen PC, Joosten LA, Netea MG, Kanneganti TD: Inflammasome is a central player in the induction of obesity and insulin resistance. Proc Natl Acad Sci USA. 2011, 108: 15324-15329. 10.1073/pnas.1100255108. PubMedCentralCrossRefPubMed
Greenwel P, Schwartz M, Rosas M, Peyrol S, Grimaud JA, Rojkind M: Characterization of fat-storing cell lines derived from normal and CCl4-cirrhotic livers. Differences in the production of interleukin-6. Lab Invest. 1991, 65: 644-653. PubMed
Greenwel P, Rubin J, Schwartz M, Hertzberg EL, Rojkind M: Liver fat-storing cell clones obtained from a CCl4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43. Lab Invest. 1993, 69: 210-216. PubMed
Borkham-Kamphorst E, Stoll D, Gressner AM, Weiskirchen R: Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis. Biochem Biophys Res Commun. 2004, 32: 413-423. CrossRef
Arias M, Sauer-Lehnen S, Treptau J, Janoschek N, Theuerkauf I, Buettner R, Gressner AM, Weiskirchen R: Adenoviral expression of a transforming growth factor-β1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats. BMC Gastroenterol. 2003, 3: 29-10.1186/1471-230X-3-29. PubMedCentralCrossRefPubMed
Liedtke C, Bangen JM, Freimuth J, Beraza N, Lambertz D, Cubero FJ, Hatting M, Karlmark KR, Streetz KL, Krombach GA, Tacke F, Gassler N, Riethmacher D, Trautwein C: Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury. Gastroenterology. 2011, 141: 2176-2187. 10.1053/j.gastro.2011.08.037. CrossRefPubMed
Xu LL, Warren MK, Rose WL, Gong W, Wang JM: Human recombinant monocyte chemotactic protein and other C-C chemokines bind and induce directional migration of dendritic cells in vitro. J Leukoc Biol. 1996, 60: 365-371. PubMed
- Expression analysis of inflammasomes in experimental models of inflammatory and fibrotic liver disease
Sorina Georgiana Boaru
- BioMed Central
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