There is no doubt that the understanding of inflammasome regulation and function will potentially offer great opportunities to interfere with the process of inflammation, fibrogenesis and tumorigenesis. Irrespectively of the inflammatory stimuli analyzed, the activation of the inflammasome machinery is a well orchestrated process in which pattern recognitions receptors recognize distinct danger signals and in turn activate signaling pathways that subsequently initiate the inflammatory response resulting in activation of different pathways such as NF-κB and MAPK and culminating in transcriptional activation of a large number of different inflammation-associated genes. It is superfluous to mention and confirmed in our study that this regulatory network is highly complex and that the individual inflammasome protein complexes might be simultaneously expressed and activated in the same cell type at the same time in the inflamed tissue. Recent studies have characterized and classified distinct molecular agents and pathways for several sensor proteins and have identified a multitude of inflammatory ligands of both endogenous and exogenous origin that drive inflammasome activities in healthy and diseased organs . However, there is only limited knowledge of inflammasome regulation and function in healthy liver and various liver diseases.
To allow quantification of mRNAs of genes that are directly linked to the activity of the inflammasomes, we have established qRT-PCR assays for mRNA quantification of NLRP-3, NLRP-1, NLRC4/NALP4, AIM2, IL-1β, IL-18, ASC, TNF-α, IL-6, IL-10 in mice and rat (Additional file 2: Figure S1, Additional file 3: Figure S2).
We have shown that the expression of NLRP-1, NLRP-3 and AIM2 in cultured primary cell population is mainly restricted to KC, LSEC and pMF, while the expression in HSC is only low and virtually absent in primary cultured hepatocytes (Figure 1). However, the challenge with LPS demonstrates that the expression of respective genes involved in formation of inflammasomes can be induced to high levels in HSC/MFB (Figure 2) and hepatocytes (Figure 6) suggesting that all liver cell types tested are in principal able to mediate inflammasome activities. Interestingly, NLRP-3, NLRP-1 and NLRC4/NALP4 are inducible in CFSC-2G in response to LPS stimulation (Figure 2) demonstrating that the different inflammasome branches can be simultaneously activated at the same time in this cell entity. However, during activation there seems to be a clear sequential order because NLRP-3 expression was found to be highest already one hour after LPS challenge, while the elevated expression of NLRP-1 and NLRC4/NALP4 followed one hour later.
During experimental liver insult induced by ligature of the common bile duct (BDL), the expression of all four core inflammasomes (i.e. NLRP-1, NLRP-3, NLRC4/NALP4, and AIM2) were simultaneously activated at the mRNA (Figures 3) and protein level (Figures 5 and 6). Also the single or repeatedly application of CCl4 resulted in a simultaneous increase of all four inflammasomes in liver (Figure 4, Figures 5 and 6) suggesting that inflammatory stimuli induce a highly complex network of biological responses in residential and infiltrating cells in which all inflammatory branches are integrated.
When mice were injected with LPS, the expression of NLRP-3 was induced at both mRNA and protein levels (Figure 5A) confirming previous reports . LPS is a prototypical ligand for the Toll like receptor 4 (TLR4) that upon activation induces the production of pro-inflammatory cytokines through activation of the NF-κB pathway [36, 37]. In addition, we observed a strong induction/activation (i. e. phosphorylation) of other genes in that disease model which are involved in inflammatory reactions and recovery from endotoxic shock including LCN2, caspase-3, NF-κB, JNK, pSTAT1, and STAT3 [35, 38]. A comparable activation pattern of all these genes was observed when normal liver was challenged with Con A (Figure 5B, 5C). Since Con A injection leads to immune-mediated liver injury and release of several cytokines (e.g. TNF-α, IFN-γ) triggering liver damage , these findings suggest that irrespectively of the stimuli triggering the inflammatory response, the subsequent changes within the liver end up in similar molecular alterations and correlate with the activation of the NLRP-3 inflammasome.
Presently, we do not know if the induction/activation of the diverse target genes and pathways occur in an orchestrated way with NLRP-3 or if the activation of these genes is mandatory to stimulate NLRP-3 expression. However, based on our experimentation, it is reasonable to speculate that the expression of inflammasome components is directly linked to the activation of NF-κB. It is known that under the condition of BDL, rats have an overall constitutive activation of NF-κB in the liver . Also carbon tetrachloride exposure in mice leads to activation of NF-κB . Likewise, the application of the lectin Con A and endotoxin LPS induces activation and nuclear translocation of NF-κB [36, 42]. Therefore, it will be interesting to test if the administration of inhibitors of canonical or non-canonical NF-κB signaling such as the thiol-reactive quinol and putative thioredoxin inhibitor PMX464 or the lack of factors necessary to activate/phosphorylate NF-κB is suitable to interfere or blunt expression of inflammasome genes.
The finding that purified hepatocytes alone do not express inflammasome components (Figure 1) but induce their transcription after appropriate challenge with inflammatory stimuli such as LPS (Figure 6) demonstrate that the presence of immune cells per se is not necessary to mediate respective responses in vitro. However, it will be essential to analyze in vivo in more detail if the initiation of inflammasome activity in inflamed liver tissue is mainly triggered by influx of neutrophils, monocytes and other immune cells, is a capacity of liver residential cells or is the outcome of both processes. Based on our findings, we suggest that infiltrating cells as well as liver residential cells have capacity to induce inflammasome expression after appropriate trigger within the liver. Most likely, all primary hepatic cell entities are capable to induce inflammasome expression and act in conjunction with infiltrating cells that may vary in the different experimental settings.
The fact that the immortalized cirrhotic fat storing cell line CFSC-2G induces the inflammasome machinery after challenge with LPS (Figure 2) further demonstrates that immortalization is not sufficient to blunt inflammasome activity.
Definitely, we are still at the beginning in understanding the regulation of inflammasomes in different disease models and far away to understand the functions of the individual components. The fact that the expression of the different inflammasome branches in liver become simultaneously activated during hepatic inflammation and their linkage to the activation of general molecular transcriptions factors (e.g. NF-κB) further strengthens the notion that there are several master key switches of inflammasome activity. It will be interesting and challenging to unravel these interactions and to identify specific regulatory control points that might be suitable for pharmacological intervention.