Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver

Abstract

Aim: To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide (LPS)-induced stimulation in the liver.

Methods: Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5 μg/g bodyweight LPS and sacrificed 2, 4, 6, 18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase (ALT) levels. To determine if LPS stimulation in the liver led to activation of the inflammasome, real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome. Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome, including caspase-1 and two cytokine targets of caspase-1, interleukin (IL)-1β and IL-18.

Results: We found that LPS injection resulted in liver damage as indicated by elevated ALT levels. This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cytokine tumor necrosis factor (TNF)-α in the liver, as well as increased levels of TNFs in serum. We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome, including Nalp3, Nalp1, pannexin-1 and the adaptor molecule apoptosis-associated speck-like, caspase recruitment domain-domain containing protein. We also found increased levels of mRNA and protein for caspase-1, a downstream target of the inflammasome. In addition, LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1, IL-1β and IL-18. Interestingly, substantial baseline expression of pre-IL-1β and pre-IL-18 was found in the liver. Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1β and IL-18 after LPS stimulation.

Conclusion: Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation.

Keywords: Caspase-1; Endotoxin; Interleukin-18; Interleukin-1β; Nod-like receptor.

Figure 1

Lipopolysaccharide stimulation results in liver damage. C57BL/6 wild-type chow-fed mice (three per group) were injected intraperitoneally with lipopolysaccharide (LPS) for 2, 4, 6, 18 or 24 h. Serum was separated from whole blood and analyzed for alanine aminotransferase (ALT), which was significantly increased after 4 h of LPS stimulation. Mean ± SD are shown. ^aP < 0.01.

Figure 2

Inflammation in the liver is seen after lipopolysaccharide stimulation. A: Tumor necrosis factor (TNF)-α mRNA in liver tissue was significantly increased at all time points after lipopolysaccharide (LPS) stimulation. TNF-α mRNA was analyzed by real-time quantitative polymerase chain reaction and normalized to 18S. The values are shown as a fold change to the non-stimulated LPS control; B: Similarly, TNF-α protein was significantly elevated in liver tissue following LPS stimulation as detected by enzyme-linked immunosorbent assay. Protein levels were normalized to total protein concentration in each tissue sample. Mean ± SD are shown. n = 3 for each group (except at 4 h LPS stimulation, where n = 2 due to an outlier), ^aP < 0.01.

Figure 3

Lipopolysaccharide stimulation increased Nalp3 inflammasome mRNA expression in liver tissue. Liver RNA analysis of Nalp3 (A), apoptosis-associated speck-like, caspase recruitment domain-domain containing protein (ASC) (B), caspase-1 (C) and pannexin-1 (D) were analyzed by real-time quantitative polymerase chain reaction. The values were normalized to 18S and are shown as a fold increase to the non-lipopolysaccharide (LPS) stimulated control. Mean ± SD are shown. n = 3 per group, ^aP < 0.01, ^bP < 0.03, ^cP < 0.05.

Figure 4

Lipopolysaccharide stimulation increased interleukin (IL)-1β and IL-18 mRNA, and IL-1RA transcription in the liver. Liver mRNA levels of IL-1β (A), IL-18 (B) and IL-1RA (C) were analyzed by real-time quantitative polymerase chain reaction and normalized to 18S. Mean ± SD are shown. n = 3 per group [except at 2 h lipopolysaccharide (LPS) stimulation, where n = 2 due to outlier], ^aP < 0.01, ^bP < 0.03.

Figure 5

Lipopolysaccharide increased interleukin-1β and interleukin-18 protein. Protein level was detected in liver tissue by enzyme-linked immunosorbent assay for total-interleukin (IL)-1β (pro-IL-1β and cleaved IL-1β) (A), cleaved IL-1β in the liver tissue (B),cleaved IL-1β in the serum (C) and total IL-18 (pro-IL-18 and cleaved IL-18) (D) in liver tissue. The values shown are the fold change compared to non-Lipopolysaccharide (LPS) stimulated control. Protein levels were normalized to total protein concentrations in each tissue sample. Mean ± SD are shown. n = 3 per group (except at 2 h LPS stimulation, where n = 2 due to an outlier), ^aP < 0.01.