Interleukin-1α and Interleukin-1β play a central role in the pathogenesis of fulminant hepatic failure in mice

<div><p>Background and aims</p><p>Fulminant hepatitis failure (FHF) is marked by the sudden loss of hepatic function, with a severe life-threatening course in persons with no prior history of liver disease. Interleukin (IL)-1α and IL-1β are key inflammatory cytokines but little is known about their role in the development of FHF. The aim of this study was to assess the involvement of IL-1α and IL-1β in the progression of LPS/GalN-induced FHF.</p><p>Methods</p><p>WT, IL-1α or IL-1β deficient mice were injected with LPS/GalN. Blood and liver tissue were collected at different time points, FHF related pathways were examined.</p><p>Results</p><p>After FHF induction the survival of both IL-1α and IL-1β KO mice was longer than that of WT mice. Lower serum liver enzyme levels, demonstrated reduced hepatic injury in the IL-1α and IL-1βKO mice. Histologically detected liver injury and apoptotic hepatocytes were significantly reduced in the IL-1αand IL-1βKO mice compared to WT mice. Reduced hepatic IkB levels and upregulated NFκB activity in WT mice remained inhibited in IL-1α and IL-1β KO mice. Hepatic expression levels of TNFα and IL-6 were significantly increased in WT mice but not in IL-1α and IL-1β KO mice.</p><p>Conclusions</p><p>IL-1α and IL-1β play a central role in the pathogenesis of LPS/GalN-induced FHF. These interleukins are associated with the activation of NFκB signaling, upregulation of the pro-inflammatory cytokines and liver damage and apoptosis. Since neither IL-1α nor IL-1β depletions completely rescued the phenotype, we believe that IL-1α and IL-1β have a similar and probably complementary role in FHF progression.</p></div

IκB protein levels in the livers of WT IL-1α and IL-1β KO mice after LPS/GalN injection.

<p>WT IL-1α and IL-1β KO mice were injected with LPS/GalN at time point 0. Mice were sacrificed at the indicated time points and proteins were purified from their livers. IκB protein levels were analyzed using western blot analysis.</p

Primers list.

<p>Primers list.</p

Deficiency of IL-1α or IL-1β prolonged survival of mice following the induction of FHF compared to WT mice.

<p>WT IL-1α and IL-1β KO mice were injected with either saline or LPS/GalN at time point 0. Mice were inspected every 30 minutes and the survival rates were calculated as percentage of mice survived from the total number of mice in the group (n = 5 in the saline injected group and n = 8 in the LPS/GalN injected groups).</p

Deficiency of IL-1α or IL-1β delayed FHF-associated liver damage compared to WT mice.

<p>Liver specimens were obtained from WT, IL-1α and IL-1β KO mice, 4 and 5 hours after the injection of LPS/GalN. Representative images of H&E-stained sections are shown. Apoptotic bodies (marked with arrows) were observed in livers of WT mice 4 hours after FHF induction (Fig 3A), while less damage and better preserved liver architecture were seen in IL1α in IL-1β KO mice at the same time post-FHF induction (Fig 3C and E). However, 5h following the injections, hepatic damage was also seen in the livers of the IL-1α and Il-1β KO mice.</p

Significant decrease in apoptosis was detected in the IL-1α or IL-1β deficient mice compared to WT mice after FHF induction.

<p>(A) WT IL-1α and IL-1β KO mice were injected with LPS/GalN at time point 0. Mice were sacrificed at the indicated time points and proteins were purified from their livers. The hepatic expression of the active, cleaved, Caspase 3 was detected using western blot analysis. (B) WT, IL-1α and IL-1β KO mice livers were harvested 5 hours after LPS/GalN injection and imbedded in paraffin blocks. Apoptotic cells were identified using the DeadEndTM flourometric TUNEL system.</p

9-cis β-carotene enriched diet inhibited foam cell formation <i>ex-vivo.</i>

<p>Cholesterol content in the cells was measured after Folch extraction and colorimetric reaction. Values are means ±SE, n = 5. * <i>P</i><0.05 as measured by student t-test (A). One representative picture of Oil Red O staining of macrophages isolated from one mouse in each group (B). Lipid droplets colored in red. </p

9-<i>cis</i> β-carotene and its metabolites inhibited foam cell formation <i>in-vitro</i>.

<p>Oil Red O staining of Raw264.7 cells loaded with minimally modified LDL. 10μM of 9-cis β-carotene, all-trans β-carotene or <i>Dunaliella</i> extract with Tween served as a control (A). 5μM of 9-cis retinoic acid, retinol or retinal with DMSO serves as a control (B). Lipid droplets colored in red. </p

Retinol production in Hepa1-6 cells.

<p>HPLC chromatogram shows retinol production in Hepa1-6 cells after 24-hour incubation with 9-cis β-carotene. Total retinol content after control treatment (A) and after 9-cis β-carotene incubation (B). Separation was conducted on C18 column and detection at 325 nm. </p

Alterations in hepatic IL-1α and IL-1β expression in IL-1α and IL-1β KO compare to WT mice after LPS/GalN injection.

<p>WT, IL-1α and IL-1β KO mice were injected with LPS/GalN at time point 0. Mice were sacrificed at the indicated time points and RNA was purified from their livers. Hepatic IL-1α and IL-1β expression levels were evaluated using qRT-PCR (n = 3 in each group at each time point). Results are expressed as mean ± standard error. Differences between groups were assessed using the analysis of variance (T-TEST).</p