Bone marrow and nonbone marrow Toll like receptor 4 regulate acute hepatic injury induced by endotoxemia.

BACKGROUND: Toll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model). METHODS: Wild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later. RESULTS: Compared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1β and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed. CONCLUSIONS: These results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury

LPS challenge to hepatocytes and resident macrophages.

<p>To investigate the role played by hepatocytes and resident macrophages following LPS challenge, we used chimeric mice that expressed TLR4 either in the liver or in peripheral blood mononuclear cells. Chimerism was confirmed 8-10 weeks following transplantation when at least 95% of the transplanted bone marrow was present. Representative samples of the 3 transplanted groups: WT/WT, TLR4KO/WT, WT/TLR4KO mice are presented in Figure A1 –A3. Following LPS challenge, TLR4 was highly expressed in the leukocyte–immune bone marrow derived cells in both chimeric mice that were transplanted with WT BM, as analyzed by FACS and presented in Figures B1 and B3. However, TLR4 expression was not induced in the leukocytes following LPS in chimeric mice that were transplanted with TLR4KO BM, since the hematopoietic system lacks TLR4 as presented in B2. TLR4 expression in the different groups are stated as mean ±SEM (n=3 in each group) and presented in Figure C. Histological analysis of chimeric mice challenged with LPS is presented in Figure D1-D2. There was diffuse hepatic congestion and focal necrosis associated with diffused neutrophil infiltration in LPS administered WT/WT, TLR4KO/WT mice compared to WT/TLR4KO mice administered LPS in which few neutrophils were observed (D3). Values are mean±SEM (n=3 mice per group) *p>0.05 compared with other groups.</p

A, D: Histological staining using H&E.

<p>The inflammatory hepatic damage following LPS-administration in WT mice (A) compared to TLR4KO mice (D) was notably higher. <b>B, E: TLR4 protein immune staining in LPS treated mice:</b> TLR4 protein expression was upregulated at 4 hours (B) in WT mice compared to (E) no TLR4 immuno staining in TLR4KO. <b>C, F: MPO activity:</b> An enzyme specific for neutrophils in WT and in TLR4KO livers was examined. A significant increase in hepatic MPO activity was observed in LPS administered WT mice. In LPS administered TLR4KO mice, a slight increase in hepatic MPO activity was observed compared to WT liver. <b>G,H: Protein expression of phosphorylated c-JUN and IkB:</b> The hepatic expression of phosphorylated c-JUN was significantly higher in LPS administered WT mice compared with saline administered mice using Western blot, p<0.05. LPS administration increased c-Jun activity in WT mice compared to LPS treated TLR4KO mice, p<0.05 (G). The activation of the NFkB signaling pathway was higher in LPS administered WT compared to TLR4KO mice (H). IkB hepatic expression was significantly lower compared with saline administered mice, (p<0.05). In LPS administered TLR4KO mice there were no significant differences in the IkB hepatic expression between the LPS administered and the non LPS administered mice. Values are mean±SEM (n=6 mice per group).</p

A: Kaplan-Meier survival curve.

<p>While 5/6 mice (83%) were treated with LPS died in less than 8 hours and the sixth died within the first 24 hours, all TLR4KO mice (6/6) survived following LPS (Kaplan-Meier, p < 0.001). <b>B: Real-time PCR analysis of hepatic TLR4 mRNA expression:</b> The results are shown as the fold-change of the relative quantity of TLR4 mRNA normalized to TATA-box mRNA values. TLR4 mRNA expression significantly increased 3-fold compared with saline administration in WT mice, 4 hours following LPS administration (*p ≤ 0.005); results are expressed as mean ±SE, n=4 in each group.</p

Serum biochemical analysis.

<p>Figs. A, D.: The serum LDH and AST level increased significantly in LPS-administered WT mice compared with saline administered mice. In LPS administered TLR4KO, the increase in serum enzyme levels was much lower compared with saline administered and compared with LPS administered WT mice (4 and 24 hours). Values are mean±SEM with n=6 mice per group *p<0.01 compared with other groups. Figs. B, E: The serum and hepatic Il-1β level increased significantly in WT mice following LPS compared with saline treated mice (p< 0.01). TLR4KO mice hepatic IL-1β level was significantly lower compared with WT LPS treated mice (p< 0.05). Values are mean±SEM (n=6 mice per group). Figs. C, F: The serum and hepatic TNFα level increased significantly in LPS WT-treated mice (p< 0.01). In TLR4KO mice the hepatic TNFα level was significantly lower compared with WT LPS treated mice (p< 0.005) and serum TNFα rose by a very small amount. Values are mean±SEM (n=6 mice per group). </p

A,C: TNFα and IL-1β protein expression in chimeras in serum and liver.

<p>In all chimeras, both TNFα and IL-1β increased significantly at 4 hours post LPS challenge. The proinflammatory cytokine levels in chimeras expressing TLR4 in the liver were found to be significantly higher than the levels expressed in TLR4KO chimera livers ( * p<0.05 vs. WT/WT LPS treated ; + p<0.01 vs. + WT/WT saline-treated). Values are mean±SEM ( n=6 mice per group). <b>B,D: Hepatic TNFα and IL-1β expression:</b> No differences were observed between hepatic TNFα and IL-1β whether the livers expressed TLR4 or KO for TLR4. Values are mean±SEM (n=3 mice per group). <b>E: TLR2 and TLR4 mRNA expression level in a Huh7 cell line:</b> Macrophages expressed a significantly higher level of TLR4 and TLR2 mRNA compared with the hepatocyte cell line (p < 0.0001). <b>F: TLR2 and TLR4 mRNA expression following LPS treatment:</b> LPS induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while there was no change in TLR2 mRNA expression. The results are shown as the fold-change of the relative quantity of TLR mRNA normalized to GAPDH mRNA values; the control cells were assigned a value of 1 and results are normalized to controls. Each point represents the mean ±SEM of a set of data determined by at least 5 experiments.</p

Gene expression of biomarkers of injury and immunostaining of TLR4 in patient auricles.

<p>(A-D) TLR4 and NOX4 are activated resulting in elevated TNF-α in the auricles. Auricles obtained during CABG surgery presented higher expression of TLR4 (P<0.03), BNP (P<0.05), NOX4 (P<0.03), and TNF-α (P = 0.135) in reduced versus ‘preserved EF’. (E) TLR2 expression was similar in both groups. (F1-F2) Representative photographs show double-immunostaining of Troponin I and TLR4 in ‘preserved EF’ auricle. (F3-F4) Representative photographs show double-immunostaining of troponin I and TLR4 in the ‘reduced EF’ auricle. TLR4 staining revealed an apparent upregulation in all ‘reduced EF’ patients examined compared to ‘preserved EF’ patients' tissue.</p

Baseline characteristics of the study population (A) and medication (B).

<p>Baseline characteristics of the study population (A) and medication (B).</p

Comparison of a 12-month post-surgical follow up to the baseline data.

<p>Legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120175#pone.0120175.t002" target="_blank">Table 2</a>:</p><p>*P = 0.03 vs baseline</p><p>Comparison of a 12-month post-surgical follow up to the baseline data.</p

miR expression.

<p>A. miR320a expression in the serum of patients undergoing CABG surgery, with reduced EF was higher than preserved EF, * P <0.001. B. The miR15a, is presented in Fig B and noted as unchanged.</p