Study Of The Mechanism And Effect Of Soluble ST2 On Mice Warm Hepatic Ischemia/Reperfusion Injury

1. IntroductionIschemia/reperfusion (I/R) injury is a complicated pathophysiologic process where- by hypoxic organ damage, tissue injury is accentuated following return of blood flow and oxygen delivery to the compromised tissue. Transient episodes of ischemia occur in procedures such as solid organ transplantation, trauma, shock, partial hepatectomy, and diverse surgical procedures, when inflow occlusion or total vascular exclusion is used to minimize blood loss. The pathophysiology of liver I/R injury includes direct cellular damage as the result of ischemic insult as well as delayed dysfunction and le- sion that results from activation of inflammatory pathways. The distal interaction el- ements in the cascade of inflammatory responses resulting in organ damage following hepatic I/R injury have been extensively studied. Activation of Kupffer cells with pr- oduction of reactive oxygen species, up-regulation of the inducible NOS in hepatocy- tes, activation of JNK, up-regualtion proinflammatiory cytokines, and neutrophil acc- umulation have all been identified as contributing events to the inflammation associa- ted damage. The extent to which the initial cellular injury contributes to propagation of the inflammatory response and leads to further tissue damage is poorly understood. Most recent studies show that a key link between the initial damage to cells and the subsequent activation of inflammatory signaling involves release of endogenous dan- ger signals from ischemic cells and disruption of the tissue matrix.TLRs are present on the surface of innate immune cells, including dendritic cells, macrophages, and natural killer cells in vertebrates. TLRs has been found to be the primary form of pattern recognition receptors for the recognition invasion of pathogenic microorganisms, mediating intracellular signal transduction pathways, leading to the activation of transcriptional factors such as NF-κB, and the expression of genes related to inflammatory and immune response. To date, the TLR family at least consists of 11 members, and is found to be an important link between innate immunity and adaptive immunity. Recent evidence suggests that the innate immune system use the TLRs for the recognition of specific molecular patterns that are present in microbial products, such as LPS, LTA, peptidoglycan, bacterial DNA, dsRNA and endogenous danger signals, such as hot shock protein (HSPs), oxygen free radicals (OFR), deoxyribonucleic acid, neurotransimitters, and interleukin too. The liver consists of parenchymal cells (hepatocytes) and nonparenchymal cells (NPC), including Kupffer cells, sinusoidal endothelial cells, stellate cells, and hepatic dendritic cells. Recent studies found that TLR4 is expressed on hepatocytes and NPC, however the levels of TLR4 expression are lower in the liver than other organs.Perhaps more than any of the other TLR family members, TLR4 located on the interface of microbial and sterile inflammation by responding to both bacterial endotoxin and multiple other endogenous ligands. Billiar and his collaborators demonstrated that the NPC, not the hepatocytes, are responsible for recognizing the initial damage including HSPs, fibrinogen, fibronectin, heparin sulfate, high mobility group box 1(HMGB1) from ischemic cells and for activating TLR4-dependent signaling required for I/R- induced injury.ST2 (also known as T1 or DER4) was originally defined in murine fibroblasts as a late response gene induced by serum, and is a member of the interleukin-1 (IL-1) rec- eptor superfamily. ST2 gene is tightly linked to the IL-1R genes region on mouse chr- omosome 1 and human chromosome 2. Despite its similarity to the IL-1R, ST2 does not bind IL-1α, IL- 1β, or IL- 1R antagonist. In the mouse, differential mRNA proce- ssing within the ST2 gene produces two mRNAs of 2.7 Kb and 5 Kb, corresponding to shorter secreted form (soluble ST2 or sST2) and longer, membrane anchored form (ST2L) of the protein. Hence, sST2 is identical with the extracelluar region of ST2L except for an additional nine amino acids, which are present at the C terminus of the molecule. sST2 is expressed in embryonic tissues, mammary tumors and fibroblasts, while ST2L expressed on mast cells and on T helper 2 (Th2) cells but not on T helper 1 (Th1) cells. Recently, another alternatively spliced transcript that is expressed in the stomach, small intestine, and colon, and encodes a novel membrane form of ST2 has been identified in humans. However, its functional role remains to be identified. In addition to the proposed involvement of ST2 in cell growth regulation and embryoge- nesis, the presence of ST2 in hematopoetic cell lines and its homology with the mem- bers of Toll/IL-1 receptor family indicates its potential functional role in immune res- ponses. Treatment with anti-ST2 antibody could induce resistance to L.major infecti- on in the susceptible BABL/C mouse by swithching a detrimental Th2 to a protective Th1 response. However, Anti-ST2 antibody pretreatment exacerbated murine collag- en-induced arthritis (CIA), in which Th1 responses predominate within rheumatoid arthritis synovial T cell subsets. In the conditions of allergic airway inflammation and infection with Shistosoma mansoni eggs or respiratory syncytial virus, anti-ST2 anti- bodies, ST2 fusion proteins or ST2-deficient mice were used to show that ST2L is re- quired for development of effective Th2 responses, characterised by eosinophil recru- itment and production of proinflammatory cytokines IL- 4, IL- 5, and IL-13.Basis on the study of the influence of soluble ST2-IgG fusion protein on LPS res- ponses of mouse macrophages, Xu and his collaborators found that soluble ST2 play an important role in down-regulation macrophage proinflammatory activity. Treatm- ent with ST2 fusion protein before LPS stimulation markedly suppressed the produc- tion of the proinflammatory cytokines TNF-α, IL- 6 and IL-12 from macrophages. Moreover, administration of sST2 significantly reduced LPS-mediated mortality. Re- cent studies showed that through the ST2 binding protein, ST2 was able to directly bind to the surface of murine macrophages and subsequently inhibited the production of inflammatory cytokines. The molecular mechanism of this down-regulation rema- ins to be investigated, however, it appears likely that ST2 serves as a negative regul- ation factor that acts at the cell surface. Among the well-known Toll-like receptors (TLRs), TLR4 has been found to accept LPS as a ligand and subsequently triggers LPS-mediated signaling. Moreover, it also has been found that TLR4 participates in the recognition of endogenous ligands from damaged/ stressed cells, such as heat sh- ock proteins, hyaluronic acid, heparin sulfate, and high-mobility group box 1. Increa- sing evidence shows that ischemia/reperfusion-induced liver inflammation and hepat- ocellular damage is partially dependent on TLR4 signaling.These above findings suggested that sST2 may also be an important negative regu- lation factor of sterile inflammation and cell injury in warm hepatic I/R and the action of sST2 may be also related to TLR4 signaling. To test this hypothesis, we construct- ed a recombinant eukaryotic expression plasmid encoding murine sST2- human IgG1 Fc (sST2-Fc) fusion protein, and investigated its effect on hepatic I/R injury. We de- monstrated here that the expression of this plasmid in vivo effectively prevents hepa- tocellular damage and suppresses the activation of inflammatory cascades. Furtherm- ore, we found that the protection of sST2-Fc in hepatic I/R was also involved in dow- nregulation of TLR4 mRNA expression. These studies may provide a new route for the therapy of ischemic liver injury and other clinical setting involved inflammation diseases.2. Cloning of mouse soluble ST2 and human IgG1αFc gene, and construction of expression vectorThe cDNA of mouse soluble ST2 was reverse transcribed from the total RNA of mouse spleen by RT-PCR, and human IgG1αFc gene cDNA was reverse transcribed from the total RNA of human PBMC by RT-PCR too. The fragment of hIg gene was cloned into the eukaryotic expression system pcDNA 3.1 to construct pcDNA 3.1-Fc vector, then the fragment of soluble ST2 gene was inserted into pcDNA 3.1-Fc vector upstream hIg Fc to construct eukaryotic expression vector pcDNA 3.1- sST2- Fc (psST2- Fc). The recombinant plasmid psST2-Fc is identified by restriction endoniclease digestion and sequence analysis.3. Preparation and biological function of soluble ST2-Fc fusion proteinThe expression and identification of soluble ST2-Fc fusion proteinBy using LipofectamineTM 2000, psST2-Fc plasmid was transfected into CHO cells, and the expression of soluble sST2-Fc fusion protein in CHO cells was confirmed by ELISA and Western blot analysis. The relative amount of soluble ST2-Fc was determined with ELISA. mRNA for recombinant sST2-Fc expression was analyzed by RT-PCR. The soluble ST2-Fc fusion protein from the cultural supernatants was purified by protein A-Sepharose affinity chromatography. The results showed that CHO cells transfected with psST2-Fc secreted sST2-Fc fusion protein into cultural medium. Western blot analysis using a monoclonal antibody specific for ST2 detected a 100 kDa protein, which is in agreement with the recombinant molecular weight of sST2 and hIg Fc. A 929 bp fragment was amplified from the total RNA of psST2-Fc transfected CHO cells by RT-PCR corresponding to anticipated size. The fusion protein display a single band at 100 kDa in SDS-PAGE analysis after purified by protein A Sepharose 4B affinity chromatography. The results showed that the quality of soluble ST2-Fc fusion is suitable for further experiment.Soluble ST2 binds to the surface of peritoneal macrophagesUsing sST2-Fc fusion protein, we analyzed the ability of peritoneal macrophages to bind ST2. The results show that cells incubated with sST2-Fc were markedly stained with anti-hIgG. However, cells incubated with normal hIgG had a staining pattern identical with the isotype control. Importanly, this binding activity was significantly enhanced by overnight exposure to peritoneal macrophages to LPS. Soluble ST2-Fc protein inhibits TNF-a and IL- 6 production from LPS- indu- ced macrophagesBasis on binding of sST2-Fc to peritoneal macrophages was upregulated by LPS tr- eatment, we investigated the reciprocal involvement of sST2-Fc in regulation of the LPS response. By using ELISA, we found that production of TNF-αand IL- 6 in res- pond to LPS in the peritoneal macrophages was markedly suppressed by sST2-Fc tre- atment.4. The warm hepatic I/R injury was attenuated by pretreatment with soluble ST2-FcThe expression of psST2-Fc in vivoBy using a specific ELISA, the concentration of serum sST2-Fc protein was analy- zed in pcDNA 3.1-treated mice and psST2-Fc- transfected mice. Results show that sST2-Fc fusion protein was not detected in non-transfected mice, nor detected in pcDNA3.1- treated mice. However, the serum from sST2-Fc-transfected mice showed a significant increase in the sST2-Fc level (84.8±11.1 ng/ml) at 36 h after psST2-Fc transfection.The effect of psST2-Fc on warm hepatic I/R injurySerum ALT levels and necrotic areas in liver tissue were measured on BALB/C mice subjected to 60 min of liver ischemia and 6 h of reperfusion. Results show that serum ALT, TNF-α, and IL- 6 levels were significantly increased in control mice treated with pcDNA 3.1 or saline underwent ischemia/reperfusion injury. In contrast, mice pretreated with psST2-Fc exhibited lower levels of serum ALT compared with control animals subjected to liver I/R. Liver histological examination was consistent with the serum ALT estimation of liver damage.In vivo expression of psST2-Fc inhibits the activation of Kupffer cells To determine the effect of sST2 on the activation of Kupffer cells in the sinusoidal space of liver after I/R, we first observed its influence on the infiltration of Kupffer cells. By staining Kupffer cells with F4/80 on liver sections, results show that the nu- mber of F4/80+ Cells in psST2-Fc- treated group (156±30 positive cells per high power field ) was significantly reduced as compared with that in pcDNA3.1- treated group (193±26) or saline group (189±28) (P < 0.05). By using real-time RT-PCR, we found that compared with pcDNA 3.1 and salined-treated animals, the expression of TNF-α, IL- 6 was significantly suppressed in treatment with psST2-Fc.TLR4 levels were suppressed in the I/R liver by sST2TLR4 mRNA levels were examined by using real-time RT-PCR in liver tissues after I/R. Compared with sham-treated animals, liver I/R in pcDNA 3.1 or saline- treated animals resulted in an enhanced expression of TLR4 mRNA 3 h after reper- fusion. However, psST2-Fc- treated mice showed minimal increase in TLR4 mRNA levels compared with sham-treated mice after I/R.sST2 modulates inflammatory signaling pathwayTo investigate the possible molecular mechanism of sST2 to down-regulate the gene expression of TNF- a and IL- 6, levels of NF-κB activation were measured by EMSA. We found that NF-κB DNA-binding activity was increased in the ischemic liver 3 h after reperfusion in pcDNA 3.1 or saline-treated control mice, when compa- red with sham- treated mice. Mice pretreated with psST2-Fc exhibited less NF-κB DNA-binding activity.All these results show that soluble ST2 could directly bind to the surface of macro- phages, and then represses the production of proinflammatory cytokine from LPS- induced macrophages. Moreover, we successfully attenuated hepatic reperfusion in- jury after short term warm ischemia by in vivo transfection of plasmid encoding sST2 gene. The protective effect is associated with inactivation of Kupffer cells and the pr- oduction of TNF- a and IL- 6, possibly through the down-regulation of TLR4 expres- sion.5. ConclusionsST2 effectively inhibit the activation of Kupffer cells and the production of TNF-a and IL- 6, possibly through the down-regulation of TLR4 expression. Furthermore, the hepatic reperfusion injury after short term warm ischemia was significantly atten- uated by in vivo transfection of plasmid encoding sST2 gene. It was suggested that pretreatment with sST2 may be effective in settings of ischemic liver injury to mini- mize organ damage and may be useful in other clinical settings related to inflamma- tion diseases.