Hepatic Ischemia and Reperfusion Injury in the Absence of Myeloid Cell-Derived COX-2 in Mice

Cyclooxygenase-2 (COX-2) is a mediator of hepatic ischemia and reperfusion injury (IRI). While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2^flox conditional knockout mouse (COX-2^−M/−M) to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2^−M/−M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2^−M/−M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2^−M/−M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2^−M/−M and WT mice. COX-2^−M/−M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9) expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2^−M/−M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s) other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.     

阿斯匹林对T H P-1来源巨噬细胞M M P g的表达及其活性的影响

目的：探讨阿司匹林对基质金属蛋白酶9（Matrix Metalloproteinases,MMPs）表达及其活性的影响。方法：应用MTT法观察阿司匹林对THP-1来源巨噬细胞增殖的影响;逆转录聚合酶链反应检测阿司匹林对体外培养的THP-1细胞基质金属蛋白酶9mRNA表达的影响,应用明胶酶谱法观察阿司匹林对体外培养的THP-1来源巨噬细胞基质金属蛋白酶9活性的影响。结果：与对照组相比,不同浓度的阿司匹林（150、3000、600、1200μmol／l）对THP-1采源巨噬细胞增殖无影响;不同浓度的阿司匹林可以抑制PMA诱导的THP-1细胞基质金属蛋白酶9mRNA的水平及其活性水平,且呈浓度依赖性。结论：阿司匹林可以抑制PMA诱导的THP-1巨噬细胞基质金属蛋白酶9的表达,并可能通过这一机制影响动脉粥样硬化斑块的稳定性．     

Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.Matrix metalloproteinases (MMP)² are extracellular hydrolytic enzymes involved in a variety of processes including connective tissue cleavage and remodeling (1–3). All 23 members of the family are able to cleave simple peptides derived from connective tissue components such as collagen, gelatin, elastin, etc. A subset of MMPs is able to hydrolyze more resistant polymeric substrates, such as cross-linked elastin, and partially degraded collagen forms, such as gelatin and type IV collagens (4). Intact triple helical type I–III collagen is only attacked by collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14 (5–12). Although the detailed mechanism of cleavage of single chain peptides by MMP has been largely elucidated (13–19), little is known about the process of hydrolysis of triple helical collagen. In fact, triple helical collagen cannot be accommodated in the substrate-binding groove of the catalytic site of MMPs (9).All MMPs (but MMP-7) in their active form are constituted by a catalytic domain (CAT) and a hemopexin-like domain (HPX) (20–22). The CAT domain contains two zinc ions and one to three calcium ions. One zinc ion is at the catalytic site and is responsible for the activity, whereas the other metal ions have structural roles. The isolated CAT domains retain full catalytic activity toward simple peptides and single chain polymeric substrates such as elastin, whereas hydrolysis of triple helical collagen also requires the presence of the HPX domain (9, 23–25). It has been shown that the isolated CAT domain regains a small fraction of the activity of the full-length (FL) protein when high amounts of either inactivated full-length proteins or isolated HPX domains are added to the assay solution (9). Finally, it has been shown that the presence of the HPX domain alone alters the CD spectrum of triple helical collagen in a way that suggests its partial unwinding (26, 27). It is tempting to speculate that full-length collagenases attack collagen by first locally unwinding the triple helical structure with the help of the HPX domain and then cleaving the resulting, exposed, single filaments (9, 28).Until 2007, three-dimensional structures of full-length MMPs had been reported only for collagenase MMP-1 (29–31) and gelatinase MMP-2 (32). The structures of the two proteins are very similar and show a compact arrangement of the two domains, which are connected by a short linker (14 and 20 amino acids, respectively). It is difficult to envisage that rigid and compact molecules of this type can interact with triple helical collagen in a way that can lead to first unwinding and then cleavage of individual filaments. It has been recently suggested that such concerted action could occur much more easily if the two domains could enjoy at least a partial conformational independence (9). Slight differences in the reciprocal orientation of the CAT and HPX domains of MMP-1 in the presence (29) and absence (30, 31) of the prodomain were indeed taken as a hint that the two domains could experience relative mobility (29).Two recent solution studies have shown that conformational independence is indeed occurring in gelatinase MMP-9 (33) and elastase MMP-12 (34), whereas the x-ray structure of the latter (34) is only slightly less compact than those of MMP-1 (29–31) and MMP-2 (32). Among MMPs, MMP-9 features an exceptionally long linker (68 amino acid) (33, 35), which in fact constitutes a small domain by itself (the O-glycosylated domain) (33), and therefore, this inspiring observation can hardly be taken as evidence that conformational freedom is a general characteristic of the two-domain MMPs. MMP-12 features a much more normal 16-amino acid linker, thereby making more probable a general functional role for this conformational freedom (34). However, both MMP-9 and MMP-12 retain their full catalytic activity against their substrates even when deprived of the HPX domain (9). Therefore, the question remains of whether conformational freedom is also a required characteristic for those MMPs that are only active as full-length proteins, i.e. collagenases. Interestingly, the three collagenases (MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all MMPs. Demonstrating or negating the presence of conformational freedom in one of these collagenases would therefore constitute a significant step forward to formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution (34) have shown that a combination of NMR relaxation studies and small angle x-ray scattering (SAXS) is enough to show the presence and the extent of the relative conformational freedom of the two domains of MMPs. Here we apply the same strategy to full-length MMP-1 and show that sizable conformational freedom is indeed experienced even by this prototypical collagenase, although somewhat less pronounced than that observed for MMP-12.     

Matrix Metalloproteinase-9 Gene Polymorphisms in South-West Iranian Multiple Sclerosis (MS) Patients

Russian Journal of Genetics - Background: Matrix metalloproteinases (MMPs) play important role in Multiple Sclerosis (MS), an autoimmune and neuro-inflammatory disease. It has been demonstrated...     

基质金属蛋白酶-9及金属蛋白酶组织抑制剂-1在孕鼠胎盘形成中期的表达模式

目的探讨基质金属蛋白酶-9(MMP-9)及金属蛋白酶组织抑制剂-1(TIMP-1)在孕鼠胎盘形成中期的表达模式。方法 HE染色观察孕鼠第9天至第14天(D9~D14)胎盘形态结构的变化,同时免疫组织化学法检测相应天数胎盘中MMP-9和TIMP-1的表达情况。结果 HE染色结果显示孕鼠D11时胎盘的3层结构开始形成,包括蜕膜区(Dec)、海绵滋养细胞层(Sp)和迷路区(Lab);随着妊娠天数的增加,Dec区域逐渐缩小,Sp和Lab区域增大;免疫组化表明,MMP-9和TIMP-1均表达于细胞浆和细胞核中,MMP-9强表达于D9和D10胎盘的外胎盘锥中,D10~D13的Dec、Sp和D11~D13的滋养巨细胞中。MMP-9在D11的Lab,D12的Dec和Lab,D13的Dec、Sp、Lab区域中的阳性细胞的积分光密度值与D14胎盘中对应值比较,差异有统计学意义(P0.05);TIMP-1在D9~D12的Dec区域中的阳性细胞的积分光密度值与D14胎盘中对应值比较,差异有统计学意义(P0.05);MMP-9/TIMP-1在D9、D11的Dec及D12的Lab区域中的比值与D14胎盘中对应值比较,差异有统计学意义(P0.05),在D10、D12的Dec和D13的Lab区域中的比值与D14胎盘中对应值比较,差异有高度统计学意义(P0.01)。结论 MMP-9和TIMP-1在孕鼠胎盘中的协同表达可能参与调节胎盘的形成。     

Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca2+

Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly¹⁵⁹ to Val, Ser²⁰⁸ to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca²⁺ (10 mm) was comparable with MMP-1 (wild type), but in low Ca²⁺ (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca²⁺. Varying the concentration of Zn²⁺ had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca²⁺ concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca²⁺ concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca²⁺ both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.     

Expression of Suppressor of Cytokine Signaling 1 (SOCS1) Impairs Viral Clearance and Exacerbates Lung Injury during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1^−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1^−/−IFN-γ^−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1^−/−IFN-γ^−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ^−/− or WT animals. Enhanced viral clearance in SOCS1^−/−IFN-γ^−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1^−/−IFN-γ^−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8⁺ splenocytes from SOCS1^−/−IFN-γ^−/− mice were sufficient to rescue RAG1^−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1^−/− mice reconstituted with SOCS1^−/−IFN-γ^−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.     

Molecular Design of a Highly Selective and Strong Protein Inhibitor against Matrix Metalloproteinase-2 (MMP-2)

Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (K_i^app = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC₅₀ > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t_½ = 30 min), APP-IP-TIMP-2 (t_½ ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.     

Matrix metalloproteinase (MMP-2 and MMP-9) and steroid receptor expressions in feline mammary tumors

We evaluated the presence of estrogen (ER) and progesterone (PR) receptors, and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) enzymes in 18 feline mammary tubulopapillary carcinomas. Immunohistochemistry was performed to localize ER, PR, MMP-2 and MMP-9 in situ. Western blotting and zymographic analyses also were performed to investigate the presence and activities of MMP-2 and MMP-9 enzymes in fresh tissue homogenates. ER immune expression was detected in five samples (27.7%) and PR was positive in sixteen (88.8%) samples. Diffuse cytoplasmic staining of MMP-2 and MMP-9 in neoplastic mammary epithelial cells, stromal fibroblasts and inflammatory cell was evident. MMP-2 and MMP-9 staining was observed also in metastasizing neoplastic cells within lymphatic vessels. MMP-2 and MMP-9 enzymes and their activities in fresh tumor homogenates were demonstrated by zymography. Comparison of MMP-9 gelatinolytic bands from tumor samples and controls revealed a statistically significant difference. We demonstrated elevated MMP-9 and MMP-2 levels in tumor samples by Western blotting; analysis of protein bands revealed 1.9-to-3 fold increase in MMP-9 in tumor samples and the difference was statistically significant. Our results suggest that the expression of MMP-9 can be an important indicator for tumor progression and the possible metastatic nature of feline tubulopapillary carcinomas.     

Matrix metalloproteinase (MMP-2 and MMP-9) and steroid receptor expressions in feline mammary tumors

We evaluated the presence of estrogen (ER) and progesterone (PR) receptors, and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) enzymes in 18 feline mammary tubulopapillary carcinomas. Immunohistochemistry was performed to localize ER, PR, MMP-2 and MMP-9 in situ. Western blotting and zymographic analyses also were performed to investigate the presence and activities of MMP-2 and MMP-9 enzymes in fresh tissue homogenates. ER immune expression was detected in five samples (27.7%) and PR was positive in sixteen (88.8%) samples. Diffuse cytoplasmic staining of MMP-2 and MMP-9 in neoplastic mammary epithelial cells, stromal fibroblasts and inflammatory cell was evident. MMP-2 and MMP-9 staining was observed also in metastasizing neoplastic cells within lymphatic vessels. MMP-2 and MMP-9 enzymes and their activities in fresh tumor homogenates were demonstrated by zymography. Comparison of MMP-9 gelatinolytic bands from tumor samples and controls revealed a statistically significant difference. We demonstrated elevated MMP-9 and MMP-2 levels in tumor samples by Western blotting; analysis of protein bands revealed 1.9-to-3 fold increase in MMP-9 in tumor samples and the difference was statistically significant. Our results suggest that the expression of MMP-9 can be an important indicator for tumor progression and the possible metastatic nature of feline tubulopapillary carcinomas.     

Extended Exenatide Administration Enhances Lipid Metabolism and Exacerbates Pancreatic Injury in Mice on a High Fat,High Carbohydrate Diet

This study expanded upon a previous study in mice reporting a link between exenatide treatment and exocrine pancreatic injury by demonstrating temporal and dose responses and providing an initial mechanistic hypothesis. The design of the present study included varying lengths of exenatide exposure (3, 6 weeks to 12 weeks) at multiple concentrations (3, 10, or 30 µg/kg) with multiple endpoints (histopathology evaluations, immunoassay for cytokines, immunostaining of the pancreas, serum chemistries and measurement of trypsin, amylase, and, lipase, and gene expression profiles). Time- and dose-dependent exocrine pancreatic injury was observed in mice on a high fat diet treated with exenatide. The morphological changes identified in the pancreas involved acinar cell injury and death (autophagy, apoptosis, necrosis, and atrophy), cell adaptations (hypertrophy and hyperplasia), and cell survival (proliferation/regeneration) accompanied by varying degrees of inflammatory response leading to secondary injury in pancreatic blood vessels, ducts, and adipose tissues. Gene expression profiles indicated increased signaling for cell survival and altered lipid metabolism in exenatide treated mice. Immunohistochemistry supported gene expression findings that exenatide caused and/or exacerbated pancreatic injury in a high fat diet environment potentially by further increasing high fat diet exacerbated lipid metabolism and resulting oxidative stress. Further investigation is required to confirm these findings and determine their relevance to human disease.     

Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP)

Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G_q-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.     

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor

Background

The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation.

Methods

Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence.

Results

Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells.

Conclusions

In this work we confirm that placental leukocytes from human term pregnancies are able to secrete large amounts of MMP-9, and that the production of the enzyme it is enhanced by labor. We also demonstrate for the first time that endogenous MMP-3 plays a major role in MMP-9 activation process. These findings support the contribution of placental leukocytes to create the collagenolytic microenvironment that induces the rupture of the fetal membranes during human labor.     

Expression of genes for metalloproteinases (MMP-1, MMP-2, MMP-9, and MMP-12) associated with psoriasis

Using real-time polymerase chain reaction (RT-PCR), we measured mRNA amounts of matrix metalloproteinases (MMPs): MMP-1, MMP-2, MMP-9, and MMP-12 genes in psoriatic lesions and unaffected skin of the same patients. We observed significant (about 15-fold) increase in the expression level of matrix metalloproteinase MMP-1 and MMP-12 genes associated with psoriasis. The results of our studies of MMP gene expression in cultured primary human keratinocytes treated with interleukin (IL-17) have shown upregulation of MMP gene expression both in cultured keratinocytes and in psoriatic skin lesions. Therefore, upregulation of MMP genes in the skin affected by psoriasis could result from IL-17 effects on skin cells.     

Soluble IL-2Rα (sCD25) Exacerbates Autoimmunity and Enhances the Development of Th17 Responses in Mice

A strong association exists between mutations at the IL2 receptor alpha chain (CD25) gene locus and susceptibility to a number of T cell driven autoimmune diseases. Interestingly, the presence of certain CD25 susceptibility alleles has been correlated with significantly increased levels of the soluble form of CD25 (sCD25) in the serum of patients. However, the functional consequences, if any, of this observation are unknown. We have demonstrated that elevated levels of sCD25 in vivo resulted in exacerbated experimental autoimmune encephalomyelitis (EAE) and enhanced antigen-specific Th17 responses in the periphery. sCD25 exerted its effects early during the Th17 developmental programme in vitro, through inhibiting signalling downstream of the IL-2R. Although, sCD25 did not interact with the T cell surface, it specifically bound to secreted IL-2 demonstrating its ability to act as a decoy receptor for IL-2 in the T cell microenvironment. These data identify the ability of sCD25 to promote autoimmune disease pathogenesis and enhance Th17 responses through its ability to sequester local IL-2.     

Lipopolysaccharide Preconditioning Protects Hepatocytes from Ischemia/Reperfusion Injury (IRI) through Inhibiting ATF4-CHOP Pathway in Mice

Background

Low-dose lipopolysaccharide (LPS) preconditioning-induced liver protection has been demonstrated during ischemia-reperfusion injury (IRI) in several organs but has not been sufficiently elucidated underlying causal mechanism. This study investigated the role of low-dose LPS preconditioning on ATF4-CHOP pathway as well as the effects of the pathway on tissue injury and inflammation in a mouse model of liver partial-warm IRI.

Methods

LPS (100 µg/kg/d) was injected intraperitoneally two days before ischemia. Hepatic injury was evaluated based on serum alanine aminotransferase levels, histopathology, and caspase-3 activity. The ATF4-CHOP pathway and its related apoptotic molecules were investigated after reperfusion. The role of LPS preconditioning on apoptosis and ATF4-CHOP pathway was examined in vitro. Moreover, the effects of the ATF4-CHOP pathway on apoptosis, Caspase-12, and Caspase-3 were determined with ATF4 small interfering RNA (siRNA). Inflammatory cytokine expression was also checked after reperfusion. Inflammatory cytokines and related signaling pathways were analyzed in vitro in macrophages treated by LPS preconditioning or ATF4 siRNA.

Results

LPS preconditioning significantly attenuated liver injury after IRI. As demonstrated by in vitro experiments, LPS preconditioning significantly reduced the upregulation of the ATF4-CHOP pathway and inhibited Caspase-12 and Caspase-3 activation after IRI. Later experiments showed that ATF4 knockdown significantly suppressed CHOP, cleaved caspase-12 and caspase-3 expression, as well as inhibited hepatocellular apoptosis. In addition, in mice pretreated with LPS, TNF-α and IL-6 were inhibited after reperfusion, whereas IL-10 was upregulated. Similarly, low-dose LPS significantly inhibited TNF-α, IL-6, ATF4-CHOP pathway, NF-κB pathway, and ERK1/2 in high-dose LPS-stimulated macrophages, whereas IL-10 and cytokine signaling (SOCS)-3 suppressor were induced. Importantly, ATF4 siRNA is consistent with results of LPS preconditioning in macrophages.

Conclusions

This work is the first time to provide evidence for LPS preconditioning protects hepatocytes from IRI through inhibiting ATF4-CHOP pathway, which may be critical to reducing related apoptosis molecules and modulating innate inflammation.     

Glutaredoxin 2 (Grx2) Gene Deletion Induces Early Onset of Age-dependent Cataracts in Mice

Glutaredoxin 2 (Grx2) is an isozyme of glutaredoxin1 (thioltransferase) present in the mitochondria and nucleus with disulfide reductase and peroxidase activities, and it controls thiol/disulfide balance in cells. In this study, we investigated whether Grx2 gene deletion could induce faster age-related cataract formation and elucidated the biochemical changes effected by Grx2 gene deletion that may contribute to lens opacity. Slit lamp was used to examine the lenses in Grx2 knock-out (KO) mice and age-matched wild-type (WT) mice ages 1 to 16 months. In the Grx2 null mice, the lens nuclear opacity began at 5 months, 3 months sooner than that of the control mice, and the progression of cataracts was also much faster than the age-matched controls. Lenses of KO mice contained lower levels of protein thiols and GSH with a significant accumulation of S-glutathionylated proteins. Actin, αA-crystallin, and βB2-crystallin were identified by Western blot and mass spectroscopy as the major S-glutathionylated proteins in the lenses of 16-month-old Grx2 KO mice. Compared with the WT control, the lens of Grx2 KO mice had only 50% of the activity in complex I and complex IV and less than 10% of the ATP pool. It was concluded that Grx2 gene deletion altered the function of lens structural proteins through S-glutathionylation and also caused severe disturbance in mitochondrial function. These combined alterations affected lens transparency.