TREM2 expression promotes liver and peritoneal M2 macrophage polarization in mice infected with Schistosoma japonicum.

Schistosomiasis is a tropical parasitic disease that damages the liver and poses a serious threat to human health. Macrophages play a key role in the development of liver granulomas and fibrosis by undergoing polarization from M1 to M2 type during schistosomiasis. Therefore, regulating macrophage polarization is important for controlling pathological changes that occur during this disease. Triggering receptor expressed on myeloid cells 2 (TREM2) expressed on the surface of macrophages, dendritic cells and other immune cells has been shown to play a role in inhibiting inflammatory responses and regulating M2 macrophage polarization, however its role in macrophage polarization in schistosomiasis has not been investigated. In this study, we confirmed that TREM2 expression was upregulated in the livers and peritoneal macrophages of mice infected with Schistosoma japonicum. Moreover, the TREM2 expression trend correlated with the expression of M2 macrophage polarization-related molecules in the liver tissues of S. japonicum-infected mice. Using Trem2-/- mice, we also showed that Trem2 deletion inhibited Arg1 and Ym1 expression in liver tissues. Trem2 deletion also increased the number of F4/80 + CD86+ cells in peritoneal macrophages of infected mice. In summary, our study suggests that TREM2 may be involved in M2 macrophage polarization during schistosomiasis.

Toxoplasma gondii excreted-secreted antigens suppress Foxp3 promoter activity via a SP1-dependent mechanism.

Toxoplasma gondii excreted-secreted antigens (ESA) could result in adverse outcomes of pregnancy including abortion, stillbirth, foetal infection or teratogenesis in mice during early stage of pregnancy. Defective generation or function of regulatory T cells (Tregs) may account for those adverse pregnancy outcomes. Forkhead box p3 (Foxp3), which is the key transcriptional factor of Tregs, modulates its development and maintains inhibitory function. We previously demonstrated that ESA inhibited Foxp3 expression by attenuating transforming growth factor ß RII/Smad2/Smad3/Smad4 pathway. In this study, we propose to study the role of ESA on the activity of Foxp3 promoter and explore potential mechanisms. We demonstrated that ESA suppressed Foxp3 promoter activity using dual-luciferase reporter assay. ESA functioned at -443/-96 region of Foxp3 promoter to suppress its activity using truncated fragments of Foxp3 promoter. Further analysis revealed that suppressive role of ESA on Foxp3 promoter activity is related to specificity protein 1 (SP1). Transfection of expression plasmid of pcDNA3.1-SP1 could restore the down-regulation of Foxp3 induced by ESA. In conclusion, this study provides a new mechanism by which ESA could inhibit the Foxp3 promoter activity via SP1.

rSJYB1 inhibits collagen type I protein expression in hepatic stellate cells via down-regulating activity of collagen α1 (I) promoter.

YB1 is a negative regulator in liver fibrosis. We wondered whether SJYB1, a homologous protein of YB1 from Schistosoma japonicum, has an effect on liver fibrosis in vitro. Recombinant SJYB1 (rSJYB1) protein was expressed in a bacterial system and purified by Ni-NTA His·Bind Resin. A human hepatic stellate cell line, the LX-2 cell line, was cultured and treated with rSJYB1. The role of rSJYB1 on LX-2 cells was then analysed by Western blot and luciferase assay. We succeeded in expressing and purifying SJYB1 in a bacterial system and the purified rSJYB1 could be recognized by S japonicum-infected rabbit sera. Western bolt analysis showed that rSJYB1 inhibited the expression of collagen type I, but had little effect on α-smooth muscle actin (α-SMA). Further analysis revealed that rSJYB1 inhibited the activity of collagen α1 (I) (COL1A1) promoter and functioned at -1592/-1176 region of COL1A1 promoter. Our data demonstrate that rSJYB1-mediated anti-fibrotic activity involves inhibiting the activity of COL1A1 promoter and subsequently suppressing the expression of collagen type I in hepatic stellate cells.

Toxoplasma gondii excreted-secreted antigens suppress Foxp3 via PI3K-AKT-mTOR signaling pathway.

Toxoplasma gondii excreted-secreted antigens (ESA) cause spontaneous abortion or fetal teratogenesis during the pregnancy in mice, especially in the early stage. Those adverse pregnancy outcomes are due to the deficit in regulatory T cells (Tregs). Forkhead box P3 (Foxp3), a critical transcription factor, modulates Tregs differentiation and its function. Besides, phosphatidylinositol 3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) signaling network is implicated in interfering with Foxp3 induction. We previously demonstrated that ESA diminished the number of Tregs and inhibited its function. And ESA suppressed Foxp3 expression via the attenuation of transforming growth factor ß RII/Smad2/Smad3/Smad4 pathway. The current study aimed to investigate whether the PI3K-AKT-mTOR signaling network is involved in Foxp3 downregulation induced by ESA. We found that ESA upregulated PI3K, P-AKT, mTOR, and P-mTOR. Knockdown of PI3K cooperated with ESA to restore Foxp3 expression mediated by ESA. This suppressive role of ESA on Foxp3 expression was abrogated by AKT inhibitor. In addition, neutralization of Toll-like receptor 4 could restore the expression of Foxp3, PI3K, and its downstream effectors induced by ESA. Collectively, the findings indicated that ESA inhibited Foxp3 expression via the upregulation of PI3K-AKT-mTOR signaling pathway.

rSjP40 protein promotes PPARγ expression in LX-2 cells through microRNA-27b.

miR-27b is reported to participate in the proliferation and differentiation of hepatic stellate cells (HSCs) and to regulate fat metabolism of rat HSCs by targeting retinoid X receptor α. Our previous study also indicated that the recombinant P40 protein from Schistosoma japonicum (rSjP40) inhibited the activation of HSCs. In this study, we observed the expression of miR-27b in rSjP40-treated LX-2 cells and explored its potential mechanisms. Quantitative real-time PCR showed that rSjP40 inhibits the expression of miR-27b in LX-2 cells. Further results obtained by Western blot and dual-luciferase reporter assay confirmed that miR-27b regulates peroxisome proliferator-activated receptor γ (PPARγ) expression in rSjP40-treated LX-2 cells by targeting the 3'-UTR of PPARγ. 5-AZA-2'-deoxycytidine (5-AZA-dC), which inhibits methylation of HSCs, partially reversed rSjP40-induced down-regulation expression of miR-27b in LX-2 cells. 5-AZA-dC also partially reversed rSjP40-induced up-regulation expression of PPARγ in LX-2 cells. The increased expression of PPARγ in rSjP40-treated LX-2 cells may be partially due to miR-27b methylation. Therefore, our study provides further insight into the mechanism by which rSjP40 inhibits HSC activation and provides a basis for future study of the blocking effect of rSjP40 in liver fibrosis.-Zhu, D., Lyu, L., Shen, P., Wang, J., Chen, J., Sun, X., Chen, L., Zhang, L., Zhou, Q., Duan, Y. rSjP40 protein promotes PPARγ expression in LX-2 cells through microRNA-27b.

Schistosoma japonicum soluble egg antigen inhibits TNF-α-induced IL-34 expression in hepatic stellate cells.

Hepatic fibrosis is characterized by the activation of the main collagen-producing cells of the liver, hepatic stellate cells, and is associated with inflammation. Although the involvement of numerous inflammatory cytokines has been reported, IL-34 in particular has recently been identified as a profibrotic factor in the development of hepatic fibrosis. Previous studies have found that schistosome eggs can lead to transcriptional downregulation of fibrosis-associated genes, and based on this evidence, we attempted to investigate whether or not IL-34 is regulated by soluble egg antigen (SEA). Our findings testified that SEA inhibited TNF-α-induced expression of IL-34 at both the mRNA and protein levels. Furthermore, results from reporter assays and qPCR experiments demonstrated that SEA impaired the activation of NF-κB triggered by TNF-α, as well as the transcription of downstream genes. More importantly, SEA decreased the phosphorylation and degradation of IκBα induced by TNF-α, two events that are hallmarks of canonical NF-κB activation. In conclusion, our results suggest that, in hepatic stellate cells, SEA impairs NF-κB activation and thereby inhibits TNF-α-induced IL-34 expression. These findings reveal a previously unidentified target and signaling pathway that support SEA's involvement in hepatic fibrosis and provide a new clue to guide ongoing research into the anti-fibrotic effects of SEA.

rSjP40 suppresses hepatic stellate cell activation by promoting microRNA-155 expression and inhibiting STAT5 and FOXO3a expression.

Activation of hepatic stellate cells (HSCs) is the central event of the evolution of hepatic fibrosis. Schistosomiasis is one of the pathogenic factors which could induce hepatic fibrosis. Previous studies have shown that recombinant Schistosoma japonicum egg antigen P40 (rSjP40) can inhibit the activation and proliferation of HSCs. MicroRNA-155 is one of the multifunctional noncoding RNA, which is involved in a series of important biological processes including cell development, proliferation, differentiation and apoptosis. Here, we try to observe the role of microRNA-155 in rSjP40-inhibited HSC activation and explore its potential mechanisms. We found that microRNA-155 was raised in rSjP40-treated HSCs, and further studies have shown that rSjP40 enhanced microRNA-155 expression by inhibiting STAT5 transcription. Up-regulated microRNA-155 can down-regulate the expression of FOXO3a and then participate in rSjP40-inhibited expression of α-smooth muscle actin (α-SMA) and collagen I. Furthermore, we observed microRNA-155 inhibitor could partially restore the down-regulation of FOXO3a, α-SMA and collagen I expression in LX-2 cells induced by rSjP40. Therefore, our research provides further insight into the mechanism by which rSjP40 could inhibit HSC activation via miR-155.

microRNA-146a is involved in rSjP40-inhibited activation of LX-2 cells by targeting Smad4 expression.

Previous studies have demonstrated that the recombinant Schistosoma japonicum protein P40 (rSjP40) could inhibit activation of hepatic stellate cells (HSCs) through the TGF-ß1/Smads signaling pathway. Since multiple microRNAs could play essential roles in HSC activation and in the process of hepatic fibrosis through targeting Smads, we attempted to seek the potential microRNAs that could be involved in rSjP40-induced inhibition of HSC activation. Using the method of quantitative real-time PCR, we found that rSjP40 could induce miR-146a expression in LX-2 cells. The down-regulated expression levels of Smad4 and α-SMA in LX-2 cells induced by rSjP40 were partially restored by an miR-146a inhibitor. miR-146a can be involved in rSjP40-induced inhibition of HSC activation through targeting Smad4. These findings provide us a new idea to explore the potential mechanisms by which rSjP40 could regulate the process of hepatic fibrosis.

Excreted-secreted antigens of Toxoplasma gondii inhibit Foxp3 via IL-2Rγ/JAK3/Stats pathway.

Toxoplasma gondii excreted-secreted antigens (ESA) could lead to the fetal abortion especially in the early stage of pregnancy. Deficit in regulatory T cells is a critical event in the fetal abortion. Transcription factor forkhead box p3 (Foxp3) mediates differentiation and functional roles on regulatory T cells. Previously, we revealed that ESA inhibited Foxp3 through the suppression of transforming growth factor-ß type II receptor, phosphorylation of Smad2, Smad3, and Smad4. Knockdown of Smad2 collaborated with ESA to further inhibit Foxp3. The decrease in Foxp3 caused by ESA reversed via forced expression of Smad2, Smad3, and Smad4, respectively. In this study, we investigate whether other signaling pathways are implicated in ESA-induced Foxp3 downregulation. EL4 cells were cultured and stimulated with ESA. Interleukin-2 receptor γ (IL-2Rγ) chain, Janus kinase 3 (JAK3), signal transducer and activator of transcription 5 (Stat5), Stat3, phosphorylation of Stat5 and Stat3 were assayed by Western blot analysis. Phosphorylation of Stat5 and Stat3 was further measured by cellular immunofluorescence. The expression plasmid of pcDNA3.1-Stat3 and pcDNA3.1-Stat5b was constructed, respectively. The concentration of interleukin-2 (IL-2) in the culture supernatants was detected by enzyme-linked immunosorbent assay. ESA inhibited the level of JAK3, phosphorylation of Stat5 and Stat3, and Foxp3 in EL4 cells. The suppressive effects of ESA on Foxp3 were attenuated by forced expression of Stat5 and Stat3. In addition, ESA suppressed IL-2Rγ in EL4 cells, while IL-2Rγ agonist could markedly reverse the diminished Foxp3 caused by ESA. Furthermore, ESA directly influenced the expression of IL-2Rγ, rather than the availability of IL-2 indirectly. ESA suppressed the level of Foxp3 via inhibiting IL-2Rγ/JAK3/Stats signaling pathway in EL4 cells.

Chinese 1 strain of Toxoplasma gondii excreted-secreted antigens negatively modulate Foxp3 via inhibition of the TGFßRII/Smad2/Smad3/Smad4 pathway.

Toxoplasma gondii is an opportunistic intracellular parasite and is considered an important aetiological factor in the process of abortion, especially as occurs in early gestation. Chinese 1 strain of T. gondii is a dominant genotype prevalent in China. Although it is known that early foetal resorption triggered by RH strain of T. gondii is attributable to immune mechanisms rather than its direct effect in uterus, the underlying mechanism of the abortion caused by Chinese 1 strain remains unclear. This study was designed to investigate the effect of excreted-secreted antigens (ESA) of Chinese 1 strain of T. gondii on the expression of forkhead box transcription factor (Foxp3) as it pertains to early pregnancy and abortion. ESA caused a marked inhibition in the expression of Foxp3 both in vivo and in vitro. In addition, ESA negatively modulated Smad2 and Smad3 at the posttranslational level. Smad2 siRNA cooperated with ESA to further suppress the level of Foxp3. This inhibitory effect on Foxp3 expression was partially abrogated by overexpression of Smad2, Smad3 and Smad4. Additionally, ESA attenuated the expression of TGFßRII, whereas TGFßRII agonist could profoundly reversed the decreased Foxp3 triggered by ESA. Collectively, the findings suggested that ESA restricted Foxp3 expression by inhibiting TGFßRII/Smad2/Smad3/Smad4 signalling, ultimately resulting in abortion.

The N-terminal pY33XL motif of CaPsy2 is critical for the function of protein phosphatase 4 in CaRad53 deactivation, DNA damage-induced filamentation and virulence in Candida albicans.

Protein phosphatase PP4 is composed of one catalytic subunit and one or two regulatory subunits and conserved in eukaryotic cells. The catalytic subunit CaPph3 forms a complex with the regulatory subunit CaPsy2, which dephosphorylates activated CaRad53 during adaptation to and recovery from MMS-mediated DNA damage. We show here that the N-terminal Y33A mutation of CaPsy2 blocks the interaction between CaPph3 and CaRad53, the deactivation of CaRad53 and the morphologic switch in recovery from genotoxic stress. In Saccharomyces cerevisiae, the ScPph3-ScPsy2-ScPsy4 complex functions to dephosphorylate γH2A. In this study, we show that CaPsy4 is a functional homolog of ScPsy4 and not involved in the deactivation of CaRad53 or CaHta, the ortholog of H2A. However, deletion of CaPSY4 causes C. albicans cells a sensitivity to genotoxic reagents and a defect in DNA damage-induced filamentation. CaPsy4 interacts with both CaPph3 and CaPsy2, but the function of CaPsy4 is independent of CaPph3 and CaPsy2 in response to genotoxic stress. C. albicans cells lacking CaPPH3, CaPSY2 or CaPSY4, and C. albicans cells carrying the Y33A mutation of CaPSY2, show increased virulence to mice. Therefore, PP4 plays a negative role in regulating the DNA damage-induced filamentation and the virulence in C. albicans.

Lipopolysaccharide Preconditioning Induces an Anti-inflammatory Phenotype in BV2 Microglia.

Increasing evidence indicates that endotoxin tolerance is an essential immune-homeostatic response to repeated exposure to lipopolysaccharide (LPS) that induces a state of altered responsiveness in macrophage, resulting in repression of pro-inflammatory gene expression and increased expression of factors that mediate the resolution of inflammation. In this study, quantitative real-time polymerase chain reaction and Western blot for M1 and M2 markers were performed to characterize phenotypic changes of BV2 microglia. We found that the cytokine and chemokine expression during endotoxin tolerance were mostly similar to those found during M2 polarization. We further examined the expression of M1 and M2 markers in CD11b+ BV2 by double immunofluorescent staining. The expression of M2 markers (CD206) increased, whereas the expression of M1 (CD54) markers reduced during endotoxin tolerance. Moreover, expression of different transcription factor, known for their function in the regulation of pro- and anti-inflammatory reaction, was also different. Our data demonstrate that repeat LPS treatment activates a differentiation program that leads to microglial polarization toward M2-like phenotype.

CaTip41 regulates protein phosphatase 2A activity, CaRad53 deactivation and the recovery of DNA damage-induced filamentation to yeast form in Candida albicans.

Phosphorylation and dephosphorylation of the checkpoint kinase CaRad53 is crucial for fungal cells in response to genotoxic stresses. The protein phosphatase 2A (PP2A) CaPph3/CaPsy2 phosphatase complex is involved in CaRad53 dephosphorylation in Candida albicans. In view of the role of ScTip41/ScTap42 in regulating PP2A phosphatases in Saccharomyces cerevisiae, we have explored the function of CaTip41 in C. albicans. Here, we show that CaTIP41 is a functional ortholog of ScTIP41 in the sensitivity of S. cerevisiae cells to rapamycin. Deletion of CaTIP41 causes C. albicans cells to be sensitive to DNA damaging agents, methylmethane sulfonate (MMS) and cisplatin, and resistant to both rapamycin and caffeine. Accordingly, expression of CaTip41 increases in response to MMS and cisplatin. In addition, C. albicans cells lacking CaTIP41 show a delay in the recovery from MMS-induced filamentation to yeast form, decreased PP2A activity and a defect in deactivation of CaRad53 during recovery from DNA damage. Through yeast two-hybrid assay we show that CaTip41 interacts with either CaPph3, CaPsy2 or CaTap42. Therefore, CaTip41 plays regulatory roles in both the CaRad53 deactivation during recovery from DNA damage and the target of rapamycin signaling pathway.

Recombinant T2 RNase protein of Schistosoma japonicum inhibits expression of α-SMA in LX-2 cells.

Recombinant T2 RNase glycoprotein, which showed a certain degree of homology to Omega-1 from Schistosoma mansoni eggs, was expressed in adult worms of Schistosoma japonicum, but not in eggs of S. japonicum. The direct biological role of the recombinant T2 RNase protein in activation of hepatic stellate cells (HSCs) remains unknown. In the present study, the immortalized human HSC line (LX-2 cells) was treated with the recombinant T2 RNase protein at indicated concentrations for various time points in vitro. The expression levels of α-smooth muscle actin (α-SMA) and Smad4 were detected by Western blot. The results showed that the recombinant T2 RNase protein significantly diminished the expression levels of α-SMA and Smad4 in LX-2 cells. The upregulated expression levels of α-SMA and Smad4 by TGF-ß1 in LX-2 cells were both suppressed by the recombinant T2 RNase protein. These data suggest that the recombinant T2 RNase protein may be a potential target of therapeutic strategy for the treatment of hepatic fibrosis.

Inhibition of IFN-γ-Induced Nitric Oxide Dependent Antimycobacterial Activity by miR-155 and C/EBPß.

miR-155 (microRNA-155) is an important non-coding RNA in regulating host crucial biological regulators. However, its regulatory function in mycobacterium infection remains unclear. Our study demonstrates that miR-155 expression is significantly increased in macrophages after Mycobacterium marinum (M.m) infection. Transfection with anti-miR-155 enhances nitric oxide (NO) synthesis and decreases the mycobacterium burden, and vice versa, in interferon γ (IFN-γ) activated macrophages. More importantly, miR-155 can directly bind to the 3'UTR of CCAAT/enhancer binding protein ß (C/EBPß), a positive transcriptional regulator of nitric oxide synthase (NOS2), and regulate C/EBPß expression negatively. Knockdown of C/EBPß inhibit the production of nitric oxide synthase and promoted mycobacterium survival. Collectively, these data suggest that M.m-induced upregulation of miR-155 downregulated the expression of C/EBPß, thus decreasing the production of NO and promoting mycobacterium survival, which may provide an insight into the function of miRNA in subverting the host innate immune response by using mycobacterium for its own profit. Understanding how miRNAs partly regulate microbicidal mechanisms may represent an attractive way to control tuberculosis infectious.

Enhancement of immune response to a hepatitis C virus E2 DNA vaccine by an immunoglobulin Fc fusion tag.

Neutralizing antibodies and cellular immune response both play essential roles in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for producing neutralizing antibodies against HCV. Here, we constructed a recombinant plasmid, termed pcDNA3.1-E2-Fc, to express HCV E2 with an immunoglobulin Fc fusion tag (E2-Fc). Importantly, we found that the titers of E2-specific IgG from mice immunized with pcDNA3.1-E2-Fc were significantly higher than that from mice immunized with pcDNA3.1-E2. Moreover, pcDNA3.1-E2-Fc immunization could boost E2-specific lymphocyte proliferation and enhance the secretion of IFN-γ by lymphocytes upon in vitro stimulation with soluble E2 compared to pcDNA3.1-E2 immunization. Neutralization assays showed that serum from pcDNA3.1-E2-Fc immunized mice exhibited more effective neutralizing capacity of HCVpp entry into Huh-7 cells compared with that from pcDNA3.1-E2 immunized mice, although both of the sera could inhibit the virus entry. Taken together, our results imply that pcDNA3.1-E2-Fc immunization could enhance E2-specific humoral and cellular immune response in mice and thus provide a promising candidate for the development of an HCV vaccine.

Schistosoma japonicum protein SjP40 inhibits TGF-ß1-induced activation of hepatic stellate cells.

SjP40 is a major egg antigen of Schistosoma japonicum. In the present study, the authors investigated the effect of SjP40 in vitro on transforming growth factor-ß1 (TGF-ß1)- stimulated hepatic stellate cells (HSCs). LX-2, an immortalized human HSC line, was treated with purified recombinant SjP40 (rSjP40) in the presence or absence of TGF-ß1. Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine messenger ribonucleic acid and protein of fibrogenic genes and TGF-ß signaling pathway. The results showed that expression of fibrogenic genes was significantly reduced by rSjP40. Furthermore, rSjP40 also suppressed the TGF-ß1-induced upregulation of Smads and ERK proteins. We also found that the effect of rSjP40 on HSCs was similar to SB431542, an inhibitor of type I TGF-ß receptor. In conclusion, the data suggest that SjP40 attenuates HSC activation, which might be, at least in part, mediated by inhibiting the TGF-ß and ERK signaling pathways.

Genetic interactions between protein phosphatases CaPtc2p and CaPph3p in response to genotoxins and rapamycin in Candida albicans.

In Saccharomyces cerevisiae cells, both of the two PP2C protein phosphatases ScPtc2p and ScPtc3p and the PP4 protein phosphatase ScPph3 are responsible for ScRad53p dephosphorylation after the DNA methylation agent methylmethane sulfonate (MMS)-induced DNA damage. In this study, we show that CaPtc2p is not required for the CaRad53p dephosphorylation during the recovery from DNA damage, as is CaPph3p in Candida albicans. However, deletion of CaPPH3 has an additive effect on the sensitivity of C. albicans cells lacking CaPTC2 to MMS and the DNA synthesis inhibitor hydroxyurea (HU). In addition, deletion of CaPPH3 promotes in vitro filamentation of C. albicans cells. Furthermore, mutation of CaPTC2 is epistatic to that of CaPPH3 in the sensitivity of C. albicans cells to rapamycin. Therefore, CaPtc2p and CaPph3p might play a role in the target of rapamycin (TOR) signaling in C. albicans cells.

TNF-α expression in Schwann cells is induced by LPS and NF-κB-dependent pathways.

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 and activates mitogen-activated protein kinase, which leads to the induction of proinflammatory cytokine gene expression. In vivo, Schwann cells (SCs) at the site of injury may also produce tumor necrosis factor-α (TNF-α). However, the precise mechanism that regulates TNF-α synthesis is still not clear. The nuclear transcription factor-κB (NF-κB) is an important transcription factor which is involved in the regulation of host immune responses. In the present study, we found that LPS possessed a comparable specific activity for activation of NF-κB-dependent gene expression in SCs. We also observed IκB-α/IκB-ß degradation and the nuclear translocation of P65 due to LPS treatments. LPS-elicited TNF-α production in SCs was also drastically suppressed by SN50 (NF-κB inhibitor).