Tristetraprolin Regulates TH17 Cell Function and Ameliorates DSS-Induced Colitis in Mice.

TH17 cells have been extensively investigated in inflammation, autoimmune diseases, and cancer. The precise molecular mechanisms for TH17 cell regulation, however, remain elusive, especially regulation at the post-transcriptional level. Tristetraprolin (TTP) is an RNA-binding protein important for degradation of the mRNAs encoding several proinflammatory cytokines. With newly generated T cell-specific TTP conditional knockout mice (CD4CreTTPf/f), we found that aging CD4CreTTPf/f mice displayed an increase of IL-17A in serum and spontaneously developed chronic skin inflammation along with increased effector TH17 cells in the affected skin. TTP inhibited TH17 cell development and function by promoting IL-17A mRNA degradation. In a DSS-induced colitis model, CD4CreTTPf/f mice displayed severe colitis and had more TH17 cells and serum IL-17A compared with wild-type mice. Furthermore, neutralization of IL-17A reduced the severity of colitis. Our results reveal a new mechanism for regulating TH17 function and TH17-mediated inflammation post-transcriptionally by TTP, suggests that TTP might be a novel therapeutic target for the treatment of TH17-mediated diseases.

Tristetraprolin inhibits macrophage IL-27-induced activation of antitumour cytotoxic T cell responses.

IFN-γ-producing cytotoxic T lymphocytes are essential for host defense against viral infection and cancer. Here we show that the RNA-binding tristetraprolin, encoded by Zfp36, is needed for CD8+ T-cell production of IFN-γ in vivo. When activated in vitro, however, IFN-γ production by naive wild type and tristetraprolin-deficient CD8+ T-cells is comparable. IL-27 is overproduced by tristetraprolin-deficient macrophages and increased systemically in tristetraprolin-deficient mice. Tristetraprolin suppresses IL-27 production by promoting p28 mRNA degradation. Importantly, deletion of IL-27 receptor WSX-1 in tristetraprolin-deficient mice (WSX-1/tristetraprolin double knockout) leads to a reduction in cytotoxic T lymphocyte numbers. Moreover, tumor growth is accelerated, not only in tristetraprolin-deficient mice after cytotoxic T lymphocyte depletion, but also in WSX-1/tristetraprolin double knockout mice, with substantial reduction in the number of tumor cytotoxic T lymphocytes. This study describes a regulatory pathway for IL-27 expression and cytotoxic T lymphocyte function mediated by tristetraprolin, contributing to regulation of antitumour immunity.IL-27 is one of a number of cytokines that can induce antitumour CD8+ T cell responses. Here the authors show that TTP, encoded by Zfp36, degrades p28 to inhibit IL-27 production by macrophages and is thereby a negative regulator of the antitumour response.

MCPIP1 Selectively Destabilizes Transcripts Associated with an Antiapoptotic Gene Expression Program in Breast Cancer Cells That Can Elicit Complete Tumor Regression.

The ability of cancer cells to evade apoptosis is dictated by a shift in the balance between proapoptotic and antiapoptotic gene expression programs. Monocyte chemotactic protein-induced protein 1 (MCPIP1) is a zinc-finger RNA binding protein with important roles in mediating inflammatory responses. Overexpression of MCPIP1 in different cancer cell types has been implicated in eliciting an antitumor response, but a direct role of MCPIP1 in apoptosis has not been established. In this study, we demonstrate that MCPIP1 functions as a potent tumor suppressor that induces apoptosis of breast tumor cells by selectively enhancing mRNA decay of antiapoptotic gene transcripts, including Bcl2L1, Bcl2A1, RelB, Birc3, and Bcl3. Mechanistically, MCPIP1 physically interacted with a stem-loop structure in the 3' untranslated region of these transcripts through its PIN domain, causing mRNA destabilization. Furthermore, we found that MCPIP1 expression was repressed in breast tumor cells, and overexpression of MCPIP1 induced apoptosis, whereas its depletion enhanced cancer cell proliferation. Moreover, MCPIP1 induction in vivo resulted in complete regression of established tumors and a significant reduction in metastatic disease. Notably, low MCPIP1 expression in tumor samples from breast cancer patients was strongly associated with poor survival over 13 years of follow-up. Collectively, our results highlight that MCPIP1 is a new tumor suppressor in breast cancer that induces cell death by tipping the balance in favor of proapoptotic gene expression.

Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-κB pathway.

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Suppression of IL-12 production by tristetraprolin through blocking NF-kcyB nuclear translocation.

Tristetraprolin (TTP), an mRNA-binding protein, plays a significant role in regulating the expression of adenylate-uridylate-rich elements containing mRNAs. Mice deficient of TTP (TTP(-/-)) develop a systemic autoimmune inflammatory syndrome characterized by cachexia, conjunctivitis, and dermatitis. IL-12 plays a crucial role in immune defense against infectious and malignant diseases. In this study, we found increased production of IL-12 during endotoxic shock and enhanced Th1 cells in TTP knockout mice. The levels of IL-12 p70 and p40 protein as well as p40 and p35 mRNA were also increased in activated macrophages deficient of TTP. In line with these findings, overexpression of TTP suppressed IL-12 p35 and p40 expression at the mRNA and promoter level, whereas it surprisingly had little effects on their mRNA stability. Our data showed that the inhibitory effects of TTP on p35 gene transcription were completely rescued by overexpression of NF-кB p65 and c-Rel but not by the p50 in activated macrophages. Our data further indicated that TTP acquired its inhibition on IL-12 expression through blocking nuclear translocation of NF-кB p65 and c-Rel while enhancing p50 upon stimulation. In summary, our study reveals a novel pathway through which TTP suppresses IL-12 production in macrophages, resulting in suppression of Th1 cell differentiation. This study may provide us with therapeutic targets for treatment of inflammatory and autoimmune disorders.

Increased Th17 cells in the tumor microenvironment is mediated by IL-23 via tumor-secreted prostaglandin E2.

Tumor cell-derived molecules such as cytokines and lipid mediators play a critical role in inducing chronic inflammation in the tumor microenvironment. We found that Th17 cells were increased in the peripheral blood, spleen, and tumor tissues of mammary gland tumor-bearing mice. The Th17 cell survival factor, IL-23, was also overexpressed in tumor tissues isolated from mice and human breast cancer patients. Soluble molecules secreted from breast tumor cells, but not normal breast epithelial cells, induced IL-23 protein secretion in dendritic cells via induction of p19 mRNA expression. Our data further indicate that tumor-secreted PGE2 through EP2 and EP4 receptors enhanced IL-23 p19 gene transcription through binding to the cAMP-response element in the p19 promoter. Blocking PGE2 synthesis by NS398, a COX2 inhibitor, abrogated the enhancement of p19 expression both in vitro and in vivo. Furthermore, blocking protein kinase A (PKA) by H89 completely abrogated the inductive effects of tumor-conditioned medium and PGE2 on p19 transcription, whereas the cAMP active analog, Forskolin, mimics the PGE2 effect. Taken together, our results indicate that tumor-secreted PGE2 induces IL-23, but not IL-12, production in the tumor microenvironment, leading to Th17 cell expansion. This inductive effect of PGE2 on IL-23 p19 transcription is mediated through cAMP/PKA signaling transduction pathway.

[Effects of cypermethrin exposure during lactation of female mice on testicular steroidogenesis in male baby mice].

OBJECTIVE: To discuss the influence on the testicular steroidogenesis synthesation during baby mice cut-milking period and adult period of male mice by the cypermethrin exposure during lactation of the female mice. METHODS: Fourteen dams of new-born mice were randomly divided into two groups, cypermethrin poisoning group and solution control group. Since the first day after baby delivery, the aternal mice in the cypermethrin poisoning group were given stomach cypermethrin poisoning, using the corn oil as the solution and the poison dose is 25mg! kg, until the 21st day after the delivery when the baby mice has cut-milking. While the control group is given the gavagy of the corn oil of the same volume. In each group, 15 male baby mice were killed at 21st day and 70th day after delivery, respectively. Taking blood from the eye-balls, and separate the testes. Use radioimmunoassay (LIlA) method to measure serum testosterone (T) and estrogen (E2) levels. Use RT-PCR method to measure StAR in the testes and the mRNA expression level of testosterone synthetic enzymes. Use Western blot to measure StAR in the testes and the protein expression level of testosterone synthetic enzymes. RESULTS: Cypermethrin exposure during lactation of the female mice significantly leads to the decrease of serum testosterone of the male baby mice (P < 0.01) and the decrease of the testosterone in the testes (P < 0.05), having no influence of the female hormones (P < 0.05). And the cypermethrin exposure during lactation makes the significant decrease of the expression levels of the mRNA and the protein, of P450scc in the male baby mice testes, compared with the control group. And the mRNA expression levels of StAR, 17beta-HSD, and P450 17alpha decrease somewhat (P < 0.05), compared with the control group, but the protein expression levels of the above almost suffer no influence. Cypermethrin exposure during lactation of the female mice has almost no influence on the serum testosterone level, testosterone level in the testes, the StAR in the testes, and the expression levels of mRNA and protein of the testosterone synthetic enzymes (P > 0.05). CONCLUSION: Cypermethrin exposure during lactation may influence testosterone biosynthesis in the testes of the 21 days male baby mice, mainly by down-modulating mRNA and protein expression levels of testicular P450scc.

Transcriptional suppression of IL-27 production by Mycobacterium tuberculosis-activated p38 MAPK via inhibition of AP-1 binding.

Mycobacterium tuberculosis remains a major global challenge to human health care, and the mechanisms of how M. tuberculosis evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that bacillus Calmette-Guérin (BCG) and viable M. tuberculosis as well as M. tuberculosis lysates could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and M. tuberculosis lysate stimulation of macrophages induced activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. M. tuberculosis lysate-induced p28 transcription was dependent on MyD88 signaling pathway. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after M. tuberculosis lysate stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to M. tuberculosis lysate stimulation. Furthermore, blockade of p38 by SB203580 enhanced M. tuberculosis-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhanced the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective tuberculosis immunity.

Posttranscriptional regulation of IL-23 expression by IFN-gamma through tristetraprolin.

IL-23 plays an essential role in maintenance of IL-17-producing Th17 cells that are involved in the pathogenesis of several autoimmune diseases. Regulation of Th17 cells is tightly controlled by multiple factors such as IL-27 and IFN-γ. However, the detailed mechanisms responsible for IFN-γ-mediated Th17 cell inhibition are still largely unknown. In this study, we demonstrate that IFN-γ differentially regulates IL-12 and IL-23 production in both dendritic cells and macrophages. IFN-γ suppresses IL-23 expression by selectively targeting p19 mRNA stability through its 3'-untranslated region (3'UTR). Furthermore, IFN-γ enhances LPS-induced tristetraprolin (TTP) mRNA expression and protein production. Overexpression of TTP suppresses IL-23 p19 mRNA expression and p19 3'UTR-dependent luciferase activity. Additionally, deletion of TTP completely abolishes IFN-γ-mediated p19 mRNA degradation. We further demonstrate that IFN-γ suppresses LPS-induced p38 phosphorylation, and blockade of p38 MAPK signaling pathway with SB203580 inhibits IFN-γ- and LPS-induced p19 mRNA expression, whereas overexpression of p38 increases p19 mRNA expression via reducing TTP binding to the p19 3'UTR. Finally, inhibition of p38 phosphorylation by IFN-γ leads to TTP dephosphorylation that could result in stronger binding of the TTP to the adenosine/uridine-rich elements in the p19 3'UTR and p19 mRNA degradation. In summary, our results reveal a direct link among TTP, IFN-γ, and IL-23, indicating that IFN-γ-mediated Th17 cell suppression might act through TTP by increasing p19 mRNA degradation and therefore IL-23 inhibition.

Activation of IL-27 p28 gene transcription by interferon regulatory factor 8 in cooperation with interferon regulatory factor 1.

Interferon regulatory factor (IRF) family members, especially interferon regulatory factor-1 (IRF-1) and interferon regulatory factor-8 (IRF-8 or ICSBP), play important roles in interferon signaling in a wide range of host responses to infection and tumor growth. Interleukin-27 (IL-27), as a member of the IL-12 cytokine family, not only acts as a proinflammatory cytokine that regulates the differentiation of naive T helper cells but also possesses anti-inflammatory properties. IL-27 consists of EBI3 (Epstein-Barr virus-induced gene 3) and p28 subunits. Our previous work has shown that IRF-1 regulates IL-27 p28 gene transcription by specifically binding to the IRF-1 response element in the p28 promoter. In this study, we found that IRF-8-deficient macrophages were highly defective in the production of IL-27 p28 at both mRNA and protein levels. Circulating IL-27 p28 in serum was also decreased in IRF-8(-/-) mice in a septic shock model. Lipopolysaccharide, as a potent inducer of IL-27 p28 expression, could activate IRF-8 expression in a MyD88-dependent pathway, which in turn induced p28 gene transcription through NF-kappaB and/or IRF-8. Transcriptional analyses revealed that IRF-8 activated p28 gene transcription through binding to a site located at -57 to -48 in the p28 promoter overlapping the IRF-1 binding site. Consistent with this observation, overexpression of both IRF-8 and IRF-1 additively activated IL-27 p28 promoter. This study provides further mechanistic information regarding how signals initiated during innate and adaptive immune responses synergize to yield greater IL-27 production and sustained cellular immunity.

Melatonin pretreatment attenuates 2-bromopropane-induced testicular toxicity in rats.

2-Bromopropane (2-BP) was used as an alternative for ozone-depleting solvents, which caused reproductive disorders in male workers and laboratory animals. A recent study indicated that 2-BP impaired antioxidant cellular defences and enhanced lipid peroxidation (LPO). Melatonin is a powerful endogenous antioxidant. We hypothesized that reactive oxygen species (ROS) and lipid peroxidation are involved in 2-BP-induced testicular toxicities. To test the hypothesis, we investigated the effects of melatonin on 2-BP-induced testicular toxicities. Rats were intraperitoneally injected with 2-BP (1g/kg) with or without melatonin (5mg/kg), then sacrificed on 7th day after 2-BP injection. Epididymal and testicular tissues were examined for biochemical and histopathological changes. Apoptotic cells in testis were detected by TUNEL staining and immunohistochemistry for active caspase-3. Exposure to 2-BP significantly decreased epididymal sperm count and morphological normal sperms. 2-BP also induced vacuolation and atrophy of the seminiferous tubules, reduction of spermatogonia and apoptosis of germ cells. 2-BP significantly increased TBARS levels in plasma and epididymis, and decreased GSH content in testis and epididymis. Pretreatment with melatonin counteracted 2-BP-induced oxidative stress, ameliorated apoptosis in testis and attenuated histopathological damage in testis. In addition, pretreatment with melatonin significantly attenuated 2-BP-induced sperm morphological changes. We conclude that pretreatment with melatonin attenuates 2-BP-induced testicular toxicity through its ROS scavenging and anti-apoptotic effects.

Differential effects of pyrrolidine dithiocarbamate on TNF-alpha-mediated liver injury in two different models of fulminant hepatitis.

BACKGROUND/AIMS: Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of nuclear factor kappa B (NF-kappaB) activation. The present study aimed to investigate the effects of PDTC on lipopolysaccharide (LPS)-induced liver injury in two different models of fulminant hepatitis. METHODS: Mice infected with Bacillus Calmette Guerin (BCG) were challenged with LPS (0.2 mg/kg) to induce the model of inflammatory liver injury. Mice were injected with D-galactosamine (GalN, 600 mg/kg) and LPS (20 microg/kg) to induce the model of apoptotic liver injury. In the treatment groups, mice were pre-treated with PDTC (100 mg/kg), initiated 24 h prior to LPS. RESULTS: PDTC pretreatment reduced the infiltration of inflammatory cells, inhibited NF-kappaB activation and the expression of tumor necrosis factor alpha (TNF-alpha), attenuated nitric oxide production, and alleviated hepatic glutathione depletion. Correspondingly, PDTC reduced serum alanine aminotransferase, improved hepatic necrosis, and prolonged the survival in the BCG/LPS model. Conversely, PDTC accelerated death and aggravated liver apoptosis in the GalN/LPS model, although it reduced nitric oxide production, attenuated glutathione depletion, and inhibited the expression of TNF-alpha in liver. CONCLUSIONS: PDTC protects mice against BCG/LPS-induced inflammatory liver injury through the repression of NF-kappaB-mediated TNF-alpha release, while it seems to be detrimental in GalN/LPS-induced apoptotic liver damage.

Maternally-administered lipopolysaccharide (LPS) increases tumor necrosis factor alpha in fetal liver and fetal brain: its suppression by low-dose LPS pretreatment.

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and neurological injury. In the LPS model, tumor necrosis factor alpha (TNF-alpha) is the major mediator leading to IUFD, IUGR and neurological injury. In the present study, we investigated the effect of maternally-administered LPS on TNF-alpha in maternal serum, amniotic fluid, fetal liver and fetal brain. The timed pregnant mice were intraperitoneally (i.p.) injected with a single dose of LPS (500microg/kg) on gestational day 17. As expected, TNF-alpha was obviously increased in maternal serum and amniotic fluid in response to LPS. Although maternally-administered LPS also increased the level of TNF-alpha protein in fetal liver and brain, no significant difference in TNF-alpha mRNA level in fetal liver and brain was observed among different groups, suggesting that the increased TNF-alpha protein in fetal liver and brain may be transferred from either the maternal circulation or amniotic fluid or placenta. When the pregnant mice were pretreated with a low-dose LPS (10microg/kg, i.p.) at 4, 12, 24 or 48h before LPS (500microg/kg, i.p.), LPS-evoked TNF-alpha in maternal serum and amniotic fluid was significantly inhibited. Importantly, low-dose LPS pretreatment also greatly attenuated LPS-induced increases in TNF-alpha protein in fetal liver and fetal brain. Taken together, these results indicate that perinatal exposure to low-dose LPS induces a reduced sensitivity to subsequent LPS challenge.

Maternally administered melatonin differentially regulates lipopolysaccharide-induced proinflammatory and anti-inflammatory cytokines in maternal serum, amniotic fluid, fetal liver, and fetal brain.

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including intra-uterine fetal death and intra-uterine growth retardation. In the LPS model, tumor necrosis factor alpha (TNF-alpha) is the major mediator leading to intra-uterine fetal death and intra-uterine growth retardation. Interleukin (IL)-10 protects rodents against LPS-induced intra-uterine fetal death and intra-uterine growth retardation. Melatonin is an immunomodulator. In the present study, we investigated the effect of maternally administered melatonin on LPS-induced proinflammatory and anti-inflammatory cytokines in maternal serum, amniotic fluid, fetal liver and fetal brain. The time pregnant mice were injected with melatonin [5.0 mg/kg, intraperitoneal (i.p.)] 30 min before LPS (500 microg/kg, i.p.) on gestational day 17. As expected, TNF-alpha, IL-1beta, IL-6 and IL-10 were obviously increased in maternal serum and amniotic fluid in response to LPS. In addition, maternal LPS exposure significantly increased the levels of TNF-alpha, IL-1beta, IL-6 and IL-10 in fetal liver, and TNF-alpha and IL-10 in fetal brain. Melatonin pretreatment significantly attenuated LPS-evoked elevation of TNF-alpha in maternal serum. On the contrary, melatonin aggravated LPS-induced increase in IL-10 in maternal serum. Melatonin had no effect on LPS-evoked IL-1beta and IL-6 in maternal serum and amniotic fluid. Interestingly, maternally administered melatonin also significantly attenuated LPS-evoked elevation of TNF-alpha in fetal brain, whereas the indole aggravated LPS-induced increase in IL-10 in fetal liver. Taken together, these results indicate that maternally administered melatonin differentially regulates LPS-induced proinflammatory and anti-inflammatory cytokines in maternal serum, amniotic fluid, fetal liver, and fetal brain.

Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in D-galactosamine-sensitized mice.

D-Galactosamine (GalN) depletes UTP primarily in liver, resulting in decreased RNA synthesis in hepatocytes. When given together with a sublethal dose of lipopolysaccharide (LPS), GalN highly sensitizes animals to produce apoptotic liver injury with severe hepatic congestion, resulting in rapid death. Melatonin is a cytokine modulator, antioxidant and anti-apoptotic agent. In the present study, we investigated the effect of melatonin on LPS-induced apoptotic liver damage in GalN-sensitized mice. Female CD-1 mice were intraperitoneally (i.p.) injected with melatonin (5.0mg/kg) 30min before GalN/LPS (700mg10microg/kg, i.p.), another two doses of melatonin (2.5mg/kg, i.p.) being administered 1 and 2h after GalN/LPS. Results showed that serum alanine aminotransferase (ALT) activities were markedly increased 8h after GalN/LPS treatment, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. Melatonin significantly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, melatonin distinctly improved GalN/LPS-induced congestion. Additional experiment showed that melatonin significantly attenuated GalN/LPS-induced hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA laddering. Furthermore, melatonin markedly increased hepatic Se-dependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic glutathione (GSH) depletion in GalN/LPS-treated mice. Increases in serum tumor necrosis factor alpha (TNF-alpha), which were observed in GalN/LPS-treated mice, were significantly reduced by melatonin. However, melatonin had no effect on LPS-evoked nitric oxide production in GalN-sensitized mice. Taken together, these results indicate that melatonin protected against LPS-induced liver damage in GalN-sensitized mice through its strong ROS-scavenging, antiinflammatory and antiapoptotic effects.

Effects of low-dose lipopolysaccharide (LPS) pretreatment on LPS-induced intra-uterine fetal death and preterm labor.

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and preterm delivery in rodents. The purpose of the present study was to investigate whether administration of a low-dose LPS to the pregnant mice induce a reduced sensitivity to subsequent high-dose LPS-induced IUFD and preterm labor. We found that LPS-induced IUFD was obviously attenuated when the pregnant mice were pretreated with low-dose LPS (10 microg/kg, i.p.) 24h before high-dose LPS (120 microg/kg, i.p.). Consistent with its protective effect, when administered 24h before high-dose LPS, low-dose LPS pretreatment obviously inhibited the releases of tumor necrosis factor alpha (TNF-alpha) in maternal serum and amniotic fluid and attenuated LPS-induced placental lipid peroxidation and GSH depletion. However, when administered 4h before high-dose LPS, low-dose LPS pretreatment did not induced a reduced sensitivity to subsequent high-dose LPS-induced release of TNF-alpha in maternal serum and amniotic fluid. Actually, low-dose LPS pretreatment 4h before high-dose LPS worsened LPS-induced oxidative stress in mouse placenta and increased nitric oxide production in maternal serum and amniotic fluid. Correspondingly, low-dose LPS pretreatment 4h before high-dose LPS aggravated LPS-induced IUFD. Taken together, these results indicate that whether a low-dose LPS exposure during pregnancy produce LPS hyporesponsiveness depends on the interval between the two doses of LPS. When administered 24h before high-dose LPS, a low-dose LPS pretreatment induces a reduced sensitivity to subsequent high-dose LPS-induced IUFD, TNF-alpha production and oxidative stress.