Splenocyte Apoptosis Research Articles

Effects of Antihypertensive and Triglyceride Lowering Agents on Splenocyte Apoptosis in Rats with Fatty Liver

Individuals with the metabolic syndrome (MS) and non-alcoholic fatty liver disease (NAFLD) have an increased incidence of infection and infection-related mortality. Rats given fructose-enriched diet (FED) develop the MS including NAFLD. In this study, we characterized changes in splenocyte apoptosis in FED rats given medications to treat various components of the MS. Apoptosis of splenocytes may induce immunosuppression. Splenocyte apoptosis was evaluated by activated caspase-3 immunohistochemistry in the periarterial sheath (PALS), (a T cell area), follicles (B cell area), marginal (B cell area) and in the red pulp zones. FED administration caused an enormous increase in splenocyte apoptosis in all of the spleen zones: PALS (+2966%), follicles (+3025%), marginal (+5228%) and red pulp (+7000%). Administration of captopril to the FED rats was associated with a further increase in the splenocyte apoptosis only in the marginal (150%), PALS (+105%) and red pulp (+67%) zones. Bezafibrate administration to the FED rats was associated with no further increase in apoptosis rates. Amlodipine administration to the FED rats was associated with almost complete amelioration of the splenocyte apoptosis that was induced by the FED diet. These pharmacological manipulations were also associated with changes in the hepatic lipids composition, and oxidative milieu that did not correlate to the changes in splenocyte apoptosis. NAFLD in FED rats is associated with an increase in splenic apoptosis. Agents administered to treat components of the MS in FED rats may lead to divergent changes in the splenic histology and splenocyte apoptosis.

Geraniin down regulates gamma radiation-induced apoptosis by suppressing DNA damage

Gamma ray irradiation triggers DNA damage and apoptosis of proliferating stem cells and peripheral immune cells, resulting in the destruction of intestinal crypts and lymphoid system. Geraniin is a natural compound extracts from an aquatic plant Nymphaea tetragona and possesses good antioxidant property. In this study, we demonstrate that geraniin rescues radiosensitive splenocytes and jejunal crypt cells from radiation-induced DNA damage and apoptosis. Isolated splenocytes from C57BL/6 mice treated with geraniin were protected against radiation injury of 2 Gy irradiation through the enhancement of the proliferation and attenuation of DNA damage. Also, geraniin inhibited apoptosis in radiosensitive splenocytes by reducing the expression level and immunoreactivity of proapoptotic p53 and Bax and increasing those of anti-apoptotic Bcl-2. In mice exposed to radiation, geraniin treatment protected splenocytes and intestinal crypt cells from radiation-induced cell death. Our results suggest that geraniin presents radioprotective effects by regulating DNA damage on splenocytes, exerting immunostimulatory capacities and inhibiting apoptosis of radiosensitive immune cells and jejunal crypt cells. Therefore, geraniin can be a radioprotective agent against γ-irradiation exposure.

Preliminary phytochemical and biological activities study of solvent extracts from a cold-field fruit—Malus baccata (Linn.) Borkh

Radiation can lead to oxidative stress in organism. Defense against oxidative stress is important in preventing the development of diseases. This study investigated in vitro antioxidant and immunomodulation potential of Malus baccata (Linn.) Borkh., also the effectiveness of different solvent for extracting active compounds was obtained by HPLC. Of all the solvents employed, acetone extract possessed the highest amounts of total phenolic (975.7±29.2mg GAE/100g) and total flavonoids (824.9±27.7mg CE/100g), with the highest capacities for scavenging ABTS+· (136.49±1.16μmol TE/g), DPPH· (18.60±0.28μmol TE/g) and OH· (32.37±0.7mg VCE/g). The extracts all dose-dependently promoted splenocyte proliferation and inhibited splenocyte apoptosis induced by γ-radiation. HPLC analyses of the solvent extracts tentatively revealed the presence of chlorogenic acid, quercetin 3-galactoside, phloridzin and cinnamic acid. The extract could be source of natural antioxidant and immunomodulation agents for applications in functional foods.

Autophagy is involved in the effects of resveratrol on prevention of splenocyte apoptosis caused by oxidative stress in restrained mice

Resveratrol, a powerful natural compound for human health, is widely reported for its immunity-related beneficial properties. However, few works have studied its effect mechanism on immunity. The present study was conducted to investigate the effects of resveratrol on splenic immunity in restraint stressed mice and the mechanism was further studied as autophagy induction. Mice were administered with resveratrol for 7 days consecutively, fixed in restraint cages for 18 h, and recovered for 12 h after the last administration. Data showed that restraint led to spleen damages, including declined spleen index, decreased CD4(+) T-cell number, increased mitochondrial oxidative damage, and apoptosis of splenocytes. Resveratrol, vitamin C (antioxidant), and rapamycin (autophagy agonist) protected spleen functions. Meanwhile, rapamycin augmented the effects of resveratrol that were abolished by chloroquine (autophagy antagonists). Further studies showed that expressions of Beclin 1 and LC3β required in autophagy development were significantly upregulated by resveratrol but not by vitamin C. This study demonstrated that resveratrol preserved splenic immunity of restraint stressed mice. It is meaningful to find that autophagy, apart from reactive oxygen species clearance, is included as a potential mechanism via which resveratrol ameliorated the state of oxidative stress and thus protected splenocytes in mice.

Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage‘

Honey isolate Bacillus subtilis M was cultivated in shake flasks and in 16-l bioreactor cultures to investigate cell growth, bio-metabolites production kinetics and bioprocess scalability. The respective maximal levan and levansucrase productions of 59.5g/l and 74.1U/ml were achieved in bioreactor cultures under pH controlled condition (pH=7.0) after only 24h. Crude levan (levE) was isolated, characterized and fractionated into F1, F2, and F3 with different molecular weight (21.8, 13.118, 9.53kDa). 1H NMR and 13C NMR spectroscopy proved that LevE and their fractions were mainly β-(2, 6)-linked levan-type polysaccharide. The cancer chemo-preventive activity indicated that the levE and its fraction 3 were promising inhibitors of cytochrome P-450 1A activity, inducers of glutathione-S-transferase activity in Murine hepatomaHepa1c1c7cells and possessed highest radical scavenging affinity to both ROO and OH. They inhibited the induced-DNA fragmentation. None of the tested samples triggered apoptosis or necrosis in splenocytes, except F2.

Adenovirus mediated CTLA4Ig transgene therapy alleviates abortion by inhibiting spleen lymphocyte proliferation and regulating apoptosis in the feto-placental unit

Pregnancy is thought to be a state of immunological tolerance. The mechanisms underlying this phenomenon are still poorly understood. In our previous study, adenovirus mediated CTLA4Ig transgene (Ad-CTLA4Ig) therapy was demonstrated to improve pregnancy outcome in an abortion-prone mouse model by skewing the Th2/Th1 cytokine balance, expanding peripheral CD4+ CD25+ regulatory T cell populations and inducing indoleamine 2,3 dioxygenase (IDO) mRNA expression at the maternal–fetal interface. However, it is still not clear whether other mechanisms are involved in the protective effect of CTLA-4 on pregnancy outcome in abortion-prone matings. In this study, we focused on the effect of CTLA4Ig on spleen lymphocyte proliferation and apoptosis at the maternal–fetal interface. We demonstrated that Ad-CTLA4Ig therapy inhibited the proliferation of CBA/J splenocytes and IL-2 secretion in response to DBA/2 stimulator cells in the abortion-prone mice model. Ad-CTLA4Ig therapy also skewed cytokine production toward a Th2 bias and regulated the expression of anti-apoptosis factor Bcl-2 and pro-apoptosis factor Bax at the maternal–fetal interface. However, it did not influence the apoptosis and cell cycles of splenocytes in pregnant CBA/J mice. On the whole, these findings indicated that Ad-CTLA4Ig therapy could ameliorate the outcome of spontaneous abortion by inhibiting proliferation of maternal spleen lymphocytes and regulating apoptosis in the feto-placental unit.

Splenocyte apoptosis in Plasmodium berghei ANKA infection: possible role of TNF‐α and TGF‐β

Cerebral malaria is associated with the circulating levels of tumour necrosis factor alpha (TNF-α) and transforming growth factor β (TGF-β), but association between these two cytokines and implications in splenocyte apoptosis remain largely obscured. We have evaluated the outcome of TGF-β and TNF-α production in the context of splenocyte apoptosis during Plasmodium berghei ANKA (PbA) infection. Blood-stage PbA infection confirmed blood-brain barrier disruption, disarray of white pulp, increase in percentage of sub-G0/G1 and splenocyte apoptosis. Flow cytometric analysis reveals up-regulation of Fas-L followed by caspase-8 and caspase-3 activation and signifies possible involvement of Fas-L-mediated splenocyte apoptosis. We have observed down-regulation of TGF-β and up-regulation of TNF-α in tissue and serum level, respectively, during PbA infection. Association between the production of TGF-β and the severity of malaria infection in splenocytes was verified with TGF-β inhibitor that exacerbated the apoptotic process. In contrary, TNF-α inhibitor causes significant delay in apoptotic process, but could not alter the lethality of parasite. Thus, results from this study suggest that the critical balance between TGF-β and TNF-α might have a key role on Fas-L-mediated splenocyte apoptosis during experimental cerebral malaria.

Zymomonas mobilis culture protects against sepsis by modulating the inflammatory response, alleviating bacterial burden and suppressing splenocyte apoptosis

Microorganisms with immunomodulating effects beneficially affect the host organism by improving the microbial equilibrium and balancing the immune system. Zymomonas mobilis is reported to have antagonistic properties against yeast and other pathogenic microorganisms in humans and animals. This study assessed the effects of Z. mobilis UFPEDA 202 (109CFU/mL) cultures on polymicrobial sepsis induced by cecal ligation and puncture (CLP). The survival of animals subjected to lethal sepsis was evaluated after pre-treatment, post-treatment or a combination of both. 6h after the induction of sepsis, neutrophil migration, the number of bacteria, myeloperoxidase, TNF-α, MCP-1, and IL-10 were performed in the peritoneal lavage of animals. Histopathological changes in the spleen of animals were evaluated by light microscopy, and apoptosis of splenocytes was analyzed by transmission electron microscopy. The results showed that the combination of prophylactic and therapeutic treatment with Z. mobilis increased the survival of animals by 50% at 96h after the induction of sepsis. There was a reduction in the levels of TNF-α and myeloperoxidase (MPO) in lung tissue. There was also a reduction in the number of viable bacteria in peritoneal fluid. However, increases in neutrophil migration and IL-10 levels were observed. The observed levels of MCP-1 remained similar to the control. Histopathology analysis showed a decrease in acute lung injury. The group pre-treated with the Z. mobilis culture demonstrated a marked decrease in the number of apoptotic cells in the spleen (24%). This study demonstrates that Z. mobilis cultures increased the survival of animals with severe sepsis. This survival was mediated by improvement of neutrophil migration, enhanced activity against pathogenic enteric bacteria and reduced lung injury.

Scavenger Receptor Class A Plays a Central Role in Mediating Mortality and the Development of the Pro-Inflammatory Phenotype in Polymicrobial Sepsis

Sepsis is a frequent complication in critical illness. The mechanisms that are involved in initiation and propagation of the disease are not well understood. Scavenger receptor A (SRA) is a membrane receptor that binds multiple polyanions such as oxidized LDL and endotoxin. Recent studies suggest that SRA acts as a pattern recognition receptor in the innate immune response. The goal of the present study was to determine the role of SRA in polymicrobial sepsis. SRA deficient (SRA−/−) and C57BL/6JB/6J (WT) male mice were subjected to cecal ligation and puncture (CLP) to induce polymicrobial sepsis. NFκB activity, myeloperoxidase activity, and co-association of SRA with toll like receptor (TLR) 4 and TLR2 was analyzed in the lungs. Spleens were analyzed for apoptosis. Serum cytokines and chemokines were assayed. Blood and peritoneal fluid were cultured for aerobic and anaerobic bacterial burdens. Long term survival was significantly increased in SRA−/− septic mice (53.6% vs. 3.6%, p<0.05) when compared to WT mice. NFκB activity was 45.5% lower in the lungs of SRA−/− septic mice versus WT septic mice (p<0.05). Serum levels of interleukin (IL)-5, IL-6, IL-10 and monocyte chemoattractant protein −1 were significantly lower in septic SRA−/− mice when compared to septic WT mice (p<0.05). We found that SRA immuno-precipitated with TLR4, but not TLR2, in the lungs of WT septic mice. We also found that septic SRA−/− mice had lower bacterial burdens than WT septic mice. SRA deficiency had no effect on pulmonary neutrophil infiltration or splenocyte apoptosis during sepsis. We conclude that SRA plays a pivotal, and previously unknown, role in mediating the pathophysiology of sepsis/septic shock in a murine model of polymicrobial sepsis. Mechanistically, SRA interacts with TLR4 to enhance the development of the pro-inflammatory phenotype and mediate the morbidity and mortality of sepsis/septic shock.

Mechanism of perfluorooctanesulfonate (PFOS)-induced apoptosis in the immunocyte

As a new type of persistent organic pollutant, perfluorooctane sulfonate (PFOS) has raised great concern in recent years due to its ubiquitous distribution in the general environment and its long elimination half-life in humans. PFOS has toxic and carcinogenic effects in animals and humans, but the effects of PFOS on apoptosis are still not clear. The present study aimed to determine the mode of cell death and its mechanism in splenocytes and thymocytes from adult male C57BL/6 mice administered 0, 1, 5, or 10 mg PFOS/kg/day by gavage daily for 7 days. The results showed that more apoptotic cells were present in PFOS-treated mice than in control mice. PFOS induced production of reactive oxygen species (ROS), dissipation of mitochondria membrane potential, and apoptosis of splenocytes and thymocytes. Moreover, activities of superoxide dismutase, catalase, and glutathione reductase were increased, whereas activities of glutathione-S-transferase and glutathione peroxidase were decreased, in splenocytes. Glutathione contents were reduced as well. Differential expressions of proteins such as p53, Bax, caspase-3, and caspase-9 were significantly up-regulated in PFOS-exposed hosts, whereas Bcl-2 expression was significantly down-regulated. One possible mechanism for the findings here was that PFOS could overwhelm homeostasis of anti-oxidative systems, boost ROS generation, impact on mitochondria, and affect protein expression of apoptotic regulators, the latter of which resulted in initiation of the apoptosis program. Results from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans, at the cellular level.

Induction of p53-mediated apoptosis in splenocytes and thymocytes of C57BL/6 mice exposed to perfluorooctane sulfonate (PFOS)

Perfluorooctane sulfonate (PFOS) is a persistent environmental contaminant found in human and wildlife tissues. It has been reported that PFOS can cause atrophy of the immune organs and apoptosis of immunocytes in rodents. However, the mechanism behind such cause is still unclear. To understand the model of cell death and its mechanism on lymphoid cells in vivo, we conducted a dose/response experiment in which 4 groups of male adult C57BL/6 mice (12 mice per group) were dosed daily by oral gavage with PFOS at 0, 0.0167, 0.0833, or 0.8333mg/kg/day, yielding targeted Total Administered Dose (TAD) of 0, 1, 5, or 50mg PFOS/kg, respectively, over 60days. The results showed that spleen and thymus weight were significantly reduced in the highest PFOS-dose-group (TAD 50mg PFOS/kg) compared to the control group, whereas liver weight was significantly increased. We analyzed the cell death via apoptosis with an annexin-V/propidium iodide assay by flow cytometry, and observed that both the percentage of apoptosis and the expression of the pro-apoptotic proteins p53 in splenocytes and thymocytes increased in a dose-related manner after PFOS treatment. We also observed that PFOS induced p53-dependent apoptosis through the cooperation between the Bcl-xl down regulation without changing the Bcl-2 and Bax expression. The down regulation of Bcl-xl was strongly indicating mitochondrial involvement in apoptosis. It is confirmed by the release of cytochrome c and activation of caspase-3. All of these findings establish an important role of p53 and mitochondrial function in PFOS induced toxic environment in the host.

Platelets Induce Apoptosis during Sepsis in a Contact-Dependent Manner That Is Inhibited by GPIIb/IIIa Blockade

PurposeEnd-organ apoptosis is well-described in progressive sepsis and Multiple Organ Dysfunction Syndrome (MODS), especially where platelets accumulate (e.g. spleen and lung). We previously reported an acute sepsis-induced cytotoxic platelet phenotype expressing serine protease granzyme B. We now aim to define the site(s) of and mechanism(s) by which platelet granzyme B induces end-organ apoptosis in sepsis.MethodsEnd-organ apoptosis in murine sepsis (i.e. polymicrobial peritonitis) was analyzed by immunohistochemistry. Platelet cytotoxicity was measured by flow cytometry following 90 minute ex vivo co-incubation with healthy murine splenocytes. Sepsis progression was measured via validated preclinical murine sepsis score.Measurements and Main ResultsThere was evident apoptosis in spleen, lung, and kidney sections from septic wild type mice. In contrast, there was a lack of TUNEL staining in spleens and lungs from septic granzyme B null mice and these mice survived longer following induction of sepsis than wild type mice. In co-incubation experiments, physical separation of septic platelets from splenocytes by a semi-permeable membrane reduced splenocyte apoptosis to a rate indistinguishable from negative controls. Chemical separation by the platelet GPIIb/IIIa receptor inhibitor eptifibatide decreased apoptosis by 66.6±10.6% (p = 0.008). Mice treated with eptifibatide in vivo survived longer following induction of sepsis than vehicle control mice.ConclusionsIn sepsis, platelet granzyme B-mediated apoptosis occurs in spleen and lung, and absence of granzyme B slows sepsis progression. This process proceeds in a contact-dependent manner that is inhibited ex vivo and in vivo by the platelet GPIIb/IIIa receptor inhibitor eptifibatide. The GPIIb/IIIa inhibitors and other classes of anti-platelet drugs may be protective in sepsis.

Apolipoprotein E-knockout mice show increased titers of serum anti-nuclear and anti-dsDNA antibodies

Apolipoprotein E-knockout (ApoE−/−) mice, atherosclerosis-prone mice, show an autoimmune response, but the pathogenesis is not fully understood. We investigated the pathogenesis in female and male ApoE−/− mice. The spleens of all ApoE−/− and C57BL/6 (B6) mice were weighed. The serum IgG level and titers of anti-nuclear antibody (ANA) and anti-double-stranded DNA (anti-dsDNA) antibody were assayed by ELISA. Apoptosis of spleen tissue was evaluated by TUNEL. TLR4 level in spleen tissue was tested by immunohistochemistry and Western blot analysis. Levels of MyD88, p38, phosphorylated p38 (pp38), interferon regulatory factor 3 (IRF3) and Bcl-2-associated X protein (Bax) in spleen tissue were detected by Western blot analysis. We also survey the changes of serum autoantibodies, spleen weight, splenocyte apoptosis and the expressions of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue in male ApoE−/− mice after 4weeks of lipopolysaccharide (LPS), Toll-like receptor 4 ligand, administration. ApoE−/− mice showed splenomegaly and significantly increased serum level of IgG and titers of ANA and anti-dsDNA antibody as compared with B6 mice. Splenocyte apoptosis and the expression of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue were significantly lower in ApoE−/− than B6 mice. The expression of TLR4, MyD88, IRF3, pp38, and Bax differed by sex in ApoE−/− spleen tissue. The down-regulation of TLR4 signal molecules induced by LPS led to decreased expression of Bax and increased serum titers of ANA and anti-dsDNA antibody. Therefore, the TLR4 signal pathway may participate in maintaining the balance of splenocyte apoptosis and autoantibody production in ApoE−/− mice.

Mesenchymal stem cells are conditionally therapeutic in preclinical models of rheumatoid arthritis

ObjectiveThe role of mesenchymal stem cells (MSC) in experimental arthritis is undoubtedly conflicting. This study explored the effect of bone marrow-derived MSC in previously untested and pathogenetically different models of...

Role of microRNA in 3,3-diindolylmethane (DIM)-mediated amelioration of SEB-induced acute lung injury (173.6)

Abstract Staphylococcal enterotoxin B (SEB) is a CDC select agent of bioterrorism and a public health threat during nosocomial infections. A deleterious effect of SEB exposure is acute lung injury (ALI) and acute respiratory distress syndrome characterized by pulmonary infiltration. 3,3-diindolylmethane (DIM) found in cruciferous vegetables, has been shown to have anti-inflammatory properties. We studied the mechanism underlying the ameliorative effects of DIM on SEB-induced ALI. Mice were exposed to SEB [50 mg; intranasal] and administered DIM [100 mg/kg] via oral gavage. Forty-eight hours after SEB exposure, serum, lung tissue and lung infiltrating cells were collected. DIM treatment led to a decrease in lymphocytic infiltration, specifically CD4+, CD8+ and Vβ8+ T cells as well as NKT cells. DIM also led to a reduction of fluid leak in lungs. DIM [10, 25, 50 µM] induced apoptosis in splenocytes and CD3+ T cells treated in vitro with SEB in a dose-dependent manner. DIM treatment led to downregulation in microRNA, miR-706 and miR-494 that target pro-apoptotic genes, and upregulation of miR-22 and miR-34a targeting anti-apoptotic genes in lung-infiltrating cells. Thus, studies are underway to examine whether in T cells, DIM treatment leads to modulation in the expression of genes involved in apoptosis. Together, these studies demonstrate that DIM treatment may suppress SEB-mediated ALI by induction of apoptosis in T cells via alteration in expression of miRNA.

Particles such as an asian sand dust activate autophagic and apoptotic key molecules in CD11b positive cells. (106.6)

Abstract Asian sand dust (ASD) is derived from air pollutants in East China and has a long history of appearing in Japan. Recently, it is knowing that ASD contains microbial materials, sulfate (SO(4)(2-)) and nitrate (NO(3)(-)), therefore there is increasing concern for its possible adverse health effects. Although it has been reported that ASD induces inflammatory responses in the lung tissue and causes respiratory diseases such as eosinophilia in mice, the effect of ASD on the peripheral lymphoid organ are not fully understood. In this study, we examine the effect of ASD on immune system. ICR mice were administered intratracheal ASD (0, 0.2, 0.02 mg/mice), and were sacrificed at 4 weeks (1 time per week and total 4 times) after administration. Spleen cells were prepared and cell lysates of splenocytes were resolved in western blotting. Immuno-blotting demonstrated that ASD induced autophagic-maker LC3 and p62 expression and also induced PARP cleavage suggesting caspase-dependent apoptosis in splenocytes. In addition, ASD could increase the expression of TNF-α mRNA analyzed by Real-time PCR. As same as in vivo, we observed ASD enhanced TNF-α production induced by ConA in splenocytes. Taken together, ASD induced systemic inflammation resulting in autophagic and apoptotic phenomena in splenocytes, suggesting that ASD administration not only triggered pulmonary inflammation but also affected peripheral lymphoid tissue.

Splenocyte Apoptosis and Autophagy Is Mediated by Interferon Regulatory Factor 1 During Murine Endotoxemia

Sepsis-induced lymphocyte and dendritic cell apoptosis contributes to immunosuppression, which results in an inability to eradicate the primary infection as well as a propensity to acquire new, secondary infections. Another cellular process, autophagy, is also activated in immune cells and plays a protective role. In the present study, we demonstrate that interferon regulatory factor 1 (IRF-1) regulates both immune cell apoptosis and autophagy in a murine endotoxemia model. Interferon regulatory factor 1 is activated at an early phase through a Toll-like receptor 4-dependent, myeloid differentiation primary response gene 88-independent manner in splenocytes. Furthermore, IRF-1 knockout (KO) mice are protected from a lethal endotoxemia model. This protection is associated with decreased apoptosis and increased autophagy in splenocytes. Interferon regulatory factor 1 KO mice experience decreased apoptotic cell loss, especially in CD4⁺ T lymphocytes and myeloid antigen-presenting cells. Meanwhile, IRF-1 KO mice demonstrate increased autophagy and improved mitochondrial integrity. This increased autophagy in KO mice is attributable, at least in part, to deactivation of mammalian target of rapamycin/P70S6 signaling--a main negative regulator of autophagy. Therefore, we propose a novel role for IRF-1 in regulating both apoptosis and autophagy in splenocytes in the setting of endotoxemia with IRF-1 promoting apoptosis and inhibiting autophagy.

Lipocalin 2 mediated hypoferremia protects against endotoxin- induced sepsis in mice (112.11)

Abstract Sepsis is characterized by hypoferremia, a primitive protective mechanism to limit iron-induced oxidative stress. Lipocalin-2 (Lcn2) which is highly upregulated during inflammation and has been shown to transport iron under physiologic conditions. Accordingly, we hypothesized that such upregulation of Lcn2 serves to protect against sepsis associated oxidative stress. Lcn2 deficient mice (Lcn2KO) and their WT littermates were given E. coli LPS (20mg/kg BW) i.p. and monitored for mortality. Serum TNFα, IL-18, LDH, aminotransferases, iron, glutathione and cysteine were analyzed. Splenocyte apoptosis was assayed by TUNEL, caspase 3 cleavage and flow cytometry. In WT mice, LPS-induced systemic Lcn2 peaked at 24h and returned to basal levels by 48h. All Lcn2KO died by day 8 while only 20% mortality was observed in WT mice. In addition, Lcn2KO exhibited substantial elevation of every parameter tested including both pro- and anti-inflammatory cytokines. Further,Lcn2KO splenocytes exhibited extensive apoptosis as measured by caspase 3 cleavage, TUNEL staining and flow cytometry. More importantly, when compared to WT mice, Lcn2KO mice failed to exhibit LPS-induced hypoferremia at early time points and exhibited severe hepatic oxidative stress. Our data demonstrate that Lcn2 facilitates LPS-induced hypoferremia and serves as a host protective factor by reducing oxidative stress. Given Lcn2 multifunctionality, it may be developed as a potential therapeutic agent to treat sepsis.

Lipocalin 2 confers protection against endotoxin‐induced sepsis in mice

Sepsis is characterized by elevated systemic proinflammatory cytokines and agents that can act as amplifiers (HMGB1) or dampeners (sIL‐1Ra) of inflammation and thus play a decisive role in sepsis. Lipocalin 2 (Lcn2), which is upregulated by log orders of magnitude during inflammation but its role in sepsis, is largely unknown. Lcn2 deficient mice (Lcn2KO) and their WT littermates were given E. coli LPS (20mg/kg BW) i.p. and monitored for mortality. Serum TNFα, IL‐18 and aminotransferases were analyzed. Splenocyte apoptosis was assayed 24h after LPS by TUNEL staining, caspase 3 and flow cytometry. Peritoneal cells from WT and Lcn2KO mice were stimulated with LPS and cytokines, COX2 and iNOS were analyzed. In WT mice, LPS‐induced systemic Lcn2 peaked at 24h and returned to basal levels by 48h. All Lcn2KO died by day 8 while only 20% mortality was observed in WT mice. In addition, Lcn2KO exhibited substantial elevation of every parameter tested including both pro and anti‐inflammatory cytokines. Further, splenocytes exhibited extensive apoptosis as measured by caspase 3 and TUNEL staining. Flow cytometry revealed that apoptosis of neutrophils, CD3+CD4+ and dendritic cells were significantly increased in Lcn2KO. Our data demonstrate that Lcn2 is a host protective factor during sepsis and, given its multifunctionality, small size, and simple structure it may be developed as potential therapeutic agent to treat sepsis.

Low Selenium Diet Alters Cell Cycle Phase, Apoptotic Population and Modifies Oxidative Stress Markers of Spleens in Broilers

The purpose of this 42-day study was to investigate the effects of low selenium (Se) on histopathological changes of spleen, cell cycle and apoptosis of splenocytes, and oxidative status of the spleen. One hundred twenty 1-day-old avian broilers were randomly divided into two groups of 60 each and were fed on a low Se diet (0.0342 mg/kg Se) or a control diet (0.2 mg/kg Se), respectively. The weight and relative weight of the spleen were significantly decreased in the low Se group when compared with those of the control group. Histopathologically, splenic lesions in low-Se chicken were characterized by lymphocyte depletion and congestion of red pulp. As measured by flow cytometry, splenocytes in G(0)/G(1) phase were significantly increased, while splenocytes in S phase and G(2) + M phase were obviously decreased in the low Se group. The percentage of apoptotic splenocytes was greatly increased in the low Se group when compared with that of control group. At the same time, the occurrence frequencies of apoptotic splenocytes was markedly increased in the low Se group with the appearance of condensed nucleus ultrastructurally. Oxidative stress in the spleens of the low Se group was evidenced by decrease in glutathione peroxidase and catalase activities and increase in malondialdehyde contents. The results showed that low Se diet intake caused increased apoptosis, arrested cell cycle, and obvious oxidative stress, which provided a possible pathway for the injured structure and immune function of the spleen in chickens.