Increase In Spleen Weight Research Articles

Caffeine Decreases Hepcidin Expression to Alleviate Aberrant Iron Metabolism under Inflammation by Regulating the IL-6/STAT3 Pathway.

Caffeine is well-known as a psychostimulant, and it can also be beneficial in numerous diseases such as diabetes and different types of cancer. Previous studies have shown that caffeine can have a protective role in bacterial infection-induced inflammation and hyperoxia-mediated pulmonary inflammation. Hepcidin, which is regulated by the IL-6/STAT3 inflammation pathway, is a peptide hormone that maintains systemic iron homeostasis. We hypothesized that caffeine’s effects on inflammation may also influence hepcidin production and therefore systemic iron metabolism. To this end, we treated 2-month-old mice with caffeine by daily intragastric administration for 7 days, administering intraperitoneal LPS after the final caffeine treatment. Twelve hours after LPS treatment the mice were euthanized, and tissues were collected. We found that caffeine decreased hepatic hepcidin expression and attenuated LPS-induced hepatic hepcidin overexpression. IL-6 expression and STAT3 phosphorylation were also reduced upon caffeine administration. Additionally, hepatic and splenic FPN1 levels increased after caffeine treatment, leading to lower iron levels in liver and spleen tissues and higher iron levels in serum. Caffeine also prevented the increase in spleen weight and decrease in body weight after LPS treatment. Together, our findings suggest that caffeine decreases hepcidin expression via inhibiting inflammation and the activation of the IL-6/STAT3 pathway, thus presenting an attractive, potential therapeutic for the treatment of anemia of inflammation.

Assessment of antimalarial medicinal plants used in Nigerian ethnomedicine reveals antimalarial potential of Cucurbita pepo leaf extract

Medicinal plants are often used to treat malaria in different parts of Nigeria and exploiting these can unravel new therapeutic leads. This study evaluated the antiplasmodial potential of selected plants used to treat malaria in Nsukka, Enugu state, Nigeria. Leaves of three different plants (Cucurbita pepo, Hibiscus rosa-sinensis and Pennisetum purpureum) were collected for screening and two extracts viz., 70%v/v ethanol and dichloromethane/methanol (1:1 v/v), were prepared for each. An acute toxicity test was done in mice and cytotoxicity was assessed using human hepatoma cell line (HUH). The extracts were screened against chloroquine-sensitive P. falciparum (Pf3D7) in vitro, and chloroquine-resistant P. berghei ANKA in vivo using a 4 day-suppressive test in mice. Cucurbita pepo ethanol extract was further tested for hemolytic effect on human erythrocytes and in established infection in mice. Parameters assessed were post-treatment parasitemia, hematological indices, organ (brain, kidney, liver, and spleen) weights, and survival. The extracts were non-cytotoxic up to a test dose of 100 μg/ml and 2000 mg/kg fed - mice did not show acute or delayed toxicity. Cucurbita pepo ethanol extract (CpE) displayed excellent in vitro antiplasmodial activity with IC50 of 3.05 μg/ml. At an oral dose of 500 mg/kg, mice were observed to display significant (p < 0.01) ∼51% suppression of parasitemia. The extract did not produce any significant hemolytic effect up to a test concentration of 1 mg/ml. In established infection, a dose of 300 mg/kg significantly (p < 0.01) protected mice from anemia caused by low hematocrit. The extract produced significant (p < 0.05) elevation in red blood cells and platelet counts, and an increase in hemoglobin was evident at 100 and 300 mg/kg. Further, CpE in a dose-dependent manner, reversed liver and spleen weight increase seen in untreated, infected mice. These findings show C. pepo as a potential candidate for further studies to identify its bioactive principle(s) and possible mechanism(s) of antimalarial action.

S252: M2-POLARIZED MACROPHAGES CONTROL LEUKEMIC STEM CELL FATE BY PROMOTING METABOLIC REPROGRAMMING

Background: Being a crucial part of the tumor microenvironment (TME) in solid tumors, tumor-associated macrophages are often associated with poor prognosis (Bruni et al. Nat Rev Cancer 2020). Yet, in acute myeloid leukemia (AML) the role of macrophages remains unclear. Aims: Here, we evaluated the impact of M2 macrophages in AML using a patient derived xenograft (PDX) model. Methods: To do so, we first injected 1x105 peripheral blood (PB) derived M2 macrophages into NSGS mice and next transplanted notoriously difficult to engraft primary Acute Promyelocytic Leukemia (APL) cells (1 x 106 cells - n=7 different patient samples) via the retro-orbital vein. Results: As a result, mice with co-injected human M2-macrophages developed full-blown leukemia with increased spleen weight in comparison with the control. Perhaps even more strikingly, ex vivo culture of APL and other favorable AML subtypes such as inv (16) or NPM1 mutant leukemic cells on M2-macrophages for 48h was sufficient to “train” these cells to engraft and induce fatal leukemia. Maintenance of self-renewal was shown in a secondary transplant assay and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays (LSC frequency: Control: 1/6x106 cells vs M2 pre-culture: 1/7.2x104 cells). To better understand the biological changes induced on leukemic blast when exposed to M2 macrophages, we performed an RNA sequencing analysis comparing AML/APL samples at diagnosis to cells that were “trained” (48h) on M2-macrophages or on MS5 mesenchymal bone marrow stromal cells, as a control. Gene ontology and gene set enrichment analysis on the genes up regulated upon M2 co-culture were significantly enriched for oxidative phosphorylation (OxPhos) signatures. Evaluation of functional respiration using seahorse measurements, confirmed the increase of oxygen consumption rate (OCR, basal and maximum) in primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5. The increase of basal and maximum OCR suggested enhanced mitochondrial metabolism, which prompted us to determine whether macrophages, similar to MSC cells, can transfer mitochondria to primary AML cells (van der Vlist et al. Neuron 2022). Flow cytometry analysis revealed an efficient mitochondrial transfer from M2 macrophages to leukemic blasts, which was more efficient compared to mitochondrial exchange from MS5 cells. Treatment with the CPT1A inhibitor Etomoxir (50 µM), prevented the gain in functional respiration and enhanced proliferation of AML cells when those were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures. These results suggest increased fatty acid oxidation to drive the OXPHO-like state in AML. Summary/Conclusion: Overall, we revealed that M2 macrophages can support leukemic growth of favorable AML subtypes that are commonly difficult to engraft in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter the biology of AML cells transforming a non-engraftable cell into a cell with high leukemic potential. In vitro we show that AML blast cells cultured on M2 macrophage adapt a more OxPhos-like state linked to intrinsic changes associated with increased uptake of mitochondria. In summary, our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions.

Mebendazole, an anti-helminth drug, suppresses inflammation, oxidative stress and injury in a mouse model of ulcerative colitis

Mebendazole (MBZ) is an efficacious anthelmintic with known anti-inflammatory and fibrinolytic properties. In this study, we aimed to explore the protective effects of this FDA-approved drug against DSS-induced colitis in a murine model either alone or in combination with Sulfasalazine (SSZ), a standard therapy for ulcerative colitis. We found that MBZ significantly improved colitis disease activity index as assessed by changes in body weight, degree of stool consistency, rectal bleeding, and prolapse. We also found that MBZ ameliorated the colon histopathological score by attenuating crypt loss, mucosal damage, and inflammation score in colitis tissues. Similarly, DSS-induced colon shortening, colon weight loss, and increase in spleen weight were all abrogated in the presence of MBZ. Moreover, MBZ decreased inflammation, possibly by reducing oxidative stress markers, suppressing inflammatory cell infiltration, and down-regulation of inflammatory genes in colon tissues. Furthermore, MBZ potently reduced fibrosis by decreasing collagen deposition and down-regulating pro-fibrotic genes including Col 1a1 and Col 1a2 in colitis tissue homogenates. In conclusion, our study showed that this broad-spectrum anthelminthic could be repurposed as a novel therapy for ulcerative colitis without any observed side effects, however, regarding the concerns about the potential toxicity of MBZ in UC patients, future experiments on MBZ therapy in other models of UC is needed to completely address the toxicity concerns.

EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice.

Previous studies have reported that deletion of the transcription factor, early growth response protein 2 (EGR2), in normal C57BL/6 (B6) resulted in the development of lupus-like autoimmune disease. However, increased EGR2 expression has been noted in human and murine lupus, which challenges the notion of the autoimmune suppressive role of EGR2 in B6 mice. In this study, we derived both conditional EGR2-/-B6/lpr and EGR2-/-B6 mice to elucidate the immune and autoimmune regulatory roles of EGR2 in autoinflammation (B6/lpr) versus physiologically normal (B6) conditions. We found that conditional EGR2 deletion increased spleen weight, enhanced T cell activation and IFNγ production, and promoted germinal center B cells and LAG3+ regulatory T cells development in both B6/lpr and B6 mice. Nevertheless, EGR2 deletion also showed strikingly differential effects in these two strains on T lymphocyte subsets profile, Foxp3+ Tregs and plasma cell differentiation, anti-dsDNA autoantibodies and immunoglobulins production, and on the induction of IL-17 in in vitro activated splenocytes. Specifically, EGR2 deletion in B6/lpr mice significantly decreased serum levels of anti-dsDNA autoantibodies, total IgG, IgM, IgG1, and IgG2a with reduced plasma cells differentiation. Furthermore, EGR2 deletion in B6/lpr mice had no obvious effect on IgG immunocomplex deposition, medium caliber vessel, and glomeruli inflammation but increased complement C3 immunocomplex deposition and large caliber vessel inflammation in the kidneys. Importantly, we demonstrated that EGR2 deletion in B6/lpr mice significantly reduced pathogenic CD4-CD8-CD3+B220+ double negative T cells, which correlated with the reduced anti-dsDNA autoantibodies in serum and decreased IL-17 production in splenocytes of EGR2-/-B6/lpr mice. Together, our data strongly suggest that the role of EGR2 is complex. The immunoregulatory role of EGR2 varies at normal or autoinflammation conditions and should not be generalized in differential experimental settings.

Experimental Colitis and Visceral Hypersensitivity Are Attenuated by GABA Treatment in Mice

Ulcerative colitis (UC) is characterized by a relapsing mucosal inflammation of the large intestine that appears to be due to an overactive response of the colon- immune system provoking rectal bleeding, diarrhea, and abdominal pain. Based on recent data indicating that 1) GABA suppresses immune cell activity, 2) there is a decrease in GABA in the colon associated with the active phase of UC, 3) that restoring GABA tone has beneficial effects such as ameliorating immune and inflammatory response. We aimed to investigate the effects of GABA treatment on colon persistent inflammation following induction of colitis in mice. Persistent inflammation was induced with seven days of dextran sodium sulfate (DSS, 3%) in the drinking water. GABA (40 mg/Kg) was administered orally for the same period as DSS, and the clinical progression of colitis (DAI) was scored each day. A day after the last GABA treatment the visceral sensitivity was assessed with the visceromotor response (VMR) evoked with balloon distention of the colon and then colon samples were collected for measurement of proinflammatory cytokines. Treatment with GABA reduced changes associated with the presence of visceral inflammation including the decrease in colon length, increase in spleen weight, histological damage to the colon mucosa, and the DAI. Furthermore, GABA markedly inhibited the DSS-induced increase in the proinflammatory cytokines TNF-α, IL-12, and IFN-gamma in the colon tissue. Importantly, GABA decreased DSS-induced visceral hypersensitivity. The data suggest that GABA treatment may be an effective therapy for the management of symptoms associated with UC.

Endogenous NRG4 promotes ST3GAL4 expression, leukocyte infiltration, and inflammation in IL‐10R neutralization colitis

BackgroundThe ErbB family of receptor tyrosine kinases are essential for the regulation of colonic epithelial cell proliferation, differentiation, survival, and wound healing. We previously showed that ErbB4 is induced as a compensatory response by intestinal inflammation; treatment with exogenous NRG4 (an ErbB4 ligand) protects colonocytes against cytokine‐induced apoptosis and ameliorates murine experimental colitis. Exogenous NRG4 can further alleviate intestinal inflammation by suppressing the activity of pro‐inflammatory macrophages. However, the role of endogenous NRG4 in the intestine has not been described.HypothesisBased on our previous studies, we hypothesized that NRG4 deletion would exacerbate intestinal inflammation.MethodsColitis was induced in 5‐week‐old wild‐type (WT) and NRG4 knockout (KO) mice by i.p. injection with 1 mg/kg IL‐10R neutralizing antibody, weekly for four weeks. Inflammation was assessed by fecal lipocalin‐2 ELISA, plasma myeloperoxidase ELISA, and total spleen weight. Disease was assessed by histological scoring of paraffin embedded colon rolls. Cytokine (TNF, IL‐1b, IFNg, and IL‐6) expression was determined by qPCR on RNA from full thickness distal colon homogenates. ST3GAL4 expression was assessed by qPCR and immunohistochemistry. Intestinal immune cell populations were identified and analyzed by mass cytometry (CyTof) using FlowJo software.ResultsFecal lipocalin expression was elevated in all mice after IL‐10R antibody treatment. Surprisingly, WT mice displayed significantly more fecal lipocalin than NRG4 KO cage mates. Similarly, after 4 weeks, WT mice had significantly higher plasma myeloperoxidase (2‐fold, p&lt;0.001), increased spleen weight (57% more, p&lt;0.01), and more histological injury and inflammation (72% higher score, p&lt;0.05) versus their KO cage mates. Furthermore, mRNA levels for IFNg (3.7‐fold difference, p&lt;0.01) and IL‐6 (1.6‐fold difference, p&lt;0.05) were significantly higher in WT versus KO cage mates. CyTof analysis identified a reduction in CD8+ T cell populations, as well as alterations in overall T cell populations. Expression of ST3GAL4, a sialyltransferase that contributes to inflammatory leukocyte recruitment, was significantly reduced in NRG4‐null colons and epithelia (&gt;90% reduction, p&lt;0.01 and p&lt;0.001 respectively), suggesting a mechanism for altered T cell populations.ConclusionIn contrast to the protective effects of exogenous NRG4, endogenous NRG4 appears to participate in recruitment of CD8+ T cells and intestinal inflammation, possibly through regulation of ST3GAL4.

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enablesthe endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitonealP. gingivalis infection.

RETRACTED ARTICLE: Anti-inflammatory Effects of Novel P2X4 Receptor Antagonists, NC-2600 and NP-1815-PX, in a Murine Model of Colitis

The pharmacological blockade of P2X4 receptors has shown potential benefits in the management of several immune/inflammatory diseases. However, data regarding the involvement of P2X4 receptors in the pathophysiological mechanisms of action in intestinal inflammation are not well defined. We aimed to evaluate the anti-inflammatory effects of two novel and selective P2X4 receptor antagonists, NC-2600 and NP-1815-PX, and characterize the molecular mechanisms of their action in a murine model of 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis. These two drugs and dexamethasone (DEX) were administered orally for 6 days, immediately after the manifestation of DNBS. The body weight decrease, resulting from colitis, was attenuated by NC-2600 and NP-1815-PX, but not DEX. However, all three drugs attenuated the increase in spleen weight and ameliorated macroscopic and microscopic colonic tissue damage. Furthermore, all three compounds decreased tissue IL-1β levels and caspase-1 expression and activity. Colonic tissue increase of tumor necrosis factor was downregulated by DEX, while both NC-2600 and NP-1815-PX were ineffective. The reduction of occludin associated with colitis was ameliorated by NC-2600 and NP-1815-PX, but not DEX. In THP-1 cells, lipopolysaccharide and ATP upregulated IL-1β release and NLRP3, caspase-1, caspase-5, and caspase-8 activity, but not of caspase-4. These changes were prevented by NC-2600 and NP-1815-PX treatment. For the first time, the above findings show that the selective inhibition of P2X4 receptors represents a viable approach to manage bowel inflammation via the inhibition of NLRP3 inflammasome signaling pathways.

A144 CHRONIC DSS LEADS TO ALTERED EXPRESSION OF IRAK4/TPL2 PATHWAYS IN UC-COLONIZED MICE

BackgroundUlcerative colitis (UC) is one of two forms of inflammatory bowel disease (IBD). The exact cause of IBD is unknown but altered host-microbe interactions and genetic susceptibility are involved in its pathogenesis. Many patients with IBD do not respond to biological therapies targeting single cytokines, therefore new therapies that target common immune pathways are being developed and need to be tested in relevant preclinical models. Previously we have shown that mice colonized with UC microbiota upregulated genes related to inflammation without induction of colitis compared to healthy volunteer-colonized mice. We thus investigated whether IRAK4 and TPL2-induced pathways, new therapeutic targets in development upstream of inflammatory cytokine gene activation, are upregulated in mice colonized with UC microbiota and chronic colitis.AimsOur aim was to characterize TPL2 and IRAK4 signalling pathways and T cell phenotypes in UC-colonized mice following chronic low-dose dextran sodium sulfate (DSS) colitis.Methods10-16-week-old germ-free C57BL/6 mice were colonized with fecal microbiota from a patient with UC experiencing a flare (n=16). Mice were housed in a gnotobiotic facility during the experiment. Three weeks following colonization, colitis was induced in half of the mice by three cycles (5 d each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a 5-d wash-out period between cycles. All mice were fed a control diet (7004, Teklad). Fecal samples were collected weekly. At sacrifice, disease activity (colon length, occult blood in feces, stool consistency, and spleen weight) was measured and colon tissue was collected for histological analysis and RNA sequencing. Mesenteric lymph nodes (MLNs) were acquired for flow cytometry to analyze T cell phenotypes.ResultsIn UC-colonized mice, chronic low-dose DSS induced softer feces (p<0.0001), shorter colon length (p<0.0001), and increased spleen weight (p<0.0001), compared with water treated mice. This was paralleled by detectable blood in stool, development of moderate colitis (DSS: 1.9+/-1.4 vs H2O: 0.5+/-0.2; p<0.02) and higher proportion of IL-17 (p=0.003) and IFN-γ-producing T cells (p=0.06) in MLNs compared with water treated mice. RNA sequencing revealed that inflammatory genes, mainly related to the IRAK4/TPL2 pathway (e.g., Gadd45b, Socs3, Il1b), were significantly increased (p≤0.05) in the colon of mice treated with DSS compared with water.ConclusionsWhen challenged with a chronic low-dose chemical injury agent, mice colonized with UC microbiota develop clinical and histological signs of colitis and upregulation of genes involved in inflammation like Gadd45b, Socs3, and Il1b. Thus, this model represents a new valuable tool for preclinical testing of new drug candidates, such as those related to the modulation of IRAK4/TPL2 pathways.Funding AgenciesCCC

Bone traits and gastrointestinal tract parameters of piglets fed cholecalciferol and 1,25-dihydroxycholecalciferol glycoside

ABSTRACT The objective of this study was to evaluate the cholecalciferol and 1,25-dihydroxycholecalciferol glycoside supplementation in piglet diet on organ biometry and physicochemical composition, pH of the digestive tract contents, and bone traits. A total of 128 entire male piglets (21 days-old, 6.82±0.38 kg body weight) were distributed in a completely randomized block design with eight replications and four animals per experimental unit. The treatments were composed of a diet 100/0 = 100% of the vitamin D supplemented with cholecalciferol; 50/50 = 50% supplemented with cholecalciferol + 0.25 μg of 1,25(OH)2D3 glycoside; 25/75 = 25% supplemented with cholecalciferol + 0.375 μg of 1,25(OH)2D3 glycoside; and 0/100 = 0.50 μg of 1,25(OH)2D3 glycoside. The results indicated that piglets fed 100/0 showed an increase in spleen weight compared with those fed 0/100 and greater heart dry matter than piglets fed the 50/50 diet. Phosphorus concentration in the spleen was higher in piglets that received 25/75 when compared with those that received the 100/0 and 0/100 diets. Piglets that received the 0/100 diet showed higher pH of the stomach contents, but showed reduced pH of the contents of the jejunum and ileum. The width of the epiphysis and diaphysis was greater when piglets consumed the 50/50 treatment; however, a lower epiphysis height was observed. The use of 1,25(OH)2D3 glycoside alone in piglet diets does not negatively influence the pH of the gastrointestinal tract and organ physicochemical composition. In addition, the use of diets with 1,25(OH)2D3 glycoside as a replacement for cholecalciferol reduces spleen weight and promotes improvements in bone development.

Antitumor activity of solamargine in mouse melanoma model: relevance to clinical safety

ABSTRACT Melanoma is the most aggressive type of skin cancer, and thus it is important to develop new drugs for its treatment. The present study aimed to examine the antitumor effects of solamargine a major alkaloid heteroside present in Solanum lycocarpum fruit. In addition solamargine was incorporated into nanoparticles (NP) of yttrium vanadate functionalized with 3-chloropropyltrimethoxysilane (YVO4:Eu3+:CPTES:SM) to determine antitumor activity. The anti-melanoma assessment was performed using a syngeneic mouse melanoma model B16F10 cell line. In addition, systemic toxicity, nephrotoxic, and genotoxic parameters were assessed. Solamargine, at doses of 5 or 10 mg/kg/day administered subcutaneously to male C57BL/6 mice for 5 days, decreased tumor size and frequency of mitoses in tumor tissue, indicative of a decrease in cell proliferation. Treatments with YVO4:Eu3+:CPTES:SM significantly reduced the number of mitoses in tumor tissue, associated with no change in tumor size. There were no apparent signs of systemic toxicity, nephrotoxicity, and genotoxicity initiated by treatments either with solamargine alone or plant alkaloid incorporated into NP. The animals treated with YVO4:Eu3+:CPTES:SM exhibited significant increase in spleen weight accompanied by no apparent histological changes in all tissues examined. In addition, animals treated with solamargine (10 mg/kg/day) and YVO4:Eu3+:CPTES:SM demonstrated significant reduction in hepatic DNA damage which was induced by tumor growth. Therefore, data suggest that solamargine may be considered a promising candidate in cancer therapy with no apparent toxic effects.

Soluble CD127 potentiates IL-7 activity in vivo in healthy mice.

IntroductionSoluble forms of cytokine receptors can be involved in the endogenous regulation of cytokine activity. Soluble interleukin 7 receptor α (sCD127) naturally binds IL‐7, therefore there is interest in its potential application as an immunotherapeutic agent to regulate IL‐7. With the hypothesis that sCD127 enhances IL‐7 activity, thus promoting T‐cell proliferation in vivo, we sought to assess the effect of sCD127, IL‐7 or IL‐7 + sCD127 treatment on CD4+ and CD8+ T‐cells in the blood and spleen of mice.MethodsPeripheral blood mononuclear cells and splenocytes were prepared, and analyzed for T‐cell number, phenotype and proliferation (Ki67+) by flow cytometry.ResultsIL‐7 treatment induced T‐cell proliferation, increased T‐cell number, and triggered T‐cell differentiation each of which was enhanced with the addition of sCD127. IL‐7 + sCD127 treatment significantly increased spleen weight over that seen with IL‐7 treatment alone. More pronounced proliferation and a greater increase in cell number was observed in CD8+ T‐cells relative to the effect on CD4+ T‐cells.ConclusionsThese findings suggest that the addition of sCD127 enhances IL‐7‐mediated T‐cell proliferation and suggests a potential therapeutic use for sCD127.

Thrombopoietin-Receptor Agonist Romiplostim Attenuates Hematopoietic System Injury By Promoting the Recovery of Early Hematopoiesis and the Hematopoietic Environment in the Bone Marrow and Spleen of Mice Exposed to Lethal Total Body γ-Irradiation

High-dose radiation exposure, usually caused by radiological accidents or nuclear disasters, can cause lead to acute radiation syndrome (ARS), which is a life-threatening condition. The International Atomic Energy Agency recommends the use of a combination of various cytokines or bone marrow (BM) transplant for treating high-dose exposure, depending on the degree of radiation exposure and ARS. Therefore, drugs that respond quickly, which can be used to treat a large number of radiation-associated casualties represent a good treatment option. However, certain hematopoietic cytokines, such as the granulocyte macrophage-colony stimulating factor and interleukin-3, which are effective against hematopoietic dysfunction and have been used in previous accidents, are not approved as pharmaceutical drugs in Japan. Although granulocyte-colony stimulating factor is only approved domestically for the treatment of ARS, it has been found that a single dose is insufficient to improve the survival rate of individuals exposed to lethal ionizing radiation. We have previously demonstrated that an effective protocol of drug therapy, based on approved pharmaceutical drugs in Japan, is the administration of romiplostim (RP), a thrombopoietin-receptor myeloproliferative leukemia virus proto-oncogene (c-mpl) agonist, at a dose of 50 μg/kg body weight/day for 3 consecutive days. This regimen achieved 100% survival at 30-days in 8-weekold female C57BL/6JJcl mice when administered immediately following exposure to a lethal dose (7 Gy) of 137Cs γ-rays. In the present study, we assessed the mechanisms by which RP ameliorated radiation-induced hematopoietic damage in mice after lethal total body γ-irradiation (TBI) by focusing on the recovery and regeneration of BM and spleen. Mice were divided into 4 groups: control group, RP group, TBI group, and TBI + RP group. On days 4, 10, 18, and 30 after TBI, morphological and histological changes of BM and spleen and antigen expression changes of cells isolated from both tissues were assessed. While the body weight of animals in the TBI group decreased significantly by day 18 compared to controls, that of mice receiving RP remained comparable. Similarly, on day 18, leukocyte, platelet, erythrocyte, and hemoglobin levels were the lowest in TBI mice. Conversely, these values markedly recovered in the TBI + RP group. Splenic atrophy was assessed by weighing the spleen after TBI, and it was observed that while the spleen contracted significantly following TBI, mice in the RP + TBI group showed a 3 to 4-fold increase in spleen weight and in cell numbers on day 18. Endogenous colonies, which are indicators of hematopoiesis, were also observed in the spleen after day 10. In addition, TBI induced severe myelosuppression that resulted in the loss of hematopoietic function. On day 18 after irradiation, while the BM exhibited obvious myeloablation, that of the RP + TBI treated mice was comparable to controls. Further, the number of BM cells in mice RP + TBI treated mice continued to increase till 30 days after TBI but did not attain pre-TBI levels. Additionally, analysis of cell-surface antigen expression in BM and spleen cells showed that, compared to the TBI group, RP administration promoted the recovery of hematopoietic stem cells, multipotent progenitor cells, and common lymphocyte precursor cells. In particular, a dramatic increase in the number of common myelo-erythroid progenitors were observed in the spleen on day 18 in the TBI + RP treated mice, accompanied with the disappearance of splenic cyst structures and acceleration of megakaryocyte hematopoiesis. These results suggest that thrombopoietin-receptor agonist RP promotes the recovery of early hematopoiesis and hematopoietic environment in both BM and spleen of lethal TBI mice, especially the promoting megakaryocytopoiesis in the spleen. As a result, treating lethally-irradiated mice with RP results in 100% survival. DisclosuresNo relevant conflicts of interest to declare.

Ulk1 Kinase Mediates Clearance of Free α-Globin in β-Thalassemia

Normal erythroid maturation is highly regulated to maximize the production of HbA heterotetramer (α2β2) and minimize the accumulation of potentially toxic free α- or β-globin subunits. In β-thalassemia, β-globin gene (HBB) mutations cause buildup of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Endogenous mechanisms exist to detoxify free α-globin, as evidenced by clinical observations that most individuals with HBB haploinsufficiency (β-thalassemia trait) are asymptomatic. Previously, we showed that free α-globin is eliminated in erythroid precursors by the ubiquitin-proteasome (UPS) system and by a lysosomal-dependent process presumed to be autophagy (Khandros et al., Blood 2012;119:5265). Our current study investigates the latter.Atg5 is required for basal autophagy and autophagosome formation via conjugation with Atg12 in most cell types. We investigated whether Atg5 played a selective role in eliminating free α-globin during unbalanced globin chain synthesis. β-thalassemic mice (HbbTh3/+) carrying a conditional Atg5 allele (Atg5fl) in which exon 3 is flanked by loxP sites were interbred with mice expressing erythroid-specific Cre recombinase (EpoR-Cre). Loss of Atg5 in β-thalassemic erythroid precursors inhibited autophagy, as evidenced by the accumulation of the markers p62/SQSTM1 and LC3. However, there was minimal effect on the β-thalassemic phenotype as measured by RBC count, Hb, hematocrit, reticulocyte count, and spleen weight. Moreover, loss of Atg5 did not alter the level of insoluble α-globin in β-thalassemic RBC fractions, as assessed by triton-acetic acid-urea (TAU) gel electrophoresis to resolve the α- and β-globin chains. Thus, Atg5, a key component of canonical autophagy, is not required for the elimination of free α-globin in β-thalassemia.The Ulk1 (unc-51−like kinase 1) protein kinase participates in canonical and alternative autophagy pathways and mediates mitophagy during erythropoiesis. To determine the role of Ulk1 in a-globin degradation, we introduced a null allele into HbbTh3/+ mice. Ulk1−/− HbbTh3/+ mice were born at normal Mendelian ratio, but exhibited a high rate of perinatal death. Only 20% of double-mutant mice survived for 30 days (n=5), compared to 70% of Ulk1+/+ HbbTh3/+ mice (n=13) (P <0.05). To examine the consequences of Ulk1 loss in β-thalassemic erythroblasts, we transplanted whole bone marrow cells from double mutants and controls (CD45.2) into lethally irradiated wild-type hosts (CD45.1; >87% engraftment after 30 days). Loss of Ulk1 exacerbated the β-thalassemic phenotype, as evidence by reduced Hb (10.85 ± 0.18 vs 10.21 ± 0.18 g/dL, n=14 vs 8; P <0.01), reduced RBC number (7.37 ± 0.13 vs 6.35 ± 0.09 × 106/mL; P <0.0001), increased reticulocyte count (17.99 ± 2.76 vs 39.41 ± 2.14%; P <0.0001), and increased spleen weight (0.24 ± 0.01 vs 0.34 ± 0.03 g; P <0.01). Moreover, insoluble α-globin was increased by approximately 2-fold in Ulk1−/− HbbTh3/+ reticulocytes, ascompared to Ulk1+/+ HbbTh3/+ cells (Figure). To assess the turnover of newly synthesized free α-globin, we pulse-labeled reticulocytes with 35S amino acids and chased with or without a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine or bafilomycin A1). Analysis of control (Ulk1+/+ HbbTh3/+) reticulocytesshowed that newly synthesized insoluble free α-globin was cleared by separate proteasomal and lysosomal pathways. In marked contrast, the lysosomal-dependent α-globin degradation pathway was completely eliminated in Ulk1−/− HbbTh3/+ reticulocytes. Thus, we conclude that Ulk1 mediates the degradation of free α-globin in β-thalassemia.Our findings illustrate a new mechanism through which protein quality-control pathways can modulate the severity of β-thalassemia by eliminating unstable free α-globin, and they open up possibilities for new therapeutic approaches, as Ulk1 activity can be modulated pharmacologically. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preclinical Development of FA5, a Novel AMP-Activated Protein Kinase (AMPK) Activator as an Innovative Drug for the Management of Bowel Inflammation.

Acadesine (ACA), a pharmacological activator of AMP-activated protein kinase (AMPK), showed a promising beneficial effect in a mouse model of colitis, indicating this drug as an alternative tool to manage IBDs. However, ACA displays some pharmacodynamic limitations precluding its therapeutical applications. Our study was aimed at evaluating the in vitro and in vivo effects of FA-5 (a novel direct AMPK activator synthesized in our laboratories) in an experimental model of colitis in rats. A set of experiments evaluated the ability of FA5 to activate AMPK and to compare the efficacy of FA5 with ACA in an experimental model of colitis. The effects of FA-5, ACA, or dexamethasone were tested in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis to assess systemic and tissue inflammatory parameters. In in vitro experiments, FA5 induced phosphorylation, and thus the activation, of AMPK, contextually to the activation of SIRT-1. In vivo, FA5 counteracted the increase in spleen weight, improved the colon length, ameliorated macroscopic damage score, and reduced TNF and MDA tissue levels in DNBS-treated rats. Of note, FA-5 displayed an increased anti-inflammatory efficacy as compared with ACA. The novel AMPK activator FA-5 displays an improved anti-inflammatory efficacy representing a promising pharmacological tool against bowel inflammation.

Immune-Enhancing Effects of Lactobacillus plantarum 200655 Isolated from Korean Kimchi in a Cyclophosphamide-Induced Immunocompromised Mouse Model.

In this study, we evaluated the immune-enhancing activity of kimchi-derived Lactobacillus plantarum 200655 on immune suppression by cyclophosphamide (CP) in ICR mice. Animals were fed distilled water or 1×109 colony-forming unit/kg B.W. 200655 or Lactobacillus rhamnosus GG as a positive control for 14 days. An in vivo model of immunosuppression was induced using CP 150 and 100 mg/kg B.W. at 7 and 10 days, respectively. Body weight, spleen index, spleen weight, and gene expression were measured to estimate the immune-enhancing effects. The dead 200655 (D-200655) group showed an increased spleen weight compared to the sham control (SC) group. Similarly, the spleen index was significantly higher than that in the CP-treated group. The live 200655 (L-200655) group showed an increased mRNA expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 in splenocytes. Also, the iNOS and COX-2 mRNA expression was upregulated in the L-200655 group compared to the CP-only (SC) group. The phosphorylation of ERK and MAPK was also upmodulated in the L-200655 group. These results indicate that L. plantarum 200655 ameliorated CP-induced immune suppression, suggesting that L. plantarum 200655 may have the potential to enhance the immune system.

Polysaccharide Isolated From Tetrastigma hemsleyanum Activates TLR4 in Macrophage Cell Lines and Enhances Immune Responses in OVA-Immunized and LLC-Bearing Mouse Models.

Tetrastigma hemsleyanum Diels et Gilg is a valuable Chinese medicinal herb with a long history of clinical application. Our previous study isolated and characterized a purified polysaccharide from the aerial part of Tetrastigma hemsleyanum (SYQP) and found it having antipyretic and antitumor effects in mice. A preliminary mechanistic study suggests these effects may be related to the binding of toll-like receptor (TLR4). The objective of this study is to further explore the detailed stimulating characteristics of SYQP on TLR4 signaling pathway and its in vivo immune regulating effect. We use HEK-BLUE hTLR4, mouse and human macrophage cell lines, as research tools. In vitro results show SYQP activated HEK-BLUE hTLR4 instead of HEK-BLUE Null cells. The secretion and the mRNA expression of cytokines related to TLR4 signaling significantly increased after SYQP treatment in both PMA-induced THP-1 and RAW264.7 macrophage cell lines. The TLR4 antagonist TAK-242 can almost completely abolish this activation. Furthermore, molecules such as IRAK1, NF-κB, MAPKs, and IRF3 in both the MyD88 and TRIF branches were all activated without pathway selection. In vivo results show SYQP enhanced antigen-specific spleen lymphocyte proliferation and serum IgG levels in OVA-immunized C57BL/6 mice. Orally administered 200 mg/kg SYQP induced obvious tumor regression, spleen weight increase, and the upregulation of the mRNA expression of TLR4-related cytokines in Lewis lung carcinoma–bearing mice. These results indicate SYQP can act as both a human and mouse TLR4 agonist and enhance immune responses in mice (p < 0.05). This study provides a basis for the development and utilization of SYQP as a new type of TLR4 agonist in the future.

Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice.

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.

Protective effect of exopolysaccharide fraction from Bacillus subtilis against dextran sulfate sodium-induced colitis through maintenance of intestinal barrier and suppression of inflammatory responses

We previously reported that an exopolysaccharide-enriched fraction from Bacillus subtilis J92 (B-EPS) could improve immune functions by regulating the immunological parameters of IFN-γ-primed macrophages, CD3/CD28-stimulated splenocytes, and in cyclophosphamide-induced immunosuppressed mice. In the present study, we investigated whether B-EPS contributes to the maintenance of intestinal barrier integrity in a dextran sodium sulfate (DSS)-induced colitis mouse model that mimics human inflammatory bowel disease (IBD). B-EPS treatment improved histological characteristics and common features including a high disease activity index (DAI), an increased spleen weight, and colon shortening in DSS-induced colitis. B-EPS also effectively restored intestinal barrier function by modulating tight junction-related proteins (claudin-1, claudin-2, and occludin) and epithelial-mesenchymal transition (EMT) marker proteins (E-cadherin, N-cadherin, and vimentin). Moreover, B-EPS downregulated immune cell infiltration and inflammatory responses including the production of inflammatory cytokines, such as IL-6 and IL-1β, and activation of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Taken together, these results suggest that B-EPS could serve as a functional food ingredient for improving intestinal barrier function and alleviating colonic inflammation in IBD.