Necroptosis Mediates Muscle Protein Degradation in a Cachexia Model of Weanling Pig with Lipopolysaccharide Challenge.

Necroptosis, an actively researched form of programmed cell death closely related to the inflammatory response, is important in a variety of disorders and diseases. However, the relationship between necroptosis and muscle protein degradation in cachexia is rarely reported. This study aimed to elucidate whether necroptosis played a crucial role in muscle protein degradation in a cachexia model of weaned piglets induced by lipopolysaccharide (LPS). In Experiment 1, the piglets were intraperitoneally injected with LPS to construct the cachexia model, and sacrificed at different time points after LPS injection (1, 2, 4, 8, 12, and 24 h). In Experiment 2, necrostatin-1 (Nec-1), a necroptosis blocker, was pretreated in piglets before the injection of LPS to inhibit the occurrence of necroptosis. Blood and longissimus dorsi muscle samples were collected for further analysis. In the piglet model with LPS-induced cachexia, the morphological and ultrastructural damage, and the release of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 were dynamically elicited in longissimus dorsi muscle. Further, protein concentration and protein/DNA ratio were dynamically decreased, and protein degradation signaling pathway, containing serine/threonine kinase (Akt), Forkhead box O (FOXO), muscular atrophy F-box (MAFbx), and muscle ring finger protein 1 (MuRF1), was dynamically activated in piglets after LPS challenge. Moreover, mRNA and protein expression of necroptosis signals including receptor-interacting protein kinase (RIP)1, RIP3, and mixed lineage kinase domain-like pseudokinase (MLKL), were time-independently upregulated. Subsequently, when Nec-1 was used to inhibit necroptosis, the morphological damage, the increase in expression of pro-inflammatory cytokines, the reduction in protein content and protein/DNA ratio, and the activation of the protein degradation signaling pathway were alleviated. These results provide the first evidence that necroptosis mediates muscle protein degradation in cachexia by LPS challenge.

Effect of flaxseed oil on muscle protein loss and carbohydrate oxidation impairment in a pig model after lipopolysaccharide challenge.

Flaxseed oil is rich in α-linolenic acid (ALA), which is the metabolic precursor of EPA and DHA. The present study investigated the effect of flaxseed oil supplementation on lipopolysaccharide (LPS)-induced muscle atrophy and carbohydrate oxidation impairment in a piglet model. Twenty-four weaned pigs were used in a 2 × 2 factorial experiment including dietary treatment (5 % maize oil v. 5 % flaxseed oil) and LPS challenge (saline v. LPS). On day 21 of treatment, the pigs were injected intraperitoneally with 100 µg/kg body weight LPS or sterile saline. At 4 h after injection, blood, gastrocnemius muscle and longissimus dorsi muscle were collected. Flaxseed oil supplementation increased ALA, EPA, total n-3 PUFA contents, protein:DNA ratio and pyruvate dehydrogenase complex quantity in muscles (P < 0·05). In addition, flaxseed oil reduced mRNA expression of toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) 2 and their downstream signalling molecules in muscles and decreased plasma concentrations of TNF-α, IL-6 and IL-8, and mRNA expression of TNF-α, IL-1ß and IL-6 (P < 0·05). Moreover, flaxseed oil inclusion increased the ratios of phosphorylated protein kinase B (Akt) 1:total Akt1 and phosphorylated Forkhead box O (FOXO) 1:total FOXO1 and reduced mRNA expression of FOXO1, muscle RING finger (MuRF) 1 and pyruvate dehydrogenase kinase 4 in muscles (P < 0·05). These results suggest that flaxseed oil might have a positive effect on alleviating muscle protein loss and carbohydrates oxidation impairment induced by LPS challenge through regulation of the TLR4/NOD and Akt/FOXO signalling pathways.

Medium-Chain Triglycerides Attenuate Liver Injury in Lipopolysaccharide-Challenged Pigs by Inhibiting Necroptotic and Inflammatory Signaling Pathways.

This study was conducted to investigate whether medium-chain triglycerides (MCTs) attenuated lipopolysaccharide (LPS)-induced liver injury by down-regulating necroptotic and inflammatory signaling pathways. A total of 24 pigs were randomly allotted to four treatments in a 2 × 2 factorial design including diet (0 and 4% MCTs) and immunological challenge (saline and LPS). After three weeks of feeding with or without 4% MCTs, pigs were challenged with saline or LPS. MCTs led to a significant increase in eicosapentaenoic acid, docosahexaenoic acid and total (n-3) polyunsaturated fatty acid concentrations. MCTs attenuated LPS-induced liver injury as indicated by an improvement in liver histomorphology and ultrastructural morphology of hepatocytes, a reduction in serum alanine aminotransferase and alkaline phosphatase activities as well as an increase in claudin-1 protein expression. In addition, MCTs also reduced serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 concentrations, liver TNF-α and IL-1ß mRNA expression and protein concentrations and enhanced liver heat shock protein 70 protein expression in LPS-challenged pigs. Moreover, MCTs decreased mRNA expression of receptor-interacting serine/threonine-protein kinase (RIP) 3, mixed-lineage kinase domain-like protein (MLKL) and phosphoglycerate mutase 5 and inhibited MLKL phosphorylation in the liver. Finally, MCTs decreased liver mRNA expression of toll-like receptor (TLR) 4, nucleotide-binding oligomerization domain protein (NOD) 1 and multiple downstream signaling molecules. MCTs also suppressed LPS-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and increased extracellular signal-related kinase 1/2 phosphorylation in the liver. These results indicated that MCTs are capable of attenuating LPS-induced liver damage by suppressing hepatic necroptotic (RIP1/RIP3/MLKL) and inflammatory (TLR4/NOD1/p38 MAPK) signaling pathways.

Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89.

Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.

Definitive hematopoiesis requires Runx1 C-terminal-mediated subnuclear targeting and transactivation.

Runx1 is a key hematopoietic transcription factor required for definitive hematopoiesis and is a frequent target of leukemia-related chromosomal translocations. The resulting fusion proteins, while retaining DNA binding activity, display loss of subnuclear targeting and associated transactivation functions encoded by the C-terminus of the protein. To define the precise contribution of the Runx1 C-terminus in development and leukemia, we created a knock-in mouse with a C-terminal truncation by introducing a single nucleic acid substitution in the native Runx1 locus. This mutation (Runx1(Q307X)) models genetic lesions observed in patients with leukemia and myeloproliferative disorders. The Runx1(Q307X) homozygous mouse exhibits embryonic lethality at E12.5 due to central nervous system hemorrhages and a complete lack of hematopoietic stem cell function. While able to bind DNA, Runx1(Q307X) is unable to activate target genes, resulting in deregulation of various hematopoietic markers. Thus, we demonstrate that the subnuclear targeting and transcriptional regulatory activities of the Runx1 C-terminus are critical for hematopoietic development. We propose that compromising the C-terminal functions of Runx1 is a common mechanism for the pathological consequences of a variety of somatic mutations and Runx1-related leukemic fusion proteins observed in human patients.

Glutamate attenuates lipopolysaccharide induced intestinal barrier injury by regulating corticotropin-releasing factor pathway in weaned pigs.

OBJECTIVE: The purpose of this study was to evaluate the protection of glutamate (GLU) against the impairment in intestinal barrier function induced by lipopolysaccharide (LPS) stress in weaned pigs. METHODS: Twenty-four weaned pigs were divided into four treatments containing: i) non-challenged control, ii) LPS-challenged control, iii) LPS+1.0% GLU, and iv) LPS+2.0% GLU. On day 28, pigs were treated with LPS or saline. Blood samples were collected at 0, 2, and 4 h post-injection. After blood samples collection at 4 h, all pigs were slaughtered, and spleen, mesenteric lymph nodes, liver and intestinal samples were obtained. RESULTS: Dietary GLU supplementation inhibited the LPS-induced oxidative stress in pigs, as demonstrated by reduced malondialdehyde level and increased glutathione level in jejunum. Diets supplemented with GLU enhanced villus height, villus height/crypt depth and claudin-1 expression, attenuated intestinal histology and ultrastructure impairment induced by LPS. Moreover, GLU supplementation reversed intestinal intraepithelial lymphocyte number decrease and mast cell number increase induced by LPS stress. GLU reduced serum cortisol concentration at 4 h after LPS stress and downregulated the mRNA expression of intestinal corticotropin-releasing factor signal (corticotrophin-releasing factor [CRF], CRF receptor 1 [CRFR1], glucocorticoid receptor, tryptase, nerve growth factor, tyrosine kinase receptor A), and prevented mast cell activation. GLU upregulated the mRNA expression of intestinal transforming growth factor ß. CONCLUSION: These findings indicate that GLU attenuates LPS-induced intestinal mucosal barrier injury, which is associated with modulating CRF signaling pathway.

Necroptosis is active and contributes to intestinal injury in a piglet model with lipopolysaccharide challenge.

Necroptosis, a newly discovered form of programmed cell death that combines the features of apoptosis and necrosis, is important in various physiological and pathological disorders. However, the role of necroptosis on intestinal injury during sepsis has been rarely evaluated. This study aimed to investigate the presence of necroptosis in intestinal injury, and its contribution to intestinal injury in a piglet model challenged with Escherichia coli lipopolysaccharide (LPS). Firstly, a typical cell necrotic phenomenon was observed in jejunum of LPS-challenged pigs by transmission electron microscope. Protein expression of necroptosis signals including receptor-interacting protein kinase (RIP) 1, RIP3, and phosphorylated mixed-lineage kinase domain-like protein (MLKL), mitochondrial proteins including phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (DRP1), and cytoplasmic high-mobility group box 1 (HMGB1) were time-independently increased in jejunum of LPS-challenged piglets, which was accompanied by the impairment of jejunal morphology, and digestive and barrier function indicated by lower activities of jejunal disaccharidases and protein expression of jejunal tight junction proteins claudin-1 and occludin. Pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were also dynamically induced in serum and jejunum of piglets after LPS challenge. Moreover, pretreatment with necrostatin-1 (Nec-1), an specific inhibitor of necroptosis, inhibited necroptosis indicated by decreased necrotic ultrastructural changes and decreased protein expression of RIP1, RIP3, and phosphorylated MLKL as well as PGAM5, DRP1, and cytoplasmic HMGB1. Nec-1 pretreatment reduced jejunal morphological injury, and improved digestive and barrier function. Nec-1 pretreatment also decreased the levels of serum and jejunal pro-inflammatory cytokines and the numbers of jejunal macrophages and monocytes. These findings indicate for the first time that necroptosis is present and contributes to LPS-induced intestinal injury. Nec-1 may have a preventive effect on intestinal injury during sepsis.

Kinetics of changes in gene and microRNA expression related with muscle inflammation and protein degradation following LPS-challenge in weaned piglets.

To test the dynamic changes of the expression of genes and microRNA in the gastrocnemius muscle after LPS challenge, 36 piglets were assigned to a control group (slaughtered 0 h after saline injection) and LPS groups (slaughtered at 1 h, 2 h, 4 h, 8 h, and 12 h after LPS treatment, respectively). After LPS treatment, the mRNA expression of IL-1ß, IL-6, and TNF-α reached maximal levels at 1 h, 2 h, and 1 h, respectively (P < 0.05), and mRNA expression of TLR4, NODs, muscle-specific ring finger 1, and muscle atrophy F-box peaked at 12 h (P < 0.05). Moreover, the expression of miR-122, miR-135a, and miR-370 reduced at 1 h, 1 h, and 2 h, respectively (P < 0.05), and miR-34a, miR-224, miR-132, and miR-145 reached maximum expression levels at 1 h, 1 h, 2 h, and 4 h, respectively (P < 0.05). These results suggested that mRNA expression of pro-inflammatory cytokines was elevated in the early stage, mRNA expression of genes related to TLR4 and NODs signaling pathways and protein degradation increased in the later phase, and the expression of microRNA related to muscle inflammation and protein degradation changed in the early stage after LPS injection.

Necroptosis Underlies Hepatic Damage in a Piglet Model of Lipopolysaccharide-Induced Sepsis.

Background: Necroptosis is a newly recognized form of programmed cell death with characteristics of both necrosis and apoptosis. The role of necroptosis in hepatic damage during sepsis is poorly understood. In this study, we investigated the occurrence of necroptosis in hepatic damage, and its contribution to hepatic damage in a piglet model of lipopolysaccharide (LPS)-induced sepsis. Methods: Two animal experiments were conducted. In trial 1, piglets were challenged with LPS and sacrificed at different time points after LPS challenge. In trial 2, piglets were pretreated with necrostatin-1, a specific inhibitor of necroptosis, prior to LPS challenge. Alterations in the hepatic structure and function, pro-inflammatory cytokine expression, and the necroptosis signaling pathway were investigated. Typical ultrastructural characteristics of cell necrosis was observed in the liver of LPS-challenged piglets. Results: Expressions of critical components of necroptosis including kinases (RIP1, RIP3, and MLKL), mitochondrial proteins (PGAM5 and DRP1), and an intracellular damage-associated molecular pattern (HMGB1) were increased in the liver in a time-dependent manner, followed by hepatic inflammation, morphological damage, and dysfunction as manifested by elevated hepatic expression of IL-1ß, IL-6 and TNF-α as well as increased serum AST and AKP activities and the AST/ALT ratio. Pretreatment with necrostatin-1 significantly reduced the expression of RIP1, RIP3 and MLKL as well as PGAM5, DRP1 and HMGB1, which subsequently led to obvious attenuation of hepatic inflammation and damage. Conclusions: Our study demonstrates that necroptosis occurs in the liver during sepsis and contributes to septic hepatic injury.

Prophylactic orthosteric inhibition of leukocyte integrin CD11b/CD18 prevents long-term fibrotic kidney failure in cynomolgus monkeys.

Ischaemic acute kidney injury (AKI), an inflammatory disease process, often progresses to chronic kidney disease (CKD), with no available effective prophylaxis. This is in part due to lack of clinically relevant CKD models in non-human primates. Here we demonstrate that inhibition of the archetypal innate immune receptor CD11b/CD18 prevents progression of AKI to CKD in cynomolgus monkeys. Severe ischaemia-reperfusion injury of the right kidney, with subsequent periods of the left ureter ligation, causes irreversible right kidney failure 3, 6 or 9 months after AKI. Moreover, prophylactic inactivation of CD11b/CD18, using the orthosteric CD11b/CD18 inhibitor mAb107, improves microvascular perfusion and histopathology, reduces intrarenal pro-inflammatory mediators and salvages kidney function long term. These studies reveal an important early role of CD11b+ leukocytes in post-ischaemic kidney fibrosis and failure, and suggest a potential early therapeutic intervention to mitigate progression of ischaemic AKI to CKD in humans.

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

Subnuclear targeting of Runx1 is required for synergistic activation of the myeloid specific M-CSF receptor promoter by PU.1.

Many types of acute myelogenous leukemia involve chromosomal translocations that target the C-terminus of Runx1/AML1 transcription factor, a master regulator of hematopoiesis. The C-terminus of Runx1/AML1 that includes the nuclear matrix targeting signal (NMTS) is essential for embryonic development, hematopoiesis, and target gene regulation. During the onset and normal progression of hematopoiesis, several lineage-specific factors such as C/EBPalpha and PU.1 interact with Runx1 to regulate transcription combinatorially. Here we addressed the functional interplay between subnuclear targeting of Runx1 and gene activation during hematopoiesis. Point mutations were generated in the NMTS of the human Runx1 protein and tested for their effect on transcriptional cooperativity with C/EBPalpha and PU.1 at myeloid-specific promoters. We characterized five mutants that do not alter nuclear import, DNA binding or C/EBPalpha-dependent synergistic activation of the target gene promoters. However a critical tyrosine in the NMTS is required for subnuclear targeting and activation of the granulocyte-macrophage colony stimulating factor (GM-CSF) promoter. Furthermore, this point mutation is defective for transcriptional synergism with PU.1 on the macrophage colony stimulating factor (MCSF) receptor c-FMS promoter. Our results indicate that the NMTS region of Runx1 is required for functional interactions with PU.1. Taken together, our findings establish that subnuclear targeting of Runx1 is a critical component of myeloid-specific transcriptional control.