Inflammation-induced hepcidin-25 is associated with the development of anemia in septic patients: an observational study

Introduction  

Anemia is a frequently encountered problem during inflammation. Hepcidin is an interleukin-6 (IL-6)-induced key modulator of inflammation-associated anemia. Human sepsis is a prototypical inflammatory syndrome, often complicated by the development of anemia. However, the association between inflammation, hepcidin release and anemia has not been demonstrated in this group of patients. Therefore, we explored the association between hepcidin and sepsis-associated anemia.     

Anemia of inflammation: the cytokine-hepcidin link

The anemia of inflammation, commonly observed in patients with chronic infections, malignancy, trauma, and inflammatory disorders, is a well-known clinical entity. Until recently, we understood little about its pathogenesis. It now appears that the inflammatory cytokine IL-6 induces production of hepcidin, an iron-regulatory hormone that may be responsible for most or all of the features of this disorder.     

重组人红细胞生成素对人多发性骨髓瘤患者铁调节蛋白Hepcidin mRNA表达的影响

本研究探讨人多发性骨髓瘤血清对肝腺瘤细胞系Hep-3b细胞铁调节蛋白hepcidin表达的影响,及白介素-6(IL-6)单克隆抗体或重组人红细胞生成素(rhEPO)对其的抑制作用。在Hep-3b细胞培养液中按10%终浓度加入人多发性骨髓瘤患者血清,共培养后用逆转录聚合酶链式反应半定量方法检测Hep-3b细胞hepcidin mRNA的表达水平。在上述培养体系中,加入人IL-6单克隆抗体或rhEPO观察hepcidin mRNA表达的变化。结果表明,未治疗的多发性骨髓瘤组hepcidin mRNA表达量高于正常对照组及缺铁性贫血组,这种升高作用能被IL-6单克隆抗体或rhEPO完全抑制。对多发性骨髓瘤患者短期随访中,规律治疗能稳定血红蛋白,降低患者血清对Hep-3b细胞hepcidin mRNA的影响。结论:未治疗的多发性骨髓瘤患者血清能促进肝腺瘤细胞系Hep-3b细胞hepcidin表达,这种促进作用可被人IL-6单克隆抗体拮抗,提示IL-6可能导致Hep-3b细胞hepcidin表达量升高,从而造成慢性病性贫血(anemia of chronic disease,ACD)。这种促进作用也能被rhEPO抑制,提示rhEPO可能对ACD有治疗作用。在多发性骨髓瘤患者短期随访中,血红蛋白水平稳定,患者血清对Hep-3b细胞hepcidin mRNA的影响减小,这表明治疗使病情稳定,改善了ACD。     

EPO对铁调节蛋白Hepcidin表达影响的研究

本研究探讨红细胞生成素（EPO）在正常小鼠、急性炎症小鼠和慢性病性贫血（ACD）小鼠模型中对hepcidin mRNA表达的影响。通过小鼠背部皮下双肩胛骨间脂肪垫内注射松节油的方法建立急性炎症模型和ACD模型。正常小鼠、急性炎症期小鼠和ACD小鼠腹腔注射EPO后用逆转录聚合酶链式反应（RT—PCR）半定量法检测肝脏hepcidin mRNA的表达水平。结果显示，长期慢性炎症并未使小鼠肝脏hepcidin mRNA表达显著升高，但单次注射松节油可以使小鼠肝脏hepcidin mRNA水平一过性显著升高。腹腔注射EPO能够抑制正常小鼠、ACD小鼠与急性炎症小鼠肝脏hepcidin mRNA的表达。ACD小鼠在注射EPO后血红蛋白水平与肝脏hepcidinmRNA表达水平呈线性负相关。结论：皮下多次注射松节油可以建立ACD模型。在ACD发病过程早期肝脏hepcidin mRNA升高，在此之后恢复正常。EPO在正常条件、急性炎症条件和慢性炎症条件下均可抑制hepeidin mRNA的表达。     

Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion

TLRs are recognized as promoters of tissue damage, even in the absence of pathogens. TLR binding to damage-associated molecular patterns (DAMPs) released by injured host cells unleashes an inflammatory cascade that amplifies tissue destruction. However, whether TLRs possess the reciprocal ability to curtail the extent of sterile inflammation is uncertain. Here, we investigated this possibility in mice by studying the role of conventional DCs (cDCs) in liver ischemia/reperfusion (I/R) injury, a model of sterile inflammation. Targeted depletion of mouse cDCs increased liver injury after I/R, as assessed by serum alanine aminotransferase and histologic analysis. In vitro, we identified hepatocyte DNA as an endogenous ligand to TLR9 that promoted cDCs to secrete IL-10. In vivo, cDC production of IL-10 required TLR9 and reduced liver injury. In addition, we found that inflammatory monocytes recruited to the liver via chemokine receptor 2 were downstream targets of cDC IL-10. IL-10 from cDCs reduced production of TNF, IL-6, and ROS by inflammatory monocytes. Our results implicate inflammatory monocytes as mediators of liver I/R injury and reveal that cDCs respond to DAMPS during sterile inflammation, providing the host with protection from progressive tissue damage.     

Corticotropin-releasing hormone regulates IL-6 expression during inflammation

Stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by proinflammatory cytokines results in increased release of glucocorticoid that restrains further development of the inflammatory process. IL-6 has been suggested to stimulate the HPA axis during immune activation independent of the input of hypothalamic corticotropin-releasing hormone (CRH). We used the corticotropin-releasing hormone-deficient (Crh(-/-)) mouse to elucidate the effect of CRH deficiency on IL-6 expression and IL-6-induced HPA axis activation during turpentine-induced inflammation. We demonstrate that during inflammation CRH is required for a normal adrenocorticotropin hormone (ACTH) increase but not for adrenal corticosterone rise. The paradoxical increase of plasma IL-6 associated with CRH deficiency suggests that IL-6 release during inflammation is CRH-dependent. We also demonstrate that adrenal IL-6 expression is CRH-dependent, as its basal and inflammation-induced expression is blocked by CRH deficiency. Our findings suggest that during inflammation, IL-6 most likely compensates for the effects of CRH deficiency on food intake. Finally, we confirm that the HPA axis response is defective in Crh(-/-)/IL-6(-/-) mice. These findings, along with the regulation of IL-6 by CRH, support the importance of the interaction between the immune system and the HPA axis in the pathophysiology of inflammatory diseases.     

Pathogenesis of anemia of chronic disease

Anemia of chronic disease (ACD) is a mild to moderate anemia seen with many infections and inflammatory disorders. These patients have low serum iron, but high serum ferritin levels. As for the pathogenesis of ACD, previous studies have reported several abnormalities, such as insufficient EPO production, impaired growth response of erythroid progenitors to EPO, and shortened survival of erythrocytes, due to the inflammatory cytokines. However, recent analyses have clearly shown that hepcidin, of which expression is induced by inflammatory cytokines such as IL-1beta and IL-6, suppresses the expression of the iron transporter, ferroportin-1, thereby inhibiting the absorption of iron from the duodenum, the release of iron from the reticulo-endothelial system. So, the abnormal expression of hepcidin alone may be able to explain the unique iron metabolism in ACD.     

Balance of pro- and anti-inflammatory cytokines in liver surgery.

Inflammatory response in surgery is associated with the release of cytokines. Many cytokines are produced by macrophages; therefore surgical injuries to the liver may have great influence on the release of cytokines. Ischemia creates tissue injury and may contribute to the cytokine release. A balanced ratio of pro- and anti-inflammatory cytokines is important for appropriate immune response; excessive inflammation or hypo-responsiveness can lead to post-operative complications. To determine the magnitude of the cytokine response caused by liver surgery and to evaluate the balance of pro- and anti-inflammatory cytokines released during the operation, we measured levels of tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-1beta, IL-6 and IL-10 in 19 patients undergoing liver resection. The results showed a continuous rise of IL-6 and a transient elevation of IL-10. Levels of TNFalpha remained low; IL-1beta was not detected at any sampling time. We conclude that liver surgery induces cytokine response characterized predominantly by an early appearance of IL-6 and IL-10, the elevation of IL-6 may be mainly caused by splanchnic ischemia. The IL-6/IL-10 ratio could possibly reflect the balance of pro- and anti-inflammatory cytokines in liver surgery better than the TNFalpha/IL-10 ratio, which can well represent inflammatory status in sepsis.     

Defective inflammatory response in interleukin 6-deficient mice

Systemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interleukin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor alpha (TNF-alpha). The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL- 6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-alpha than wild-type controls, suggesting that increased TNF-alpha production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothalamic-pituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.     

Serum hepcidin in haemodialysis patients: associations with iron status and microinflammation

Hepcidin plays a key role in iron homeostasis. This cross-sectional study measured the serum hepcidin levels of 48 maintenance haemodialysis patients and 20 age-matched healthy control subjects using a competitive enzyme-linked immunosorbent assay (C-ELISA). Serum hepcidin, interleukin (IL)-6 and high-sensitivity C-reactive protein levels were significantly higher in maintenance haemodialysis patients compared with control subjects. In all patients, there was a positive correlation between serum hepcidin levels and ferritin, transferrin saturation and IL-6, and an inverse correlation between serum hepcidin and unsaturated iron-binding capacity, total iron-binding capacity (TIBC) and transferrin. Linear regression analyses showed that ferritin and TIBC were independently associated with serum hepcidin levels. In conclusion, serum hepcidin levels are associated with iron status and microinflammation (defined as hsCRP < 15 mg/l, without clinical manifestation of inflammation) in maintenance haemodialysis patients. The C-ELISA method for measuring serum hepcidin should facilitate the routine measurement of hepcidin in clinical practice.     

The gene encoding the iron regulatory peptide hepcidin is regulated by anemia,hypoxia, and inflammation

The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders.     

Elimination of innate immune responses and liver inflammation by PEGylation of adenoviral vectors and methylprednisolone

Improvement of the therapeutic index of adenoviral gene transfer requires the development of strategies to abrogate adenoviral capsid-induced inflammation and cytokine production. The effect of monomethoxypolyethylene glycol (MPEG) conjugation to adenoviral vectors and of methylprednisolone (MP) on innate immunity, liver inflammation, and thrombocyte counts was evaluated after transfer of 1011 particles of E1/E3/E4- deleted adenoviral vector expressing human apolipoprotein A-I (apoA-I). Gene transfer with unPEGylated vectors induced peak interleukin-6 (IL-6) plasma levels that were 66-fold above baseline levels in C57BL/6 mice. PEGylation combined with 4 mg of MP 6 hr before and at the time of gene transfer suppressed IL-6 plasma levels to baseline values at all time points. This combination resulted in 24-, 28-, 5.9-, 42-, 26-, and 2.5- fold reduced mRNA expression in the liver of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interferon-inducible protein-10, macrophage inflammatory protein-1 beta, lipopolysaccharide-induced CXC chemokine, and keratinocyte-derived chemokine, respectively; abrogated neutrophil infiltration in the liver; and reduced alanine aminotransferase levels. PEGylation reduced vector uptake in the spleen and in nonparenchymal liver cells. PEGylation also inhibited the development of thrombocytopenia. In conclusion, PEGylation of adenoviral vectors combined with MP administration improves the therapeutic index of adenoviral gene transfer.     

多发性骨髓瘤患者体内C/EBPα可能导致Hepcidin升高

本研究探讨多发性骨髓瘤患者外周血单核细胞hepcidin表达升高的可能机制.收集符合条件的初治多发性骨髓瘤患者的临床资料及采集外周静脉血,用ELISA法测定血清IL-6浓度,CD14＋磁珠分选外周血单核细胞,实时定量PCR测定外周血单核细胞hepcidin,IL-6和C/EBPα mRNA.结果显示,入选17例初治多发性骨髓瘤患者的血红蛋白水平降低（97.8±27.5 g/L）,呈现慢性病贫血特点,与对照者相比其外周血单核细胞hepcidin和C/EBPα表达明显升高（分别为P =0.0002,P=0.001）,血清IL-6水平也明显高于正常对照（P=0.00003）.初始患者血清IL-6水平与单核细胞hepcidin,C/EBPα表达呈正相关（P＜0.05）,单核细胞C/EBPα表达与单核细胞hepcidin表达呈正相关（P＜0.05）.结论：初治多发性骨髓瘤患者体内升高的IL-6可能通过上调C/EBPα诱导hepcidin表达.     

红细胞生成素对人单核细胞前炎症因子的影响及机理

红细胞生成素(erythropoietin,EPO)是治疗慢性病贫血(anemia of chronic disease,ACD)的主要药物,通过刺激造血、抑制hepcidin和前炎症因子而改善贫血。近来发现单核细胞是hepcidin的另一来源,EPO能降低IL-6诱导的hepcidin,推测EPO可能通过降低IL-6进而抑制hepcidin的间接途径。然而,EPO降低单核细胞IL-6相应的分子生物学机制还不清楚。本研究探讨EPO对单核细胞前炎症因子的作用及其分子学机制。采用实时定量PCR检测IL-6 mRNA和TNF-αmRNA表达,Western blot方法检测PARP-1信号分子的蛋白水平。采用1μg/ml脂多糖(lipopolysaccharide,LPS)刺激THP-1单核细胞,观察EPO不同浓度(0.5,1,2,5,10 U/ml)和作用时间(0,3,6,12,24小时)对THP-1单核细胞IL-6 mRNA、TNF-αmRNA以及PARP-1蛋白的抑制作用。加用1μg/ml或5μg/mlEPO受体(EPOR)抗体和/或3-氨基苯甲酰胺(3AB,PARP-1抑制剂),观察其对EPO的拮抗作用以及...     

Extrahepatic hepcidin production: The intriguing outcomes of recent years

Hepcidin is the hyposideremic hormone regulating iron metabolism. It is a defensin-like disulfide-bonded peptide with antimicrobial activity. The main site of hepcidin production is the liver where its synthesis is modulated by iron, inflammation and erythropoietic signaling. However, hepcidin locally produced in several peripheral organs seems to be an important actor for the maintenance of iron homeostasis in these organs. This review highlights the presence of peripheral hepcidin and its potential functions. Understanding the role of extrahepatic hepcidin could be of great physiological and therapeutic importance for several specific pathologies.     

Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: a microdialysis study

Animal studies have documented a critical role for cytokines in cell signaling events underlying inflammation and pain associated with tissue injury. While clinical reports indicate an important role of cytokines in inflammatory pain, methodological limitations have made systematic human studies difficult. This study examined the utility of a human in vivo bioassay combining microdialysis with multiplex immunoassay techniques for measuring cytokine arrays in tissue. The first experiment measured cytokines in interstitial fluid collected from non-inflamed and experimentally inflamed skin (UVB). The effects of noxious heat on cytokine release were also assessed. The second experiment examined whether anti-hyperalgesic effects of the COX-inhibitor ibuprofen were associated with decreased tissue levels of the pro-inflammatory cytokines IL-1 beta and IL-6. In the first experiment, inflammation significantly increased IL-1 beta, IL-6, IL-8, IL-10, G-CSF, and MIP-1 beta. Noxious heat but not experimental inflammation significantly increased IL-7 and IL-13. In the second experiment, an oral dose of 400 and 800 mg ibuprofen produced similar anti-hyperalgesic effects suggesting a ceiling effect. Tissue levels of IL-1 beta and IL-6 were not affected after the 400mg dose but decreased significantly (44+/-32% and 38+/-13%) after the 800 mg dose. These results support the utility of explored method for tracking cytokines in human tissue and suggest that anti-hyperalgesic and anti-inflammatory effects of ibuprofen are at least partially dissociated. The data further suggest that high clinical doses of ibuprofen exert anti-inflammatory effects by down-regulating tissue cytokine levels. Explored human bioassay is a promising tool for studying the pathology and pharmacology of inflammatory and chronic pain conditions.