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Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice Shengnan Wang a, 1, Caizhi Liu a, 1, Shuhong Pan a, Qing Miao a, Jianqi Xue a, Jingna Xun a, Yuling Zhang a, Yanhong Gao b, Xianglin Duan a, **, Yumei Fan a, * a
Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Science, Hebei Normal University, Shijiazhuang 050024, PR China Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Science, Nanjing Normal University, Nanjing 210046, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2015 Accepted 9 August 2015 Available online xxx
To examine the role of the intracellular labile iron pool (LIP) in the induction of inflammatory responses, we investigated the anti-inflammatory effect of the iron chelator deferoxamine (DFO) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophage cells and endotoxic shock in mice in the present study. Our data showed that DFO significantly decreased LPS-induced LIP and ROS upregulation. We then found that DFO inhibited phosphorylation of MAP kinases such as ERK and p38 and also inhibited the activation of NF-kB induced by LPS. Furthermore, the production of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), nitric oxide (NO) and prostaglandin E2 (PGE2) induced by LPS was inhibited by DFO in RAW264.7 macrophages. Administration of DFO significantly decreased the mortality and improved the survival of septic mice with lethal endotoxemia in LPS-injected mice. These results demonstrate that iron plays a pivotal role in the induction of inflammatory responses and against septic shock. DFO has effective inhibitory effect on the production of inflammatory mediators via suppressing activation of MAPKs and NF-kB signaling pathways; it also has a protective effect on LPSinduced endotoxic shock in mice. Our findings open doors to further studies directed at exploring a new class of drugs against septic shock or other inflammatory diseases by modulating cellular chelatable iron. © 2015 Elsevier Inc. All rights reserved.
Keywords: Deferoxamine Inflammation Reactive oxygen species Labile iron pool MAPKs NF-kB
1. Introduction Iron is an essential trace element utilized in almost every aspect of normal cell function and body growth [1]. Most iron is stored in ferritin, which is a ubiquitously expressed cytosolic iron-storage protein that forms a hetero-oligomeric protein shell composed of
Abbreviations: DFO, deferoxamine; LPS, lipopolysaccharide or endotoxin; TNF-a, tumor necrosis factor a; IL-1b, interleukin 1b; NO, nitric oxide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; ERK, extracellular-signal-related kinase; NF-kB, nuclear factor-kB; LIP, labile iron pool; ROS, reactive oxidative species; DCFH-DA, 20 , 70 -dichlorfluorescein-diacetate. * Corresponding author. College of Life Science, Hebei Normal University, No.20 2nd South Ring Eastern Road, Shijiazhuang, Hebei Province 050024, PR China. ** Corresponding author. College of Life Science, Hebei Normal University, No.20 2nd South Ring Eastern Road, Shijiazhuang, Hebei Province 050024, PR China. E-mail addresses: [email protected] (X. Duan), [email protected] (Y. Fan). 1 Contributed equally to this work.
ferritin heavy chain (FTH) and ferritin light chain (FTL) [2]. The cellular labile iron pool (LIP), also named free iron, is defined as a cellular chelatable iron pool that consists of both ionic forms of iron. Free iron acts as a catalytic agent for Fenton reactions and participates in the generation of free radicals that possess unpaired electrons such as HO, which are generally known as reactive oxygen species (ROS), resulting in oxidative damage [3e5]. Upregulation of iron-dependent ROS mediates cell death and a new form of iron-dependent cell death termed ferroptosis [6]. In addition, ROS play important roles in the pathogenesis of inflammation [7,8]. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and it activates the mitogen-activated protein kinases (MAPKs) and NF-kB pathway involves the production of ROS in monocytes/macrophages [9e13]. Severe infections result in septic shock and systemic inflammatory response syndrome (SIRS), etc [14e16]. The production of pro-inflammatory cytokines and expression of inflammatory mediators are closely related to the LPS-activation of MAPKs and NF-kB pathways, such as
http://dx.doi.org/10.1016/j.bbrc.2015.08.032 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Wang, et al., Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.08.032
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tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [17e19]. Recently, we have reported that LPS induced LIP increases in macrophage cells and that ferritin light chain suppresses LPSinduced inflammatory responses by inhibiting ROS-activated MAPKs and NF-kB pathways through iron sequestration [17]. We hypothesized that LIP, which acts as a ROS source, facilitates LPSinduced inflammatory responses. In the current study, we assessed the protective effect of the iron chelator deferoxamine (DFO) against LPS-induced inflammation in macrophages and endotoxic shock in mice to assess the mechanisms underlying the role of iron during inflammatory responses that occur after LPS stimulation. Our results demonstrated the role of LIP in the development of the inflammatory process and in the LPS-induced activation of signaling pathways. Furthermore, we analyzed and elucidated the molecular mechanisms of LPS-stimulated inflammatory mediator production and signal transduction activation. Our findings provide new insights into potential novel mechanisms for the treatment of inflammation by modulating cellular chelatable iron. 2. Materials and methods 2.1. Cell culture RAW264.7 macrophage cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, People's Republic of China). The cells were cultured in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 C in a humidified atmosphere of 5% CO2. 2.2. Antibodies and reagents For western blot, the following antibodies were used: rabbit polyclonal antibodies against p38, p44/42 (ERK1/2), p-p38 (Thr180/ Tyr182), p-p44/42 (p-ERK1/2) (Thr202/Tyr204), and IkBa (Cell Signaling Technology, Danvers, MA, USA), polyclonal antibody against iNOS (Cayman chemical), polyclonal antibody against COX2 (ABCAM, Burlingame, CA, USA), and polyclonal antibody against NF-kB (p65 subunit) (ABCAM, Hong Kong, China). All secondary antibodies were purchased from Cell Signaling Technology. LPS, calcein-AM, DCFH-DA and DFO were purchased from Sigma. 2.3. Labile iron pool measurements The LIP of the cells was assessed by the calcein-based method as described in a previous report [17]. Calcein fluorescence was measured in a fluorescence plate reader (excitation 488 nm, emission 517 nm). Triplicate wells were used for each condition. The cell viability (assayed as Trypan Blue dye exclusion) was >95% and unchanged during the assay. 2.4. Measurement of intracellular ROS levels Intracellular ROS was measured by detecting the fluorescence intensity of DCF through DCFH-DAeDCFH-DCF conversion, as previously described [17]. Briefly, the experimental cells were collected and washed with PBS and then incubated with DCFH-DA (1 mM) at 37 C for 30 min in the dark. The cells were washed and suspended in PBS. DCF fluorescence images and cell morphology images were examined using an Olympus fluorescence microscope (Tokyo, Japan).
2.5. Reverse transcription-PCR analysis Total RNA was extracted with the Trizol reagent (Invitrogen), according to the manufacturer's instructions. Reverse transcription reaction of 2 mg of each total RNA was performed at 42 C for 1 h. PCR was performed using the Mastercycler Gradient RT-PCR System (Eppendorf) with the following primers: TNF-a: sense 50 agcacagaaagcatga-30 , antisense 50 -cagagcaatgactcca-30 ; IL-1b: sense 50 -aagctctccacctc-30 , antisense 50 -ctgatgtaccagttg-30 ; iNOS: sense 50 -cccttccgaagtttctggcagc-30 , antisense 50 -ggctgtca0 0 gagcctcgtggctt-3 ; COX-2: sense 5 -tctccaacctctcctatcac-30 , antisense 50 -gcacgtagtcttcgatcact-30 ; and b-actin: sense 50 agccatgtacgtagccatcc-30 , antisense 50 -tttgatgtcacgcacgattt-30 . The PCR products were resolved on 1.2% agarose gels and stained with ethidium bromide. b-actin was used as a housekeeping gene where indicated. 2.6. Western blot Cells were lysed in the buffer described in a previous report [17]. Cell lysates were subjected to SDS-PAGE, and proteins were transferred to PVDF membranes (Roche, Indianapolis, IN). Immunoblot (IB) analysis was performed as described previously [5]. The antibodyeantigen complexes were visualized using Super Signal West Pico (Pierce). The total density of the protein bands was detected with the LAS4000 System (FujiFilm). 2.7. ELISA assay RAW264.7 cells were seeded in 24-well plates at 2.5 105 cells/ well 1 day before the experiment. After LPS treatment, media were collected and centrifuged at 10,000 rpm for 5 min. The TNF-a, IL-1b and PGE2 contents were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using the mouse ELISA kits for TNF-a (Roche), IL-1b (Roche) and PGE2 (Cayman) according to the manufacturer's instructions. 2.8. Nitrite analysis RAW264.7 cells were seeded in 24-well plates at 2.5 105 cells/ well 1 day before the experiment. Cells were treated with LPS (1 mg/ ml) for 14 h; NO synthesis was then determined by assaying the culture supernatants for nitrite using the Griess Reagent System (Promega, Madison, USA) according to the manufacturer's instructions. Absorbance was measured at 550 nm. 2.9. Mice Male Kunming mice weighing 28e32 g were purchased from the Experimental Animal Center of Hebei Medical University. All mice were housed in a temperature- and humidity-controlled environment with free access to food and water. They were maintained on a reverse 12 h/12 h light/dark cycle (lights off at 8:00 A.M.). All procedures were performed in accordance with the requirements of the provisions and general recommendation of the Chinese Experimental Animals Administration Legislation and were approved by the Science and Technology Department of Hebei Province. 2.10. Immunofluorescence assay Immunofluorescence was performed using as described [17]. For the present study, cells were treated by LPS 1 mg/ml for 0.5 h.
Please cite this article in press as: S. Wang, et al., Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.08.032
S. Wang et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
2.11. Statistical analysis All experiments were repeated at least three times. The data are expressed as the mean ± SEM. Statistical analysis was performed by t-test and was considered significant when p < 0.05. 3. Results 3.1. DFO suppresses the LPS-induced increase of intracellular LIP and ROS Previously, we reported that LIP is upregulated by LPS, as well as ROS [17]. Here, we hypothesized that LPS-induced ROS formation and subsequent inflammatory responses might be controlled by LIP level as well. In the present study, RAW264.7 cells were treated directly with LPS (1 mg/ml) with or without a 0.5 h pretreatment with the iron chelator DFO (25 mM). The LIP of RAW264.7 cells was assessed by the calcein-based method as described in the Materials and Methods. As shown in Fig. 1A, pretreatment with DFO reduced the LPS-induced LIP upregulation. Then we determined the role of iron during ROS generation induced by LPS. The results presented in Fig. 1B indicated that LPS-treated cellular DCF fluorescence was increased whereas the fluorescence was attenuated in the presence of DFO. These data confirmed that the LPS-induced ROS production was a result of an increase in LIP and indicated that DFO inhibited LPS-induced ROS production through iron sequestration. 3.2. DFO suppresses LPS-induced activation of MAPKs and NF-kB To investigate whether LIP is involved in the LPS-induced activation of MAPKs and NF-kB signaling pathways, we first tested the effect of DFO on the LPS-induced phosphorylation of p38 and ERK
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via western blot analysis. RAW264.7 cells were treated directly with LPS (1 mg/ml) with or without a 0.5 h pretreatment with the iron chelator DFO (25 mM). Pretreatment with DFO significantly suppressed the phosphorylation of p38 and ERK (p-p44/42) (Fig. 2A). We next examined the effect of DFO on the intracellular localization of the NF-kB p65 subunit and the level of IkBa because NF-kB transcription activity is proceeded by the phosphorylation and proteolytic degradation of IkBa [20]. Our present study showed that pretreatment of DFO could significantly restore the decreased IkBa protein level induced by LPS (Fig. 2B). Additionally, pretreatment with DFO inhibited the LPS-induced nuclear translocation of NF-kB p65 in RAW264.7 cells (Fig. 2C). Thus, these observations indicated that LIP participates in LPS-induced activation of p38 and ERK, degradation of IkBa and subsequent activation of NF-kB. 3.3. DFO inhibits LPS-induced production of TNF-a IL-1b, NO and PGE2 It is well known that MAPKs and NF-kB activation are essential for the expression of various pro-inflammatory genes [17e19]. To explore the role of iron in LPS-induced inflammation, we examined the effect of DFO on the LPS-induced production of TNF-a and IL-1b, as well as NO and PGE2. We found that after 14 h of LPS (1 mg/ml) treatment of cells, the mRNA levels of TNF-a and IL-1b increased. However, pretreatment with DFO significantly inhibited the LPSinduced upregulation of TNF-a and IL-1b mRNA expression as shown in Fig. 3A. Followed ELISA experiment confirmed this result. As shown in Fig. 3B and C, DFO significantly reduced the release of TNF-a and IL-1b from RAW264.7 cells. NO and PGE2 are critical inflammatory mediators produced by activated macrophages via induction of iNOS and COX-2. We next tested the effect of DFO on the mRNA and protein expression level of iNOS and COX-2. As displayed in Fig. 3D and E, upon exposure to LPS (1 mg/ml) for 14 h, the mRNA and protein levels of iNOS and COX-2 increased markedly up to 5-fold. DFO attenuated the mRNA and protein level increase of iNOS and COX-2 significantly. Furthermore, we tested the effect of DFO on the secretion of NO and PGE2 by using ELISA. As shown in Fig. 3F and G, compared with untreated control cells, the secretion of NO and PGE2 from RAW264.7 cells increased dramatically after 14 h of LPS incubation. However, DFO inhibited the LPS-induced secretion of NO and PGE2. These results strongly demonstrated the direct correlation between LPS-induced upregulation of cytosolic LIP and the inflammatory responses in macrophages. 3.4. DFO protects mice against LPS-induced mortality
Fig. 1. DFO suppresses the LPS-induced increase of intacellular LIP and ROS. RAW264.7 cells were treated with LPS 1 mg/ml for 0.5 h, pretreated with or without 25 mM DFO for 0.5 h. (A) The mean calcein fluorescence intensity was detected by a fluorescence plate reader at an excitation wavelength of 488 nm and emission of 517 nm. The amount of intracellular iron bound to calcein was assessed. The data are the mean ± SEM (n ¼ 5). ***, p < 0.001, compared with PBS-treated control cells; ###, p < 0.001, compared with LPS-treated cells. Similar results were observed in replicate experiments. (B) ROS production was determined through the rise in DCF fluorescence as described in the Materials and Methods. The figure shows a representative result of three repeated experiments.
LPS-induced sepsis is one of the leading causes of death around the world. To determine whether iron plays a role in the regulation of innate immune responses, we further investigated the role of DFO in a murine model of endotoxic shock. The mice were injected intravenously (i.v.) with DFO (100 mg/kg) 0.5 h or 4 h after an LPS (30 mg/kg) challenge, and mouse survival was monitored. As shown in Fig. 4, 60% of the mice died from endotoxin shock 50 h after the injection of LPS. DFO treatment (4 h after LPS instillation) raised the survival rate to 80% in this animal model of endotoxin shock. The data indicated that DFO suppresses endotoxic shock from LPS stimulation. Thus, collectively, our observations suggested that iron may be a promising therapeutic target of antiinflammatory responses. 4. Discussion In this study, we showed that DFO suppresses the production of proinflammatory cytokines from RAW264.7 macrophage cells by
Please cite this article in press as: S. Wang, et al., Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.08.032
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Fig. 2. DFO suppresses the LPS-induced activation of MAPKs and NF-kB. RAW264.7 cells were treated with LPS 1 mg/ml for 0.5 h, pretreated with or without 25 mM DFO for 0.5 h. (A) The protein levels of p-p38, p38, p-p44/p42 and p44/p42 were determined by western blot analysis. (B) The protein level of IkBa was determined by western blot analysis. (C) The localization of NF-kB was analyzed by double immunofluorescence staining of the NF-kB p65 subunit (green) and nucleus (blue). Immunofluorescence images were acquired using an Axio Imager A2 microscope. All magnifications are 200. The experiments were repeated three times, and similar results were obtained. ***, p < 0.001, compared with PBStreated control cells; ###, p < 0.001, ##, p < 0.01, compared with LPS-treated cells. The results are representative of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
inhibiting both MAPKs and the NF-kB pathway upon LPS stimulation. Finally, we also showed that DFO inhibits LPS-induced endotoxic shock. These results suggest that iron is a potential therapeutic target that might help to diminish inflammation and protect against endotoxic shock. It is well established that iron is essential to organism survival and is involved in a wide range of metabolic pathways. The intracellular level of iron is tightly controlled through regulation of the cellular uptake of iron and the sequestering of low molecular labile iron by iron chelators. An excess of free iron also donates electrons for the generation of superoxide radicals (known as ROS) and can participate in the regulation of hydroxyl radicals via the Fenton reaction [3]. ROS facilitates LPS-induced inflammatory processes, eventually leading to tissue damage and organ failure. Although LPS-induced oxidative stress is well documented [21e23], the source of ROS induced by LPS and the role of iron in LPS-induced inflammatory responses remain elusive. The study presented here showed that DFO inhibited the increase in ROS formation induced by LPS (Fig. 1). DFO functions to limit Fe (II) that is available to participate in the generation of ROS. Thus, it appears that intracellular iron is a key mediator of ROS production induced by LPS, in addition to NADPH oxidase. This interpretation is consistent with previous reports that iron level reduction can attenuate tissue oxidative stress by downregulating iron-catalyzed radical production and the activity of NADPH oxidase activity [24]. Our results demonstrate that iron may play important roles in subsequent inflammatory responses. MAPKs and NF-kB signaling pathways activated by LPS, which are important for the production of inflammatory cytokines and mediators, are mediated by ROS production [25,26]. Not surprisingly, we further found that DFO suppressed the LPS-induced
phosphorylation of ERK and p38, as well as the degradation of IkBa and the nuclear translocation of NF-kB (Fig. 2). Inflammation and oxidation contribute to the pathophysiology of grand global health challenges. Pro-inflammatory cytokines such as TNF-a and IL-1b play a pivotal role in the LPS-induced pathogenesis of many clinical disorders such as LPS-induced sepsis [17,27e29]. When cytokine production increases to such an extent that it escapes the local infection, sepsis or SIRS ensues. NO is synthesized from arginine by iNOS. A high concentration of NO is responsible for severe cell damage and tissue destruction in inflammation. The COX-2-PGE2 cascade also plays critical roles in modulating many physiological and pathological actions in different organs [30]. Massive overproduction of the proinflammatory cytokines and mediators is tightly linked to proinflammatory and proapoptotic pathways, which can be lethal, as seen in septic shock induced by LPS. The present results first revealed that DFO reduced the LPS-upregulated mRNA levels of TNF-a and IL-1b, as well as the protein and mRNA levels of iNOS and COX-2. Furthermore, DFO reduced the LPS-induced secretion of TNF-a, IL-1b, NO and PGE2 from RAW264.7 cells (Fig. 3). We also found that DFO protected mice against LPS-induced lethal endotoxemia (Fig. 4). This increased resistance to endotoxic shock illustrates that a decreased cellular iron level is important to achieve greater protection to LPS challenge. Our results demonstrate, both in vivo mouse and in vitro macrophage experiments, that iron is a potential therapeutic target that might help to diminish inflammation. Previous reports showed an increase in LIP by TNF-a [31] and NO [32], but the balance of the free iron level and inflammatory mediator production remains to be investigated during the activation of RAW264.7 cells.
Please cite this article in press as: S. Wang, et al., Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.08.032
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Fig. 3. DFO inhibits the LPS-induced production of TNF-a, IL-1b, NO and PGE2. RAW264.7 cells were treated with LPS 1 mg/ml for 14 h, pretreated with or without 25 mM DFO for 0.5 h. (A) The mRNA levels of TNF-a and IL-1b were determined by RT-PCR. (B and C) The secretion of TNF-a and IL-1b was measured by ELISA. (D) The mRNA levels of iNOS and COX-2 were determined by RT-PCR. (E) The protein levels of iNOS and COX-2 were determined by immunoblot analysis. (F and G) The secretion of NO and PGE2 was measured by ELISA. ***, p < 0.001, **, p < 0.01, *, p < 0.05, compared with PBS-treated control cells; ###, p < 0.001, ##, p < 0.01, compared with LPS-treated cells. These results are representative of three independent experiments.
Please cite this article in press as: S. Wang, et al., Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.08.032
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Fig. 4. DFO protects against LPS-induced mortality. Mice were i.v. injected with LPS (30 mg/kg), followed by i.v. injecting with saline (-, n ¼ 10), DFO (100 mg/kg) for 0.5 h (:, n ¼ 10) or 4 h (A, n ¼ 10) after LPS treatment, and the survival rates were monitored continuously.
In summary, we show for the first time that the iron chelator DFO inhibits LPS-induced activation of the MAPKs and NF-kB signaling pathways by reducing cellular LIP and ROS production and subsequently decreasing the production of pro-inflammatory factors such as TNF-a and IL-1b and inflammatory mediators including NO and PGE2. Thus, iron may be an important regulator of inflammatory responses. Our study indicates a potential treatment for inflammatory diseases by modulating cellular chelatable iron. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 31000632, 31371073 and 31401004), Scientific Research Foundation for Returned Overseas Chinese Scholars (No. 20141685), State Education Ministry of China (No. 211100BC151), Program of Natural Science Research of Jiangsu Higher education Institution of China (No. 14KJB180010), Research Foundation for High-level Talents of Nanjing Normal University of China (No. 184130060403). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.08.032. References [1] R.G. Sangani, A.J. Ghio, Iron, human growth, and the global epidemic of obesity, Nutrients 5 (2013) 4231e4249. [2] P. Arosio, S. Levi, Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage, Biochim. Biophys. Acta 1800 (2010) 783e792. [3] A. Al-Qenaei, A. Yiakouvaki, O. Reelfs, P. Santambrogio, S. Levi, N.D. Hall, R.M. Tyrrell, C. Pourzand, Role of intracellular labile iron, ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide, Free Radic. Biol. Med. 68 (2014) 87e100. [4] N.E. Piloni, V. Fermandez, L.A. Videla, S. Puntarulo, Acute iron overload and oxidative stress in brain, Toxicology 314 (2013) 174e182. [5] K.L. Kuo, S.C. Hung, T.S. Lee, D.C. Tarng, Iron sucrose accelerates early atherogenesis by increasing superoxide production and upregulating adhesion molecules in CKD, J. Am. Soc. Nephrol. 25 (2014) 2596e2606. [6] S.J. Dixon, B.R. Stockwell, The role of iron and reactive oxygen species in cell death, Nat. Chem. Biol. 10 (2014) 9e17. [7] J. Deng, X. Wang, F. Qian, S. Vogel, L. Xiao, R. Ranjan, H. Park, M. Karpurapu, R.D. Ye, G.Y. Park, J.W. Christman, Protective role of reactive oxygen species in endotoxin-induced lung inflammation through modulation of IL-10
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Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice Shengnan Wang a, 1, Caizhi Liu a, 1, Shuhong Pan a, Qing Miao a, Jianqi Xue a, Jingna Xun a, Yuling Zhang a, Yanhong Gao b, Xianglin Duan a, **, Yumei Fan a, * a
Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Science, Hebei Normal University, Shijiazhuang 050024, PR China Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Science, Nanjing Normal University, Nanjing 210046, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2015 Accepted 9 August 2015 Available online xxx
To examine the role of the intracellular labile iron pool (LIP) in the induction of inflammatory responses, we investigated the anti-inflammatory effect of the iron chelator deferoxamine (DFO) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophage cells and endotoxic shock in mice in the present study. Our data showed that DFO significantly decreased LPS-induced LIP and ROS upregulation. We then found that DFO inhibited phosphorylation of MAP kinases such as ERK and p38 and also inhibited the activation of NF-kB induced by LPS. Furthermore, the production of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), nitric oxide (NO) and prostaglandin E2 (PGE2) induced by LPS was inhibited by DFO in RAW264.7 macrophages. Administration of DFO significantly decreased the mortality and improved the survival of septic mice with lethal endotoxemia in LPS-injected mice. These results demonstrate that iron plays a pivotal role in the induction of inflammatory responses and against septic shock. DFO has effective inhibitory effect on the production of inflammatory mediators via suppressing activation of MAPKs and NF-kB signaling pathways; it also has a protective effect on LPSinduced endotoxic shock in mice. Our findings open doors to further studies directed at exploring a new class of drugs against septic shock or other inflammatory diseases by modulating cellular chelatable iron. © 2015 Elsevier Inc. All rights reserved.
Keywords: Deferoxamine Inflammation Reactive oxygen species Labile iron pool MAPKs NF-kB
1. Introduction Iron is an essential trace element utilized in almost every aspect of normal cell function and body growth [1]. Most iron is stored in ferritin, which is a ubiquitously expressed cytosolic iron-storage protein that forms a hetero-oligomeric protein shell composed of
Abbreviations: DFO, deferoxamine; LPS, lipopolysaccharide or endotoxin; TNF-a, tumor necrosis factor a; IL-1b, interleukin 1b; NO, nitric oxide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; ERK, extracellular-signal-related kinase; NF-kB, nuclear factor-kB; LIP, labile iron pool; ROS, reactive oxidative species; DCFH-DA, 20 , 70 -dichlorfluorescein-diacetate. * Corresponding author. College of Life Science, Hebei Normal University, No.20 2nd South Ring Eastern Road, Shijiazhuang, Hebei Province 050024, PR China. ** Corresponding author. College of Life Science, Hebei Normal University, No.20 2nd South Ring Eastern Road, Shijiazhuang, Hebei Province 050024, PR China. E-mail addresses: [email protected] (X. Duan), [email protected] (Y. Fan). 1 Contributed equally to this work.
ferritin heavy chain (FTH) and ferritin light chain (FTL) [2]. The cellular labile iron pool (LIP), also named free iron, is defined as a cellular chelatable iron pool that consists of both ionic forms of iron. Free iron acts as a catalytic agent for Fenton reactions and participates in the generation of free radicals that possess unpaired electrons such as HO, which are generally known as reactive oxygen species (ROS), resulting in oxidative damage [3e5]. Upregulation of iron-dependent ROS mediates cell death and a new form of iron-dependent cell death termed ferroptosis [6]. In addition, ROS play important roles in the pathogenesis of inflammation [7,8]. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and it activates the mitogen-activated protein kinases (MAPKs) and NF-kB pathway involves the production of ROS in monocytes/macrophages [9e13]. Severe infections result in septic shock and systemic inflammatory response syndrome (SIRS), etc [14e16]. The production of pro-inflammatory cytokines and expression of inflammatory mediators are closely related to the LPS-activation of MAPKs and NF-kB pathways, such as
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tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [17e19]. Recently, we have reported that LPS induced LIP increases in macrophage cells and that ferritin light chain suppresses LPSinduced inflammatory responses by inhibiting ROS-activated MAPKs and NF-kB pathways through iron sequestration [17]. We hypothesized that LIP, which acts as a ROS source, facilitates LPSinduced inflammatory responses. In the current study, we assessed the protective effect of the iron chelator deferoxamine (DFO) against LPS-induced inflammation in macrophages and endotoxic shock in mice to assess the mechanisms underlying the role of iron during inflammatory responses that occur after LPS stimulation. Our results demonstrated the role of LIP in the development of the inflammatory process and in the LPS-induced activation of signaling pathways. Furthermore, we analyzed and elucidated the molecular mechanisms of LPS-stimulated inflammatory mediator production and signal transduction activation. Our findings provide new insights into potential novel mechanisms for the treatment of inflammation by modulating cellular chelatable iron. 2. Materials and methods 2.1. Cell culture RAW264.7 macrophage cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, People's Republic of China). The cells were cultured in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 C in a humidified atmosphere of 5% CO2. 2.2. Antibodies and reagents For western blot, the following antibodies were used: rabbit polyclonal antibodies against p38, p44/42 (ERK1/2), p-p38 (Thr180/ Tyr182), p-p44/42 (p-ERK1/2) (Thr202/Tyr204), and IkBa (Cell Signaling Technology, Danvers, MA, USA), polyclonal antibody against iNOS (Cayman chemical), polyclonal antibody against COX2 (ABCAM, Burlingame, CA, USA), and polyclonal antibody against NF-kB (p65 subunit) (ABCAM, Hong Kong, China). All secondary antibodies were purchased from Cell Signaling Technology. LPS, calcein-AM, DCFH-DA and DFO were purchased from Sigma. 2.3. Labile iron pool measurements The LIP of the cells was assessed by the calcein-based method as described in a previous report [17]. Calcein fluorescence was measured in a fluorescence plate reader (excitation 488 nm, emission 517 nm). Triplicate wells were used for each condition. The cell viability (assayed as Trypan Blue dye exclusion) was >95% and unchanged during the assay. 2.4. Measurement of intracellular ROS levels Intracellular ROS was measured by detecting the fluorescence intensity of DCF through DCFH-DAeDCFH-DCF conversion, as previously described [17]. Briefly, the experimental cells were collected and washed with PBS and then incubated with DCFH-DA (1 mM) at 37 C for 30 min in the dark. The cells were washed and suspended in PBS. DCF fluorescence images and cell morphology images were examined using an Olympus fluorescence microscope (Tokyo, Japan).
2.5. Reverse transcription-PCR analysis Total RNA was extracted with the Trizol reagent (Invitrogen), according to the manufacturer's instructions. Reverse transcription reaction of 2 mg of each total RNA was performed at 42 C for 1 h. PCR was performed using the Mastercycler Gradient RT-PCR System (Eppendorf) with the following primers: TNF-a: sense 50 agcacagaaagcatga-30 , antisense 50 -cagagcaatgactcca-30 ; IL-1b: sense 50 -aagctctccacctc-30 , antisense 50 -ctgatgtaccagttg-30 ; iNOS: sense 50 -cccttccgaagtttctggcagc-30 , antisense 50 -ggctgtca0 0 gagcctcgtggctt-3 ; COX-2: sense 5 -tctccaacctctcctatcac-30 , antisense 50 -gcacgtagtcttcgatcact-30 ; and b-actin: sense 50 agccatgtacgtagccatcc-30 , antisense 50 -tttgatgtcacgcacgattt-30 . The PCR products were resolved on 1.2% agarose gels and stained with ethidium bromide. b-actin was used as a housekeeping gene where indicated. 2.6. Western blot Cells were lysed in the buffer described in a previous report [17]. Cell lysates were subjected to SDS-PAGE, and proteins were transferred to PVDF membranes (Roche, Indianapolis, IN). Immunoblot (IB) analysis was performed as described previously [5]. The antibodyeantigen complexes were visualized using Super Signal West Pico (Pierce). The total density of the protein bands was detected with the LAS4000 System (FujiFilm). 2.7. ELISA assay RAW264.7 cells were seeded in 24-well plates at 2.5 105 cells/ well 1 day before the experiment. After LPS treatment, media were collected and centrifuged at 10,000 rpm for 5 min. The TNF-a, IL-1b and PGE2 contents were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using the mouse ELISA kits for TNF-a (Roche), IL-1b (Roche) and PGE2 (Cayman) according to the manufacturer's instructions. 2.8. Nitrite analysis RAW264.7 cells were seeded in 24-well plates at 2.5 105 cells/ well 1 day before the experiment. Cells were treated with LPS (1 mg/ ml) for 14 h; NO synthesis was then determined by assaying the culture supernatants for nitrite using the Griess Reagent System (Promega, Madison, USA) according to the manufacturer's instructions. Absorbance was measured at 550 nm. 2.9. Mice Male Kunming mice weighing 28e32 g were purchased from the Experimental Animal Center of Hebei Medical University. All mice were housed in a temperature- and humidity-controlled environment with free access to food and water. They were maintained on a reverse 12 h/12 h light/dark cycle (lights off at 8:00 A.M.). All procedures were performed in accordance with the requirements of the provisions and general recommendation of the Chinese Experimental Animals Administration Legislation and were approved by the Science and Technology Department of Hebei Province. 2.10. Immunofluorescence assay Immunofluorescence was performed using as described [17]. For the present study, cells were treated by LPS 1 mg/ml for 0.5 h.
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2.11. Statistical analysis All experiments were repeated at least three times. The data are expressed as the mean ± SEM. Statistical analysis was performed by t-test and was considered significant when p < 0.05. 3. Results 3.1. DFO suppresses the LPS-induced increase of intracellular LIP and ROS Previously, we reported that LIP is upregulated by LPS, as well as ROS [17]. Here, we hypothesized that LPS-induced ROS formation and subsequent inflammatory responses might be controlled by LIP level as well. In the present study, RAW264.7 cells were treated directly with LPS (1 mg/ml) with or without a 0.5 h pretreatment with the iron chelator DFO (25 mM). The LIP of RAW264.7 cells was assessed by the calcein-based method as described in the Materials and Methods. As shown in Fig. 1A, pretreatment with DFO reduced the LPS-induced LIP upregulation. Then we determined the role of iron during ROS generation induced by LPS. The results presented in Fig. 1B indicated that LPS-treated cellular DCF fluorescence was increased whereas the fluorescence was attenuated in the presence of DFO. These data confirmed that the LPS-induced ROS production was a result of an increase in LIP and indicated that DFO inhibited LPS-induced ROS production through iron sequestration. 3.2. DFO suppresses LPS-induced activation of MAPKs and NF-kB To investigate whether LIP is involved in the LPS-induced activation of MAPKs and NF-kB signaling pathways, we first tested the effect of DFO on the LPS-induced phosphorylation of p38 and ERK
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via western blot analysis. RAW264.7 cells were treated directly with LPS (1 mg/ml) with or without a 0.5 h pretreatment with the iron chelator DFO (25 mM). Pretreatment with DFO significantly suppressed the phosphorylation of p38 and ERK (p-p44/42) (Fig. 2A). We next examined the effect of DFO on the intracellular localization of the NF-kB p65 subunit and the level of IkBa because NF-kB transcription activity is proceeded by the phosphorylation and proteolytic degradation of IkBa [20]. Our present study showed that pretreatment of DFO could significantly restore the decreased IkBa protein level induced by LPS (Fig. 2B). Additionally, pretreatment with DFO inhibited the LPS-induced nuclear translocation of NF-kB p65 in RAW264.7 cells (Fig. 2C). Thus, these observations indicated that LIP participates in LPS-induced activation of p38 and ERK, degradation of IkBa and subsequent activation of NF-kB. 3.3. DFO inhibits LPS-induced production of TNF-a IL-1b, NO and PGE2 It is well known that MAPKs and NF-kB activation are essential for the expression of various pro-inflammatory genes [17e19]. To explore the role of iron in LPS-induced inflammation, we examined the effect of DFO on the LPS-induced production of TNF-a and IL-1b, as well as NO and PGE2. We found that after 14 h of LPS (1 mg/ml) treatment of cells, the mRNA levels of TNF-a and IL-1b increased. However, pretreatment with DFO significantly inhibited the LPSinduced upregulation of TNF-a and IL-1b mRNA expression as shown in Fig. 3A. Followed ELISA experiment confirmed this result. As shown in Fig. 3B and C, DFO significantly reduced the release of TNF-a and IL-1b from RAW264.7 cells. NO and PGE2 are critical inflammatory mediators produced by activated macrophages via induction of iNOS and COX-2. We next tested the effect of DFO on the mRNA and protein expression level of iNOS and COX-2. As displayed in Fig. 3D and E, upon exposure to LPS (1 mg/ml) for 14 h, the mRNA and protein levels of iNOS and COX-2 increased markedly up to 5-fold. DFO attenuated the mRNA and protein level increase of iNOS and COX-2 significantly. Furthermore, we tested the effect of DFO on the secretion of NO and PGE2 by using ELISA. As shown in Fig. 3F and G, compared with untreated control cells, the secretion of NO and PGE2 from RAW264.7 cells increased dramatically after 14 h of LPS incubation. However, DFO inhibited the LPS-induced secretion of NO and PGE2. These results strongly demonstrated the direct correlation between LPS-induced upregulation of cytosolic LIP and the inflammatory responses in macrophages. 3.4. DFO protects mice against LPS-induced mortality
Fig. 1. DFO suppresses the LPS-induced increase of intacellular LIP and ROS. RAW264.7 cells were treated with LPS 1 mg/ml for 0.5 h, pretreated with or without 25 mM DFO for 0.5 h. (A) The mean calcein fluorescence intensity was detected by a fluorescence plate reader at an excitation wavelength of 488 nm and emission of 517 nm. The amount of intracellular iron bound to calcein was assessed. The data are the mean ± SEM (n ¼ 5). ***, p < 0.001, compared with PBS-treated control cells; ###, p < 0.001, compared with LPS-treated cells. Similar results were observed in replicate experiments. (B) ROS production was determined through the rise in DCF fluorescence as described in the Materials and Methods. The figure shows a representative result of three repeated experiments.
LPS-induced sepsis is one of the leading causes of death around the world. To determine whether iron plays a role in the regulation of innate immune responses, we further investigated the role of DFO in a murine model of endotoxic shock. The mice were injected intravenously (i.v.) with DFO (100 mg/kg) 0.5 h or 4 h after an LPS (30 mg/kg) challenge, and mouse survival was monitored. As shown in Fig. 4, 60% of the mice died from endotoxin shock 50 h after the injection of LPS. DFO treatment (4 h after LPS instillation) raised the survival rate to 80% in this animal model of endotoxin shock. The data indicated that DFO suppresses endotoxic shock from LPS stimulation. Thus, collectively, our observations suggested that iron may be a promising therapeutic target of antiinflammatory responses. 4. Discussion In this study, we showed that DFO suppresses the production of proinflammatory cytokines from RAW264.7 macrophage cells by
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Fig. 2. DFO suppresses the LPS-induced activation of MAPKs and NF-kB. RAW264.7 cells were treated with LPS 1 mg/ml for 0.5 h, pretreated with or without 25 mM DFO for 0.5 h. (A) The protein levels of p-p38, p38, p-p44/p42 and p44/p42 were determined by western blot analysis. (B) The protein level of IkBa was determined by western blot analysis. (C) The localization of NF-kB was analyzed by double immunofluorescence staining of the NF-kB p65 subunit (green) and nucleus (blue). Immunofluorescence images were acquired using an Axio Imager A2 microscope. All magnifications are 200. The experiments were repeated three times, and similar results were obtained. ***, p < 0.001, compared with PBStreated control cells; ###, p < 0.001, ##, p < 0.01, compared with LPS-treated cells. The results are representative of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
inhibiting both MAPKs and the NF-kB pathway upon LPS stimulation. Finally, we also showed that DFO inhibits LPS-induced endotoxic shock. These results suggest that iron is a potential therapeutic target that might help to diminish inflammation and protect against endotoxic shock. It is well established that iron is essential to organism survival and is involved in a wide range of metabolic pathways. The intracellular level of iron is tightly controlled through regulation of the cellular uptake of iron and the sequestering of low molecular labile iron by iron chelators. An excess of free iron also donates electrons for the generation of superoxide radicals (known as ROS) and can participate in the regulation of hydroxyl radicals via the Fenton reaction [3]. ROS facilitates LPS-induced inflammatory processes, eventually leading to tissue damage and organ failure. Although LPS-induced oxidative stress is well documented [21e23], the source of ROS induced by LPS and the role of iron in LPS-induced inflammatory responses remain elusive. The study presented here showed that DFO inhibited the increase in ROS formation induced by LPS (Fig. 1). DFO functions to limit Fe (II) that is available to participate in the generation of ROS. Thus, it appears that intracellular iron is a key mediator of ROS production induced by LPS, in addition to NADPH oxidase. This interpretation is consistent with previous reports that iron level reduction can attenuate tissue oxidative stress by downregulating iron-catalyzed radical production and the activity of NADPH oxidase activity [24]. Our results demonstrate that iron may play important roles in subsequent inflammatory responses. MAPKs and NF-kB signaling pathways activated by LPS, which are important for the production of inflammatory cytokines and mediators, are mediated by ROS production [25,26]. Not surprisingly, we further found that DFO suppressed the LPS-induced
phosphorylation of ERK and p38, as well as the degradation of IkBa and the nuclear translocation of NF-kB (Fig. 2). Inflammation and oxidation contribute to the pathophysiology of grand global health challenges. Pro-inflammatory cytokines such as TNF-a and IL-1b play a pivotal role in the LPS-induced pathogenesis of many clinical disorders such as LPS-induced sepsis [17,27e29]. When cytokine production increases to such an extent that it escapes the local infection, sepsis or SIRS ensues. NO is synthesized from arginine by iNOS. A high concentration of NO is responsible for severe cell damage and tissue destruction in inflammation. The COX-2-PGE2 cascade also plays critical roles in modulating many physiological and pathological actions in different organs [30]. Massive overproduction of the proinflammatory cytokines and mediators is tightly linked to proinflammatory and proapoptotic pathways, which can be lethal, as seen in septic shock induced by LPS. The present results first revealed that DFO reduced the LPS-upregulated mRNA levels of TNF-a and IL-1b, as well as the protein and mRNA levels of iNOS and COX-2. Furthermore, DFO reduced the LPS-induced secretion of TNF-a, IL-1b, NO and PGE2 from RAW264.7 cells (Fig. 3). We also found that DFO protected mice against LPS-induced lethal endotoxemia (Fig. 4). This increased resistance to endotoxic shock illustrates that a decreased cellular iron level is important to achieve greater protection to LPS challenge. Our results demonstrate, both in vivo mouse and in vitro macrophage experiments, that iron is a potential therapeutic target that might help to diminish inflammation. Previous reports showed an increase in LIP by TNF-a [31] and NO [32], but the balance of the free iron level and inflammatory mediator production remains to be investigated during the activation of RAW264.7 cells.
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Fig. 3. DFO inhibits the LPS-induced production of TNF-a, IL-1b, NO and PGE2. RAW264.7 cells were treated with LPS 1 mg/ml for 14 h, pretreated with or without 25 mM DFO for 0.5 h. (A) The mRNA levels of TNF-a and IL-1b were determined by RT-PCR. (B and C) The secretion of TNF-a and IL-1b was measured by ELISA. (D) The mRNA levels of iNOS and COX-2 were determined by RT-PCR. (E) The protein levels of iNOS and COX-2 were determined by immunoblot analysis. (F and G) The secretion of NO and PGE2 was measured by ELISA. ***, p < 0.001, **, p < 0.01, *, p < 0.05, compared with PBS-treated control cells; ###, p < 0.001, ##, p < 0.01, compared with LPS-treated cells. These results are representative of three independent experiments.
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Fig. 4. DFO protects against LPS-induced mortality. Mice were i.v. injected with LPS (30 mg/kg), followed by i.v. injecting with saline (-, n ¼ 10), DFO (100 mg/kg) for 0.5 h (:, n ¼ 10) or 4 h (A, n ¼ 10) after LPS treatment, and the survival rates were monitored continuously.
In summary, we show for the first time that the iron chelator DFO inhibits LPS-induced activation of the MAPKs and NF-kB signaling pathways by reducing cellular LIP and ROS production and subsequently decreasing the production of pro-inflammatory factors such as TNF-a and IL-1b and inflammatory mediators including NO and PGE2. Thus, iron may be an important regulator of inflammatory responses. Our study indicates a potential treatment for inflammatory diseases by modulating cellular chelatable iron. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 31000632, 31371073 and 31401004), Scientific Research Foundation for Returned Overseas Chinese Scholars (No. 20141685), State Education Ministry of China (No. 211100BC151), Program of Natural Science Research of Jiangsu Higher education Institution of China (No. 14KJB180010), Research Foundation for High-level Talents of Nanjing Normal University of China (No. 184130060403). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.08.032. References [1] R.G. Sangani, A.J. Ghio, Iron, human growth, and the global epidemic of obesity, Nutrients 5 (2013) 4231e4249. [2] P. Arosio, S. Levi, Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage, Biochim. Biophys. Acta 1800 (2010) 783e792. [3] A. Al-Qenaei, A. Yiakouvaki, O. Reelfs, P. Santambrogio, S. Levi, N.D. Hall, R.M. Tyrrell, C. Pourzand, Role of intracellular labile iron, ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide, Free Radic. Biol. Med. 68 (2014) 87e100. [4] N.E. Piloni, V. Fermandez, L.A. Videla, S. Puntarulo, Acute iron overload and oxidative stress in brain, Toxicology 314 (2013) 174e182. [5] K.L. Kuo, S.C. Hung, T.S. Lee, D.C. Tarng, Iron sucrose accelerates early atherogenesis by increasing superoxide production and upregulating adhesion molecules in CKD, J. Am. Soc. Nephrol. 25 (2014) 2596e2606. [6] S.J. Dixon, B.R. Stockwell, The role of iron and reactive oxygen species in cell death, Nat. Chem. Biol. 10 (2014) 9e17. [7] J. Deng, X. Wang, F. Qian, S. Vogel, L. Xiao, R. Ranjan, H. Park, M. Karpurapu, R.D. Ye, G.Y. Park, J.W. Christman, Protective role of reactive oxygen species in endotoxin-induced lung inflammation through modulation of IL-10
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Please cite this article in press as: S. Wang, et al., Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.08.032