Epub ahead of print April 2, 2014 - doi:10.1189/jlb.3A1113-582R
Article
Leishmanial lipid suppresses the bacterial endotoxin-induced inflammatory response with attenuation of tissue injury in sepsis Nabanita Chatterjee,* Subhadip Das,* Dipayan Bose,* Somenath Banerjee,* Tarun Jha,† and Krishna Das Saha*,1 *Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, West Bengal, India; and †Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India RECEIVED NOVEMBER 6, 2013; REVISED FEBRUARY 14, 2014; ACCEPTED MARCH 4, 2014. DOI: 10.1189/jlb.3A1113-582R
ABSTRACT
Introduction
The use of live, attenuated, or genetically modified microbes or their cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver biologically active entities. Thus, advancing our knowledge of such microbe-mediated therapy may suggest new avenues for therapeutic intervention in many diseases. We had earlier reported that the total lipid of attenuated Leishmania donovani suppressed the inflammatory responses in rheumatoid arthritis patients. Our present study reveals that the pLLD, isolated from pathogenic L. donovani, decreases the inflammatory level of bacterial endotoxin in stimulated mouse macrophages, as also in the in vivo murine system. It exerts the activity by reducing the level of different mediators, such as cytokine-chemokines. It also suppresses the expression of the ubiquitous transcription factor NF-Bp65 in stimulated macrophage cells, improves the endotoxin-associated liver damage, reduces the vascular permeability factors, such as VEGF, and suppresses the expression of cell adhesion molecules, including ICAM-1, VCAM-1, PECAM-1, P-selectin, and E-selectin, in liver of septic mice. These findings indicate that pLLD may prove to be a potential anti-inflammatory agent and protect from endotoxininduced sepsis in hepatic impairment. J. Leukoc. Biol. 96: 000 – 000; 2014.
Inflammation is a fundamental adaptation to the loss of cellular and tissue homeostasis with a physiological response, including host defense, tissue remodeling and repair, and the regulation of metabolism. In response to infection, a cascade of signals leads to the recruitment of inflammatory cells, particularly innate immune cells, such as neutrophils and macrophages [1]. Macrophages act through the pathogen-associated molecular patterns mediated signaling pathway that phagocytoses infectious agents and releases a series of cytokines, chemokines, and other mediators, such as TNF-␣, IL-1, IL-6, IL-17, IL-12p40, IFN-␥, MIP-2, RANTES, KC, and different regulatory enzymes, etc. Any dysregulation of these inflammatory factors can promote the outburst of inflammatory responses, and this turns on the initial insult of different disease conditions, including sepsis, atherosclerosis, diabetes, obstructive pulmonary disease, asthma, arthritis, infectious diseases, and cancer [2, 3]. The pathological consequences of sepsis represent an overexuberant inflammatory response, in which an unbridled, cytokine-mediated host-defense mechanism induces significant cell organ injury [4] and septic shock, leading to lethal multiple organ failure and death [5]. Liver is identified as the major affected organ in sepsis. It participates in host defense and a tissue repair process through the involvement of different cells, including Kupffer cells, hepatocytes, and endothelial sinusoidal cells that regulate most of the inflammatory processes for bacterial scavenging, bacterial product inactivation, and inflammatory mediator clearance [6]. If the regulatory activities of hepatic cells prove inadequate, a secondary hepatic dysfunction may develop that may lead to bacterial product spillover and enhanced inflammatory processes; in turn, this endorses the morbidity frequency [7]. Our aim is to prevent the
Abbreviations: ALT⫽alanine aminotransferase, AST⫽aspartate aminotransferase, BCIP⫽5-bromo-4- chloro-3-indolyl phosphate, COX-2⫽cyclooxygenase 2, CSIR⫽Council of Scientific and Industrial Research, DAPI⫽4=,6diamidino-2-phenylindole, EMCCD⫽electron multiplying charge-coupled device, H2DCFDA⫽2=,7=-dichlorodihydrofluorescein diacetate, HIF-1␣⫽ hypoxia-inducible factor 1-␣, KC⫽keratinocyte-derived chemokine(s), MPO⫽myeloperoxidase, PGE2⫽prostaglandin E2, PIGF⫽placental growth factor, pLLD⫽pathogenic leishmanial lipid, PMN⫽polymorphonuclear, ROS⫽reactive oxygen species, SFMC⫽synovial fluid mononuclear cell, VEGF⫽vascular endothelial growth factor The online version of this paper, found at www.jleukbio.org, includes supplemental information.
0741-5400/14/0096-0001 © Society for Leukocyte Biology
1. Correspondence: Cancer Biology & Inflammatory Disorder Division, CSIRIndian Institute of Chemical Biology, 4 Raja S.C. Mullick Rd., Kolkata-700 032, West Bengal, India. E-mail: [email protected]
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Copyright 2014 by The Society for Leukocyte Biology.
physiologic and biochemical changes that initiate systemic inflammation activities, promote hepatic dysfunction during sepsis, and participate in the potential evolution to multiple organ dysfunction syndromes. Although the eukaryote protozoan microbe Leishmania is a member of the Trypanosomatida group, it circumvents the classical host innate immune response in macrophage [8]. The lipids isolated from Leishmania major are reported to act by inhibiting NO production and exert leishmanicidal activity in stimulated macrophages [9]. Recently, we have shown that the lipid from a strain of L. donovani promastigotes (MHO/IN/ 1978/UR6), developed by long-term in vitro culture, suppresses several inflammatory mediators by inducing apoptosis in adherent SFMCs isolated from rheumatoid arthritis patients [10]. In the present study, we demonstrate that the anti-inflammatory role of pLLD in bacterial endotoxin (LPS) induced sepsis and its inflammatory progression toward acute hepatic damage.
MATERIALS AND METHODS
Materials Escherichia coli LPS (0111:B4), MTT, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA); p-NBT-BCIP systems were from Amresco (Solon, OH, USA); DMEM, FBS, penicillin–streptomycin–neomycin, trypsin, and EDTA were from Gibco-BRL (Grand Island, NY, USA); tissue-culture plasticware was from Nunc (Roskilde, Denmark); Bradford protein assay reagent was from Fermentas (Pittsburgh, PA, USA); DAPI was from Invitrogen (Carlsbad, CA, USA); rabbit and goat anti-TNF-␣, IL-1, IL-6, NF-B/p65, IB␣, HIF-1␣, COX-2, P-selectin, E-selectin, VEGF, iNOS polyclonal and secondary antibodies in alkaline phosphatase, and FITC, PE-conjugated, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and a nuclear extraction kit was from Cayman Chemical (Ann Arbor, MI, USA).
L. donovani cell culture and isolation of lipid L. donovani strain AG83 (MHOM/IN/1983/AG83) was used for the present experiments. AG83 was obtained originally from Indian kala-azar patients and was maintained in golden hamsters [11]. Promastigotes obtained after transforming amastigotes from infected hamster spleen were maintained in M199 (Invitrogen), supplemented with antibiotics and 10% FCS at 22°C. The Bligh and Dyer method [12] of lipid extraction was used to isolate the total lipid from Leishmania cells (1⫻1010). The total lipid, obtained from the lower organic phase after evaporation to dryness at 40°C, was then stored at 4°C in vacuum desiccators until used.
TLC pLLD was dissolved in two:one chloroform:methanol. TLC was performed in chloroform:methanol:water (30:60:10), and lipid spots were visualized using iodine spray [10].
Cell culture Murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM containing 25 mmol/L glucose, supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, and 10% heat-inactivated FBS at 37°C in a humidified atmosphere containing 5% CO2.
NO assay Cells (1⫻106 cells/well) were plated onto six-well plates, pretreated with the concentrations of pLLD for 1 h, and stimulated with of LPS (1 g/
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mL) for 6, 12, and 24 h. The sample supernatants were mixed with equal volumes of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm on a plate reader. Nitrite concentration was determined using a dilution of sodium nitrite as a standard. Concentration values were determined for two wells of each sample, and the experiment was performed in triplicate [13].
Cell viability assay Cell viability was measured based on the formation of blue formazan metabolized from colorless MTT by mitochondrial dehydrogenases, which are active only in live cells. RAW264.7 macrophages were plated in 96-well plates at a density of 4 ⫻ 103 cells/well for 24 h and then washed. Cells incubated with various concentrations of pLLD were treated with LPS for 24 h and then incubated in MTT solution. Three hours later, the supernatant was removed, and the formation of formazan was measured at 595 nm using a microplate reader [14].
ELISA After several treatments of pLLD and LPS, followed by incubation, ELISA was performed, per the manufacturers’ instruction (R&D Systems, Minneapolis, MN, USA), to measure the levels of IL-1, IL-6, TNF-␣, IL-12p40, and PGE2.
ROS determination The level of intracellular ROS was determined based on the change in fluorescence, resulting from oxidation of the fluorescent probe H2DCFDA. Briefly, 5 ⫻ 105 cells/well were incubated with pLLD and LPS. After final incubation with 2.5 mM H2DCFDA for 1 h, the intracellular ROS level was determined using flow cytometry with a green (488 nm) laser [15].
Phagocytic activity RAW264.7 cells (1⫻106/mL) were plated in a cell culture dish overnight. Prior to the assay, cells were treated with pLLD and LPS. The medium was decanted, and latex beads of fluorescent red (14 l/plate, 10%, 2 m size) were added and then incubated for an additional 6 h. The supernatant was decanted; the cells were washed carefully, fixed in slide, and visualized by confocal microscope, using a 561 and 405 laser. More than 100 RAW264.7 cells and latex bead-internalized macrophages were counted from 10 different fields, and the percentage of phagocytic activity was calculated.
Confocal laser-scanning microscopy study The NF-B p65 nuclear localization was detected by indirect immunofluorescence assay using confocal microscopy. RAW264.7 cells were cultured directly on glass coverslips in six-well plates for 24 h. After stimulation with LPS and treatment with pLLD, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and blocked with 1.5% normal goat serum (Sigma Chemical Co.). Polyclonal antibodies against NF-B p65 (1 g/well) were applied overnight; this was followed by a 1-h incubation with FITC-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology). After washing with PBS, slides were mounted using DAPI to visualize the nuclei. Specimens were covered with coverslips and evaluated under a confocal laser-scanning microscope (Revolution XD Spinning Disk with an iXon 897 EMCCD camera; Andor, Belfast, UK) [16].
Extraction of nuclear proteins and assay of NF-B p65 Cells were plated onto 24-well plates (1⫻106 cells/well), pretreated in the presence or absence of pLLD for 1 h, and stimulated with LPS (1 g/mL) for 12 h. After centrifugation, these were resuspended in 400 l ice-cold hypotonic buffer for 10 min, vortexed, and centrifuged at 15,000 g at 4°C to get the supernatant-containing nuclear protein [10]. Aliquots of this were added to incubation wells, precoated with the NF-B p65 DNA-bind-
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis ing consensus sequence, and the translocated p65 subunits, present in the nuclear lysate, were assayed, according to the recommendations of the manufacturer of the NF-B assay kit (Cayman Chemical).
Western blot analysis The cells and liver tissues were washed with PBS three to five times and lysed with the lysis buffer. Equal amounts of protein were separated in 10–15% SDSpolyacrylamide minigels and transferred to immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After incubation with the appropriate primary antibody, the membrane was hybridized with a secondary antibody conjugated to alkaline phosphatase for 1 h at room temperature. Following washes with TBST, immunoreactive bands were visualized using NBTBCIP chromogenic substrates. In a parallel experiment, nuclear protein was prepared by nuclear extraction reagents (Pierce, Rockford, IL, USA), according to the manufacturer’s protocol [17].
Animals All in vivo experiments were performed using BALB/C adult female mice, weighing 18 –22 g and obtained from the Animal House of the Indian Institute of Chemical Biology (West Bengal, India). The mice were housed in micro-isolator cages at a laboratory temperature of 24 ⫾ 1°C with 40 – 80% relative humidity and received food and water ad libitum; light-dark cycle was maintained. The animals were allowed to adapt to the experimental environment for 5–7 days before experimentation. Care and maintenance of animals were done in adherence to the guidelines of the Institutional Animal Care and Use Committee.
Acute toxicity study These studies were carried out in female BALB/c mice (n⫽20). pLLD was solubilized in 0.2% Tween 80, suspended in PBS, and administered i.p. in 1–200 mg/kg doses. Then, all groups of animals were observed for signs and symptoms of toxicity, and the number of mortality was recorded from 24 h to 15 days following treatment. The LD50 was determined according to the method of Litchfield and Wilcoxon [18].
Measurement of liver function Liver dysfunction was assessed by measuring the rise in serum levels of ALT, AST, and total bilirubin. The serum samples were obtained from the mice, 24 h after challenge with LPS, and analyzed with a commercial kit, according to the manufacturer’s instructions (BioVision, Milpitas, CA, USA).
MPO estimation Neutrophil sequestration in the liver was quantified by measuring tissue MPO activity. After treatment with pLLD and LPS, mice were killed. Liver tissue samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), and centrifuged (13,000 rpm, 10 min, 4°C). The pellet was resuspended in 50 mM phosphate buffer (pH 6.0), containing 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co.). The suspension was subjected to four cycles of freezing and thawing and disrupted further by sonication for 40 s. The sample was then centrifuged (13,000 rpm, 5 min, 4°C), and the supernatant was used for the MPO assay. The reaction mixture consisted of the supernatant, 1.6 mM tetramethyl benzidine (Sigma Chemical Co.), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide. This mixture was incubated at 37°C for 110 s, and the absorbance was measured at 450 nm [19].
Immunohistochemistry For immunohistochemical study, 5 m-thickness tissue sections were deparaffinized with xylene, rehydrated with descending alcohol series, incubated with trypsin (0.05% trypsin, 0.1% CaCl2) for antigen retrieval, and subjected to blocking using 5% BSA in TBS (20 mM Tris HCl, pH 7.4, containing 150 mM NaCl) for 4 h at room temperature. This was followed by incubation overnight at 4°C in primary antibody (iNOS, TNF-␣, HIF-1␣, VEGF) solution (1:300 dilutions in TBS) using a humid chamber. The tissue sections were washed with TBST (20 mM Tris HCl, pH 7.4, containing 150 mM NaCl and 0.025% Triton X-100) and incubated with FITC- and PE-conjugated secondary antibody (Santa Cruz Biotechnology) solution (1:600 dilutions in TBS containing) for 1 h at room temperature. After mounting with DAPI, visualization was done by a confocal laser-scanning microscope (Revolution XD Spinning Disk with an iXon 897 EMCCD camera; Andor).
Survival study In the survival study, BALB/c mice were assigned randomly into groups and treated i.p. with normal saline or with pLLD at doses of 25 and 50 mg/kg, every day for 3 days. LPS (32 mg/kg), dissolved in normal saline, was injected i.p., 2 h after pLLD-i.p. treatment. Survival rate was then recorded every 12 h for 6 days.
Analysis of serum inflammatory factors For the analysis of serum cytokines, mice were assigned randomly into groups (n⫽10). The control group received the vehicle, the second group received only pLLD, the third group received LPS at 32 mg/kg i.p., and animals of the remaining groups received pLLD at the dose of 50 mg/kg i.p., 2 h before LPS administration. The sera were collected from the animals at 1st, 3rd, and 6th h to measure cytokines TNF-␣, IL-1, IL-6, IL12p40, IL-17, IFN-␥, MIP-2, KC, and RANTES, besides vascular factors VEGF and PIGF. The measurements were done at Times 6, 12, and 24 h by sandwich ELISA using commercially available reagents, according to the manufacturer’s instructions.
Histological examination For histopathological observation, the mice were killed, and lungs, liver, and kidneys were removed, 24 h after LPS or normal saline injection and administration of only pLLD. The portions of lung, liver, and kidney tissues were fixed immediately in 10% neutral-buffered formalin solution and then dehydrated. Next, the fixed tissues were embedded in paraffin, sectioned (5 m) using a microtome, mounted on glass slides, and stained with H&E for morphological examination.
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Flow cytometry in isolated hepatocyte from mice Hepatocytes were isolated from each group of mice by two-step collagenase perfusion, following the literature procedure [20, 21], with some modifications. Isolated monolayers chosen for the experiment were permeabilized in 70% alcohol and incubated with fluorochrome-conjugated antibody; the expressions of ICAM-1, VCAM-1, and PECAM-1 in hepatocytes were then analyzed by flow cytometry.
Statistical analysis Results were expressed as mean ⫾ sem. Statistical analyses were performed with ANOVA, followed by Dunnett’s test. P ⬍ 0.05 was considered significant.
RESULTS
TLC analysis of pLLD Figure 1 shows the TLC profile of the pLLD used in our study. Iodine staining showed six to seven spots of lipids in the TLC plate. Lipids from three different batches showing the same TLC profile (Fig. 1A) were used in further studies.
Effect of pLLD on LPS-induced NO production in macrophages The concentration-dependent effect of pLLD on NO release in RAW264.7 cells was determined in the presence and abVolume 96, August 2014
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Figure 1. TLC profile of pLLD (A). Effect of pLLD on production of NO in the LPS-stimulated RAW264.7 cells. Cells were treated with various concentrations (1–140 g/mL) of pLLD for 24 h, with or without LPS (1 g/mL), and the optical density (O.D.) was determined by the ELISA method (B). The cytotoxicity of pLLD on cells for 24 h in the presence or absence of LPS (1 g/mL) was determined by MTT assay and optical density measurement at 595 nm. Morphological changes in RAW264.7 cell, visualized by optical microscopy (200⫻) in the presence and absence of LPS with 120 g/mL pLLD (C). The data are reported as the mean ⫾ sem of triplicate experiments (*P⬍0.05; **P⬍0.01).
sence of LPS. Unstimulated macrophage cells constitutively produced minimal levels of NO, but the levels were higher with LPS-stimulated cells. Cells were treated with various concentrations of pLLD (1–140 g/mL) at 24 h in the presence of LPS. A significant reduction (up to 77%) was found upon treatment of the lipid at concentrations of 60 and 120 g/mL in LPS-treated macrophage cells at 24 h (Fig. 1B). Thereafter, we selected two concentrations of pLLD— 60 and 120 g/mL—for the in vitro experiments. Evaluation of the cytotoxic effects in peritoneal macrophage cells using the MTT assay revealed that pLLD did not affect cell viabilities at the concentrations used (1–140 g/mL; Fig. 1C). Figure 1D shows the control cells were round in shape whereas LPS-stimulated RAW 264.7 cells had changed to an irregular form with accelerated spreading and pseudopodia formation. Co-treatment of LPS with pLLD at 120 g/mL concentration changes the level of cell differentiation by reducing pseudopodia formation.
Suppression of LPS induced different inflammatory mediators by pLLD in macrophage cells pLLD was found to reduce the inflammatory factors up-regulated by LPS in murine macrophage cells. As measured by sandwich ELISA and shown in Fig. 2A–E, it also significantly lowered the levels of proinflammatory cytokines, including TNF-␣, IL-1, IL-6, IL-12p40, and PGE2 in the cell supernatant, cotreated with LPS and pLLD at concentrations of 60 g/mL and 120 g/mL, after incubation for 6, 12, and 24 h. Western blot results also revealed the impeding effect of 4 Journal of Leukocyte Biology
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pLLD, wherein the expression levels of inflammatory mediators, including TNF-␣, IL-1, IL-6, iNOS, and COX-2, were suppressed at 12 h upon pretreatment with pLLD at 60 and 120 g/mL (Fig. 2F).
Effect of pLLD on ROS production and phagocytosis in LPS-stimulated macrophages The effect of pLLD on intracellular ROS production in the LPS-stimulated RAW264.7 cells was measured using the fluorescent probe dichlorofluorescein diacetate. pLLD significantly inhibited the induction of intracellular ROS generation in these cells by LPS (Fig. 3A). Measurement of phagocytic activity, microscopically (Fig. 3B) using 2 m fluorescent red-bead internalization, revealed that pLLD simultaneously augmented the phagocytic activity from 90% to 40% in stimulated macrophage cells.
Effects of pLLD on LPS induced production of proinflammatory cytokines (TNF-␣, IL-1, and IL-6) and transcriptional factor NF-B in RAW264.7 cells LPS-induced inflammatory stress leads to activation of the transcriptional factor NF-B and release of the subsequent proinflammatory mediators, which are regulated by the cytoplasmic inhibitor IB. Thus, we examined whether pLLD inhibited the levels of NF-Bp65 expression in a concentration- and timedependent manner. As shown in Fig. 3D, exposure to LPS for 1 h led to increased expression of the NF-B p65 subunit in nuclear, whereas the expression of IB␣ was inhibited in LPSinduced macrophage cells. Conversely, pLLD, at 60 and 120
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis
Figure 2. Effect of pLLD on production of cytokines and mediators from stimulated RAW264.7 cells. After incubation with pLLD (60 and 120 g/mL), the production of inflammatory mediators (A–E) was measured for 6, 12, and 24 h by ELISA in the presence of LPS (1 g/mL). Cytokine and mediator expression levels were determined by Western blot analysis after 12 h incubation of RAW264.7 with LPS alone or with pLLD at 60 and 120 g/mL. The values are mean ⫾ sem of three independent experiments.
g/mL concentrations, significantly inhibited the expressions of the NF-B p65 subunit, whereas the expression of IB␣ was increased (Fig. 3D). Interestingly, pLLD also significantly inhibited the level of nuclear extraction of NF-Bp65 in a concentration- and time-dependent manner, as shown in Fig. 3C. Immunocytochemistry studies showed that the translocation of the p65 subunit of NF-B is inhibited in LPS stimulated macrophages with pLLD (Fig. 3E).
Acute toxicity study Administered i.p., pLLD was nontoxic until 500 mg/kg in mice. The experimental mice were closely observed for the first 24 h and monitored for the next 15 days. However, no toxic symptom
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or abnormal behavior was observed. Thus, 1/10th and 1/20th of this dose, i.e., 50 mg/kg i.p. and 25 mg/kg i.p., were taken as the experimental doses as per the inhibitory efficiency of inflammatory response (Supplemental Fig. 2).
Improvement in survival rate of BALB/c mice challenged with a lethal dose of LPS and the effect of pLLD on cytokine production To examine the protective effect of pLLD against LPS-induced lethality, mice were pretreated with 25 mg/kg and 50 mg/kg pLLD, once a day for 3 days, before i.p. injection of 20 mg/kg LPS, and animal survival was recorded for 6 days after LPS challenge. As shown in Fig. 4, the survival rates at 24 and 48 h Volume 96, August 2014
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Figure 3. Effect of pLLD on ROS production in RAW264.7 cells induced by LPS for 12 h at the concentrations of 60 and 120 g/mL pLLD, analyzed by flow cytometry (A). FL1-H, Fluorescence 1-height. pLLD inhibited the LPS-induced phagocytosis, as assessed by 2 m fluorescent red-bead uptake by RAW264.7 macrophages for 12 h (B). Effect of pLLD on levels of nuclear extract of NF-B p65 in RAW264.7 cells after 0.5, 1, and 3 h incubation at concentrations of 60 and 120 g/mL in the presence and absence of LPS (C). Nuclear NF-B p65 and IB-␣ protein expression levels after treatment with pLLD at concentrations of 60 and 120 g/ml were determined at 4 h after incubation in the presence of LPS (D). Effect of pLLD for 4 h on LPS-induced NF-B translocation by confocal laser-scanning microscopy (magnification 600⫻); nucleus is identified with arrows. Original scale bars, 10 m (E). Data obtained from three independent experiments. DIC, Differential interference contrast; MFI, mean fluorescence intensity (*P⬍0.05; **P⬍0.01).
after LPS challenge were 41% and 9%, respectively, in mice pretreated with Vehicle. However, in mice pretreated with 50 and 25 mg/kg pLLD, survival rates at the corresponding timepoints after LPS treatment significantly increased to 78.8% and 53.8%, respectively. The early responses generated by cytokines and chemokines, such as TNF-␣, IL-1, IL-6, IL-12p40, IL-17, IFN-␥, MIP-2, KC, and RANTES, play a critical role in moderating the physiological responses in the progression of inflammation. To evaluate the effect of pLLD on the production of cytokines in mice linked with a fatal outcome, we determined the serum concentration of proinflammatory cytokines at different time intervals. After challenging with LPS, serum concentrations of cytokines in pLLD-treated mice were measured by the ELISA method at 0, 1, 3, and 6 h (Fig. 5C–E). This showed that 50 mg/kg pLLD significantly reduced the higher levels of cytokine production in serum of LPS-challenged mice. In accordance with our findings of the serum profile in Fig. 4C–K, the histological data also demonstrated improvement of organ in6 Journal of Leukocyte Biology
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jury in mice challenged with LPS but pretreated with pLLD at 50 mg/kg (Fig. 5A). As compared with tissue sections from control group of normal mice exposed to LPS, mice pretreated with pLLD exhibited marked inflammatory alterations characterized by infiltration of the tissue and alveoli with inflammatory cells and the presence of pulmonary edema (Fig. 5A, LPS, Lung panel), considerable hemorrhaging in the renal medulla (Fig. 5A, LPS, Kidney panel) and PMN infiltration and portal injury in the liver (Fig. 5A, LPS, Liver panel) were observed. However, in LPS-injected mice treated with pLLD at the dose of 50 mg/kg prior to LPS challenge, the attenuation was found in the histopathological changes of lung, kidney and liver.
pLLD improves LPS-induced hepatic impairment When pLLD was administered i.p. at the dose of 50 mg/kg before LPS challenge, improvement in hepatic damage was observed (Fig. 5A), as brought out by histopathological scores in septic mice liver (Fig. 5B). LPS caused a large increase in the plasma levels of ALT, AST, bilirubin, and MPO at the late
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Figure 4. Effect of pLLD on (A) survival and (B) body weight of mice (nⴝ10), treated with LPS (32 mg/kg). Levels of cytokines TNF-␣, IL1, IL-6, IL-12p40, IL-17, IFN-␥, ⌴⌱P-2, KC, and RANTES (C–K) were measured by ELISA at 1, 3, and 6 h upon treatment with pLLD at doses of 50 mg/kg, after challenging with LPS. The data are reported as the mean ⫾ sem of triplicate experiments (*P⬍0.05; **P⬍0.01; ***P⬍0.001).
stage (24 h). Pretreatment with pLLD at 50 mg/kg significantly reduced the rise in plasma level of ALT, AST, and total bilirubin generated by LPS. Simultaneously, hepatic MPO was found to be significantly lowered at 24 h in treated mice with reduction of liver PMN compared with mice challenged with LPS only, as shown in Figs. 5G and 5C. To evaluate the localization of proinflammatory mediators in tissue-level expression on LPS-induced liver damage, iNOS and IL-6 localizations were checked in hepatic tissue by immunofluorescence analysis (Fig. 5H and I). Overexpression of IL-6 was observed predominantly in the epithelial region in LPS-treated tissue, whereas iNOS localization was observed in
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the LPS-treated tissue section in the epithelial and submucosal layers (Fig. 4). Reduced expressions of IL-6 and iNOS were observed in the epithelial layer, pretreated with pLLD at 50 mg/kg in LPS-induced septic liver tissue. Besides these, we have also studied the effect of pLLD at the dose of 50 mg/kg in sepsis-induced murine hepatic tissue by Western blot analysis to validate our findings (Fig. 5J).
Effects of pLLD on vascular permeability factor(s) and cell adhesion molecule(s) Hypoxia is well-known to induce the expression of different growth factors, such as VEGF and PIGF, and of cell adhesion Volume 96, August 2014
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Figure 5. Effect of pLLD on LPS-induced organ injury. (A) Mice were pretreated with pLLD at a dose of 50 mg/kg i.p., 1 h after LPS (32 mg/kg). After 24 h, liver was removed, and the histopathological changes in kidney, lungs, and liver of mice were pretreated with LPS, assessed by H&E stain (200⫻). Histopathological studies by light microscope, morphologically showing the tissue sections (kidney, lung, and liver) from mice in the control, LPS, and LPS ⫹ pLLD groups (arrows), indicate the infiltration of inflammatory cells in captured fields (scale bars, 20 m). (B) Scores of liver injury. Livers were considered injured if any of these lesions were detected. Injury was scored as 0 (none), 1⫹ (rare focal apoptosis, inflammation, or hemorrhage), 2⫹ (focal apoptosis, inflammation, and/or hemorrhage in 25–50% of the fields), 3⫹ (focal apoptosis, inflammation, and/or hemorrhage in ⬎50% of the fields). (C) PMN. Serum levels of ALT (D), AST (E), total bilirubin (F), and liver MPO (G) were also assessed to determine the localization of iNOS (H) and IL-6 (I) in liver tissues. Magnification, 100⫻; arrows indicate immunopositive cells. Approximately 200 cells were counted/field; five fields were examined/slide; five slides were examined/group; and expression of inflammatory mediator and cytokines was analyzed by Western blot (J). The data were obtained from three independent experiments. Values are presented as mean ⫾ sem (*P⬍0.05; **P⬍0.01; ***P⬍0.001).
molecules, including ICAM-1, VCAM-1, PECAM, P-selectin, and E-selectin, in inflammatory sepsis-associated hepatic injury. Flow cytometry revealed the inhibitory effect of pLLD in the expressions of cell adhesion molecules in primary hepatocytes of LPSchallenged mice (Fig. 6F–H). Significant reduction in VEGF and HIF-1␣ levels of liver parenchymal tissue compared with that in only the LPS-challenged group (Fig. 6C) was evident. Furthermore, Western blot data revealed that pretreatment with pLLD at 8 Journal of Leukocyte Biology
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50 mg/kg reduced the protein level expressions of VEGF, P-selectin, and E-selectin at 24 h, as seen in Fig. 6I.
DISCUSSION Microorganisms and their cellular component(s) may possess some degree of bioactivity, either against other microorgan-
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis
Figure 6. Effect on circulatory levels of VEGF (serum) (A) and PIGF (serum; B) and on liver tissue localization of VEGF and HIF-1␣ (C) in LPSinduced mice, administered 50 mg/kg pLLD, 1 h after LPS administration, as determined by confocal microscopy (100ⴛ). Expression of VEGF (D) and PIGF (E) by ELISA from liver tissue lysate and hepatocyte expression of ICAM-1 (F), VCAM-1 (G), and PECAM-1 (H), detected by flow cytometry, with expression of adhesion molecules, such as VEGF, in liver tissue analyzed by Western blot (I). FITC-A, FITC-area. The data are obtained from three independent experiments. Values are presented as mean ⫾ sem (*P⬍0.05; **P⬍0.01).
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ism(s) or against certain physiological states of a diseased body [22, 23]. Azurin, a protein isolated by the pathogenic bacteria Pseudomonas aeruginosa, induces apoptosis by interfering with signaling molecules and regulating the growth of cancer cells [24]. Sophorolipid, produced from the new yeast strain Wickerhamiella domercqiae, shows its potency against human pancreatic cancer cells and septicemia [25–27]. Interestingly, in spite of the potent leishmanicidal activity, L. amazonesis and its components inhibited the expression of the cytokine-inducible nitric oxide synthase (iNOS) in stimulated macrophage cells [9, 28]. Our previous study revealed that the lipid from an attenuated strain of L. donovani promastigote (MHO/IN/1978/UR6), developed by long-term in vitro culture, suppresses several inflammatory mediators by inducing apoptosis in adherent SFMCs isolated from rheumatoid arthritis patients [10]. These findings encouraged us to evaluate the anti-inflammatory role of pLLD against bacterial endotoxin-induced, sepsis-associated inflammatory progression toward acute hepatic damage. We could thus demonstrate the anti-inflammatory activities of the lipid isolated from L. donovani (MHOM/IN/1983/AG83), both in vitro using macrophages stimulated by endotoxin (LPS) from Gram-negative bacteria and in vivo using mouse models of septic inflammation. Macrophages play a critical role in cellular host defense against infection and tissue injury [29]. In response to stimuli, macrophages undergo a series of inflammatory processes, including chemotaxis, phagocytosis, intracellular killing, and release of inflammatory cytokines [30, 31]. In acute and chronic inflammation, NO plays a significant role to cause vasodilation and tissue damage. In addition, it functions in immune systems as a cytotoxic macrophage effector molecule, modulator of PMN leukocyte chemotaxis and adhesion, mediator of tissue injury caused by adhesion of immune complexes, and regulator of lymphocyte proliferation [32]. Therefore, NO production can be used as a measurement of the progression of inflammation, and its inhibition might have potential therapeutic value in the context of inflammation-associated diseases. Here, we report for the first time that the pathogenic leishmanial lipid inhibits NO production from stimulated macrophages in a dose-dependent manner (Fig. 1B). Although the formation of ROS is desirable for a host’s defense, their overproduction can jeopardize the body’s own cells, cause tissue injury, and contribute to the development of a number of serious diseases [33, 34]. As shown in Fig. 3A, pLLD significantly reduced the ROS production in stimulated macrophage cells. To elucidate the molecular approaches by which pLLD exerts anti-inflammatory activity on Gram-negative bacterial sepsis, we have evaluated the survival rate of mice in an endotoxin-induced murine sepsis model. Interestingly, pLLD-induced inhibition of the production of serum cytokines, including TNF-␣, IL-1, IL-6, IL-12, IL-17, IFN-␥, and KC, and of chemokine MIP-2 was consistent with our in vitro results (Fig. 4) on the survival status in multiple organ failure of LPS-challenged mice. IL-6 plays the pivotal role in host-defense responses to infection and inflammation by activating macrophages and inducing acute-phase proteins in liver [35]. Importantly, the autocrine/paracrine inflammatory factor, PGE2-dependent NF-B activation, is required for IL-6 production in stimulated 10 Journal of Leukocyte Biology
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macrophages [36], and pLLD inhibited the overproduction of IL-6. Bacterial endotoxins are well-known to trigger the signaling pathway of TLR4-CD14, leading to the activation of the IB kinase complex and phosphorylation of the inhibitory IB protein. This protein is necessary for ubiquitination, and its degradation leads to the release and subsequent translocation of NF-B p65 into the nucleus [37] through degradation of I-B␣ on serine subunits. This study demonstrated that pLLD inhibits the activation of NF-B that eventually induces the various inflammatory mediators (Fig 3) and other signaling molecules in inflammatory cascade (Fig. 3 and Supplemental Fig. 1). Culture of macrophages with LPS triggers the signaling cascades and leads to rapid translocation of NFBp65 subunit to nucleus, followed by the increased expression of NF-B dependent cytokines. Similarly, during sepsis or numerous disease states, the exposure to LPS may initiate a systemic inflammatory response via the activation of NF-B leading to significant morbidity and mortality [38, 39]. Furthermore, in agreement with the in vitro study, inflammatory factors were regulated by pLLD in sepsis model. In a septic environment, enhancement of PMN activation could promote cytokine production and induce tissue injury, leading to an increase in PMN accumulation in the hepatic sinusoids and development of hepatic injury [40]. Both increased PMN level and rate of mortality declined significantly with pretreatment of pLLD, as shown in Figs. 4 and 5. These cytokines induce accumulation and activation of another transcription factor, HIF-1, whereas LPS-induced HIF-1␣ accumulation is predominantly dependent on NF-B activation. In inflamed tissue, VEGF and HIF-1␣ are activated as a result of vascular damage and edema, as well as the intense metabolic activity of bacteria and numerous infiltrating cells that are generated primarily during hypoxia [41]. Specifically, TNF-␣ and IL-1 contribute to the increase in the number of infiltrating neutrophils, which play a critical role in bacterial clearance [42]. We observed that pLLD profoundly reduced the elevated serum cytokine level with concurrent reduction of VEGF and HIF-1␣ expression in hepatic tissue. Primary hepatic dysfunction refers to sepsis-induced dysfunction in the period immediately after an episode of shock and resuscitation [43, 44]. It was found that hepatic injury was reduced by the alteration of liver function parameters (AST/ALT) after administration of pLLD in septic mice. In addition to its role in promoting endothelial permeability and proliferation, VEGF may contribute to inflammation and coagulation. These proinflammatory effects of VEGF are mediated, at least in part, by the activation of NF-B transcription [45]. Furthermore, during the inflammatory changes, the process of angiogenic vascular enlargement also occurs by regulating PIGF status in vascular remodeling endothelial cell proliferation [46]. Few cytokines, such as IL-1, IL-6, and COX-2, regulate the expression level of VEGF in different cell types. Under inflamed conditions, VEGF induces the expression of cell adhesion molecules (E-selectin and P-selectin), along with ICAM-1 and VCAM-1 in endothelial cells, and promotes the adhesion of leukocytes [47]. Consequently, PECAM regulates the leukocyte migration process in the presence of cytokines, such as TNF-␣, IFN-␥, and
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis
different cell adhesion molecules [48]. The expression of these adhesion molecules, regulated by the cytokine-inducible NF-B, is altered by pLLD, as shown in Fig. 6. In conclusion, this study has revealed that pathogenic leishmanial lipid exerts anti-inflammatory responses by regulating the inflammatory factors in vitro and in vivo. In addition, it affords protection against sepsis-mediated organ injury, including hepatic damage by regulating different mediators, cytokines, chemokines, vascular permeability factors, and cellular adhesion molecules. Taken together, these findings suggest that the pLLD may be used as a novel therapeutic agent of microbial origin against inflammatory responses.
9.
10.
11.
12. 13.
AUTHORSHIP N.C. and S.D. carried out the studies and wrote the manuscript. D.B. and S.B. participated in ELISA and flow cytometry. N.C. and T.J. contributed to data and statistical analysis. K.D.S. initiated and supervised the experimental work and drafted the manuscript. All authors approved the final version of the manuscript.
ACKNOWLEDGMENTS The CSIR and Indian Council of Medical Research (ICMR; India) financially supported this work. The authors convey their sincerest thanks to Prof. Siddhartha Roy (director of CSIR-Indian Institute of Chemical Biology) for providing us the necessary support for this work. We extend our thanks to Dr. Suvendra Nath Bhattacharyya (Indian Institute of Chemical Biology) and Mr. Diptadeep Sarkar for their help in confocal laser-scanning microscopy. We are also indebted to Dr. J. Rajan Vedasiromoni and Dr. Basudeb Achari of our institute for critically reviewing the manuscript.
14. 15. 16.
17.
18. 19.
20. 21. 22.
DISCLOSURES
The authors declare that they have no competing interests.
23. 24.
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KEY WORDS: Leishmania 䡠 macrophage 䡠 lipopolysaccharide 䡠 inflammation 䡠 septic damage
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Article
Leishmanial lipid suppresses the bacterial endotoxin-induced inflammatory response with attenuation of tissue injury in sepsis Nabanita Chatterjee,* Subhadip Das,* Dipayan Bose,* Somenath Banerjee,* Tarun Jha,† and Krishna Das Saha*,1 *Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, West Bengal, India; and †Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India RECEIVED NOVEMBER 6, 2013; REVISED FEBRUARY 14, 2014; ACCEPTED MARCH 4, 2014. DOI: 10.1189/jlb.3A1113-582R
ABSTRACT
Introduction
The use of live, attenuated, or genetically modified microbes or their cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver biologically active entities. Thus, advancing our knowledge of such microbe-mediated therapy may suggest new avenues for therapeutic intervention in many diseases. We had earlier reported that the total lipid of attenuated Leishmania donovani suppressed the inflammatory responses in rheumatoid arthritis patients. Our present study reveals that the pLLD, isolated from pathogenic L. donovani, decreases the inflammatory level of bacterial endotoxin in stimulated mouse macrophages, as also in the in vivo murine system. It exerts the activity by reducing the level of different mediators, such as cytokine-chemokines. It also suppresses the expression of the ubiquitous transcription factor NF-Bp65 in stimulated macrophage cells, improves the endotoxin-associated liver damage, reduces the vascular permeability factors, such as VEGF, and suppresses the expression of cell adhesion molecules, including ICAM-1, VCAM-1, PECAM-1, P-selectin, and E-selectin, in liver of septic mice. These findings indicate that pLLD may prove to be a potential anti-inflammatory agent and protect from endotoxininduced sepsis in hepatic impairment. J. Leukoc. Biol. 96: 000 – 000; 2014.
Inflammation is a fundamental adaptation to the loss of cellular and tissue homeostasis with a physiological response, including host defense, tissue remodeling and repair, and the regulation of metabolism. In response to infection, a cascade of signals leads to the recruitment of inflammatory cells, particularly innate immune cells, such as neutrophils and macrophages [1]. Macrophages act through the pathogen-associated molecular patterns mediated signaling pathway that phagocytoses infectious agents and releases a series of cytokines, chemokines, and other mediators, such as TNF-␣, IL-1, IL-6, IL-17, IL-12p40, IFN-␥, MIP-2, RANTES, KC, and different regulatory enzymes, etc. Any dysregulation of these inflammatory factors can promote the outburst of inflammatory responses, and this turns on the initial insult of different disease conditions, including sepsis, atherosclerosis, diabetes, obstructive pulmonary disease, asthma, arthritis, infectious diseases, and cancer [2, 3]. The pathological consequences of sepsis represent an overexuberant inflammatory response, in which an unbridled, cytokine-mediated host-defense mechanism induces significant cell organ injury [4] and septic shock, leading to lethal multiple organ failure and death [5]. Liver is identified as the major affected organ in sepsis. It participates in host defense and a tissue repair process through the involvement of different cells, including Kupffer cells, hepatocytes, and endothelial sinusoidal cells that regulate most of the inflammatory processes for bacterial scavenging, bacterial product inactivation, and inflammatory mediator clearance [6]. If the regulatory activities of hepatic cells prove inadequate, a secondary hepatic dysfunction may develop that may lead to bacterial product spillover and enhanced inflammatory processes; in turn, this endorses the morbidity frequency [7]. Our aim is to prevent the
Abbreviations: ALT⫽alanine aminotransferase, AST⫽aspartate aminotransferase, BCIP⫽5-bromo-4- chloro-3-indolyl phosphate, COX-2⫽cyclooxygenase 2, CSIR⫽Council of Scientific and Industrial Research, DAPI⫽4=,6diamidino-2-phenylindole, EMCCD⫽electron multiplying charge-coupled device, H2DCFDA⫽2=,7=-dichlorodihydrofluorescein diacetate, HIF-1␣⫽ hypoxia-inducible factor 1-␣, KC⫽keratinocyte-derived chemokine(s), MPO⫽myeloperoxidase, PGE2⫽prostaglandin E2, PIGF⫽placental growth factor, pLLD⫽pathogenic leishmanial lipid, PMN⫽polymorphonuclear, ROS⫽reactive oxygen species, SFMC⫽synovial fluid mononuclear cell, VEGF⫽vascular endothelial growth factor The online version of this paper, found at www.jleukbio.org, includes supplemental information.
0741-5400/14/0096-0001 © Society for Leukocyte Biology
1. Correspondence: Cancer Biology & Inflammatory Disorder Division, CSIRIndian Institute of Chemical Biology, 4 Raja S.C. Mullick Rd., Kolkata-700 032, West Bengal, India. E-mail: [email protected]
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Journal of Leukocyte Biology 1
Copyright 2014 by The Society for Leukocyte Biology.
physiologic and biochemical changes that initiate systemic inflammation activities, promote hepatic dysfunction during sepsis, and participate in the potential evolution to multiple organ dysfunction syndromes. Although the eukaryote protozoan microbe Leishmania is a member of the Trypanosomatida group, it circumvents the classical host innate immune response in macrophage [8]. The lipids isolated from Leishmania major are reported to act by inhibiting NO production and exert leishmanicidal activity in stimulated macrophages [9]. Recently, we have shown that the lipid from a strain of L. donovani promastigotes (MHO/IN/ 1978/UR6), developed by long-term in vitro culture, suppresses several inflammatory mediators by inducing apoptosis in adherent SFMCs isolated from rheumatoid arthritis patients [10]. In the present study, we demonstrate that the anti-inflammatory role of pLLD in bacterial endotoxin (LPS) induced sepsis and its inflammatory progression toward acute hepatic damage.
MATERIALS AND METHODS
Materials Escherichia coli LPS (0111:B4), MTT, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA); p-NBT-BCIP systems were from Amresco (Solon, OH, USA); DMEM, FBS, penicillin–streptomycin–neomycin, trypsin, and EDTA were from Gibco-BRL (Grand Island, NY, USA); tissue-culture plasticware was from Nunc (Roskilde, Denmark); Bradford protein assay reagent was from Fermentas (Pittsburgh, PA, USA); DAPI was from Invitrogen (Carlsbad, CA, USA); rabbit and goat anti-TNF-␣, IL-1, IL-6, NF-B/p65, IB␣, HIF-1␣, COX-2, P-selectin, E-selectin, VEGF, iNOS polyclonal and secondary antibodies in alkaline phosphatase, and FITC, PE-conjugated, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and a nuclear extraction kit was from Cayman Chemical (Ann Arbor, MI, USA).
L. donovani cell culture and isolation of lipid L. donovani strain AG83 (MHOM/IN/1983/AG83) was used for the present experiments. AG83 was obtained originally from Indian kala-azar patients and was maintained in golden hamsters [11]. Promastigotes obtained after transforming amastigotes from infected hamster spleen were maintained in M199 (Invitrogen), supplemented with antibiotics and 10% FCS at 22°C. The Bligh and Dyer method [12] of lipid extraction was used to isolate the total lipid from Leishmania cells (1⫻1010). The total lipid, obtained from the lower organic phase after evaporation to dryness at 40°C, was then stored at 4°C in vacuum desiccators until used.
TLC pLLD was dissolved in two:one chloroform:methanol. TLC was performed in chloroform:methanol:water (30:60:10), and lipid spots were visualized using iodine spray [10].
Cell culture Murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM containing 25 mmol/L glucose, supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, and 10% heat-inactivated FBS at 37°C in a humidified atmosphere containing 5% CO2.
NO assay Cells (1⫻106 cells/well) were plated onto six-well plates, pretreated with the concentrations of pLLD for 1 h, and stimulated with of LPS (1 g/
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mL) for 6, 12, and 24 h. The sample supernatants were mixed with equal volumes of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm on a plate reader. Nitrite concentration was determined using a dilution of sodium nitrite as a standard. Concentration values were determined for two wells of each sample, and the experiment was performed in triplicate [13].
Cell viability assay Cell viability was measured based on the formation of blue formazan metabolized from colorless MTT by mitochondrial dehydrogenases, which are active only in live cells. RAW264.7 macrophages were plated in 96-well plates at a density of 4 ⫻ 103 cells/well for 24 h and then washed. Cells incubated with various concentrations of pLLD were treated with LPS for 24 h and then incubated in MTT solution. Three hours later, the supernatant was removed, and the formation of formazan was measured at 595 nm using a microplate reader [14].
ELISA After several treatments of pLLD and LPS, followed by incubation, ELISA was performed, per the manufacturers’ instruction (R&D Systems, Minneapolis, MN, USA), to measure the levels of IL-1, IL-6, TNF-␣, IL-12p40, and PGE2.
ROS determination The level of intracellular ROS was determined based on the change in fluorescence, resulting from oxidation of the fluorescent probe H2DCFDA. Briefly, 5 ⫻ 105 cells/well were incubated with pLLD and LPS. After final incubation with 2.5 mM H2DCFDA for 1 h, the intracellular ROS level was determined using flow cytometry with a green (488 nm) laser [15].
Phagocytic activity RAW264.7 cells (1⫻106/mL) were plated in a cell culture dish overnight. Prior to the assay, cells were treated with pLLD and LPS. The medium was decanted, and latex beads of fluorescent red (14 l/plate, 10%, 2 m size) were added and then incubated for an additional 6 h. The supernatant was decanted; the cells were washed carefully, fixed in slide, and visualized by confocal microscope, using a 561 and 405 laser. More than 100 RAW264.7 cells and latex bead-internalized macrophages were counted from 10 different fields, and the percentage of phagocytic activity was calculated.
Confocal laser-scanning microscopy study The NF-B p65 nuclear localization was detected by indirect immunofluorescence assay using confocal microscopy. RAW264.7 cells were cultured directly on glass coverslips in six-well plates for 24 h. After stimulation with LPS and treatment with pLLD, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and blocked with 1.5% normal goat serum (Sigma Chemical Co.). Polyclonal antibodies against NF-B p65 (1 g/well) were applied overnight; this was followed by a 1-h incubation with FITC-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology). After washing with PBS, slides were mounted using DAPI to visualize the nuclei. Specimens were covered with coverslips and evaluated under a confocal laser-scanning microscope (Revolution XD Spinning Disk with an iXon 897 EMCCD camera; Andor, Belfast, UK) [16].
Extraction of nuclear proteins and assay of NF-B p65 Cells were plated onto 24-well plates (1⫻106 cells/well), pretreated in the presence or absence of pLLD for 1 h, and stimulated with LPS (1 g/mL) for 12 h. After centrifugation, these were resuspended in 400 l ice-cold hypotonic buffer for 10 min, vortexed, and centrifuged at 15,000 g at 4°C to get the supernatant-containing nuclear protein [10]. Aliquots of this were added to incubation wells, precoated with the NF-B p65 DNA-bind-
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis ing consensus sequence, and the translocated p65 subunits, present in the nuclear lysate, were assayed, according to the recommendations of the manufacturer of the NF-B assay kit (Cayman Chemical).
Western blot analysis The cells and liver tissues were washed with PBS three to five times and lysed with the lysis buffer. Equal amounts of protein were separated in 10–15% SDSpolyacrylamide minigels and transferred to immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After incubation with the appropriate primary antibody, the membrane was hybridized with a secondary antibody conjugated to alkaline phosphatase for 1 h at room temperature. Following washes with TBST, immunoreactive bands were visualized using NBTBCIP chromogenic substrates. In a parallel experiment, nuclear protein was prepared by nuclear extraction reagents (Pierce, Rockford, IL, USA), according to the manufacturer’s protocol [17].
Animals All in vivo experiments were performed using BALB/C adult female mice, weighing 18 –22 g and obtained from the Animal House of the Indian Institute of Chemical Biology (West Bengal, India). The mice were housed in micro-isolator cages at a laboratory temperature of 24 ⫾ 1°C with 40 – 80% relative humidity and received food and water ad libitum; light-dark cycle was maintained. The animals were allowed to adapt to the experimental environment for 5–7 days before experimentation. Care and maintenance of animals were done in adherence to the guidelines of the Institutional Animal Care and Use Committee.
Acute toxicity study These studies were carried out in female BALB/c mice (n⫽20). pLLD was solubilized in 0.2% Tween 80, suspended in PBS, and administered i.p. in 1–200 mg/kg doses. Then, all groups of animals were observed for signs and symptoms of toxicity, and the number of mortality was recorded from 24 h to 15 days following treatment. The LD50 was determined according to the method of Litchfield and Wilcoxon [18].
Measurement of liver function Liver dysfunction was assessed by measuring the rise in serum levels of ALT, AST, and total bilirubin. The serum samples were obtained from the mice, 24 h after challenge with LPS, and analyzed with a commercial kit, according to the manufacturer’s instructions (BioVision, Milpitas, CA, USA).
MPO estimation Neutrophil sequestration in the liver was quantified by measuring tissue MPO activity. After treatment with pLLD and LPS, mice were killed. Liver tissue samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), and centrifuged (13,000 rpm, 10 min, 4°C). The pellet was resuspended in 50 mM phosphate buffer (pH 6.0), containing 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co.). The suspension was subjected to four cycles of freezing and thawing and disrupted further by sonication for 40 s. The sample was then centrifuged (13,000 rpm, 5 min, 4°C), and the supernatant was used for the MPO assay. The reaction mixture consisted of the supernatant, 1.6 mM tetramethyl benzidine (Sigma Chemical Co.), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide. This mixture was incubated at 37°C for 110 s, and the absorbance was measured at 450 nm [19].
Immunohistochemistry For immunohistochemical study, 5 m-thickness tissue sections were deparaffinized with xylene, rehydrated with descending alcohol series, incubated with trypsin (0.05% trypsin, 0.1% CaCl2) for antigen retrieval, and subjected to blocking using 5% BSA in TBS (20 mM Tris HCl, pH 7.4, containing 150 mM NaCl) for 4 h at room temperature. This was followed by incubation overnight at 4°C in primary antibody (iNOS, TNF-␣, HIF-1␣, VEGF) solution (1:300 dilutions in TBS) using a humid chamber. The tissue sections were washed with TBST (20 mM Tris HCl, pH 7.4, containing 150 mM NaCl and 0.025% Triton X-100) and incubated with FITC- and PE-conjugated secondary antibody (Santa Cruz Biotechnology) solution (1:600 dilutions in TBS containing) for 1 h at room temperature. After mounting with DAPI, visualization was done by a confocal laser-scanning microscope (Revolution XD Spinning Disk with an iXon 897 EMCCD camera; Andor).
Survival study In the survival study, BALB/c mice were assigned randomly into groups and treated i.p. with normal saline or with pLLD at doses of 25 and 50 mg/kg, every day for 3 days. LPS (32 mg/kg), dissolved in normal saline, was injected i.p., 2 h after pLLD-i.p. treatment. Survival rate was then recorded every 12 h for 6 days.
Analysis of serum inflammatory factors For the analysis of serum cytokines, mice were assigned randomly into groups (n⫽10). The control group received the vehicle, the second group received only pLLD, the third group received LPS at 32 mg/kg i.p., and animals of the remaining groups received pLLD at the dose of 50 mg/kg i.p., 2 h before LPS administration. The sera were collected from the animals at 1st, 3rd, and 6th h to measure cytokines TNF-␣, IL-1, IL-6, IL12p40, IL-17, IFN-␥, MIP-2, KC, and RANTES, besides vascular factors VEGF and PIGF. The measurements were done at Times 6, 12, and 24 h by sandwich ELISA using commercially available reagents, according to the manufacturer’s instructions.
Histological examination For histopathological observation, the mice were killed, and lungs, liver, and kidneys were removed, 24 h after LPS or normal saline injection and administration of only pLLD. The portions of lung, liver, and kidney tissues were fixed immediately in 10% neutral-buffered formalin solution and then dehydrated. Next, the fixed tissues were embedded in paraffin, sectioned (5 m) using a microtome, mounted on glass slides, and stained with H&E for morphological examination.
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Flow cytometry in isolated hepatocyte from mice Hepatocytes were isolated from each group of mice by two-step collagenase perfusion, following the literature procedure [20, 21], with some modifications. Isolated monolayers chosen for the experiment were permeabilized in 70% alcohol and incubated with fluorochrome-conjugated antibody; the expressions of ICAM-1, VCAM-1, and PECAM-1 in hepatocytes were then analyzed by flow cytometry.
Statistical analysis Results were expressed as mean ⫾ sem. Statistical analyses were performed with ANOVA, followed by Dunnett’s test. P ⬍ 0.05 was considered significant.
RESULTS
TLC analysis of pLLD Figure 1 shows the TLC profile of the pLLD used in our study. Iodine staining showed six to seven spots of lipids in the TLC plate. Lipids from three different batches showing the same TLC profile (Fig. 1A) were used in further studies.
Effect of pLLD on LPS-induced NO production in macrophages The concentration-dependent effect of pLLD on NO release in RAW264.7 cells was determined in the presence and abVolume 96, August 2014
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Figure 1. TLC profile of pLLD (A). Effect of pLLD on production of NO in the LPS-stimulated RAW264.7 cells. Cells were treated with various concentrations (1–140 g/mL) of pLLD for 24 h, with or without LPS (1 g/mL), and the optical density (O.D.) was determined by the ELISA method (B). The cytotoxicity of pLLD on cells for 24 h in the presence or absence of LPS (1 g/mL) was determined by MTT assay and optical density measurement at 595 nm. Morphological changes in RAW264.7 cell, visualized by optical microscopy (200⫻) in the presence and absence of LPS with 120 g/mL pLLD (C). The data are reported as the mean ⫾ sem of triplicate experiments (*P⬍0.05; **P⬍0.01).
sence of LPS. Unstimulated macrophage cells constitutively produced minimal levels of NO, but the levels were higher with LPS-stimulated cells. Cells were treated with various concentrations of pLLD (1–140 g/mL) at 24 h in the presence of LPS. A significant reduction (up to 77%) was found upon treatment of the lipid at concentrations of 60 and 120 g/mL in LPS-treated macrophage cells at 24 h (Fig. 1B). Thereafter, we selected two concentrations of pLLD— 60 and 120 g/mL—for the in vitro experiments. Evaluation of the cytotoxic effects in peritoneal macrophage cells using the MTT assay revealed that pLLD did not affect cell viabilities at the concentrations used (1–140 g/mL; Fig. 1C). Figure 1D shows the control cells were round in shape whereas LPS-stimulated RAW 264.7 cells had changed to an irregular form with accelerated spreading and pseudopodia formation. Co-treatment of LPS with pLLD at 120 g/mL concentration changes the level of cell differentiation by reducing pseudopodia formation.
Suppression of LPS induced different inflammatory mediators by pLLD in macrophage cells pLLD was found to reduce the inflammatory factors up-regulated by LPS in murine macrophage cells. As measured by sandwich ELISA and shown in Fig. 2A–E, it also significantly lowered the levels of proinflammatory cytokines, including TNF-␣, IL-1, IL-6, IL-12p40, and PGE2 in the cell supernatant, cotreated with LPS and pLLD at concentrations of 60 g/mL and 120 g/mL, after incubation for 6, 12, and 24 h. Western blot results also revealed the impeding effect of 4 Journal of Leukocyte Biology
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pLLD, wherein the expression levels of inflammatory mediators, including TNF-␣, IL-1, IL-6, iNOS, and COX-2, were suppressed at 12 h upon pretreatment with pLLD at 60 and 120 g/mL (Fig. 2F).
Effect of pLLD on ROS production and phagocytosis in LPS-stimulated macrophages The effect of pLLD on intracellular ROS production in the LPS-stimulated RAW264.7 cells was measured using the fluorescent probe dichlorofluorescein diacetate. pLLD significantly inhibited the induction of intracellular ROS generation in these cells by LPS (Fig. 3A). Measurement of phagocytic activity, microscopically (Fig. 3B) using 2 m fluorescent red-bead internalization, revealed that pLLD simultaneously augmented the phagocytic activity from 90% to 40% in stimulated macrophage cells.
Effects of pLLD on LPS induced production of proinflammatory cytokines (TNF-␣, IL-1, and IL-6) and transcriptional factor NF-B in RAW264.7 cells LPS-induced inflammatory stress leads to activation of the transcriptional factor NF-B and release of the subsequent proinflammatory mediators, which are regulated by the cytoplasmic inhibitor IB. Thus, we examined whether pLLD inhibited the levels of NF-Bp65 expression in a concentration- and timedependent manner. As shown in Fig. 3D, exposure to LPS for 1 h led to increased expression of the NF-B p65 subunit in nuclear, whereas the expression of IB␣ was inhibited in LPSinduced macrophage cells. Conversely, pLLD, at 60 and 120
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis
Figure 2. Effect of pLLD on production of cytokines and mediators from stimulated RAW264.7 cells. After incubation with pLLD (60 and 120 g/mL), the production of inflammatory mediators (A–E) was measured for 6, 12, and 24 h by ELISA in the presence of LPS (1 g/mL). Cytokine and mediator expression levels were determined by Western blot analysis after 12 h incubation of RAW264.7 with LPS alone or with pLLD at 60 and 120 g/mL. The values are mean ⫾ sem of three independent experiments.
g/mL concentrations, significantly inhibited the expressions of the NF-B p65 subunit, whereas the expression of IB␣ was increased (Fig. 3D). Interestingly, pLLD also significantly inhibited the level of nuclear extraction of NF-Bp65 in a concentration- and time-dependent manner, as shown in Fig. 3C. Immunocytochemistry studies showed that the translocation of the p65 subunit of NF-B is inhibited in LPS stimulated macrophages with pLLD (Fig. 3E).
Acute toxicity study Administered i.p., pLLD was nontoxic until 500 mg/kg in mice. The experimental mice were closely observed for the first 24 h and monitored for the next 15 days. However, no toxic symptom
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or abnormal behavior was observed. Thus, 1/10th and 1/20th of this dose, i.e., 50 mg/kg i.p. and 25 mg/kg i.p., were taken as the experimental doses as per the inhibitory efficiency of inflammatory response (Supplemental Fig. 2).
Improvement in survival rate of BALB/c mice challenged with a lethal dose of LPS and the effect of pLLD on cytokine production To examine the protective effect of pLLD against LPS-induced lethality, mice were pretreated with 25 mg/kg and 50 mg/kg pLLD, once a day for 3 days, before i.p. injection of 20 mg/kg LPS, and animal survival was recorded for 6 days after LPS challenge. As shown in Fig. 4, the survival rates at 24 and 48 h Volume 96, August 2014
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Figure 3. Effect of pLLD on ROS production in RAW264.7 cells induced by LPS for 12 h at the concentrations of 60 and 120 g/mL pLLD, analyzed by flow cytometry (A). FL1-H, Fluorescence 1-height. pLLD inhibited the LPS-induced phagocytosis, as assessed by 2 m fluorescent red-bead uptake by RAW264.7 macrophages for 12 h (B). Effect of pLLD on levels of nuclear extract of NF-B p65 in RAW264.7 cells after 0.5, 1, and 3 h incubation at concentrations of 60 and 120 g/mL in the presence and absence of LPS (C). Nuclear NF-B p65 and IB-␣ protein expression levels after treatment with pLLD at concentrations of 60 and 120 g/ml were determined at 4 h after incubation in the presence of LPS (D). Effect of pLLD for 4 h on LPS-induced NF-B translocation by confocal laser-scanning microscopy (magnification 600⫻); nucleus is identified with arrows. Original scale bars, 10 m (E). Data obtained from three independent experiments. DIC, Differential interference contrast; MFI, mean fluorescence intensity (*P⬍0.05; **P⬍0.01).
after LPS challenge were 41% and 9%, respectively, in mice pretreated with Vehicle. However, in mice pretreated with 50 and 25 mg/kg pLLD, survival rates at the corresponding timepoints after LPS treatment significantly increased to 78.8% and 53.8%, respectively. The early responses generated by cytokines and chemokines, such as TNF-␣, IL-1, IL-6, IL-12p40, IL-17, IFN-␥, MIP-2, KC, and RANTES, play a critical role in moderating the physiological responses in the progression of inflammation. To evaluate the effect of pLLD on the production of cytokines in mice linked with a fatal outcome, we determined the serum concentration of proinflammatory cytokines at different time intervals. After challenging with LPS, serum concentrations of cytokines in pLLD-treated mice were measured by the ELISA method at 0, 1, 3, and 6 h (Fig. 5C–E). This showed that 50 mg/kg pLLD significantly reduced the higher levels of cytokine production in serum of LPS-challenged mice. In accordance with our findings of the serum profile in Fig. 4C–K, the histological data also demonstrated improvement of organ in6 Journal of Leukocyte Biology
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jury in mice challenged with LPS but pretreated with pLLD at 50 mg/kg (Fig. 5A). As compared with tissue sections from control group of normal mice exposed to LPS, mice pretreated with pLLD exhibited marked inflammatory alterations characterized by infiltration of the tissue and alveoli with inflammatory cells and the presence of pulmonary edema (Fig. 5A, LPS, Lung panel), considerable hemorrhaging in the renal medulla (Fig. 5A, LPS, Kidney panel) and PMN infiltration and portal injury in the liver (Fig. 5A, LPS, Liver panel) were observed. However, in LPS-injected mice treated with pLLD at the dose of 50 mg/kg prior to LPS challenge, the attenuation was found in the histopathological changes of lung, kidney and liver.
pLLD improves LPS-induced hepatic impairment When pLLD was administered i.p. at the dose of 50 mg/kg before LPS challenge, improvement in hepatic damage was observed (Fig. 5A), as brought out by histopathological scores in septic mice liver (Fig. 5B). LPS caused a large increase in the plasma levels of ALT, AST, bilirubin, and MPO at the late
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis
Figure 4. Effect of pLLD on (A) survival and (B) body weight of mice (nⴝ10), treated with LPS (32 mg/kg). Levels of cytokines TNF-␣, IL1, IL-6, IL-12p40, IL-17, IFN-␥, ⌴⌱P-2, KC, and RANTES (C–K) were measured by ELISA at 1, 3, and 6 h upon treatment with pLLD at doses of 50 mg/kg, after challenging with LPS. The data are reported as the mean ⫾ sem of triplicate experiments (*P⬍0.05; **P⬍0.01; ***P⬍0.001).
stage (24 h). Pretreatment with pLLD at 50 mg/kg significantly reduced the rise in plasma level of ALT, AST, and total bilirubin generated by LPS. Simultaneously, hepatic MPO was found to be significantly lowered at 24 h in treated mice with reduction of liver PMN compared with mice challenged with LPS only, as shown in Figs. 5G and 5C. To evaluate the localization of proinflammatory mediators in tissue-level expression on LPS-induced liver damage, iNOS and IL-6 localizations were checked in hepatic tissue by immunofluorescence analysis (Fig. 5H and I). Overexpression of IL-6 was observed predominantly in the epithelial region in LPS-treated tissue, whereas iNOS localization was observed in
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the LPS-treated tissue section in the epithelial and submucosal layers (Fig. 4). Reduced expressions of IL-6 and iNOS were observed in the epithelial layer, pretreated with pLLD at 50 mg/kg in LPS-induced septic liver tissue. Besides these, we have also studied the effect of pLLD at the dose of 50 mg/kg in sepsis-induced murine hepatic tissue by Western blot analysis to validate our findings (Fig. 5J).
Effects of pLLD on vascular permeability factor(s) and cell adhesion molecule(s) Hypoxia is well-known to induce the expression of different growth factors, such as VEGF and PIGF, and of cell adhesion Volume 96, August 2014
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Figure 5. Effect of pLLD on LPS-induced organ injury. (A) Mice were pretreated with pLLD at a dose of 50 mg/kg i.p., 1 h after LPS (32 mg/kg). After 24 h, liver was removed, and the histopathological changes in kidney, lungs, and liver of mice were pretreated with LPS, assessed by H&E stain (200⫻). Histopathological studies by light microscope, morphologically showing the tissue sections (kidney, lung, and liver) from mice in the control, LPS, and LPS ⫹ pLLD groups (arrows), indicate the infiltration of inflammatory cells in captured fields (scale bars, 20 m). (B) Scores of liver injury. Livers were considered injured if any of these lesions were detected. Injury was scored as 0 (none), 1⫹ (rare focal apoptosis, inflammation, or hemorrhage), 2⫹ (focal apoptosis, inflammation, and/or hemorrhage in 25–50% of the fields), 3⫹ (focal apoptosis, inflammation, and/or hemorrhage in ⬎50% of the fields). (C) PMN. Serum levels of ALT (D), AST (E), total bilirubin (F), and liver MPO (G) were also assessed to determine the localization of iNOS (H) and IL-6 (I) in liver tissues. Magnification, 100⫻; arrows indicate immunopositive cells. Approximately 200 cells were counted/field; five fields were examined/slide; five slides were examined/group; and expression of inflammatory mediator and cytokines was analyzed by Western blot (J). The data were obtained from three independent experiments. Values are presented as mean ⫾ sem (*P⬍0.05; **P⬍0.01; ***P⬍0.001).
molecules, including ICAM-1, VCAM-1, PECAM, P-selectin, and E-selectin, in inflammatory sepsis-associated hepatic injury. Flow cytometry revealed the inhibitory effect of pLLD in the expressions of cell adhesion molecules in primary hepatocytes of LPSchallenged mice (Fig. 6F–H). Significant reduction in VEGF and HIF-1␣ levels of liver parenchymal tissue compared with that in only the LPS-challenged group (Fig. 6C) was evident. Furthermore, Western blot data revealed that pretreatment with pLLD at 8 Journal of Leukocyte Biology
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50 mg/kg reduced the protein level expressions of VEGF, P-selectin, and E-selectin at 24 h, as seen in Fig. 6I.
DISCUSSION Microorganisms and their cellular component(s) may possess some degree of bioactivity, either against other microorgan-
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Chatterjee et al. Protective role of pathogenic leishmanial lipid in sepsis
Figure 6. Effect on circulatory levels of VEGF (serum) (A) and PIGF (serum; B) and on liver tissue localization of VEGF and HIF-1␣ (C) in LPSinduced mice, administered 50 mg/kg pLLD, 1 h after LPS administration, as determined by confocal microscopy (100ⴛ). Expression of VEGF (D) and PIGF (E) by ELISA from liver tissue lysate and hepatocyte expression of ICAM-1 (F), VCAM-1 (G), and PECAM-1 (H), detected by flow cytometry, with expression of adhesion molecules, such as VEGF, in liver tissue analyzed by Western blot (I). FITC-A, FITC-area. The data are obtained from three independent experiments. Values are presented as mean ⫾ sem (*P⬍0.05; **P⬍0.01).
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ism(s) or against certain physiological states of a diseased body [22, 23]. Azurin, a protein isolated by the pathogenic bacteria Pseudomonas aeruginosa, induces apoptosis by interfering with signaling molecules and regulating the growth of cancer cells [24]. Sophorolipid, produced from the new yeast strain Wickerhamiella domercqiae, shows its potency against human pancreatic cancer cells and septicemia [25–27]. Interestingly, in spite of the potent leishmanicidal activity, L. amazonesis and its components inhibited the expression of the cytokine-inducible nitric oxide synthase (iNOS) in stimulated macrophage cells [9, 28]. Our previous study revealed that the lipid from an attenuated strain of L. donovani promastigote (MHO/IN/1978/UR6), developed by long-term in vitro culture, suppresses several inflammatory mediators by inducing apoptosis in adherent SFMCs isolated from rheumatoid arthritis patients [10]. These findings encouraged us to evaluate the anti-inflammatory role of pLLD against bacterial endotoxin-induced, sepsis-associated inflammatory progression toward acute hepatic damage. We could thus demonstrate the anti-inflammatory activities of the lipid isolated from L. donovani (MHOM/IN/1983/AG83), both in vitro using macrophages stimulated by endotoxin (LPS) from Gram-negative bacteria and in vivo using mouse models of septic inflammation. Macrophages play a critical role in cellular host defense against infection and tissue injury [29]. In response to stimuli, macrophages undergo a series of inflammatory processes, including chemotaxis, phagocytosis, intracellular killing, and release of inflammatory cytokines [30, 31]. In acute and chronic inflammation, NO plays a significant role to cause vasodilation and tissue damage. In addition, it functions in immune systems as a cytotoxic macrophage effector molecule, modulator of PMN leukocyte chemotaxis and adhesion, mediator of tissue injury caused by adhesion of immune complexes, and regulator of lymphocyte proliferation [32]. Therefore, NO production can be used as a measurement of the progression of inflammation, and its inhibition might have potential therapeutic value in the context of inflammation-associated diseases. Here, we report for the first time that the pathogenic leishmanial lipid inhibits NO production from stimulated macrophages in a dose-dependent manner (Fig. 1B). Although the formation of ROS is desirable for a host’s defense, their overproduction can jeopardize the body’s own cells, cause tissue injury, and contribute to the development of a number of serious diseases [33, 34]. As shown in Fig. 3A, pLLD significantly reduced the ROS production in stimulated macrophage cells. To elucidate the molecular approaches by which pLLD exerts anti-inflammatory activity on Gram-negative bacterial sepsis, we have evaluated the survival rate of mice in an endotoxin-induced murine sepsis model. Interestingly, pLLD-induced inhibition of the production of serum cytokines, including TNF-␣, IL-1, IL-6, IL-12, IL-17, IFN-␥, and KC, and of chemokine MIP-2 was consistent with our in vitro results (Fig. 4) on the survival status in multiple organ failure of LPS-challenged mice. IL-6 plays the pivotal role in host-defense responses to infection and inflammation by activating macrophages and inducing acute-phase proteins in liver [35]. Importantly, the autocrine/paracrine inflammatory factor, PGE2-dependent NF-B activation, is required for IL-6 production in stimulated 10 Journal of Leukocyte Biology
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macrophages [36], and pLLD inhibited the overproduction of IL-6. Bacterial endotoxins are well-known to trigger the signaling pathway of TLR4-CD14, leading to the activation of the IB kinase complex and phosphorylation of the inhibitory IB protein. This protein is necessary for ubiquitination, and its degradation leads to the release and subsequent translocation of NF-B p65 into the nucleus [37] through degradation of I-B␣ on serine subunits. This study demonstrated that pLLD inhibits the activation of NF-B that eventually induces the various inflammatory mediators (Fig 3) and other signaling molecules in inflammatory cascade (Fig. 3 and Supplemental Fig. 1). Culture of macrophages with LPS triggers the signaling cascades and leads to rapid translocation of NFBp65 subunit to nucleus, followed by the increased expression of NF-B dependent cytokines. Similarly, during sepsis or numerous disease states, the exposure to LPS may initiate a systemic inflammatory response via the activation of NF-B leading to significant morbidity and mortality [38, 39]. Furthermore, in agreement with the in vitro study, inflammatory factors were regulated by pLLD in sepsis model. In a septic environment, enhancement of PMN activation could promote cytokine production and induce tissue injury, leading to an increase in PMN accumulation in the hepatic sinusoids and development of hepatic injury [40]. Both increased PMN level and rate of mortality declined significantly with pretreatment of pLLD, as shown in Figs. 4 and 5. These cytokines induce accumulation and activation of another transcription factor, HIF-1, whereas LPS-induced HIF-1␣ accumulation is predominantly dependent on NF-B activation. In inflamed tissue, VEGF and HIF-1␣ are activated as a result of vascular damage and edema, as well as the intense metabolic activity of bacteria and numerous infiltrating cells that are generated primarily during hypoxia [41]. Specifically, TNF-␣ and IL-1 contribute to the increase in the number of infiltrating neutrophils, which play a critical role in bacterial clearance [42]. We observed that pLLD profoundly reduced the elevated serum cytokine level with concurrent reduction of VEGF and HIF-1␣ expression in hepatic tissue. Primary hepatic dysfunction refers to sepsis-induced dysfunction in the period immediately after an episode of shock and resuscitation [43, 44]. It was found that hepatic injury was reduced by the alteration of liver function parameters (AST/ALT) after administration of pLLD in septic mice. In addition to its role in promoting endothelial permeability and proliferation, VEGF may contribute to inflammation and coagulation. These proinflammatory effects of VEGF are mediated, at least in part, by the activation of NF-B transcription [45]. Furthermore, during the inflammatory changes, the process of angiogenic vascular enlargement also occurs by regulating PIGF status in vascular remodeling endothelial cell proliferation [46]. Few cytokines, such as IL-1, IL-6, and COX-2, regulate the expression level of VEGF in different cell types. Under inflamed conditions, VEGF induces the expression of cell adhesion molecules (E-selectin and P-selectin), along with ICAM-1 and VCAM-1 in endothelial cells, and promotes the adhesion of leukocytes [47]. Consequently, PECAM regulates the leukocyte migration process in the presence of cytokines, such as TNF-␣, IFN-␥, and
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different cell adhesion molecules [48]. The expression of these adhesion molecules, regulated by the cytokine-inducible NF-B, is altered by pLLD, as shown in Fig. 6. In conclusion, this study has revealed that pathogenic leishmanial lipid exerts anti-inflammatory responses by regulating the inflammatory factors in vitro and in vivo. In addition, it affords protection against sepsis-mediated organ injury, including hepatic damage by regulating different mediators, cytokines, chemokines, vascular permeability factors, and cellular adhesion molecules. Taken together, these findings suggest that the pLLD may be used as a novel therapeutic agent of microbial origin against inflammatory responses.
9.
10.
11.
12. 13.
AUTHORSHIP N.C. and S.D. carried out the studies and wrote the manuscript. D.B. and S.B. participated in ELISA and flow cytometry. N.C. and T.J. contributed to data and statistical analysis. K.D.S. initiated and supervised the experimental work and drafted the manuscript. All authors approved the final version of the manuscript.
ACKNOWLEDGMENTS The CSIR and Indian Council of Medical Research (ICMR; India) financially supported this work. The authors convey their sincerest thanks to Prof. Siddhartha Roy (director of CSIR-Indian Institute of Chemical Biology) for providing us the necessary support for this work. We extend our thanks to Dr. Suvendra Nath Bhattacharyya (Indian Institute of Chemical Biology) and Mr. Diptadeep Sarkar for their help in confocal laser-scanning microscopy. We are also indebted to Dr. J. Rajan Vedasiromoni and Dr. Basudeb Achari of our institute for critically reviewing the manuscript.
14. 15. 16.
17.
18. 19.
20. 21. 22.
DISCLOSURES
The authors declare that they have no competing interests.
23. 24.
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KEY WORDS: Leishmania 䡠 macrophage 䡠 lipopolysaccharide 䡠 inflammation 䡠 septic damage
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