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Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPS-induced shock
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPS-induced shock Valeriya Gyurkovska, Nina Ivanovska n Department of Immunology, Institute of Microbiology, BAS, Sofia, Bulgaria
art ic l e i nf o
a b s t r a c t
Article history: Received 1 October 2014 Received in revised form 12 January 2015 Accepted 15 January 2015
Sepsis remains a serious clinical problem despite continuous efforts to increase survival. Experimental animal models of sepsis are pointed to a great extent on blocking the activity of cytokines. A number of signal-transducing molecules are associated with the occurrence of excessive tissue inflammation. Through inhibition of tyrosine phosphorilation and thereby changing cell signaling, tyrosine kinase inhibitors can influence multiple inflammatory pathways. The purpose of the present investigation was to evaluate the effect of tyrosine kinase inhibitor tyrphostin AG490 in a mouse LPS-induced shock. Cytokine and chemokine blood levels were determined by ELISA assays. CD11b þ Ly6C þ , CD3 þ CD69 þ and C5aR positive cell populations in the peritoneal exudate were detected by flow cytometry. The expression of iNOS and Signal Transducer and Activator of Transcription (STAT) in the liver were observed by immunohistochemistry. We found that tyrphostin AG490 inhibited Regulated upon activation normal T cell expressed and secreted (RANTES), IL-6 and IL-12 serum levels, decreased the number of CD11b þ Ly6C þ and CD3 þ CD69 þ subpopulations in the peritoneal exudate and prevented thells expressing C5a receptor and TNF-alpha receptor. Tyrphostin ameliorated liver injury associated with suppressed iNOS, STAT3 and pSTAT3 expression. Our data suggest that tyrphostin AG490 diminished the degree of inflammation starting in peritoneal cavity and minimized liver dysfunction thus representing one approach for better outcome of sepsis conditions. & 2015 Published by Elsevier B.V.
Chemical compounds studied in this article: Tyrphostin AG490 (PubChem CID: 5328771) Keywords: RANTES IL-6 IL-12 iNOS STAT3 pSTAT3
1. Introduction Sepsis is a condition that results from a harmful host response to infection. Many of the components of the innate immune response that normally ensure host defences against infection can cause cell and tissue damage and lead to multiple organ failure, the clinical hallmark of sepsis. Because of the high mortality of sepsis, many efforts have been made to reveal how the host protection is deregulated. As a result, new data have been accumulated about basic principles underlying bacterial-host interactions, and new opportunities for therapeutic intervention have been elaborated (Natanson et al., 1994). Different models of sepsis are in use, one of them being LPS-induced shock. LPS elicits a broad, nonspecific cascade of events in vivo, resulting in secretion of a variety of potent cytokines through an intracellular signal amplification pathway such as IL-1, IL-6, TNF-α and IL-12. They contribute to the pathophysiology of multisystem organ failure and often lead to commonly fatal exit (Dinarello, 1991; Higgins et al., 2003). LPS is capable to activate myeloid and non-myeloid cells, as well as other innate host defense mechanisms, such as serum complement, and specific components
n
Corresponding author. Tel.: þ 359 2 979 3195. E-mail address: [email protected] (N. Ivanovska).
within the coagulation pathway (Markiewski et al., 2008). The mechanism of action of LPS involves interaction with Toll-like receptor (TLR)4 and CD14 that results in the activation of Nuclear Factor (NF)-κB pathway, the production of endogenous mediators by monocytes, macrophages and polymorphonuclear neutrophils, and the induction of antigen-specific cellular immunity (Jeyaseelan et al., 2005; Palsson-McDermott and O’Neill, 2004). Cytokine signal transduction is believed to be mediated primarily through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway which leads to the expression of certain target genes. Continuous activation of the JAK/STAT pathway has been implicated in numerous cancers and immune disorders. Recently, over 20 drugs that target kinases have been introduced in clinical practice and many are currently in preclinical studies (Cohen, 2009; Cohen and Alessi, 2013). Tyrphostin AG490 (α-cyano-(3,4-dihydroxy)-N-benzylcinnamide) is a synthetic protein tyrosine kinase inhibitor which at first has been considered as a specific JAK2 inhibitor but later its JAK3-dependent action has been established (Kirken et al., 2001). A number of tyrphostins have been investigated in LPS-induced sepsis in rats and their protective role on organ injury has been observed (Ruetten and Thiemermann, 1997). Recent studies claim that immune response suppression by AG490 prevents the lethal outcome in a mouse model of polymicrobial sepsis (Hui et al., 2009; Pena et al., 2010). However, the protective
http://dx.doi.org/10.1016/j.ejphar.2015.01.045 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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role of AG490 in the course of LPS-induced inflammation currently remains elusive. Previously, we have studied the effect of tyrphostin AG490 in zymosan-induced nonseptic shock in mice. A single dose of 5 mg/kg reduced the kidney and liver dysfunction associated with inhibition of iNOS, STAT1, STAT3 and C5a receptor expression in organs as well as with changes of various cytokines and chemokines (Dimitrova et al., 2009; Dimitrova and Ivanovska, 2008). Except, TLR-2 dependent mode of AG490 action we have demonstrated that it acts through TLR-4 and TLR-9 pathways (Dimitrova et al., 2008). The aim of the present study was to investigate the action of AG490 on the early events of LPS-induced inflammation in peritoneum and on the liver dysfunction.
2. Materials and methods 2.1. Mice Male ICR mice (CD-2) background (8–10 week old, weight 20–22 g) were bred in the Animal Facility of the Institute of Microbiology, maintained on a 12:12 h light:dark cycle and fed standard diet and tap water ad libitum. All experiments were conducted in accordance with the Bulgarian Food Safety Agency Guidelines no. 352 06.01.2012 and approved by the Animal Care Committee at the Institute of Microbiology, Sofia (Decree no. 14/19.07.2000). 2.2. LPS-induced shock Lipopolysaccharide (serotype 055:B5 from Escherichia coli; Sigma Aldrich, St. Louise, MO) was dissolved in endotoxin-free water and stored at 20 1C. Tyrphostin AG490 (2-Cyano-3-dihyroxyphenyl)–N(benzyl)-2-propenamide (Sigma-Aldrich, St. Louis, USA) was dissolved in DMSO and further diluted in PBS (final DMSO concentration was o0.05%). Mice were randomly divided into four experimental groups: a group injected with PBS (control), mice injected with 5 mg/kg AG490, mice injected i.p. with 25 mg/kg LPS (LPS) and a group injected with 25 mg/kg LPS and immediately after that injected i.p. with 5 mg/kg AG490 (LPSþAG490). Such dose resulted in 70–80% mortality and the animals expressed all signs of acute inflammation within 48 h, such as ruffled fur, diarrhea and lethargy (Supplemental Fig. 1). The dose for AG490 was chosen based on our experiments showing that lower dose of 1 mg/kg did not reduce significantly the rate of mortality and on our previous study in zymosan-induced shock with different doses of AG490 (Dimitrova et al., 2009) pointing that a higher dose of 10 mg/kg resulted in insignificant improvement of mortality. 2.3. Measurement of serum cytokine and chemokine levels Mice were killed by cervical dislocation. Blood was collected and sera were obtained after centrifugation at 350 g for 15 min at 4 1C and stored in aliquots at 80 1C until required. The levels of IL-6, IL12, MIP-1α and RANTES were measured by quantitative ELISA kits (Biolegend, USA) according to manufacture's instructions.
Isolated mouse peritoneal cells were stained with antibodies from BD Pharmingen (Erembodegem, Belgium) against mouse Ly-6C (FITCconjugated; clone AL-21), CD11b (PE-conjugated; clone M1/70), CD69 (APC-conjugated, clone H1.2F3), and CD3 (FITC conjugated, clone 145-2C11). For detection of cell surface expression of C5aR and TNF-α receptor, the cells were processed in PBS/2% FCS and incubated for 20 min at 4 1C with biotinylated antibodies against mouse C5aR or TNF-α receptor1 (2 μg/ml; Santa-Cruz Biotechnology Inc., Heidelberg, Germany) or IgG isotype controls (Biolegend). After washing with 2% FCS/ PBS, secondary avidin-FITC (4 μl/sample, Becton Dickinson,San Jose, CA, USA) was added for 15 min at 4 1C. After three washings with PBS, the samples were analyzed by a flow cytometer (BD™ LSR II) and FCS Express™ Diva Software (Becton Dickinson, San Jose, CA, USA). 2.5. Histopathologic changes in liver and evaluation of liver dysfunction Livers were fixed in 4% p-formalaldehyde (pH 7.4). The organs were embedded in paraffin and sections with thickness 5 μm were cut. The slides were then stained with hematoxylin and eosin (H&E) and examined by a light microscope (Boeco, Hamburg, Germany) connected to the device camera. Sera were collected as described above and analyzed immediately by using standard laboratory kits (Dialab GmbH, Wiener Heudorf, Austria) measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST). 2.6. Immunohistochemistry Tissue paraffin sections (5 μm) were incubated for 10 min with HRP blocking solution (3% H2O2/60% methanol for 10 min), washed with PBS and blocked for 10 min with 5% BSA/PBS followed by washing and 40 min incubation with antibodies against iNOS (1:2000 diluted), STAT3 (1:100 diluted, Santa-Cruz Biotechnology, Heidelberg, Germany) or pSTAT3 (1:100 diluted, Abcam). Isotype antibodies (Sigma-Aldrich, Munich, Germany) were used as a background staining controls. After washing with 0.01% Tween/PBS, the sections were incubated for 20 min with HRP-labeled anti-rabbit IgG antibody (1:2000, Sigma-Aldrich), washed in PBS and stained with DAB (3,3'-diaminobenzidine-tetrahydrochloride) substrate solution (Sigma-Aldrich) for 10 min and then counterstained for 2 min with Gill's hematoxylin. 2.7. Statistical analyses Statistical analyses were performed using InStat3.0 and GraphicPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Data are expressed as mean 7 S.D. or mean 7 S.E.M. The differences in mean values between groups were analyzed by two-way ANOVA. Differences were considered significant when Po0.05.
3. Results 2.4. Flow cytometry Resident peritoneal macrophages were harvested by rinsing the peritoneal cavity with 10 ml of RPMI-1640 medium. The cells were washed twice, re-suspended at a density of 2 106 cells/ml in complete RPMI-1640 medium supplemented with 5% fetal calf serum (FCS) for 1 h (37 1C, 5% CO2). The total cell population contained approximately 95% viable cells (trypan blue exclusion test). Then, the non-adherent cells were removed by two washings, the adherent population was collected and more than 90% of these cells were positive for F4/80 (marker for mature mouse macrophages).
3.1. Influence of tyrphostin AG490 on the levels of chemokines and cytokines in sera from mice with LPS-induced shock We determined the effect of AG490 on the serum concentration of some critical mediators of developing inflammation at 1, 4 and 24 h after LPS injection. The level of MIP-1α was not influenced by tyrphostin at 1 and 4 h and was increased at 24 h in a result of AG490 treatment (Fig. 1B). Tyrphostin injection of healthy mice also provoked elevation of MIP-1α concentration. A decrease of RANTES level was established in AG490-treated mice at all time points
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 1. Cytokine levels in sera of mice with LPS-induced shock. Mice (n¼10 per group) were injected with PBS (control), with 5 mg/kg AG490 (AG490), with 25 mg/kg LPS (LPS) or with 25 mg/kg LPS plus 5 mg/kg AG490 (LPS þAG490). The concentrations of RANTES (A), MIP-1α (B), IL-6 (C) and IL-12 (D) were determined by ELISA at 1, 4 and 24 h after treatment. *P o 0.05; **Po 0.01; *** Po 0.001 vs control, Po 0.05; Po 0.01 LPS vs LPS þ AG490, two-way ANOVA with Bonferroni post-test.
(Fig. 1B). Same tendency was observed for IL-6 (Fig. 1C) and for IL-12 (Fig. 1D). 3.2. Effect of tyrphostin AG490 on the number of CD11b þ Ly6C þ and CD3 þ CD69 þ peritoneal exudate cells
that such lack of C5a receptor expression on PE cells was considerably prevented by AG490 (Fig. 4A, and B). Tyrphostin administration resulted in lower TNF-α receptor expression on 4 and 24 h which suggests excessive TNF-α production (Fig. 4C, and D). 3.4. Effects of AG490 on liver injury in LPS-induced shock
The increase of peritoneal cells during inflammation is a consequence from the influx of blood monocytes into the peritoneum. We exploited CD11b and Ly6C markers to discriminate these cells in the peritoneal exudate. We found that the number of double positive CD11b þ Ly6C þ cells was increased dramatically at 24 h after LPS injection which was prevented by tyrphostinAG490 (Fig. 2A, and B). The initiation of inflammation was associated with a rapid recruitment of T cells into the peritoneal cavity which started at 1 h and was maintained during 24 h (Fig. 3). CD69 is a useful marker for quantifying T-cell and T-cell subset activation in mixed populations. The percentage of CD3 þ CD69 þ cells was significantly elevated in animals exposed to LPS. Tyrphostin AG490 significantly suppressed this elevation at 1 h after LPS injection but later on the effect disappeared (Fig. 3). 3.3. Effect of tyrphostin AG490 on C5a receptor and TNF-α receptor expression by peritoneal cells The complement activation product C5a is one of the most potent inflammatory chemoattractants and has been involved in the pathogenesis of multiple inflammatory diseases. This suggests an increase in C5a and C5a receptor production which on the other hand supposes less available free C5a receptor molecules on the cell surface. Our data were in accordance with this opinion as we established a decreased number of C5a receptor positive cells in LPS treated mice on 4 and 24 h. In AG490 treated group we observed
To find the role of AG490 in LPS shock, livers were harvested at 1, 4, 24 h and at day 21 after LPS injection. Normal liver appearance was shown in control mice (Fig. 5A). At 1 h massive inflammatory cells infiltration, causing hepatocellular necrosis and presence of acidophilic bodies were found after H&E staining (Fig. 5C). In LPS injected group we observed dilated bile ducts filled with bile at 4 h (Fig. 5E) and hemorrhages at 4 and 24 h (Fig. 5E, and G). Hepatic necrosis was seen and hepatic lesions coalesce to develop larger lesions (Fig. 5E). At day 21 after LPS administration histomorphological appearance of liver showed ballooning degeneration of hepatocytes with disintegrated pyknotic nuclei (Fig. 5I). Minor hepatocyte and parenchimal tissue changes were observed in AG490-treated animals such as less cell infiltration (Fig. 5D), lack of necrosis and large zones of injury were absent (Fig. 5F) as well as lack of hemorrhages (Fig. 5H, and J). This correlated with a little elevation of serum ALT and AST levels in AG490-treated mice while in LPSinjected group they were quite increased at 24 h after LPS injection (Fig. 6). There were no changes in mice injected with tyrphostin only. 3.5. Effect of AG490-treatment on iNOS, STAT3 and pSTAT3 expression in liver during LPS-induced shock Our results indicated that livers from mice subjected to LPS showed a strong iNOS induction at 1 and 4 h (Fig. 7C, and E). In contrast, iNOS expression in livers from mice that had been treated
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 2. Effect of AG490 on the number of CD11b þ Ly6C þ cells in peritoneal exudates (PE) LPS-induced shock. Mice (n ¼10 per group) were injected as described in Fig. 1. Representative dot-plot graphs (A). Tyrphostin significantly lowered the percentage of CD11b þ Ly6C þ cells at 24 h (B). Values are means7 S.E.M. *Po 0.05, two-way ANOVA.
with AG490 was apparently minimized at 4 h (Fig. 7F). Twenty four hour upon endotoxin administration iNOS expression was undetectable in both, treated and untreated with AG490 mice (Fig. 7G, and H). iNOS expression was shown at day 21 in mice with shock (Fig. 7I), while in mice treated with AG490 no staining was observed (Fig. 7J). No histopathological changes were observed in the livers of controls (Fig. 7A) and in AG490-treated mice (a representative picture Fig. 7B). We further investigated the STAT3 and pSTAT3 expression in hepatocytes during inflammation. Immunohistochemical analyses revealed that STAT3 protein was expressed in the liver of LPS-injected mice at each time point examined (Fig. 8C, E and G). Lower expression was observed at 1 h (Fig. 8D) and at 4 and 24 h this protein was not detected in mice treated with AG490 (Fig. 8F and H). Intensive pSTAT3 staining was established at 1 h in LPS-injected mice, not presente in AG490-treated mice with shock (Fig. 8K and L, respectively). At 4 h pSTAT3 presented in both
AG490-treated and untreated groups with shock (Fig. 8M, and N) and no expression was found at 24 h in these groups (Fig. 8O, and P). No evident staining was detected in livers of animals treated with AG490 only in regard to STAT3 and pSTAT3 (Fig. 8B, and J).
4. Discussion The incidence of sepsis is increasing despite the major advances in the development of antimicrobial agents and other supportive treatments. Septic patients often develop systemic inflammatory response, of which multiple organ failure is a main complication. Chronic inflammation arises when the acute response is not completely turned off, continuing to stimulate pro-inflammatory mediators when they may not be needed. Cytokines regulate many of the pathways involved in the host inflammatory response to sepsis. The Jak/STAT pathway is associated with synthesis of various cytokines.
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 3. Effect of AG490 on the number of CD3 þ CD69 þ cells in PE in LPS-induced shock. Mice (n¼10 per group) were injected as shown in Fig. 1. Tyrphostin inhibited the increase of the number of CD3 þ CD69 þ cells at 4 h. Values are means7 S.E.M. *Po 0.05; two-way ANOVA.
Fig. 4. Effect of tyrphostin AG490 on the expression of C5a receptor and TNF-α receptor by PE cells. Mice (n¼ 10 per group) were injected as shown in Fig. 1. AG490 prevented the decrease of the number of C5a receptor positive cells (A) and graph (B), and of TNF-α receptor (C) and graph (D). *P o 0.05; **Po 0.01; *** Po 0.001, twoway ANOVA.
They require binding with receptor-associated kinases in order to trigger a phosphorylation cascade. Regulation of Jak/STAT signaling is important in controlling the immune response to sepsis. The balance of TH1 and TH2 cytokines is central to the host response to infection. Many different models of sepsis have been used to investigate the role of STATs and Jaks for the maintenance of TH1/TH2 balance. They vary from clinically relevant models, such as polymicrobial peritonitis
to intraperitoneal injection of pathogens or their infectious components, such as LPS (Zisman et al., 1997). In this study, we sought to investigate the effect of tyrosine kinase inhibitor tyrphostin AG490 on the development of LPS-induced inflammation. We followed its effect on the early events within 24 h caused by LPS administration in blood, peritoneal cavity and liver. This time interval corresponded to an elevation of pro-inflammatory
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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1 mediators, such as RANTES, MIP-1α, IL-6 and IL-12 levels in circulation. and we did not determine whether it is definitely dose-dependant. 2 Tyrphostin diminished RANTES, IL-6 and IL-12 levels, while MIP-1α Previously, we have found that the substance applied in zymosan3 level was augmented at 24 h. The effect seemed to be time-dependant induced shock under the same dose of 5 mg/kg, expressed high 4 inhibitory action, associated with decreased cytokine/chemokine 5 levels including MIP-1α (Dimitrova and Ivanovska, 2008). It suggests 6 that there are TLR-dependent differences in tyrphostin action since 7 LPS is TLR4 ligand, while zymosan is TLR2 ligand. Also, it was 8 determined that the predominant infiltrating cells in peritoneal 9 exudate (about 50%) at 24 h being of CD11b þ Ly6C þ type. In contrast, 10 such increase was prevented in AG490-treated group. Having in view, 11 that the CD11b þ Ly6C þ population provides strong suppression of 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Fig. 5. Liver injury in mice with LPS-induced shock. Histological examination of 38 liver was performed after i.p. injection of LPS (C, E, G and I) and after LPS þAG490 39 injection (D, F, H and J), (n ¼8 per group). Tissue alterations were analyzed at 1 h (C, 40 and D), 4 h (E, and F), 24 h (G, and H) and 21 day (I, and J) post injection. Normal 41 liver appearance was shown in control group (A) and in (B) was shown mice treated with AG490 alone. In (C and D) black arrows indicate mononuclear cell 42 infiltration, hepatocellular necrosis (rectangle in C) and presence of acidophilic 43 bodies were also found (yellow arrows, C). Dilated bile ducts filled with bile (black 44 arrow, E), hemorrhages (white arrow, E) and necrotic foci which coalesce (rectan45 gles, E) were observed in LPS injected mice. At day 21 histomorphological 46 appearance of liver showed ballooning degeneration of hepatocytes (yellow Fig. 7. Immunohistochemical detection of iNOS expression in liver of LPS injected arrows, I) with disintegrated pyknotic nuclei (black arrows, I). Minor hepatocyte mice (C, E, G, and I) and LPS þ AG490 (D, F, H, and J). iNOS staining is not evident in 47 and parenchimal tissue changes in AG490-treated animals (D) and normal controls (A) and in group treated with AG490 alone (B). At 1 and 4 h and at day 21 48 appearance (F, and H) were observed. One of four representative slides from after LPS administration strong iNOS expression was detected (red arrows C, E, 49 Q5 3 separate experiments is shown (H&E, original magnification x40 A–J). (For and I), while in AG490-treated mice only at 1 h iNOS was expressed (D, F, H, and J). 50 Similar results were obtained in two independent experiments (n¼ 7 per group), interpretation of the references to color in this figure legend, the reader is referred 51 original magnification x40. to the web version of this article.) 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fig. 6. Tyrphostin AG490 reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at 24 h after LPS or LPSþ AG490 injection and no changes 66 were noticed in mice treated with tyrphostin only. Values are means7S.E.M. **Po0.05; Po0.001 vs control; Po0.05 and Po0.001 vs LPSþ AG490, two-way ANOVA. Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 8. Effect of AG490 on STAT3 and pSTAT3 expression in liver. Immunohistochemical staining for STAT3 in mice with LPS-induced shock was observed at all time points (red arrows, C, E, and G), while in AG490-treated mice STAT3 was expressed only at 1 h (D). pSTAT3 was expressed at 1 and 4 h (K, and M) and not at 24 h (O). AG490 expressed inhibitory effect at 4 h (L). No apperent staining observed in control groups (A, and I) and in AG490-treated groups at 1 or 24 h (B, and J). Similar results were obtained in two independent experiments (n¼ 7 per group), original magnification x40.
adaptive immune responses by several mechanisms, including the expression of iNOS and STAT1 (Kallberg et al., 2012), it might be expected AG490 to have influence on these processes. CD69 is a lymphoid activation antigen whose rapid expression makes it responsible for the early detection of T-cell activation. Increased CD69 expression has been noted in response to infectious agents (Terres and Pajares, 1998). Present results witnessed that about 20–30% of PE cells in LPS injected mice represent T lymphocytes. Notably, they expressed the earliest marker of T cell activation CD69 while AG490 abrogated the increase of CD3 þ CD69 þ population at the initiation of inflammation. It is considered that the rapid complement activation in a result of LPS-injection leads to a massive C5a generation and a strong proinflammatory response, including production of proinflammatory mediators such as IL-6, which can enhance C5a receptor expression in various cell types (Riedemann et al., 2003). The importance of C5a/C5a receptor-induced effects is reflected by the fact that blockade of C5a or C5a receptor has been suggested to be beneficial in a variety of inflammatory disorders (Czermak et al., 1999). Complement activation during human sepsis is associated with significantly reduced survival rates together with multi-organ failure when compared with lesssevere septic patients and survivors (Nakae et al., 1994). C5a receptor became one focus of interest due to the results, which demonstrated benefits of blockade of C5a with antibodies in cecal ligation and puncture (CLP) model. This increase was found to be strongly dependent on plasma interleukin (IL)-6 (Huber-Lang et al., 2002). A significant decrease of C5a receptor positive peritoneal cells was
detected in mice with shock which suppose generation of C5a and binding to surface C5a receptor. This was prevented by tyrhostin AG490 pointing on complement-dependant mechanism of antiinflammatory action. One of the earliest cytokines which appear after sepsis initiation is TNF-α. It causes the induction of iNOS in a variety of cells, followed by NO production and thus leading to organ injury. Immunohistochemical observation proved a significant expression of TNF-α receptor in the liver one and four hour after LPS injection, as well as in the late inflammation which was reduced by AG490 after 4 h. There is a diverse array of host mechanisms involved in cytokine network, so it could be that the positive effects mediated by AG490 are due to its action on other cytokines through TNF-α in the tissues. During sepsis the infiltration of leukocytes plays a decisive role in tissue damage through an early accumulation of polymorphonuclear leukocytes in the liver at first three hours, which is followed by an infiltration of mononuclear phagocytes (Ruetten et al., 1999). LPS provoked progressive hepatocyte damage, cell infiltration and necrosis while tyrphostin treatment resulted in a less severe liver injury with no parenchymal tissue disruption. This corresponded to diminished serum levels of liver injury markers ALT and AST in tyrphostin AG490treated animals, compared to untreated. Except, it was found that liver dysfunction was observed not only in the acute phase of inflammation but also in the late phase (day 21) when pathohistological appearance of livers in tyrphostin-treated mice resembles that of controls. During infection and other inflammatory conditions, NO production is controlled by inducible NO synthase (Nathan and Xie, 1994). LPS induced an increase of iNOS expression on J774.2 macrophages
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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powerfully prevented by tyrosine kinase inhibitors, tyrphostin AG126 and genistein (Ruetten et al., 1999). Also, an enhanced induction of iNOS by LPS in macrophages and vascular smooth muscle cells caused circulatory failure and multiple organ dysfunction syndrome associated with septic shock (Dong et al., 1993; Ruetten and Thiemermann, 1997). We demonstrated that the LPS-induced increase of iNOS expression in liver at acute and late phase was ameliorated in tyrphostin-treated mice. This contributed to the reduction of the liver pathology caused by LPS. STAT1 and STAT3 are transcription factors which are the important part of the cell signaling. JAK/STAT pathway is playing a key role in inflammation (de Prati et al., 2005; Stark and Darnell, 2012). STATs exist in an inactive form in the cytoplasm and after activation by phosphorilation they transduce a signal from a cellular receptor into the nucleus and up-regulate pro-inflammatory factors such as TNF-α, IL-1, MIP-1α (Darnell et al., 1994; Matsukawa, 2007). Various in vivo and in vitro studies convincingly define STAT3 as a principal mediator of liver acute-phase genes induction downstream of IL-6 in response to LPS (Levy et al., 2003). In acute inflammation we observed that endotoxin induced high STAT3 expression in liver during 24 h which was lowered by tyrphostin. In a CLP rat model the level of IL-6 has been associated with the final sepsis outcome. Therefore, it is supposed that in CLP hepatocytes become hyporesponsive to IL-6, thus decreasing STAT3 phosphorylation (Remick et al., 2002). Contrary to that, in a model of LPS-induced shock has been shown that higher levels of interleukin (IL)-6 correlated with higher mortality (Remick et al., 2000). Our results showed that the decrease of IL-6 blood level corresponded to inhibited STAT3 expression in liver. Activated STATs translocate to the nucleus to induce gene expressions, followed by certain cytokine secretion. In the present experiments, our results showed that AG490 reduced the expressions of STAT3 and pSTAT3 which suggests that tyrphostin inhibits the STAT3 activation at JAK2 phosphorylation level. Currently, therapeutic interventions targeting the complex network of inflammatory and immune responses appear to be a risky approach, given that inhibition of these responses may significantly impair the ability of the host to naturally fight infections (Albrecht and Ward, 2004; Eichacker et al., 2002). However, the combination of appropriate antibiotic therapy introduced early during sepsis combined with inhibition of inflammation in the late stages of the disease process appears to be a promising mode of treatment of septic patients in the near future. In conclusion, the anti-inflammatory effect of AG490 may involve: (1) a reduction of serum RANTES, IL-6 IL-12 at 24 h and the recruitment of CD11b/Ly6C and CD3/CD69 positive cells in peritoneal exudate; (2) changes of C5a receptor and TNF-α receptor expression by peritoneal cells; (3) amelioration of liver dysfunction attended with reduced iNOS, STAT3 and pSTAT3 expression. Further investigations are important in considering the ultimate goals for therapeutic application of this tyrphostin in order to reduce the shock pathology and provide novel insights in its molecular and functional complexity.
Acknowledgment This work was supported by a Grant B01/6 from the Bulgarian National Science Fund, Bulgaria.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2015.01.045.
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Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPS-induced shock Valeriya Gyurkovska, Nina Ivanovska n Department of Immunology, Institute of Microbiology, BAS, Sofia, Bulgaria
art ic l e i nf o
a b s t r a c t
Article history: Received 1 October 2014 Received in revised form 12 January 2015 Accepted 15 January 2015
Sepsis remains a serious clinical problem despite continuous efforts to increase survival. Experimental animal models of sepsis are pointed to a great extent on blocking the activity of cytokines. A number of signal-transducing molecules are associated with the occurrence of excessive tissue inflammation. Through inhibition of tyrosine phosphorilation and thereby changing cell signaling, tyrosine kinase inhibitors can influence multiple inflammatory pathways. The purpose of the present investigation was to evaluate the effect of tyrosine kinase inhibitor tyrphostin AG490 in a mouse LPS-induced shock. Cytokine and chemokine blood levels were determined by ELISA assays. CD11b þ Ly6C þ , CD3 þ CD69 þ and C5aR positive cell populations in the peritoneal exudate were detected by flow cytometry. The expression of iNOS and Signal Transducer and Activator of Transcription (STAT) in the liver were observed by immunohistochemistry. We found that tyrphostin AG490 inhibited Regulated upon activation normal T cell expressed and secreted (RANTES), IL-6 and IL-12 serum levels, decreased the number of CD11b þ Ly6C þ and CD3 þ CD69 þ subpopulations in the peritoneal exudate and prevented thells expressing C5a receptor and TNF-alpha receptor. Tyrphostin ameliorated liver injury associated with suppressed iNOS, STAT3 and pSTAT3 expression. Our data suggest that tyrphostin AG490 diminished the degree of inflammation starting in peritoneal cavity and minimized liver dysfunction thus representing one approach for better outcome of sepsis conditions. & 2015 Published by Elsevier B.V.
Chemical compounds studied in this article: Tyrphostin AG490 (PubChem CID: 5328771) Keywords: RANTES IL-6 IL-12 iNOS STAT3 pSTAT3
1. Introduction Sepsis is a condition that results from a harmful host response to infection. Many of the components of the innate immune response that normally ensure host defences against infection can cause cell and tissue damage and lead to multiple organ failure, the clinical hallmark of sepsis. Because of the high mortality of sepsis, many efforts have been made to reveal how the host protection is deregulated. As a result, new data have been accumulated about basic principles underlying bacterial-host interactions, and new opportunities for therapeutic intervention have been elaborated (Natanson et al., 1994). Different models of sepsis are in use, one of them being LPS-induced shock. LPS elicits a broad, nonspecific cascade of events in vivo, resulting in secretion of a variety of potent cytokines through an intracellular signal amplification pathway such as IL-1, IL-6, TNF-α and IL-12. They contribute to the pathophysiology of multisystem organ failure and often lead to commonly fatal exit (Dinarello, 1991; Higgins et al., 2003). LPS is capable to activate myeloid and non-myeloid cells, as well as other innate host defense mechanisms, such as serum complement, and specific components
n
Corresponding author. Tel.: þ 359 2 979 3195. E-mail address: [email protected] (N. Ivanovska).
within the coagulation pathway (Markiewski et al., 2008). The mechanism of action of LPS involves interaction with Toll-like receptor (TLR)4 and CD14 that results in the activation of Nuclear Factor (NF)-κB pathway, the production of endogenous mediators by monocytes, macrophages and polymorphonuclear neutrophils, and the induction of antigen-specific cellular immunity (Jeyaseelan et al., 2005; Palsson-McDermott and O’Neill, 2004). Cytokine signal transduction is believed to be mediated primarily through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway which leads to the expression of certain target genes. Continuous activation of the JAK/STAT pathway has been implicated in numerous cancers and immune disorders. Recently, over 20 drugs that target kinases have been introduced in clinical practice and many are currently in preclinical studies (Cohen, 2009; Cohen and Alessi, 2013). Tyrphostin AG490 (α-cyano-(3,4-dihydroxy)-N-benzylcinnamide) is a synthetic protein tyrosine kinase inhibitor which at first has been considered as a specific JAK2 inhibitor but later its JAK3-dependent action has been established (Kirken et al., 2001). A number of tyrphostins have been investigated in LPS-induced sepsis in rats and their protective role on organ injury has been observed (Ruetten and Thiemermann, 1997). Recent studies claim that immune response suppression by AG490 prevents the lethal outcome in a mouse model of polymicrobial sepsis (Hui et al., 2009; Pena et al., 2010). However, the protective
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Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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role of AG490 in the course of LPS-induced inflammation currently remains elusive. Previously, we have studied the effect of tyrphostin AG490 in zymosan-induced nonseptic shock in mice. A single dose of 5 mg/kg reduced the kidney and liver dysfunction associated with inhibition of iNOS, STAT1, STAT3 and C5a receptor expression in organs as well as with changes of various cytokines and chemokines (Dimitrova et al., 2009; Dimitrova and Ivanovska, 2008). Except, TLR-2 dependent mode of AG490 action we have demonstrated that it acts through TLR-4 and TLR-9 pathways (Dimitrova et al., 2008). The aim of the present study was to investigate the action of AG490 on the early events of LPS-induced inflammation in peritoneum and on the liver dysfunction.
2. Materials and methods 2.1. Mice Male ICR mice (CD-2) background (8–10 week old, weight 20–22 g) were bred in the Animal Facility of the Institute of Microbiology, maintained on a 12:12 h light:dark cycle and fed standard diet and tap water ad libitum. All experiments were conducted in accordance with the Bulgarian Food Safety Agency Guidelines no. 352 06.01.2012 and approved by the Animal Care Committee at the Institute of Microbiology, Sofia (Decree no. 14/19.07.2000). 2.2. LPS-induced shock Lipopolysaccharide (serotype 055:B5 from Escherichia coli; Sigma Aldrich, St. Louise, MO) was dissolved in endotoxin-free water and stored at 20 1C. Tyrphostin AG490 (2-Cyano-3-dihyroxyphenyl)–N(benzyl)-2-propenamide (Sigma-Aldrich, St. Louis, USA) was dissolved in DMSO and further diluted in PBS (final DMSO concentration was o0.05%). Mice were randomly divided into four experimental groups: a group injected with PBS (control), mice injected with 5 mg/kg AG490, mice injected i.p. with 25 mg/kg LPS (LPS) and a group injected with 25 mg/kg LPS and immediately after that injected i.p. with 5 mg/kg AG490 (LPSþAG490). Such dose resulted in 70–80% mortality and the animals expressed all signs of acute inflammation within 48 h, such as ruffled fur, diarrhea and lethargy (Supplemental Fig. 1). The dose for AG490 was chosen based on our experiments showing that lower dose of 1 mg/kg did not reduce significantly the rate of mortality and on our previous study in zymosan-induced shock with different doses of AG490 (Dimitrova et al., 2009) pointing that a higher dose of 10 mg/kg resulted in insignificant improvement of mortality. 2.3. Measurement of serum cytokine and chemokine levels Mice were killed by cervical dislocation. Blood was collected and sera were obtained after centrifugation at 350 g for 15 min at 4 1C and stored in aliquots at 80 1C until required. The levels of IL-6, IL12, MIP-1α and RANTES were measured by quantitative ELISA kits (Biolegend, USA) according to manufacture's instructions.
Isolated mouse peritoneal cells were stained with antibodies from BD Pharmingen (Erembodegem, Belgium) against mouse Ly-6C (FITCconjugated; clone AL-21), CD11b (PE-conjugated; clone M1/70), CD69 (APC-conjugated, clone H1.2F3), and CD3 (FITC conjugated, clone 145-2C11). For detection of cell surface expression of C5aR and TNF-α receptor, the cells were processed in PBS/2% FCS and incubated for 20 min at 4 1C with biotinylated antibodies against mouse C5aR or TNF-α receptor1 (2 μg/ml; Santa-Cruz Biotechnology Inc., Heidelberg, Germany) or IgG isotype controls (Biolegend). After washing with 2% FCS/ PBS, secondary avidin-FITC (4 μl/sample, Becton Dickinson,San Jose, CA, USA) was added for 15 min at 4 1C. After three washings with PBS, the samples were analyzed by a flow cytometer (BD™ LSR II) and FCS Express™ Diva Software (Becton Dickinson, San Jose, CA, USA). 2.5. Histopathologic changes in liver and evaluation of liver dysfunction Livers were fixed in 4% p-formalaldehyde (pH 7.4). The organs were embedded in paraffin and sections with thickness 5 μm were cut. The slides were then stained with hematoxylin and eosin (H&E) and examined by a light microscope (Boeco, Hamburg, Germany) connected to the device camera. Sera were collected as described above and analyzed immediately by using standard laboratory kits (Dialab GmbH, Wiener Heudorf, Austria) measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST). 2.6. Immunohistochemistry Tissue paraffin sections (5 μm) were incubated for 10 min with HRP blocking solution (3% H2O2/60% methanol for 10 min), washed with PBS and blocked for 10 min with 5% BSA/PBS followed by washing and 40 min incubation with antibodies against iNOS (1:2000 diluted), STAT3 (1:100 diluted, Santa-Cruz Biotechnology, Heidelberg, Germany) or pSTAT3 (1:100 diluted, Abcam). Isotype antibodies (Sigma-Aldrich, Munich, Germany) were used as a background staining controls. After washing with 0.01% Tween/PBS, the sections were incubated for 20 min with HRP-labeled anti-rabbit IgG antibody (1:2000, Sigma-Aldrich), washed in PBS and stained with DAB (3,3'-diaminobenzidine-tetrahydrochloride) substrate solution (Sigma-Aldrich) for 10 min and then counterstained for 2 min with Gill's hematoxylin. 2.7. Statistical analyses Statistical analyses were performed using InStat3.0 and GraphicPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Data are expressed as mean 7 S.D. or mean 7 S.E.M. The differences in mean values between groups were analyzed by two-way ANOVA. Differences were considered significant when Po0.05.
3. Results 2.4. Flow cytometry Resident peritoneal macrophages were harvested by rinsing the peritoneal cavity with 10 ml of RPMI-1640 medium. The cells were washed twice, re-suspended at a density of 2 106 cells/ml in complete RPMI-1640 medium supplemented with 5% fetal calf serum (FCS) for 1 h (37 1C, 5% CO2). The total cell population contained approximately 95% viable cells (trypan blue exclusion test). Then, the non-adherent cells were removed by two washings, the adherent population was collected and more than 90% of these cells were positive for F4/80 (marker for mature mouse macrophages).
3.1. Influence of tyrphostin AG490 on the levels of chemokines and cytokines in sera from mice with LPS-induced shock We determined the effect of AG490 on the serum concentration of some critical mediators of developing inflammation at 1, 4 and 24 h after LPS injection. The level of MIP-1α was not influenced by tyrphostin at 1 and 4 h and was increased at 24 h in a result of AG490 treatment (Fig. 1B). Tyrphostin injection of healthy mice also provoked elevation of MIP-1α concentration. A decrease of RANTES level was established in AG490-treated mice at all time points
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 1. Cytokine levels in sera of mice with LPS-induced shock. Mice (n¼10 per group) were injected with PBS (control), with 5 mg/kg AG490 (AG490), with 25 mg/kg LPS (LPS) or with 25 mg/kg LPS plus 5 mg/kg AG490 (LPS þAG490). The concentrations of RANTES (A), MIP-1α (B), IL-6 (C) and IL-12 (D) were determined by ELISA at 1, 4 and 24 h after treatment. *P o 0.05; **Po 0.01; *** Po 0.001 vs control, Po 0.05; Po 0.01 LPS vs LPS þ AG490, two-way ANOVA with Bonferroni post-test.
(Fig. 1B). Same tendency was observed for IL-6 (Fig. 1C) and for IL-12 (Fig. 1D). 3.2. Effect of tyrphostin AG490 on the number of CD11b þ Ly6C þ and CD3 þ CD69 þ peritoneal exudate cells
that such lack of C5a receptor expression on PE cells was considerably prevented by AG490 (Fig. 4A, and B). Tyrphostin administration resulted in lower TNF-α receptor expression on 4 and 24 h which suggests excessive TNF-α production (Fig. 4C, and D). 3.4. Effects of AG490 on liver injury in LPS-induced shock
The increase of peritoneal cells during inflammation is a consequence from the influx of blood monocytes into the peritoneum. We exploited CD11b and Ly6C markers to discriminate these cells in the peritoneal exudate. We found that the number of double positive CD11b þ Ly6C þ cells was increased dramatically at 24 h after LPS injection which was prevented by tyrphostinAG490 (Fig. 2A, and B). The initiation of inflammation was associated with a rapid recruitment of T cells into the peritoneal cavity which started at 1 h and was maintained during 24 h (Fig. 3). CD69 is a useful marker for quantifying T-cell and T-cell subset activation in mixed populations. The percentage of CD3 þ CD69 þ cells was significantly elevated in animals exposed to LPS. Tyrphostin AG490 significantly suppressed this elevation at 1 h after LPS injection but later on the effect disappeared (Fig. 3). 3.3. Effect of tyrphostin AG490 on C5a receptor and TNF-α receptor expression by peritoneal cells The complement activation product C5a is one of the most potent inflammatory chemoattractants and has been involved in the pathogenesis of multiple inflammatory diseases. This suggests an increase in C5a and C5a receptor production which on the other hand supposes less available free C5a receptor molecules on the cell surface. Our data were in accordance with this opinion as we established a decreased number of C5a receptor positive cells in LPS treated mice on 4 and 24 h. In AG490 treated group we observed
To find the role of AG490 in LPS shock, livers were harvested at 1, 4, 24 h and at day 21 after LPS injection. Normal liver appearance was shown in control mice (Fig. 5A). At 1 h massive inflammatory cells infiltration, causing hepatocellular necrosis and presence of acidophilic bodies were found after H&E staining (Fig. 5C). In LPS injected group we observed dilated bile ducts filled with bile at 4 h (Fig. 5E) and hemorrhages at 4 and 24 h (Fig. 5E, and G). Hepatic necrosis was seen and hepatic lesions coalesce to develop larger lesions (Fig. 5E). At day 21 after LPS administration histomorphological appearance of liver showed ballooning degeneration of hepatocytes with disintegrated pyknotic nuclei (Fig. 5I). Minor hepatocyte and parenchimal tissue changes were observed in AG490-treated animals such as less cell infiltration (Fig. 5D), lack of necrosis and large zones of injury were absent (Fig. 5F) as well as lack of hemorrhages (Fig. 5H, and J). This correlated with a little elevation of serum ALT and AST levels in AG490-treated mice while in LPSinjected group they were quite increased at 24 h after LPS injection (Fig. 6). There were no changes in mice injected with tyrphostin only. 3.5. Effect of AG490-treatment on iNOS, STAT3 and pSTAT3 expression in liver during LPS-induced shock Our results indicated that livers from mice subjected to LPS showed a strong iNOS induction at 1 and 4 h (Fig. 7C, and E). In contrast, iNOS expression in livers from mice that had been treated
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 2. Effect of AG490 on the number of CD11b þ Ly6C þ cells in peritoneal exudates (PE) LPS-induced shock. Mice (n ¼10 per group) were injected as described in Fig. 1. Representative dot-plot graphs (A). Tyrphostin significantly lowered the percentage of CD11b þ Ly6C þ cells at 24 h (B). Values are means7 S.E.M. *Po 0.05, two-way ANOVA.
with AG490 was apparently minimized at 4 h (Fig. 7F). Twenty four hour upon endotoxin administration iNOS expression was undetectable in both, treated and untreated with AG490 mice (Fig. 7G, and H). iNOS expression was shown at day 21 in mice with shock (Fig. 7I), while in mice treated with AG490 no staining was observed (Fig. 7J). No histopathological changes were observed in the livers of controls (Fig. 7A) and in AG490-treated mice (a representative picture Fig. 7B). We further investigated the STAT3 and pSTAT3 expression in hepatocytes during inflammation. Immunohistochemical analyses revealed that STAT3 protein was expressed in the liver of LPS-injected mice at each time point examined (Fig. 8C, E and G). Lower expression was observed at 1 h (Fig. 8D) and at 4 and 24 h this protein was not detected in mice treated with AG490 (Fig. 8F and H). Intensive pSTAT3 staining was established at 1 h in LPS-injected mice, not presente in AG490-treated mice with shock (Fig. 8K and L, respectively). At 4 h pSTAT3 presented in both
AG490-treated and untreated groups with shock (Fig. 8M, and N) and no expression was found at 24 h in these groups (Fig. 8O, and P). No evident staining was detected in livers of animals treated with AG490 only in regard to STAT3 and pSTAT3 (Fig. 8B, and J).
4. Discussion The incidence of sepsis is increasing despite the major advances in the development of antimicrobial agents and other supportive treatments. Septic patients often develop systemic inflammatory response, of which multiple organ failure is a main complication. Chronic inflammation arises when the acute response is not completely turned off, continuing to stimulate pro-inflammatory mediators when they may not be needed. Cytokines regulate many of the pathways involved in the host inflammatory response to sepsis. The Jak/STAT pathway is associated with synthesis of various cytokines.
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 3. Effect of AG490 on the number of CD3 þ CD69 þ cells in PE in LPS-induced shock. Mice (n¼10 per group) were injected as shown in Fig. 1. Tyrphostin inhibited the increase of the number of CD3 þ CD69 þ cells at 4 h. Values are means7 S.E.M. *Po 0.05; two-way ANOVA.
Fig. 4. Effect of tyrphostin AG490 on the expression of C5a receptor and TNF-α receptor by PE cells. Mice (n¼ 10 per group) were injected as shown in Fig. 1. AG490 prevented the decrease of the number of C5a receptor positive cells (A) and graph (B), and of TNF-α receptor (C) and graph (D). *P o 0.05; **Po 0.01; *** Po 0.001, twoway ANOVA.
They require binding with receptor-associated kinases in order to trigger a phosphorylation cascade. Regulation of Jak/STAT signaling is important in controlling the immune response to sepsis. The balance of TH1 and TH2 cytokines is central to the host response to infection. Many different models of sepsis have been used to investigate the role of STATs and Jaks for the maintenance of TH1/TH2 balance. They vary from clinically relevant models, such as polymicrobial peritonitis
to intraperitoneal injection of pathogens or their infectious components, such as LPS (Zisman et al., 1997). In this study, we sought to investigate the effect of tyrosine kinase inhibitor tyrphostin AG490 on the development of LPS-induced inflammation. We followed its effect on the early events within 24 h caused by LPS administration in blood, peritoneal cavity and liver. This time interval corresponded to an elevation of pro-inflammatory
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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1 mediators, such as RANTES, MIP-1α, IL-6 and IL-12 levels in circulation. and we did not determine whether it is definitely dose-dependant. 2 Tyrphostin diminished RANTES, IL-6 and IL-12 levels, while MIP-1α Previously, we have found that the substance applied in zymosan3 level was augmented at 24 h. The effect seemed to be time-dependant induced shock under the same dose of 5 mg/kg, expressed high 4 inhibitory action, associated with decreased cytokine/chemokine 5 levels including MIP-1α (Dimitrova and Ivanovska, 2008). It suggests 6 that there are TLR-dependent differences in tyrphostin action since 7 LPS is TLR4 ligand, while zymosan is TLR2 ligand. Also, it was 8 determined that the predominant infiltrating cells in peritoneal 9 exudate (about 50%) at 24 h being of CD11b þ Ly6C þ type. In contrast, 10 such increase was prevented in AG490-treated group. Having in view, 11 that the CD11b þ Ly6C þ population provides strong suppression of 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Fig. 5. Liver injury in mice with LPS-induced shock. Histological examination of 38 liver was performed after i.p. injection of LPS (C, E, G and I) and after LPS þAG490 39 injection (D, F, H and J), (n ¼8 per group). Tissue alterations were analyzed at 1 h (C, 40 and D), 4 h (E, and F), 24 h (G, and H) and 21 day (I, and J) post injection. Normal 41 liver appearance was shown in control group (A) and in (B) was shown mice treated with AG490 alone. In (C and D) black arrows indicate mononuclear cell 42 infiltration, hepatocellular necrosis (rectangle in C) and presence of acidophilic 43 bodies were also found (yellow arrows, C). Dilated bile ducts filled with bile (black 44 arrow, E), hemorrhages (white arrow, E) and necrotic foci which coalesce (rectan45 gles, E) were observed in LPS injected mice. At day 21 histomorphological 46 appearance of liver showed ballooning degeneration of hepatocytes (yellow Fig. 7. Immunohistochemical detection of iNOS expression in liver of LPS injected arrows, I) with disintegrated pyknotic nuclei (black arrows, I). Minor hepatocyte mice (C, E, G, and I) and LPS þ AG490 (D, F, H, and J). iNOS staining is not evident in 47 and parenchimal tissue changes in AG490-treated animals (D) and normal controls (A) and in group treated with AG490 alone (B). At 1 and 4 h and at day 21 48 appearance (F, and H) were observed. One of four representative slides from after LPS administration strong iNOS expression was detected (red arrows C, E, 49 Q5 3 separate experiments is shown (H&E, original magnification x40 A–J). (For and I), while in AG490-treated mice only at 1 h iNOS was expressed (D, F, H, and J). 50 Similar results were obtained in two independent experiments (n¼ 7 per group), interpretation of the references to color in this figure legend, the reader is referred 51 original magnification x40. to the web version of this article.) 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fig. 6. Tyrphostin AG490 reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at 24 h after LPS or LPSþ AG490 injection and no changes 66 were noticed in mice treated with tyrphostin only. Values are means7S.E.M. **Po0.05; Po0.001 vs control; Po0.05 and Po0.001 vs LPSþ AG490, two-way ANOVA. Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Fig. 8. Effect of AG490 on STAT3 and pSTAT3 expression in liver. Immunohistochemical staining for STAT3 in mice with LPS-induced shock was observed at all time points (red arrows, C, E, and G), while in AG490-treated mice STAT3 was expressed only at 1 h (D). pSTAT3 was expressed at 1 and 4 h (K, and M) and not at 24 h (O). AG490 expressed inhibitory effect at 4 h (L). No apperent staining observed in control groups (A, and I) and in AG490-treated groups at 1 or 24 h (B, and J). Similar results were obtained in two independent experiments (n¼ 7 per group), original magnification x40.
adaptive immune responses by several mechanisms, including the expression of iNOS and STAT1 (Kallberg et al., 2012), it might be expected AG490 to have influence on these processes. CD69 is a lymphoid activation antigen whose rapid expression makes it responsible for the early detection of T-cell activation. Increased CD69 expression has been noted in response to infectious agents (Terres and Pajares, 1998). Present results witnessed that about 20–30% of PE cells in LPS injected mice represent T lymphocytes. Notably, they expressed the earliest marker of T cell activation CD69 while AG490 abrogated the increase of CD3 þ CD69 þ population at the initiation of inflammation. It is considered that the rapid complement activation in a result of LPS-injection leads to a massive C5a generation and a strong proinflammatory response, including production of proinflammatory mediators such as IL-6, which can enhance C5a receptor expression in various cell types (Riedemann et al., 2003). The importance of C5a/C5a receptor-induced effects is reflected by the fact that blockade of C5a or C5a receptor has been suggested to be beneficial in a variety of inflammatory disorders (Czermak et al., 1999). Complement activation during human sepsis is associated with significantly reduced survival rates together with multi-organ failure when compared with lesssevere septic patients and survivors (Nakae et al., 1994). C5a receptor became one focus of interest due to the results, which demonstrated benefits of blockade of C5a with antibodies in cecal ligation and puncture (CLP) model. This increase was found to be strongly dependent on plasma interleukin (IL)-6 (Huber-Lang et al., 2002). A significant decrease of C5a receptor positive peritoneal cells was
detected in mice with shock which suppose generation of C5a and binding to surface C5a receptor. This was prevented by tyrhostin AG490 pointing on complement-dependant mechanism of antiinflammatory action. One of the earliest cytokines which appear after sepsis initiation is TNF-α. It causes the induction of iNOS in a variety of cells, followed by NO production and thus leading to organ injury. Immunohistochemical observation proved a significant expression of TNF-α receptor in the liver one and four hour after LPS injection, as well as in the late inflammation which was reduced by AG490 after 4 h. There is a diverse array of host mechanisms involved in cytokine network, so it could be that the positive effects mediated by AG490 are due to its action on other cytokines through TNF-α in the tissues. During sepsis the infiltration of leukocytes plays a decisive role in tissue damage through an early accumulation of polymorphonuclear leukocytes in the liver at first three hours, which is followed by an infiltration of mononuclear phagocytes (Ruetten et al., 1999). LPS provoked progressive hepatocyte damage, cell infiltration and necrosis while tyrphostin treatment resulted in a less severe liver injury with no parenchymal tissue disruption. This corresponded to diminished serum levels of liver injury markers ALT and AST in tyrphostin AG490treated animals, compared to untreated. Except, it was found that liver dysfunction was observed not only in the acute phase of inflammation but also in the late phase (day 21) when pathohistological appearance of livers in tyrphostin-treated mice resembles that of controls. During infection and other inflammatory conditions, NO production is controlled by inducible NO synthase (Nathan and Xie, 1994). LPS induced an increase of iNOS expression on J774.2 macrophages
Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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powerfully prevented by tyrosine kinase inhibitors, tyrphostin AG126 and genistein (Ruetten et al., 1999). Also, an enhanced induction of iNOS by LPS in macrophages and vascular smooth muscle cells caused circulatory failure and multiple organ dysfunction syndrome associated with septic shock (Dong et al., 1993; Ruetten and Thiemermann, 1997). We demonstrated that the LPS-induced increase of iNOS expression in liver at acute and late phase was ameliorated in tyrphostin-treated mice. This contributed to the reduction of the liver pathology caused by LPS. STAT1 and STAT3 are transcription factors which are the important part of the cell signaling. JAK/STAT pathway is playing a key role in inflammation (de Prati et al., 2005; Stark and Darnell, 2012). STATs exist in an inactive form in the cytoplasm and after activation by phosphorilation they transduce a signal from a cellular receptor into the nucleus and up-regulate pro-inflammatory factors such as TNF-α, IL-1, MIP-1α (Darnell et al., 1994; Matsukawa, 2007). Various in vivo and in vitro studies convincingly define STAT3 as a principal mediator of liver acute-phase genes induction downstream of IL-6 in response to LPS (Levy et al., 2003). In acute inflammation we observed that endotoxin induced high STAT3 expression in liver during 24 h which was lowered by tyrphostin. In a CLP rat model the level of IL-6 has been associated with the final sepsis outcome. Therefore, it is supposed that in CLP hepatocytes become hyporesponsive to IL-6, thus decreasing STAT3 phosphorylation (Remick et al., 2002). Contrary to that, in a model of LPS-induced shock has been shown that higher levels of interleukin (IL)-6 correlated with higher mortality (Remick et al., 2000). Our results showed that the decrease of IL-6 blood level corresponded to inhibited STAT3 expression in liver. Activated STATs translocate to the nucleus to induce gene expressions, followed by certain cytokine secretion. In the present experiments, our results showed that AG490 reduced the expressions of STAT3 and pSTAT3 which suggests that tyrphostin inhibits the STAT3 activation at JAK2 phosphorylation level. Currently, therapeutic interventions targeting the complex network of inflammatory and immune responses appear to be a risky approach, given that inhibition of these responses may significantly impair the ability of the host to naturally fight infections (Albrecht and Ward, 2004; Eichacker et al., 2002). However, the combination of appropriate antibiotic therapy introduced early during sepsis combined with inhibition of inflammation in the late stages of the disease process appears to be a promising mode of treatment of septic patients in the near future. In conclusion, the anti-inflammatory effect of AG490 may involve: (1) a reduction of serum RANTES, IL-6 IL-12 at 24 h and the recruitment of CD11b/Ly6C and CD3/CD69 positive cells in peritoneal exudate; (2) changes of C5a receptor and TNF-α receptor expression by peritoneal cells; (3) amelioration of liver dysfunction attended with reduced iNOS, STAT3 and pSTAT3 expression. Further investigations are important in considering the ultimate goals for therapeutic application of this tyrphostin in order to reduce the shock pathology and provide novel insights in its molecular and functional complexity.
Acknowledgment This work was supported by a Grant B01/6 from the Bulgarian National Science Fund, Bulgaria.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2015.01.045.
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Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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Please cite this article as: Gyurkovska, V., Ivanovska, N., Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPSinduced shock. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.045i
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