Repositioning of the antipsychotic drug TFP for sepsis treatment
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Sepsis is a disease responsible for the death of almost all critical patients. Once infected by virus or bacteria, patients can die due to systemic inflammation within a short period of time. Cytokine storm plays an essential role in causing organ dysfunction and septic shock. Thus, inhibition of cytokine secretion is considered very important in sepsis therapy. In this study, we found that TFP, an antipsychotic drug mainly used to treat schizophrenia by suppressing dopamine secretion, inhibited cytokine release from activated immune cells both in vitro and in vivo. Trifluoperazine (TFP) decreased the levels of pro-inflammatory cytokines without altering their transcription level. In LPS-induced endotoxemia and cecal content injection (CCI) models, TFP intraperitoneal administration improved survival rate. Thus, TFP was considered to inhibit the secretion of proteins through a mechanism similar to that of W7, a calmodulin inhibitor. Finally, we confirmed that TFP treatment relieved organ damage by estimating the concentrations of aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) in the serum. Our findings were regarded as a new discovery of the function of TFP in treating sepsis patients.
• TFP inhibits LPS-induced activation of DCs by suppressing pro-inflammatory cytokine.
• Treatment of TFP increases survival of LPS-induced endotoxemia and CCI sepsis models.
• TFP exerted a protective effect against tissue or organ damage in animal models.
KeywordsSepsis Cytokine TFP Calmodulin (CaM) Inflammation
Sepsis, which is caused by a various bacterial and viral infections, leads to a systemic activation of the innate immune system. As a result of the inflammatory response induced by infected cells, phagocytic cells are activated and release pro-inflammatory cytokine . During infection, dendritic cells (DCs) play an essential interface between innate and adaptive immunity and exist in most tissues of the reticuloendothelial system, including all lymphoid organs. They are especially prominent in tissues that are exposed to the external environment where frequent exposure to foreign antigen and microorganisms occurs . Therefore, at the early stage of inflammation, the activated DCs induce immunosuppression reducing pro-inflammatory cytokine production . In sepsis, since the release of the initial inflammatory cytokine causes cytokine storm and eventually results in death due to loss of organ functions such as lung injection, recent studies emphasized the importance of DC, which improve the aberrant immune response and prolong the life during sepsis progression, in the therapeutic strategy target [4, 5]. To date, a large number of therapeutic agents have been developed to treat sepsis, such as antibodies against lipopolysaccharides (LPS), toll-like receptor 4 (TLR4) agonists, antitumor necrosis factor (TNF) agents, drugs targeting platelet-activating factor (PAF), and drugs targeting coagulation cascades [6, 7, 8]. In a clinical trial, Xigris, which is a recombinant human form of protein C, blocks coagulation, inhibits inflammatory effects, and preserves organ function [9, 10, 11]. However, the result was unsuccessful owing to side effects, and the drug was withdrawn from the market in 2012. The specific drug was not yet approved for clinical use to treat sepsis. Once pathogenic organisms invade a host, they spread through the blood stream. In response to systemic infectious agents, innate immune cells are activated and cause cytokine emission. The rapidly increasing level of pro-inflammatory cytokines causes cytokine storm in the host , inducing organ injury or tissue damage, and this phenomenon is defined as a severe sepsis. Finally, septic shock, which is abnormal distribution of blood flow, results in inadequate blood supply to the body tissues, causing ischemia and organ dysfunction and leading to the death of patients . In general, disrupting cytokine storm is very important in determining the mortality of sepsis patients . Thus, the regulation of cytokine secretion should be studied and considered a key role in sepsis treatment.
Trifluoperazine (TFP) is a clinical antipsychotic drug approved in 2001 and primarily used to treat schizophrenia caused by excess of dopamine . TFP exerts an antipsychotic effect by blocking central dopamine and serotonin receptor in patients suffering from megalomania and hallucinations . This agent is also known to function as a calmodulin inhibitor, which prevents calcium from binding to calmodulin (CaM), thereby leading to the elevation of cytosolic calcium level. In other words, the mode of action of TFP is binding to a well-known Ca2+-binding protein, “calmodulin.” It has been reported that TFP exerts an inhibitory effect on the function of CaM by directly binding to CaM  . CaM is a multifunctional Ca2+-binding protein and involved in the activity of various target proteins, such as kinase and phosphatases, to regulate cellular processes, including cell proliferation, development, and secretion [18, 19]. Consequently, calmodulin has been reported to potentially play a role in the secretion of thyroid hormone from the thyroid . Thus, calcium balance and homeostasis is important in protein secretion  . According to a previous study, LPS-induced TNF-α production is inhibited by Ca2+ chelation and CaM inhibition, and elevating macrophage intracellular Ca2+ augments pro-inflammatory cytokine production [22, 23]. In addition, in aberrant inflammation such as sepsis, disrupted calcium homeostasis is known to exacerbate organ dysfunction and accelerate septic shock [24, 25].
In this study, we identified that TFP as a calmodulin inhibitor reduced inflammatory response by suppressing cytokine secretion in LPS-stimulated macrophages and dendritic cells. Ultimately, TFP administration increased the survival rate of LPS-induced endotoxemia model and cecal content injection (CCI) model by preventing cytokine secretion in the serum and normalizing pathogen infection-induced tissue damage and organ dysfunction. These findings suggested that TFP as a clinical drug exerts a novel therapeutic effect on sepsis by suppressing cytokine release, which meant that TFP can enhance accessibility to sepsis treatment through drug reposition.
TFP inhibits pro-inflammatory cytokine release in diverse PAMP-stimulated state
TFP administration enhances survival of LPS-induced endotoxemia and CCI-induced sepsis models
TFP impedes pathogen infection-induced cytokine secretion in vivo
TFP reduces organ dysfunction and tissue damage caused by cytokine storm
TFP influences cytokine secretion independently of the signaling and transcriptional level of MAPKs following LPS stimulation
Effect of TFP as a W7-like calcium/calmodulin inhibitor
TFP is an antipsychotic drug mainly used to treat schizophrenia patients by suppressing dopamine secretion. However, another therapeutic potential of TFP has been reported. TFP suppressed the growth of tumor and brain metastasis by inducing G0/G1 cell cycle arrest of triple-negative breast cancer (TNBC) without causing detectable side effects in vivo . TFP, a novel autophagy inhibitor, increases radiosensitivity in glioblastoma by impairing homologous recombination . Schubart et al. showed that TFP inhibits insulin secretion from transplantable hamster insulinoma cells. In accordance with this result, this study showed the ability of TFP to inhibit cytokine secretion by stimulated and activated phagocytic cells. We confirmed that TFP reduced the secretion of the pro- and anti-inflammatory cytokines TNF-α, IL-6, and IL-10 in LPS-stimulated DCs (Fig. 1a–c). Cytokine production, however, is not solely induced by LPS, which is known as the main component of the cell wall of gram-negative bacteria. Additionally, we found that TFP inhibited cytokine production induced by the single- or double-strand RNA, DNA, and lipoprotein released from virus or gram-positive bacteria (Fig. 1d). We postulated that the protective effect of TFP in models of LPS-induced endotoxemia and CCI-induced sepsis was significant and without any particular side effect (Fig. 2a, b). Our findings further showed the role of TFP as an inhibitor of systemic inflammation through blockage of cytokine secretion to the serum (Fig. 3). Thus, we have shown, both in vitro and in vivo, that TFP suppressed the levels of the pro-inflammatory cytokines TNF-α and IL-6 in a state of abnormal infection. Importantly, because multiple organ dysfunction caused by sepsis is critical for survival, we also identified that TFP treatment improved tissue damage and liver function, as well as reduce hepatotoxicity and kidney dysfunctions in sepsis models by maintaining normal AST, ALT, and BUN levels (Fig. 4a, b). Additionally, because septic shock leads to multiple organ dysfunctions, we confirmed that lung failure recovered to a normal condition, preventing infiltration of lung PMN (Fig. 4c, d). Moreover, we should consider the potential correlation between TFP and the transcriptional levels of these proteins. As shown in Fig. 5a–c, TFP appeared to have no correlation with the transcriptional levels of these proteins. TFP did not alter the regulation of the mRNA levels of the cytokines and did not exclusively engage in the secretion of cytokines in LPS-simulated DCs, RAW 264.7 cells, and peritoneal residual macrophages. Furthermore, LPS binds to the identical receptor complex (TLR4/MD2) and imposes TLR4-mediated phosphorylation of MAPKs to produce pro-inflammatory cytokines. We found that TFP did not interrupt the downstream signaling of TLR4, which is known as a receptor of LPS (Fig. 5d–f). Our additional results supported these findings in that TFP inhibited pro-inflammatory cytokine production by suppressing cytokine secretion without altering the signaling and transcriptional levels of MAPKs. Blockage of cytokine secretion, however, cannot fully explain the mechanism of TFP. Previous studies reported that dysregulated Ca2+ handling is prevalent to organ dysfunction and tissue damage in sepsis. Inhibition of calcium/calmodulin-dependent protein kinase Iα increased survival rate by reducing systemic concentrations of IL-10, IL-6, TNF-α, and HMGB1 in a CLP model of sepsis . Particularly, the current study showed that calmodulin antagonists abrogated activated immune cell–mediated cytokine secretion in DCs. In accordance with these results, we expected TFP to have an equivalent effect with that of W7, a calmodulin inhibitor. As shown in Fig. 6a, W7 pretreatment significantly decreased the secretion of each cytokine irrespective of the transcriptional level and downstream signaling of TLR4. Our results supported that TFP was involved in the regulation of cytokine secretion with the same mechanism as the calmodulin inhibitor W7.
In summary, this study reported that TFP, as a calmodulin inhibitor, inhibited cytokine secretion. This is a new approach to treat sepsis. The discovery of traditional drugs is inefficient and too costly. Recently, reposition of FDA-approved therapeutics for other diseases is regarded as a rapid, alternative approach to develop drugs . Therefore, our findings brought a new approach to treat sepsis for investigation in future clinical trials. The exact mechanism of TFP in inhibiting cytokine secretion in sepsis remains to be elucidated in future studies.
Six- to eight-week-old female C57BL/6 mice, weighing 16–18 g, were purchased from Orient Bio, Inc. All animal procedures were approved by and performed according to guidelines of the Institutional Animal Care and Use Committee (IACUC) of Konkuk University. To study survival rate, humane endpoints were used to minimize suffering. In case clinical signs of the moribund state were recognized, the animals were euthanized by CO2 euthanasia (PMID: 14676679). The animals were placed in a CO2 chamber, and a low flow CO2 gas was administered. CO2 gas (100%) was administered for another 5 min after the animals lost consciousness.
To isolate DCs, monocytes were isolated from the bone marrow of C57BL/6 mice, which were then cultured in RPMI 1640 medium (Biowest, USA) supplemented with 10% fetal bovine serum, 50 U/mL penicillin/streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 2 mmol/L nonessential amino acids, 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech), and 5 ng/mL IL4 (Peprotech) at 37 °C and 5% CO2. The monocytes were incubated for 6 days before use in the experiment. The murine macrophage RAW 264.7 cell line was purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum and 50 U/mL penicillin/streptomycin. The cells were cultured at 37 °C in a humidified atmosphere cultivator with 5% CO2. To isolate peritoneal resident macrophages, peritoneal cells were collected by washing the peritoneal cavity with 10 mL of 1× phosphate-buffered saline (PBS) and then centrifuged. Pellets were resuspended with RBC lysis buffer and washed twice using 1× PBS. The acquired cells were sorted according to the MACS CD11b MicroBeads protocol (Miltenyi Biotec, Germany) and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 50 U/mL penicillin/streptomycin.
The following reagents were used in this study: TFP (Sigma-Aldrich, USA), W-7 (calmodulin inhibitor) (Enzo Life Sciences, USA), in vitro LPS (from E. coli O111:B4; InvivoGen, USA), in vivo LPS (E. coli serotype O127:B8; Sigma Aldrich), CD11b+ cell isolation kit and LS columns (Miltenyi Biotec), RPMI 1640, DMEM, DMEM/F12, and fetal bovine serum (FBS) (Biowest). Pam3CSK4, FSL-1, poly(I:C), imiquimod, and ODN1826 (InvivoGen, USA).
DCs were stained with a fluorescein isothiocyanate (FITC)-conjugated CD11c DC surface antibody, as well as with phycoerythrin (PE)-conjugated CD40, CD80, and MHC-I (Biolegend, USA) DC maturation antibodies. The cells were analyzed using a FACS Calibur cytometer equipped with the BD CELL Quest Pro software.
CCI-induced sepsis model
A mouse model of CCI-induced sepsis was generated as previously described (23841524). The mice were euthanized by CO2 inhalation, cecectomy was performed, and cecal contents were extracted with a cotton swab into a petri dish. PBS was added to a final concentration of 20 mg/mL, which was then minced using ground glass. Cecal contents were passed through a 100-μm cell strainer to allow a smooth injection. The mice were then intraperitoneally injected with 1 mL of homogenized cecal contents. Each mouse was intraperitoneally injected with 5 mg/kg TFP 30 min before CCI.
LPS-induced endotoxemia model
Mice were intraperitoneally injected with 100 mg/kg of LPS (E. coli serotype O127:B8) (Sigma, USA) dissolved in PBS. Each mouse was intraperitoneally injected with 5 mg/kg TFP 30 min before LPS injection.
For the in vitro and in vivo cytokine analyses, the concentrations of the pro-inflammatory cytokines TNF-α and IL-6 and the anti-inflammatory cytokines IL-10 and TGF-β in cell supernatants or the serum were measured by using commercially obtained ELISA kits. The ELISA kits used to measure TNF-α, IL-6, and IL-10 were all purchased from eBioscience, whereas that used to measure TGF-β was purchased from Becton Dickinson. Each assay was carried out according to the instructions provided by the manufacturers.
In LPS-induced endotoxemia model and CCI-induced sepsis model, the serum levels of AST, ALT, and BUN were measured by the Konkuk University Hospital Automatic Hematology Analyzer.
Lungs were perfused with 4% paraformaldehyde immediately after being isolated from mice and maintained at 4 °C for 18 h. Fixed lung tissues were washed with distilled water for 2 h to remove paraformaldehyde (PFA). Tissue processing was done using an Auto Leica Tissue Processor 1020 (Leica Biosystems, Germany) which allowed automatic control of tissue infiltration, dehydration, and infiltration under vacuum. Lung tissues were perfused twice in formalin solution for 2 h each. Fixed lungs were then sequentially immersed in 70, 80, 90, and 100% ethanol. After the tissues were immersed in xylene, they were embedded in paraffin and cut into 7-μm-thick sections. Slides were stored for 18 h at 65 °C for deparaffinization. Tissues were hydrated by alcohol and rinsed with distilled water for 10 min. Tissues were then stained with hematoxylin (Merck, USA) and eosin (Merck). Images were captured with a digital camera (NikonDS-Ri1) coupled with a Nikon Eclipse Nimicroscope under × 20 magnification.
To measure the transcription level of cytokines, bone marrow DCs were incubated with LPS in the presence or absence of TFP and W7. After 4 h of LPS treatment, the cells were collected and centrifuged. Pellets were resuspended in 0.2 mL chloroform (Sigma-Aldrich) and 1 mL TRIzol Reagent (Invitrogen, USA). After 3 min of incubation, homogenized cells were centrifuged at 12,000 rpm for 15 min at 4 °C, and the top layer of aqueous phase was collected. RNA was precipitated with isopropanol and washed with 75% ethanol. Acquired RNA was subjected to reverse transcription to synthesize cDNA using PCR. PCR products were analyzed with the Light-Cycler 480 software (Roche) using SYBT Green dye to detect double-stranded DNA.
Western blotting analysis
Bone marrow DCs were incubated with LPS (50 ng/mL) in the presence or absence of TFP and W7 for 0, 10, 30, or 60 min. Cells were scraped, washed, centrifuged, and resuspended on ice in RIPA protein extraction solution [50 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 1 mmol/L phenylmethylsulphonyl fluoride (PMSF), 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40 (NP40), and 0.5 mmol/L EDTA; Elpis Biotech] for 1 h. Protein concentrations were determined by the Bradford protein assay. Proteins of an equal quantity were mixed with the SDS-PAGE loading buffer (250 mmol/L Tris-HCl, pH 6.8, 0.5 mol/L DTT, 10% SDS, 0.5% bromophenol blue, and 50% glycerol), boiled for 10 min, separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Ltd). The membranes were probed with mouse antibodies against JNK, p-JNK, p38, p-p38, ERK, p-ERK, IkB-α (Cell Signaling Technology), and β-actin (Sigma) diluted to 1:1000 in 5% bovine serum albumin and incubated with a goat anti-mouse IgG (Abbiotec) conjugated to horseradish peroxidase secondary antibodies (Enzo Life Sciences). Immunoreactive bands were visualized by enhanced chemiluminescence reaction.
DCs were incubated with TFP and W7 at various concentrations for 18 h. After treatment, the cells were stained with PE-conjugated CD11c DC surface antibody and FITC-conjugated AnnexinV with AnnexinV binding buffers for 15 min, and then analyzed using a FACS Calibur cytometer equipped with the BD CELL Quest Pro software.
[Ca2+] i measurement
BMDCs were incubated with 5 μM Fura-2 AM (intracellular Ca2+ level indicator (Thermo Fisher Scientific, Waltham, MA, USA)) for 30 min at room temperature. The incubated cells were then loaded into the patch-clamp chamber and washed in Normal Tyrode solution(143 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 0.5 mM MgCl2, 5 mM HEPES, 2 mM CaCl2, and 11 mM glucose (pH 7.4 with NaOH)) for 30 min at room temperature. For measurement for Fura-2, excitation and emission were at 340 and 510 nm (Lambda DG-4, Sutter Instrument Company, Navato, CA, USA). Origin 8.0 was used for data analysis.
Data presented in this study were obtained from one representative experiment of the two or three experiments performed. All data were presented as the mean ± standard deviation (SD) of three independent experiments. Individual data points were compared by the Student’s t test. Survival of mice was analyzed by the Kaplan–Meier method followed by the log-rank test. Analysis was performed using the SPSS software (version 22.0). Differences between groups were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2016R1A5A2012284 and NRF-2018R1A2B6008455). This study was also supported by a grant of Korea Health Technology R&D project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number HI15C2524).
Compliance with ethical standards
All animal procedures were approved by and performed according to guidelines of the Institutional Animal Care and Use Committee (IACUC) of Konkuk University.
Conflict of interest
The authors declare that they have no conflict of interest.
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