Blocking Effects Of The C-Met Inhibitors 17-AAG And Cabozantinib On Listeria Monocytogenes Infection In Mice

BackgroundListeria monocytogenes,a gram-positive facultative intracellular bacterium, was isolated from rats and rabbits with sepsis by Murray as early as 1926. L. Monocytogenes has strong resistance to the environment, so it exists in the nature of different environments widely including soil, plants, water, and contaminates food easily such as meat,milk,seafood. L.Monocytogenes infects immunocompromised patients mainly especially pregnant women and old men. It can lead to different types of human and animal listeriosis such as encephalitis, meningoencephalitis, abortion, sepsis, pneumonia, keratitis, among which the mortality incidence of infant,pregnancy and immune-tolerant patients reach up to 20%-30%.From 2001 to the present, the national China Inspection and Quarantine Bureau have detected L. Monocytogenes from cheese and meat imported from the United States, Canada, France and other countries.The main clinical manifestations of listeriosis include meningoencephalitis, septicemia,infection during pregnancy,neonatal septic granuloma.Meningoenc ephalitis and septicemia are the most common among the non-pregnant immunocompromised adults. L. Monocytogenes has been listed as one of the four foodborne pathogenic bacteria in the 1990s and the second leading cause of bacterial meningitis in patients younger than 1 month or older than 60 years. In addition, L. monocytogenes is one of the major pathogens causing community-acquired bacterial meningitis in adults in the developed countries. Human listeriosis infection of the CNS can manifest in many ways, including meningitis, rhombencephalitis, and choriomeningitis. Despite the availability of effective bactericidal antibiotics, intracellular bacterial infections including neurolisteriosis remain a severe threat. The main reasons are the inability of the drugs to reach the offending organisms and the current relative ignorance of the host antimicrobial activities. Therefore, the generation of new anti-infectives against intracellular pathogens has emerged as an urgent issue in the therapeutics of L. monocytogenes infection.c-Met, a protein product encoded by c-met proto-oncogene,is the human hepatocyte growth factor/scatter factor(HGF/SF) of mesenchymal origin with PTK activity and related to many products of oncogenes and regulator proteins.Microbial pathogens are able to invade host tissues by inducing their own uptake and transport across normally protective tissue barriers. It is possible that some pathogens engage in biologic mimicry by producing a molecule that resembles a natural host ligand, for which there is a host cell receptor. This phenomenon designated "bacteria-directed transcytosis," has been observed for the bacterial invasion of both epithelial and endothelial cells. L. monocytogenes can through three barriers of the host-intestinal barrier, blood-brain barrier and placental barrier, survive and reproduce in the professional or non-professional phagocytes after invade host. The process of infection include:internalization, escape from the phagocytic vacuoles, polarity movement and cell-to-cell transmission of L. monocytogenes within the host cell. L. monocytogenes enters the host cell under the action of virulence factors related to adhesion and invasion. Internalins,one of them, can identify relevant receptors on the host cell. InlA, InlB combine with specific receptors by LRRs. InlA interacts with E-cadherin that mediate bacteria to enter the epithelial cell; InlB mediates bacteria to cross liver cells, fibroblasts and epithelial cell by binding to complement component Clq or hepatocyte growth factor receptor (Met). InlB binding to Met in lipid rafts induces Cb1, Gab1, Shc, Grb2 protein and phosphoinositide (PI) 3-kinase of p85/p110I family aggregation, then PI-3 kinase convert PI (4,5) P2 (PIP2) to PI(3,4,5)P3(PIP3), which prompt an unknown protein aggregation to activate the Rac that activate LIM kinase indirectly, regulate polymerizing and depolymerizing of actin and mediate LM internalization.Therefore targeting the infection mediated by InlB is very important for developing new treatment against LM intracellular infections specifically.Geldanamycin(GA) is a prototype drug in the family of benzoquinone ansamycin antibioticshas, which has been shown to disrupt the c-Met-HGF/SF axis in cancer cells and specifically block tyrosine phosphorylation of c-Met, is specific inhibitors of c-Met signal signaling pathway. HGF/SF-c-Met pathways involve in the occurrence and metastasis of human tumors, and are equally important in the process of L. monocytogenes invasion of host cell. Antitumor activity of GA has been shown in cancer xenografts, but dose-dependent lethal liver toxicity and gastrointestinal damage and nephrotoxicity in vivo precluded its further development. GA analogues, such as 17-AAG and 17-DMAG, represent a class of drugs capable of affecting multiple targets in the signaling cascades involved in tumor cell proliferation and survival.This class of drug is able to specifically block tyrosine phosphorylation of c-Met and disrupt the function of the chaperone protein Hsp90, which has been shown to be involved in oncogenesis and many pathophysiological conditions including microbial infections.17-AAG and 17-DMAG exert similar antitumour activity by the same mechanism, but have higher blood levels and lesser hepatotoxicity, which are potential inhibitors of c-Met signaling pathways and were selected for drug development.Cabozantinib is an orally bioavailable tyrosine kinase inhibitor, has approved for the treatment metastatic medullary thyroid cancer by the US Food and Drug Administration(FDA). Cell and animal experiments have confirmed that cabozantinib can modulate Met abnormal signal pathway singularly which inhibiting cancer cells proliferation. Recent clinical studies have shown that cabozantinib can treat medullary thyroid carcinoma and prostate cancer and has safe clinical dose.This study intends to establish the L. monocytogenes infected mouse model,the survival curves were dramn,and the number of bacteria was determined;serum IL-10 and NF-κB p65 level were assayed, and Evans Blue content and pathological changes in brain were examined,to verify blocking effect of c-Met inhibitors 17-AAG and cabozantinib on listeria infection in vivo and explore the feasibility of c-Met inhibitors as drug for L. monocytogenes infection.ObjectiveThe goal is to define whether c-Met inhibitors can efficiently block intracellular L. monocytogenes infection in mice using animal models and explore the feasibility of c-Met inhibitors as drug for L. monocytogenes infection, and look for a new therapeutic strategy of infectious disease.MethodsBlocking effects of 17-AAG on L. monocytogenes infection in mice1. Analysis of survival curveMice were randomized into four groups with eight in each group:17-AAG group, ampicillin group,17-AAG in combination with ampicillin group,PBS control group. Mice received treatment with intraperitoneal of 17-AAG(5μg/g), intraperitoneal injection of ampicillin(20μg/g),17-AAG plus ampicillin,or intraperitoneal injection of equal PBS,6,12,24h after intraperitoneal injection of 1×106CFU L. monocytogenes. Then mice were observed for clinical symptoms and survival for five days.2. Bacterial loads in blood,brain,liver,spleen(1)The dose-dependent effect24 mice were randomly randomized into four 17-AAG(2-10μg/g body weight) dose groups with six in each group. Mice received L. monocytogenes (5×105 CFU) by intra-peritoneal injection. Then 17-AAG treatments started from 2 hours before and 24 hours after bacterial inoculation by intra-peritoneal injection.48 hours after injection, animals were anaesthetized with 200μl of 10% chloral hydrate, and 200μl of blood samples were collected from heart puncture for bacterial culture using BHI plates after double dilution. After perfusion from heart puncture, the skull was opened, brain tissues were extracted and homogenized by tissue grinders. The homogenates was serially diluted and plated on BHI agar for determination of CFUs in brain tissues. Liver and spleen were extracted and homogenized in 1 ml of PBS. The homogenates was serially diluted and plated on BHI agar for determination of CFUs. The dead mice within 48 h should dissect immediately and take its blood, brain, liver, spleen.(2)The synergy24 mice were randomized into four groups with six in each group:17-AAG group, ampicillin group,17-AAG plus ampicillin group,PBS group. Mice were injected (i.p.) with 17-AAG (5μg/g) and Amp (20μg/g) alone, or 17-AAG in combination with Amp. Drugs were injected at 2h,6h and 24h respectively after bacterial infection (5×105 CFU of L. monocytogenes). The bacterial loads in blood, brain,liver and spleen were determined.3. Count of circulating brain microvascular endothelial cells(cBMECs)(1)The dose-dependent effectMice were randomized into four 17-AAG dose groups.Different concentrations of 17-AAG were injected (i.p.) into mice twice at 2h before and at 24h after L. monocytogenes (5×105 CFU) inoculation.48 hours after injection,mice were dissected.Tke heart blood and crack red blood cells. Beads were prepared according to the manufacturer’s instructions and resuspended in Hanks’ balanced salt solution (HBSS) plus 5% fetal calf serum (HBSS+5%FCS) to a final concentration of 4×108 beads/ml. Endothelial cells from blood samples were isolated by absorption to Ulex-coated beads and detached from the beads by fucose. Then endothelial cells were adhered again to MSFD2-coated beads. These endothelial cells were positive for CD146, demonstrating their endothelial origin, and also expressed MSFD2 indicating their brain origin. To count the cBMECs from blood samples, cells adhered to MSFD2-coated beads were labeled with PE-conjugated CD 146 antibody and transferred to glass splices by cytospin for counting under a fluorescence microscope.(2)The synergyMice were randomized into four groups:17-AAG group,ampicillin group,17-AAG plus ampicillin group,PBS group. Mice were injected (i.p.) with 17-AAG (5μg/g) and Amp (20μg/g) alone, or 17-AAG in combination with Amp. Drugs were injected at 2h,6h and 24h respectively after bacterial infection (5×105CFU of L. monocytogenes).48 hours after injection,take heart blood and count cBMECs.4. The examination of Evans Blue(EB) in brain(1)The dose-dependent effectMice were randomized into different 17-AAG dose groups.The ways of infection and treatment were the same as 2(1).Mice were injected intraperitoneally with EB(50μg/g). Three hours after receiving EB, animals were anaesthetized and perfused with 30 ml pre-cooling PBS by heart puncture. Then mouse skull was opened and the brain sample was put into a tube with 1 ml formamide to extract the EB. After incubation at 50℃ for 48h, all the tubes were centrifuged at 5200rpm for 10 min. Then the supernatants were collected, and measured at 620 nm by spectrophotometry to determine the OD of the extracted EB. The EB contents of the samples were quantified by comparing to the standard curve.(2)The synergyMice were randomized into four groups:17-AAG group,ampicillin group,17-AAG plus ampicillin group,PBS group.The ways of infection and treatment were the same as 2(2).Mice were injected intraperitoneally with EB three hours before dissection. The EB contents of the samples were quantified.5. The assay of NF-κB p65 in CSF(1)The dose-dependent effectMice were randomized into different 17-AAG dose groups.The ways of infection and treatment were the same as 2(1).The brain tissues in mice were removed after dissection. CSF samples were collected by washing the brain tissues with 200μl of PBS. CSF samples containing more than 10 erythrocytes per μl were discarded as contaminated samples and then p65 in CSF samples were determined using the NF-κB p65 ELISA kit.(2)The synergyMice were randomized into four groups:17-AAG group,ampicillin group,17-AAG plus ampicillin group,PBS group.The ways of infection and treatment were the same as 2(2).48 hours after infection,p65 in CSF samples were determined.6. IL-10 assayMice were randomized into four groups:17-AAG group,ampicillin group,17-AAG plus ampicillin group,PBS group.The ways of infection and treatment were the same as 2(2).48 hours after infection, blood were collected and centrifuged at 5200rpm for 15 min. Serum were collected within 30 minutes.IL-10 level were determined using the IL-10 ELISA kit.7. Histological analysis of liverLiver of mice in PBS group and 10μg/g 17-AAG group were collected,and fixed in 10% formaldehyde. After washing, dehydration, transparent, paraffin, embedding, block, slicing, patch, baking sheets,HE dyeing, sealing,the liver were observed histopathologically under microscope.Blocking effects of cabozantinib on L. monocytogenes infection in mice1.Survival curve analysisMice were randomized into four groups with nine in each group:cabozantinib group,ampicillin group,cabozantinib plus ampicillin group,PBS group.Mice received treatment with intragastric administration of cabozantinib(20μg/g), intraperitoneal injection of ampicillin(20μg/g),cabozantinib plus ampicillin,or intraperitoneal injection of equal PBS,6,12,24 hours after intraperitoneal injection of 1×106 CFU L. monocytogenes. Then mice were observed for clinical symptoms and survival for five days.2.Bacterial loads in blood and brain(1)The dose-dependent effectMice were randomly randomized into four cabozantinib dose groups with five in each group. Different concentrations of cabozantinib were given twice at 2h before and at 24h after L. monocytogenes (5×105CFU) inoculation. Animals were anaesthetized with 200μl of 10% chloral hydrate, and 200μl of blood samples were collected from heart puncture for bacterial culture using BHI plates after double dilution at 48h after infection. After perfusion from heart puncture, the skull was opened, brain tissues were extracted and homogenized by tissue grinders. The homogenates was serially diluted and plated on BHI agar for determination of CFUs in brain tissues.(2)The synergy24 mice were randomized into cabozantinib group, ampicillin group, cabozantinib plus ampicillin group,PBS group. Drugs were injected at 2h,6h and 24h respectively after bacterial infection (5×105 CFU of L. monocytogenes). Mice received treatment with intragastric administration of cabozantinib(20μg/g), intraperitoneal injection of ampicillin(20μg/g),cabozantinib plus ampicillin,or intraperitoneal injection of equal PBS.48h after infection,animals were dissected.Blood and brain were collected for bacterial culture using BHI plates.3.Count of circulating brain microvascular endothelial cellsMice were randomized into cabozantinib group,ampicillin group, group and PBS control group. The ways of infection and treatment were the same as 2(2).48 hours after infection,mice were dissected. Red blood cells in blood were disrupted. Beads were prepared according to the manufacturer’s instructions and resuspended in Hanks’ balanced salt solution (HBSS) plus 5% fetal calf serum (HBSS+5%FCS) to a final concentration of 4×108 beads/ml. Endothelial cells from blood samples were isolated by absorption to Ulex-coated beads and detached from the beads by fucose. Then endothelial cells were adhered again to MSFD2-coated beads. These endothelial cells were positive for CD146, demonstrating their endothelial origin, and also expressed MSFD2 indicating their brain origin. To count the cBMECs from blood samples, cells adhered to MSFD2-coated beads were labeled with PE-conjugated CD146 antibody and transferred to glass splices by cytospin for counting under a fluorescence microscope.4. IL-10 assayMice were randomized into four groups:cabozantinib group,ampicillin group, cabozantinib plus ampicillin group,PBS group.The ways of infection and treatment were the same as 2(2).48 hours after infection, blood were collected and centrifuged at 5200rpm for 15 min. Serum were collected within 30 minutes.IL-10 level were determined using the IL-10 ELISA kit.5.The assay of NF-κB p65 in CSFMice were randomized into cabozantinib group,ampicillin group, cabozantinib plus ampicillin group and PBS control group. The ways of infection and treatment were the same as 2(2).The brain tissues in mice were removed after dissection. CSF samples were collected by washing the brain tissues with 200μl of PBS. CSF samples containing more than 10 erythrocytes per μl were discarded as contaminated samples and then p65 in CSF samples were determined using the NF-κB p65 ELISA kit.6.The examination of Evans Blue in brainMice were randomized into cabozantinib group,ampicillin group, group and PBS control group. The ways of infection and treatment were the same as 2(2).Three hours after receiving EB, animals were anaesthetized and perfused with 30 ml pre-cooling PBS by heart puncture. Then mouse skull was opened and the brain sample was put into a tube with 1 ml formamide to extract the EB. After incubation at 50℃ for 48h. all the tubes were centrifuged at 5200rpm for 10 min. Then the supernatants were collected, and measured at 620 nm by spectrophotometry to determine the OD of the extracted EB. The EB contents of the samples were quantified by comparing to the standard curve.7.Histological analysis of the brain tissuesThe brain tissues were collected,and fixed in 10% formaldehyde. After washing, dehydration, transparent, paraffin, embedding, block, slicing, patch, baking sheets,HE dyeing, sealing,the brain tissues were observed histopathologically under microscope.Statistical analysisData were expressed as mean±standard deviation. Comparation between two groups was treated with t-test. Data were analyzed using GraphPad Prism 5 software. P< 0.05 was considered statistically significant differences. The level of test is alpha =0.05.ResultsBlocking effects of 17-AAG on L. monocytogenes infection in mice1.Compared with PBS-treated mice,the mice treated with 17-AAG,ampicillin, 17-AAG plus ampicillin showed a higher survival rate.2.17-AAG could dose-dependently suppress proliferation of LM in blood, brain, liver, spleen and decrease cBMECs quantity in blood, EB content in brain tissue and NF-κBp65 content in CSF.3.The blood,brain,liver and spleen bacteria counts, cBMECs quantity,the EB level in brain,the content of NF-κB p65 in CSF were all significantly lower in mice treated with the combination of drugs than in mice treated with one drug alone.4.Compared with PBS-treated mice,the pathological changes of liver in mice treated with 17-AAG were milder.Blocking effects of cabozantinib on L. monocytogenes infection in mice1.Compared with PBS-treated mice,the mice treated with cabozantinib,ampicillin, cabozantinib plus ampicillin showed a higher survival rate.2.Cabozantinib could dose-dependently suppress proliferation of LM in blood and brain.3.The blood and brain bacteria counts, cBMECs quantity,the EB level in brain,the content of NF-κB p65 in CSF were all significantly lower in mice treated with the combination of drugs than in mice treated with one drug alone.4.Compared with PBS-treated mice,the pathological changes of brain in mice treated with the combination of drugs or one drug alone were milder. The brain in mice treated with the combination of drugs only have little inflammatory cells.ConclusionIn a word,the tyrosine kinase inhibitors 17-AAG and cabozantinib could decrease bacterial loads of L. monocytogenes in mice to some extent.17-AAG and cabozantinib could block bacteremia and meningitis and may be synergistic to some extent in combination with ampicillin.