Inhibition of Inducible Nitric Oxide Controls Pathogen Load and Brain Damage by Enhancing Phagocytosis of Escherichia coli K1 in Neonatal Meningitis
2010; Elsevier BV; Volume: 176; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.090851
ISSN1525-2191
AutoresRahul Mittal, Ignacio González-Gómez, Kerstin Goth, Nemani V. Prasadarao,
Tópico(s)Neonatal and fetal brain pathology
ResumoEscherichia coli K1 is a leading cause of neonatal meningitis in humans. In this study, we sought to determine the pathophysiologic relevance of inducible nitric oxide (iNOS) in experimental E. coli K1 meningitis. By using a newborn mouse model of meningitis, we demonstrate that E. coli infection triggered the expression of iNOS in the brains of mice. Additionally, iNOS−/− mice were resistant to E. coli K1 infection, displaying normal brain histology, no bacteremia, no disruption of the blood–brain barrier, and reduced inflammatory response. Treatment with an iNOS specific inhibitor, aminoguanidine (AG), of wild-type animals before infection prevented the development of bacteremia and the occurrence of meningitis. The infected animals treated with AG after the development of bacteremia also completely cleared the pathogen from circulation and prevented brain damage. Histopathological and micro-CT analysis of brains revealed significant damage in E. coli K1–infected mice, which was completely abrogated by AG administration. Peritoneal macrophages and polymorphonuclear leukocytes isolated from iNOS−/− mice or pretreated with AG demonstrated enhanced uptake and killing of the bacteria compared with macrophages and polymorphonuclear leukocytes from wild-type mice in which E. coli K1 survive and multiply. Thus, NO produced by iNOS may be beneficial for E. coli to survive inside the macrophages, and prevention of iNOS could be a therapeutic strategy to treat neonatal E. coli meningitis. Escherichia coli K1 is a leading cause of neonatal meningitis in humans. In this study, we sought to determine the pathophysiologic relevance of inducible nitric oxide (iNOS) in experimental E. coli K1 meningitis. By using a newborn mouse model of meningitis, we demonstrate that E. coli infection triggered the expression of iNOS in the brains of mice. Additionally, iNOS−/− mice were resistant to E. coli K1 infection, displaying normal brain histology, no bacteremia, no disruption of the blood–brain barrier, and reduced inflammatory response. Treatment with an iNOS specific inhibitor, aminoguanidine (AG), of wild-type animals before infection prevented the development of bacteremia and the occurrence of meningitis. The infected animals treated with AG after the development of bacteremia also completely cleared the pathogen from circulation and prevented brain damage. Histopathological and micro-CT analysis of brains revealed significant damage in E. coli K1–infected mice, which was completely abrogated by AG administration. Peritoneal macrophages and polymorphonuclear leukocytes isolated from iNOS−/− mice or pretreated with AG demonstrated enhanced uptake and killing of the bacteria compared with macrophages and polymorphonuclear leukocytes from wild-type mice in which E. coli K1 survive and multiply. Thus, NO produced by iNOS may be beneficial for E. coli to survive inside the macrophages, and prevention of iNOS could be a therapeutic strategy to treat neonatal E. coli meningitis. Bacterial meningitis is a fatal and serious infection of the central nervous system (CNS).1Somand D Meurer W Central nervous system infections.Emerg Med Clin North Am. 2009; 27: 89-100Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 2Dawson KG Emerson JC Burns JL Fifteen years of experience with bacterial meningitis.Pediatr Infect Dis J. 1999; 18: 816-822Crossref PubMed Scopus (106) Google Scholar The underlying pathophysiological mechanisms implicated in the development of meningitis are quite complex and remain poorly understood. 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In this study, we established a mouse of model of E. coli meningitis using C57BL/6 mice and used iNOS−/− mice to determine the role of NO in experimental E. coli K1 meningitis. A compelling observation of these studies is that lack of iNOS expression is protective against E. coli–induced meningitis, and treating the animals with iNOS inhibitor completely prevented E. coli–induced brain damage by enhancing the killing of bacteria by macrophages and reducing the pathogen load. E. coli E44 (OmpA+ E. coli) is a rifampicin-resistant mutant of E. coli K1 strain RS 218 (serotype O18:K1:H7), which was isolated from the cerebrospinal fluid of a neonate with meningitis and invades human brain microvascular endothelial cells in vitro.37Prasadarao NV Wass CA Weiser JN Stins MF Huang SH Kim KS Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells.Infect Immun. 1996; 64: 146-153Crossref PubMed Google Scholar Bacteria were grown in Luria broth (LB; DIFCO Laboratories) with appropriate antibiotics. Antibodies to iNOS, eNOS, and β actin were obtained from BD Biosciences (San Diego, CA) and Santa Cruz Biotechnology (San Diego, CA), respectively. All other reagents were purchased from Sigma. Antibodies to CD64, CR3 were obtained from BD Biosciences (San Diego, CA) whereas TLR4 and TLR2 were obtained from Imgenex (San Diego, CA). Anti-gp96 antibodies were generated in our laboratory as described earlier.37Prasadarao NV Wass CA Weiser JN Stins MF Huang SH Kim KS Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells.Infect Immun. 1996; 64: 146-153Crossref PubMed Google Scholar Breeding pairs of C57BL/6 iNOS−/− mice and wild-type C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). The colony was maintained by intercrossing iNOS−/− male and female (homozygous) mice. Offspring was tested by PCR of tail DNA for the presence or absence of iNOS gene. The animal studies were approved by the IACUC of CHLA and followed National Institutes of Health guidelines for the performance of animal experiments. Three-day-old mouse pups were randomly divided into various groups and infected intranasally with 103 cfu of bacteria. Control mice received pyrogen free saline through the same route. To assess the effect of aminoguanidine (AG) on E. coli meningitis, AG (10 mg/kg body weight) was injected intraperitoneally (IP) 24 hours postinfection and two more similar doses at 6-hour intervals after initial dose. In another group, mice were pretreated with AG via IP injection (10 mg/kg body weight). A total of four doses were given at an interval of 12 hours in a two-day period before infection. Blood was collected from the tail or facial vein at different time periods of postinfection, dilutions were made, and plated on rifampicin LB agar plates to ensure bactremia and success of infection. CSF samples were collected aseptically under anesthesia by cis-ternal puncture and directly inoculated into broth containing antibiotics. Mice were perfused by intracardiac route with 0.9% saline to remove blood and contaminating intravascular leukocytes. Brain, liver, spleen, lung, kidney, and intestine were aseptically removed and homogenized in sterile PBS. Bacterial counts in these organs were determined by plating ten-fold serial dilutions on rifampicin LB agar plates. Growth of E. coli in rifampicin containing LB broth from CSF samples was considered positive for meningitis. Half of the brain was fixed in 10% buffered formalin, routinely processed, and embedded in paraffin.38Mittal R Wang Y Hunter CJ Gonzalez-Gomez I Prasadarao NV Brain damage in newborn rat model of meningitis by Enterobacter sakazakii: a role for outer membrane protein A.Lab Invest. 2009; 89: 263-277Crossref PubMed Scopus (70) Google Scholar Four- to 5-μm sections were cut using Leica microtome and stained with hematoxylin and eosin (H&E) or prepared for immunohistochemistry. Pictures were taken using Zeiss Axiovert Microscope connected to a JVC 3-chip color video camera and read by the pathologist in a blinded fashion. For immunohistochemistry staining, the paraffin was extracted from the sections with a 5-minute wash in 100% xylene. Samples were treated for 10 minutes with 70% ethanol, followed by 5-minute treatments each with 95% ethanol, 100% ethanol, and distilled water. Sections were microwaved at high temperature in antigen unmasking solution for 2 to 3 minutes (Vector Laboratories, Burlingame, CA) and then allowed to cool for antigen retrieval. Endogenous peroxidases were quenched by incubation with 7.5% hydrogen peroxidase in water for 5 minutes. Samples were incubated with PBST (0.1 M PBS plus 0.01% triton X100) containing 5% normal donkey serum to block non-specific binding followed by incubation with a 1:1000 dilution of iNOS antibody for 30 minutes. Samples were then rinsed with PBST containing 1% normal donkey serum, incubated with a 1:500 dilution of goat anti-rabbit IgG (IgG)-biotin for 30 minutes and then again, rinsed with PBST. The bound antibody was visualized by probing with streptavadin-horseradish peroxidase followed by diaminobenzidine and hydrogen peroxide, which yields a brown reaction product. Counterstaining was performed using Mayer hematoxylin. Concentration of nitrite in the brain tissues was measured as an index for NO production. Equal weights of the brains of infected mice were homogenized in sterile PBS (1 ml), supernatants were collected, and analyzed for NO production by modified Greiss method as described earlier.39Mittal R Sharma S Chhibber S Harjai K Contribution of free radicals to Pseudomonas aeruginosa induced acute pyelonephritis.Microb Pathog. 2008; 45: 323-330Crossref PubMed Scopus (21) Google Scholar Briefly, nitrate was converted to nitrites with β-nicotinamide adenine dinucleotide phosphate (NADPH; 1.25 mg/ml) and nitrate reductase followed by addition of Griess reagent. The reaction mixture was incubated at room temperature for 20 minutes followed by addition of TCA. Samples were centrifuged, clear supernatants were collected, and optical density was recorded at 550 nm. The amounts of NO produced were determined by calibrating a standard curve using sodium nitrite. To detect the expression of iNOS by flow cytometry, brains were removed, transferred to ice-cold Hanks buffer/3% fetal calf serum, homogenized using a glass potter, and passed through a stainless-steel sieve. The dissociated sample was collected by centrifugation and was digested for 60 minutes at 37°C with 1.4 ml of 0.75% (w/v) with type II collagenase (0.95 unit/mg, Sigma) and 104 units of DNase I (Sigma) in dissociation buffer (42 mmol/L MgCl2/23 mmol/L CaCl2/50 mmol/L KCI/153 mmol/L NaCl). The digested sample was pelleted and resuspended in PBS. The cells were first preincubated for 30 minutes with IgG blocking buffer to mask nonspecific binding sites, fixed with 2% paraformaldehyde, and permeabilized using BD cytofix and cytoperm kit. Cells were then incubated with iNOS or an isotype control antibody for 30 minutes at 4°C and then washed with BD permwash buffer. FITC-conjugated secondary antibody was then added, incubated for 20 minutes at 4°C, and washed with permwash buffer. The stained cells were then analyzed by flow cytometry using FACS calibur Cell Quest Pro software (BD Biosciences, San Diego, CA), and at least 10,000 events were collected for analysis. Results are expressed as mean fluorescence intensity subtracted from isotype matched control. For detection of iNOS and eNOS by RT-PCR, frozen brains from infected wild-type, iNOS−/−, and control mice were cut with a cryostat. Total RNA was isolated from frozen sections containing lateral ventricles and hippocampal tissue with TRIZOL-LS-reagent (Gibco BRL, Gaithersburg, MD). RNA was quantified using a nanodrop machine. RT-PCR for iNOS and eNOS was performed using the following primer sequences: 5′ (sense) 5′-GCCTCGCTCTGGAAAGA-3′; 3′ (antisense) 5′-TCCATGCA GACAACCTT-3′ and 5′ (sense) 5′-CAGTGTCCAACATGCTGCTGGAAATTG-3′; 3′(antisense) 5′-TAAAGGTCTTCTTCCTGGTGATGCC-3′, respectively. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, using the following primer sequences: GAPDH sense, 5′-CATCACCATCTTC CAGGAGCG-3′ and GAPDH antisense, 5′-GAGGGGCCATCCACAGTCTTC-3′. Negative controls without RT were performed in parallel for every reaction, to exclude amplification of contaminating DNA. The amplified products were separated on a 1% agarose gel and were stained with ethidium bromide. The gels were photographed and optical densities were determined by using a computer imaging analysis system (Visitron Systems GmbH). RT-PCR was performed in duplicates, and the mean density of PCR products was determined, to calculate the ratio to GAPDH. The BBB permeability was quantitatively evaluated by detection of extravasated Evans blue dye.40Lu B Figini M Emanueli C Geppetti P Grady EF Gerard NP Ansell J Payan DG Gerard C Bunnett N The control of microvascular permeability and blood pressure by neutral endopeptidase.Nat Med. 1997; 3: 904-907Crossref PubMed Scopus (140) Google Scholar Briefly, 2% Evans blue dye in saline was injected intraperitoneally, and after 4 hours mice were deeply anesthetized with Nembutal and transcardially perfused until colorless perfusion fluid was obtained from the right atrium. After decapitation, brain tissue was removed, weighed, and homogenized. The supernatant was obtained by centrifugation, and protein concentration was determined. Evans blue intensity was determined by a microplate reader at 550 nm. Calculations were based on the external standards dissolved in the same solvent. The amount of extravasated Evans blue dye was quantified as micrograms per milligram protein. To perform bactericidal assays using whole blood, 12 μl of 100 mmol/L CaCl2 in the presence of heparin (2 U/ml) was added to 1 ml of citrated blood and aliquots of blood were incubated with bacteria (106 cells) at 37°C for varying periods. The experiments were repeated for at least five times in triplicates. Peritoneal macrophages were isolated from mice according to the method of Mittal et al.41Mittal R Sharma S Chhibber S Harjai K Iron dictates the virulence of Pseudomonas aeruginosa in urinary tract infections.J Biomed Sci. 2008; 15: 731-741Crossref PubMed Scopus (27) Google Scholar Briefly, mice peritoneal cavity was exposed carefully without disrupting blood vessels, and 2 to 3 ml RPMI was slowly injected. The lavage was sucked back and cultured in tissue culture flasks for 2 hours at 37°C under 5% CO2 to allow adherence of macrophages. The nonadherent cells were removed and flasks were washed three times with Hanks solution. The adherent cells were harvested from the flask by using a rubber policeman and were resuspended in 10% FCS-RPMI 1640 medium. Macrophages were then positively selected using Miltenyi biotech kit and percentage purity was examined by FACS analysis using F4/80 antibody, which was >95%. Viability of macrophages after interaction with bacteria was assessed by Annexin V kit according to manufacturer's instructions (BD Biosciences, San Diego, CA). Polymorphonuclear leukocytes (PMNs) were isolated from mice as described previously.42Luo Y Dorf ME Isolation of mouse neutrophils.Curr Protoc Immunol. 2001; Chapter 3: Unit 3.20PubMed Google Scholar The purity of PMNs was examined by flow cytometry using Gr-1 antibody, which was >95%. The expression of TLR2, TLR4, CR3, FcγI (CD64), and GP96 on the surface of wild-type, iNOS−/− and AG pretreated macrophages (0.5 mmol/L) or isolated from infected mice was determined by flow cytometry. In brief, macrophages were infected with bacteria for six hours and then washed. The cells were first preincubated for 30 minutes with IgG blocking buffer to mask nonspecific binding sites and then fixed using BD cytofix reagent. Cells were then incubated with antibodies to TLR2, TLR4, CR3, CD64, and GP96 or an isotype control antibody for 30 minutes at 4°C and then washed with PBS. Then respective FITC conjugated secondary antibodies were added, incubated for 20 minutes at 4°C, and washed with PBS. The stained cells were then analyzed by flow cytometry using FACS calibur Cell Quest Pro software (BD Biosciences, San Diego, CA), and at least 10,000 events were collected for analysis. Macrophages were gated using F4/80 as a gating marker. Results are expressed as mean fluorescence intensity subtracted from isotype matched and respective uninfected controls. The formalin-fixed specimens were sent to Numira Biosciences (Salt Lake City, UT) for micro-CT imaging. Specimens were stained with a proprietary contrast agent. A high-resolution volumetric micro-CT scanner (μCT40 ScanCo Medical, Zurich, CH) was used to scan the tissue with the following parameters: 10 μm isometric voxel resolution at 200 ms exposure time, 2000 views and 10 frames per view. The micro-CT generated DICOM files were converted into a file format compatible with the image processing software applications. Using Teem (Version 1.10.0; http://teem.sourceforge.net/), SCIRun (Scientific and Computing Imaging Institute, University of Utah), and Numira proprietary software tools, images of the samples were generated. The levels of TNF-α, IL-1β, IL-6, and IL-12p70 were measured in blood and brain homogenates of mice pups infected with E. coli using ELISA kits from Biosource (Carlsbad, CA) according to the manufacturer's instructions. For statistical analysis of the data, two-tailed Fisher test, Wilcoxon signed rank test, and Student t test was applied, and P values <0.05 were considered statistically significant. Previous studies from our lab have utilized a newborn rat model of meningitis by infecting the rat pups by intracardiac route in which the pathology of the brain mimics human brain pathology attributable to E. coli meningitis.43Prasadarao NV Wass CA Kim KS Endothelial cell GlcNAc beta 1–4GlcNAc epitopes for outer membrane protein A enhance traversal of Escherichia coli across the blood-brain barrier.Infect Immun. 1996; 64: 154-160Crossref PubMed Google Scholar Because the rat model circumvents several bacterial interactions with host tissues during the initial stages of infection, we set out to establish a mouse model of meningitis by intranasal infection in this study, such as to take advantage of various knockout animals for subsequent investigations. The brain pathology of wild-type mice infected with E. coli revealed inflammatory infiltrates of PMNs in the leptomeningeal and ventricular spaces (Figure 1A). Hippocampus showed severe inflammation and apoptosis of neurons in the Ammon horn. Acute hemorrhage, most prominent in the white matter of the brain, was observed. Cortex and molecular layer showed increased cellularity attributable to inflammatory exudates. Similar results were also observed with rat pups infected with E. coli intranasally (see Supplemental Figure S1 at http://ajp.amjpathol.org), indicating that mouse and rat brain damage induced by E. coli is very similar. All animals infected with E. coli were positive for meningitis by 72 hours postinfection and succumbed between 96 and 120 hours, whereas animals infected with a nonvirulent strain HB101 showed no meningitis and survived (Table 1). In addition, significantly higher NO production in infected brains was observed in comparison with the brains of uninfected mice or animals inoculated with saline as assessed by Griess reagent (Figure 1B; P < 0.01 by Student t test). NO levels increased from 23 μg/g brain tissue at 12 hours to 80 μg/g brain tissue by 72 hours postinfection. Flow cytometry analysis of brain tissue homogenates revealed a fourfold increase in the expression of iNOS at 72 hours compared with the levels at 12 hours postinfection (Figure 1C). To examine whether there was an increase in iNOS transcription during the infection, RNA was extracted from brain tissues and subjected to
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