Receptor Blockade: A Novel Approach to Protect the Brain From Pneumococcal Invasion

Receptor Blockade: A Novel Approach to Protect the Brain From Pneumococcal Invasion Abstract Background Pneumococci are the major cause of bacterial meningitis globally. To cause meningitis pneumococci interact with the 2 endothelial receptors, polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), to penetrate the blood-brain barrier (BBB) and invade the brain. Methods C57BL/6 mice were infected intravenously with bioluminescent pneumococci, and treated with ceftriaxone (1 hour postinfection) and anti-pIgR and PECAM-1 antibodies (1 or 5 hours postinfection), then monitored for 5 and 10 days. Bacterial brain invasion was analyzed using IVIS imaging and bacterial counts. Results Ceftriaxone, given early after pneumococcal challenge, cleared pneumococci from the blood but not from the brain. After combining ceftriaxone with receptor blockade, using anti-pIgR and PECAM-1 antibodies, we found 100% survival after 5 and 10 days of infection, in contrast to 60% for ceftriaxone alone. Combined antibiotic and antibody treatment resulted in no or few viable bacteria in the brain and no microglia activation. Antibodies remained bound to the receptors during the study period. Receptor blockade did not interfere with antibiotic permeability through the BBB. Conclusions We suggest that adjunct treatment with pIgR and PECAM-1 antibodies to antibiotics may prevent pneumococcal meningitis development and associated brain damages. However, further evaluations are required. antibody treatment, PECAM-1, pIgR, pneumococcal meningitis, Streptococcus pneumoniae Streptococcus pneumoniae (the pneumococcus) is a major cause of bacterial meningitis globally, and approximately 100 000 cases are reported annually [1–3]. Pneumococcal meningitis is mainly caused by bacteria that penetrate the blood-brain barrier (BBB) and invade the brain [4, 5]. Despite access to antimicrobial agents and introduction of conjugated pneumococcal vaccines (PCVs), mortality remains high at 10%–40% [1, 3]. Pneumococcal meningitis is treated with antibiotics, preferably β-lactams. In the pre-antibiotic era, mortality rates were close to 100% [6]. Introduction of PCVs has decreased the incidence of pneumococcal meningitis caused by vaccine-type strains, but the incidence of invasive pneumococcal disease caused by nonvaccine-types has increased [7–9]. Meningitis symptoms, such as rash, severe headache, and impaired consciousness, usually develop late when the disease is already at an advanced stage [10, 11]. Among survivors of childhood meningitis, approximately 50% develop chronic sequelae such as behavioral and/or intellectual disorders, hearing loss, or other neurological deficiencies [12]. Hence, early interventions that minimize bacterial entry into the brain are crucial to enhance survival of the patients and to minimize brain damage of survivors. Pneumococci penetrate the BBB by binding to the polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1) expressed on the brain endothelium [5, 13]. Using a bacteremia-derived meningitis model, we recently reported that blocking pIgR, and/or PECAM-1, using knockout mice and/or antibodies to the 2 receptors, led to a significant reduction of bacteria in the brain compared with untreated wild-type mice after 14 hours of systemic infection [13]. In this study, we investigated the therapeutic effect of one-time treatment with antibodies against the 2 receptors, in a long-term pneumococcal infection for up to 10 days. METHODS Pneumococcal Strains and Growth Conditions The bioluminescent S pneumoniae strain TIGR4 Xenogen 35 (PerkinElmer) was used in the in vivo mouse experiments. Pneumococci were grown in Todd-Hewitt broth with 0.5% yeast extract at 37°C, as previously described [13, 14]. Mouse Experiments Animal experiments were approved by the local ethical committee (Stockholms Norra djurförsöksetiska nämnd). The bacteremia-derived meningitis model was performed as previously described [13, 14]. We used C57BL/6 mice, 6–7 weeks old (obtained from Charles River, Sulzfeld, Germany), for all experiments. A total of 200 µL 5 × 107 colony-forming units (CFU) pneumococci was injected intravenously into the tail vein, and survival of mice was monitored for either 5 or 10 days according to the local approved ethical regulations. After sacrifice, unattached bacteria in the blood stream were removed by perfusion with sterile phosphate-buffered saline (PBS) [13, 14]. Bioluminescent signals from the brain of mice were then imaged with the IVIS Spectrum Imaging System. For antibody treatment in mice, 20 µg/mL antibodies (anti-pIgR and PECAM-1 antibodies were obtained from Abcam, catalog numbers ab170321 and ab28364, respectively) were administered intravenously, in a volume of 200 µL, either 1 hour or 5 hours after pneumococcal challenge. Ceftriaxone ([Sigma-Aldrich] 100 mg/kg) was administered intravenously, either alone or in combination with antibodies, 1 hour after pneumococcal challenge. Antibodies and ceftriaxone were diluted in sterile PBS as previously described [13]. To test the possible interference of anti-pIgR and PECAM-1 antibodies in antibiotic penetration of the BBB, C57BL/6 mice, 6–7 weeks old, were either systemically treated with ceftriaxone only or ceftriaxone combined with anti-pIgR and PECAM-1 antibodies (same dose of antibiotic and antibody concentrations as stated above). Clinical symptoms were monitored for 12 hours after treatment (either with ceftriaxone or with ceftriaxone combined with antibodies) according to the local approved ethical regulations. Quantification of IVIS-Bioluminescent and Iba-1-Fluorescent Signals Bioluminescence signals from IVIS imaging analysis and fluorescent signal of the microglial marker Iba-1 were quantified using Image J as previously described [13]. In brief, bioluminescent signals were selected using the function Image-Adjust-Color Threshold. For the bioluminescence signal of the IVIS imaging, RGB Profile Plot was generated for each image taken with the IVIS Spectrum System, and intensities of blue/green/red colors, mild/severe/very severe infection, respectively, were measured separately. For the fluorescence signal of Iba-1, RGB Profile Plot was generated for each image taken in each group and in each time-point postinfection, and intensities of the red colors were measured separately. Antibodies, Lectin, and Isotype Controls Immunofluorescence detection was performed using antibodies diluted in sterile PBS with 5% fetal calf serum (Biochrom). For detection of the BBB, endothelium DyLight 594-labeled LEL lectin (Vector Laboratories, catalog number DL-1177) diluted 1:200 was used [13–15]. For detection of mouse PECAM-1 and pIgR, a rabbit antimouse PECAM-1 antibody (Abcam, catalog number ab28364) and a rat antimouse pIgR antibody (Abcam, catalog number ab170321) both diluted 1:50 were used. For the detection of microglia, a goat anti-Iba-1 antibody (Abcam, catalog number ab5076) diluted 1:100 was used. The primary antibodies to detect pIgR and PECAM-1 were used in combination with the secondary antibodies Alexa Fluor 350 Goat anti-Rabbit (for the detection of PECAM-1) and Alexa Fluor 488 Goat anti-Rat (for the detection of pIgR) both diluted 1:500 (Thermo Fisher Scientific, catalog numbers A-11046 and A-11006, respectively). The primary antibody to detect Iba-1 was used in combination with the secondary antibody Alexa Fluor 488 Donkey anti-Goat (Thermo Fisher Scientific, catalog number A-11055). As isotype controls, rabbit IgG (Innovative Research), rat IgG (Sigma-Aldrich), and goat IgG (Santa Cruz Biotechnology, catalog number sc-2028) were used at the same dilution as those for the primary antibodies, no fluorescent signal was detected. Immunofluorescence Stainings and High-Resolution Microscopy Imaging Slides with 5-µm brain cryosections were fixed with acetone for 10 minutes and dried. Incubations with the antibodies and lectin were performed for 1 hour, in the dark in case of fluorophore-labeled antibodies. Slides were washed in PBS twice for 5 minutes between each incubation step. At the end of the staining, a 5 µL drop of Vectashild solution (Vector Laboratories) was added to each section and a coverslip was applied. For Iba-1 staining of microglia (results shown in Figure 3A and C), 10 µm brain cryosections were used. Microscopic analysis was performed using a Delta Vision Elite high-resolution microscope (Applied Precision). For the imaging analysis shown in Figure 3A, a 100× objective was used to gain the highest magnification combined with the highest resolution possible. For the imaging analysis shown in Figures 3C and 4A, a 20× objective was used to gain a general comprehensive overview of microglia and receptor signals. For each microscopy analysis, brain sections were imaged using the same imaging setup (percentage laser intensity and exposure time). The z-stacks images were acquired using a scientific complementary metal-oxide-semiconductor camera and processed with Softworx imaging program (Applied Precision). Quantification of Receptor Signal Intensity From Immunofluorescence Images Signal intensity from immunofluorescence images was performed as previously described [14]. In brief, using the Threshold function of Image J, we measured the area covered by the DyLight 594-labeled LEL lectin, brain endothelium marker, and the area of either pIgR or PECAM-1 receptors to determine the area occupied by the 488 nm (receptors) signal and 594 nm (brain vasculature) signals, respectively. We calculated the receptor to brain endothelium ratio by dividing the surface of the 488 nm signal by the total area of the brain endothelium of the 59 nm signal. Tissue sections from 3 mice were analyzed for each group, and we used 6 brain sections per mice. Ten images were taken from each brain section. The averages of each group were calculated for the final quantification and statistical analysis. Antibiotic-Susceptibility Testing of Brain Homogenates and Minimum Inhibitory Concentration Analysis for Ceftriaxone Streptococcus pneumoniae strain TIGR4 Xenogen 35 was streaked onto blood-agar plates. Whatman paper discs (Sigma-Aldrich) were applied on the agar. Brain homogenates from mice treated either with ceftriaxone or ceftriaxone combined with anti-pIgR and PECAM-1 antibodies were pipetted on the paper discs, and, as positive control, 2 mL ceftriaxone solution (100 mg/kg, the same concentration as that used in the mouse experiments [see the “Mouse Experiments” section]) was used; PBS was used as negative control. Plates were incubated overnight at 37°C with 5% CO2, and zones of bacterial growth inhibition were analyzed. For minimum inhibitory concentration (MIC) analysis using E-test, a bacterial suspension in PBS made by harvesting fresh colonies of S pneumoniae Xenogen 35 was streaked on a blood-agar plate (14 cm diameter), and an E-strip of the antibiotic ceftriaxone was placed on the agar surface. The plate was incubated overnight at 37°C with 5% CO2 and then analyzed. Statistical Analysis For multiple comparisons, the nonparametric analysis of variance test was used to assess differences between groups, then the Dunn’s test was used to make pairwise comparisons. For 2 group comparisons, the non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as the Mann-Whitney test) was used (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001). RESULTS Receptor Blockade Confers 100% Protection Towards Systemic Pneumococcal Infection and Prevents Bacterial Invasion of the Brain To study the therapeutic effect of antibody treatment after pneumococcal challenge, we first used our bacteremia-derived meningitis model [13, 14] with an infection lasting for 5 days, using the pneumococcal strain of serotype 4, TIGR4 Xenogen 35, and gave antibodies to pIgR and PECAM-1 one time, 1 hour post pneumococcal challenge (Figure 1A). Bacterial invasion of the brain was monitored by calculating the number of CFU in the brain tissue and detection and quantification of the bioluminescent signal generated by luminescent bacteria in the brain (Figure 2A and B). Mice treated with antibodies alone showed a longer survival than mice without treatment, but they reached high bacteremia levels at the second day of infection and succumbed (Figure 1A and Supplementary Figure S1A). At the time of sacrifice, mice that only received antibodies had a 10-fold lower number of bacteria (CFU) in the brain compared with untreated mice (Figure 2A) and lower bioluminescent signals (Figure 2B). Figure 1. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies combined with ceftriaxone treatment of mice infected with pneumococci intravenously led to 100% survival. Survival of mice infected with pneumococci intravenously and treated with anti-pIgR and PECAM-1 antibodies 1 hour postinfection after (A) 5 days and (B) 10 days of infection. Ten mice in each experimental group were used in 2 experiments with 5 mice/group. Figure 1. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies combined with ceftriaxone treatment of mice infected with pneumococci intravenously led to 100% survival. Survival of mice infected with pneumococci intravenously and treated with anti-pIgR and PECAM-1 antibodies 1 hour postinfection after (A) 5 days and (B) 10 days of infection. Ten mice in each experimental group were used in 2 experiments with 5 mice/group. Figure 2. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies prevent pneumococcal invasion of the brain. Measurements of pneumococcal invasion of the brain tissue after intravenous pneumococcal challenge (survival experiment in Figure 1) after (A–C) 5 days and (D–F) 10 days of infection. (A and C) Amount of pneumococci (colony-forming units [CFU]/mL) in the brain of mice at time of sacrifice, (A) 5 days postinfection, and (C) 10 days after infection, in red mice that did not survive and in green mice that survived. The non-parametric analysis of variance test was used to assess differences between the groups, and the Dunn’s test was used to make pairwise comparisons (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001). (B and D) IVIS bioluminescent signals of pneumococci in the brain of mice at the time of sacrifice and relative quantification (B) 5 days and (D) 10 days postinfection. Within red borders mice that did not survive, and within green borders mice that survived. Abbreviation: n. d., not detected. Figure 2. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies prevent pneumococcal invasion of the brain. Measurements of pneumococcal invasion of the brain tissue after intravenous pneumococcal challenge (survival experiment in Figure 1) after (A–C) 5 days and (D–F) 10 days of infection. (A and C) Amount of pneumococci (colony-forming units [CFU]/mL) in the brain of mice at time of sacrifice, (A) 5 days postinfection, and (C) 10 days after infection, in red mice that did not survive and in green mice that survived. The non-parametric analysis of variance test was used to assess differences between the groups, and the Dunn’s test was used to make pairwise comparisons (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001). (B and D) IVIS bioluminescent signals of pneumococci in the brain of mice at the time of sacrifice and relative quantification (B) 5 days and (D) 10 days postinfection. Within red borders mice that did not survive, and within green borders mice that survived. Abbreviation: n. d., not detected. Ceftriaxone is a third-generation cephalosporin that has been used previously in mice models to treat meningitis [13, 16, 17]. We next studied the effect of ceftriaxone treatment using the pneumococcal strain TIGR4, which is susceptible to ceftriaxone and has an MIC value of 0.032 µg/mL. A single ceftriaxone dose of 100 mg/kg, the same concentration as used previously in experimental meningitis mouse models [13, 17], given 1 hour after intravenous challenge with TIGR4, cleared pneumococci from the blood in 6 of 10 animals that survived after 5 days. However, at sacrifice, these mice all had viable bacteria in the brain (5 × 103 to 104 CFU/ mL), and a luminescence signal from the brain was detected (Figure 2A and B). Four of the antibiotic only treated mice succumbed before 5 days of infection with high bacteremia levels (Supplementary Figure S1A), and high numbers of bacteria were found in the brain (1–5 × 105 CFU/mL) (Figure 2A and B). We then administered anti-pIgR and PECAM-1 antibodies together with ceftriaxone and observed that all 10 mice survived (Figure 1A). Six of the mice showed no signs of pneumococcal invasion of the brain (Figure 2A and B). The remaining 4 animals had bacteria in the brain but in lower numbers compared with the surviving mice treated with only ceftriaxone (Figure 2A). Notably, when anti-pIgR and PECAM-1 antibodies were administered 5 hours postinfection (4 hours post ceftriaxone), all mice survived, but the numbers of bacteria in the brain were slightly higher compared with when the antibodies were given together with ceftriaxone 1 hour postinfection (Figure 2A and B). The result suggests that the time of administration will influence the amount of bacteria invading the brain and the effect of antibody treatment. Furthermore, we investigated the effect of antibody treatment in a longer term infection of 10 days. Untreated mice and mice that received only antibodies showed similar survival rates to that observed after the 5-day infection (Figure 1B). Four of the 10 ceftriaxone only treated mice succumbed between day 3 and 4 with high bacteremia levels (Figure 1B and Supplementary Figure S1B), and high numbers of pneumococci were found in the brain (3–9 × 105 CFU/mL) (Figure 2C and D). The 6 ceftriaxone-treated mice that survived also showed viable bacteria in the brain (5 × 102 to 5 × 103 CFU/mL) at sacrifice, and a high luminescence signal from the brain was detected (Figure 2C and D). All 10 mice that received ceftriaxone combined with anti-PECAM-1 and pIgR antibodies survived (Figure 1B). Eight mice had no viable bacteria, and 2 mice showed a very low number of bacteria in the brain (1–3 × 101 CFU/ mL) (Figure 2C), which were not detected using the IVIS Imaging System (Figure 2D). Receptor Blockade Reduces Neuroinflammation We then asked whether blockade of the 2 endothelial receptors prevents neuroinflammation. Microglia, the resident macrophages of the brain, quickly react towards invading pathogens undergoing activation [18]. Activated microglia change their morphology in comparison to resting microglia; their central soma becomes rounder and bigger, and the long branches become thicker and shorter [14]. Using high-resolution and high-magnification immunofluorescence microscopy, we found that at 14 hours postinfection, the brain of untreated mice showed severe neuroinflammation, and all microglia detected displayed a round soma with all cellular branches completely retracted (Figure 3A). At the same time point, brains of mice treated with antibodies showed some activated microglia but also microglia that displayed long and thin cell processes, indicating a less pronounced neuroinflammation compared with untreated mice (Figure 3A). Forty percent of the mice (4 out of 10) that were treated with ceftriaxone only, and that succumbed on day 3–4, showed severe neuroinflammation (Figure 3B). Moreover, at the same time point, the brains of mice treated with ceftriaxone combined with anti-pIgR and PECAM-1 antibodies showed normal microglia, indicating no major signs of neuroinflammation (Figure 3A). After the 10-day infection, we found among survivors some activated microglia in the brains of mice that were treated only with ceftriaxone, whereas mice treated with ceftriaxone combined with anti-pIgR and PECAM-1 antibodies showed normal microglia with no signs of activation (Figure 3A). Figure 3. View largeDownload slide Receptor blockade reduces microglia activation and neuroinflammation. Brain sections were collected after intravenous challenge of mice with pneumococci and used for high-magnification (100× objective) microscopy imaging to analyze the morphological changing during microglial activation. (A) The panel “14 hours” shows brain sections of untreated mice and of mice treated with antibodies to polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1, 14 hours postinfection. Untreated mice displayed activated microglia with round soma and complete retraction of cellular branches (red arrows), whereas brains of mice treated with antibodies showed both activated (red arrows) and normal microglia (blue arrows). The panel “Day 3–4” shows brain tissue section of mice treated with ceftriaxone only (nonsurvivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 3–4 days postinfection. Mice treated with ceftriaxone displayed severe microgliosis (red arrows), whereas the brain of mice that received ceftriaxone combined with antibodies showed normal microglia with no major signs of neuroinflammation (blue arrows). The panel “Day 10” shows brain tissue section of mice treated with ceftriaxone only (survivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 10 days postinfection. Mice treated with ceftriaxone only showed some activated microglia (red arrows), whereas no signs of microgliosis (blue arrows) were observed in brains from mice treated with ceftriaxone combined with antibodies. The black scale bar represents 5 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (B) Brain sections collected after intravenous challenge of mice with pneumococci at the same time points as in A and used for low magnification (20× objective) microscopy imaging to analyze Iba-1 signal intensity as marker of microglial activation. Low magnification imaging was performed to gain a comprehensive overview of microglial activation of the mice. The black scale bar represents 25 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (C) Quantification of Iba-1 signal intensity in the brain of the mice in the groups and time points shown in B. Each column and bar in the graph represent the average and standard deviation values calculated using all Iba-1 signal intensities measured in each image taken for each group and each time point postinfection, respectively. For 2-group comparison, the non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used (*, P < .05; **, P < .01). Abbreviation: n.s., nonsignificant. Figure 3. View largeDownload slide Receptor blockade reduces microglia activation and neuroinflammation. Brain sections were collected after intravenous challenge of mice with pneumococci and used for high-magnification (100× objective) microscopy imaging to analyze the morphological changing during microglial activation. (A) The panel “14 hours” shows brain sections of untreated mice and of mice treated with antibodies to polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1, 14 hours postinfection. Untreated mice displayed activated microglia with round soma and complete retraction of cellular branches (red arrows), whereas brains of mice treated with antibodies showed both activated (red arrows) and normal microglia (blue arrows). The panel “Day 3–4” shows brain tissue section of mice treated with ceftriaxone only (nonsurvivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 3–4 days postinfection. Mice treated with ceftriaxone displayed severe microgliosis (red arrows), whereas the brain of mice that received ceftriaxone combined with antibodies showed normal microglia with no major signs of neuroinflammation (blue arrows). The panel “Day 10” shows brain tissue section of mice treated with ceftriaxone only (survivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 10 days postinfection. Mice treated with ceftriaxone only showed some activated microglia (red arrows), whereas no signs of microgliosis (blue arrows) were observed in brains from mice treated with ceftriaxone combined with antibodies. The black scale bar represents 5 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (B) Brain sections collected after intravenous challenge of mice with pneumococci at the same time points as in A and used for low magnification (20× objective) microscopy imaging to analyze Iba-1 signal intensity as marker of microglial activation. Low magnification imaging was performed to gain a comprehensive overview of microglial activation of the mice. The black scale bar represents 25 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (C) Quantification of Iba-1 signal intensity in the brain of the mice in the groups and time points shown in B. Each column and bar in the graph represent the average and standard deviation values calculated using all Iba-1 signal intensities measured in each image taken for each group and each time point postinfection, respectively. For 2-group comparison, the non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used (*, P < .05; **, P < .01). Abbreviation: n.s., nonsignificant. To quantify microglia activation, we performed low-magnification imaging of microglia using brain sections of mice from the same groups and time points postinfection, as shown in Figure 3A. As previously described, activation of microglia is also characterized by upregulation of the microglial marker Iba-1 [14, 19]. Detection of Iba-1 (Figure 3B) and quantification of the intensity of the Iba-1 signal (Figure 3C) confirmed the results obtained using high-magnification imaging (Figure 3A). Fourteen hours postinfection, antibody-treated mice showed a lower Iba-1 signal intensity than untreated mice (Figure 3B and C). Mice that did not survive after ceftriaxone treatment 3–4 days postinfection showed a very high Iba-1 signal intensity compared with mice treated with ceftriaxone combined with antibodies (Figure 3B and C), and all of these mice survived. The Blockade of the Receptors In Vivo Is Long Lasting The low number of bacteria in the brain after 1 single dose of antibody treatment, despite high numbers of bacteria in the blood stream, suggests that the receptor blockade is long lasting. To investigate whether both anti-pIgR and PECAM-1 antibodies remained bound to the brain endothelium at sacrifice, we performed immunofluorescence staining on brain sections from the mice used in the 5-day infection experiment. Brain sections from mice that had been treated with antibodies were stained with only the secondary antibodies to detect bound PECAM-1 and pIgR antibodies, respectively. As positive control, brain sections from untreated mice were stained with primary antibodies to detect pIgR and PECAM-1, followed by the respective secondary antibodies. Finally, negative control brain sections from untreated mice were stained with only secondary antibodies. Staining with secondary antibodies showed similar pIgR and PECAM-1 signals with only a minor decrease of the signal intensity to the level that was observed for both receptors in the positive control sections (Figure 4A and B), indicating that both receptors were still blocked after 5 days treatment with only 1 single dose of antibodies. As expected, staining with the secondary antibodies of brain sections from untreated mice resulted in no fluorescent signal (Figure 4A and B). Figure 4. View largeDownload slide Blockade of the 2 endothelial receptors with antibodies is long lasting. To study whether anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies remained bound to the brain endothelium at sacrifice, brain sections from uninfected mice (positive control) and from mice treated with antibodies were stained only with secondary antibodies, supposing that the primary antibodies were already bound to PECAM-1 and pIgR. (A) Signals of PECAM-1 (blue) and pIgR (green) were detected on the blood-brain barrier endothelium (red) 5 days after antibody treatment (antibody treatment + secondary antibodies). Mice that did not receive antibodies showed no fluorescent signal (negative control). The black scale bar represents 25 µm. Nine brain sections from 5 mice that received ceftriaxone combined with antibodies and 9 brain sections from 5 untreated mice were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken. (B) Quantification of the intensity of the receptor signal shows that the signal was comparable to the one observed for both receptors in the positive control. Each column and bar in the graph represent the average and standard deviation values calculated using the intensity signal measured in each image taken (among all brain tissue sections analyzed for each group), respectively. Only a minor decrease was detected, which was not statistically significant (n. s.).The non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used. Figure 4. View largeDownload slide Blockade of the 2 endothelial receptors with antibodies is long lasting. To study whether anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies remained bound to the brain endothelium at sacrifice, brain sections from uninfected mice (positive control) and from mice treated with antibodies were stained only with secondary antibodies, supposing that the primary antibodies were already bound to PECAM-1 and pIgR. (A) Signals of PECAM-1 (blue) and pIgR (green) were detected on the blood-brain barrier endothelium (red) 5 days after antibody treatment (antibody treatment + secondary antibodies). Mice that did not receive antibodies showed no fluorescent signal (negative control). The black scale bar represents 25 µm. Nine brain sections from 5 mice that received ceftriaxone combined with antibodies and 9 brain sections from 5 untreated mice were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken. (B) Quantification of the intensity of the receptor signal shows that the signal was comparable to the one observed for both receptors in the positive control. Each column and bar in the graph represent the average and standard deviation values calculated using the intensity signal measured in each image taken (among all brain tissue sections analyzed for each group), respectively. Only a minor decrease was detected, which was not statistically significant (n. s.).The non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used. Receptor Blockade Does Not Interfere With Antibiotic Penetration Across the Blood-Brain Barrier Furthermore, we assessed whether administration of anti-pIgR and PECAM-1 antibodies interferes with the penetration of antibiotics across the BBB. Uninfected mice were either treated with ceftriaxone only or ceftriaxone combined with anti-pIgR and PECAM-1 antibodies and sacrificed after 12 hours, and brain homogenates were used in an antibiotic-sensitivity test. Brain homogenates from mice that received only ceftriaxone were able to inhibit pneumococcal growth on blood-agar plates (Supplementary Figure S2A). Brain homogenates from mice that received ceftriaxone combined with antibodies resulted in similar-sized inhibition zones, suggesting that receptor blockade does not interfere with the ceftriaxone permeability of the BBB (Supplementary Figure S2B and S2C). DISCUSSION Our previous findings suggest that pneumococcal entry from the blood to the brain is a relatively slow process, because only a fraction of circulating small single cocci in the blood stream seems capable of invading the BBB [14]. In this study, we found that ceftriaxone treatment combined with antibodies led to a lower number of bacteria in the brain in the 10-day infection than in the 5-day infection. This lower number could partly be explained by the poor nutrient environment in the brain that reduced bacterial growth rates, but it could also be explained by the action of microglia, which, similar to macrophages, kill invading pathogens. Ceftriaxone, similar to other β-lactam antibiotics, only act on growing bacteria [20, 21]. This may explain why we obtained clearance of bacteria from the blood with only 1 single dose of ceftriaxone, without eradicating those that had entered into the brain. Our data show that a single-dose treatment with anti-pIgR and PECAM-1 antibodies in combination with ceftriaxone led to 100% survival and abolished bacterial invasion of the brain and development of lethal meningitis, which potentially could reduce the risk for future chronic sequelae. It is interesting to note that the same dose of ceftriaxone was more successful in reducing the bacteremia levels when administered in combination with anti-pIgR and PECAM-1 antibodies (Supplementary Figure S1A and B). Pneumococcal trafficking across the BBB leads to inflammatory responses in the brain [22]. Inflammation in the central nervous system leads the recruitment of immune cells (such as leukocytes) to the sites of infection [22]. We hypothesize that when the pIgR and PECAM-1 receptors are not blocked, pneumococci in the blood stream may invade the brain and other organs together with recruited immune cells, thereby potentially reducing the number of immune cells in the circulation and therefore leading to less pneumococcal clearance from the blood. In contrast, we speculate that when the receptors are blocked, pneumococcal brain invasion is reduced, and the immune cells remain in the blood, thereby potentially clearing the infection. CONCLUSIONS In a clinical perspective, receptor blockade may be an approach to reduce bacterial invasion into the brain, because the antibody blockade appears to be long lasting. Early antibody blockade may protect the brain while providing a longer time window for proper choice of antibiotic and antibiotic eradication of bacteria from the blood stream, and thereby increase survival and potentially reduce the risk of neurological sequelae. However, because interventions were administered rapidly after infection in this study, implications for preventing meningitis progression and sequelae will require further evaluation in animal models using antibody administration repeatedly and at later time points. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We thank professor Staffan Normark for scientific discussions. We also thank Kenth Andersson for technical assistance during the mouse experiments and Anna Granlund (veterinary) for medical consult concerning the use of ceftriaxone in the mouse experiments. Finanical support. This work was funded by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Swedish Foundation for Strategic Research, and Stockholm County Council. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. 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Lutsar I, Ahmed A, Friedland IR, et al.   Pharmacodynamics and bactericidal activity of ceftriaxone therapy in experimental cephalosporin-resistant pneumococcal meningitis. Antimicrob Agents Chemother  1997; 41: 2414– 7. Google Scholar PubMed  21. Satta G, Cornaglia G, Foddis G, Pompei R. Evaluation of ceftriaxone and other antibiotics against Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae under in vitro conditions simulating those of serious infections. Antimicrob Agents Chemother  1998; 32: 552– 60. Google Scholar CrossRef Search ADS   22. Prager O, Friedman A, Nebenzahl YM. Role of neural barriers in the pathogenesis and outcome of Streptococcus pneumoniae meningitis. Exp Ther Med  2017; 13: 799– 809. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Receptor Blockade: A Novel Approach to Protect the Brain From Pneumococcal Invasion

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Abstract

Abstract Background Pneumococci are the major cause of bacterial meningitis globally. To cause meningitis pneumococci interact with the 2 endothelial receptors, polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), to penetrate the blood-brain barrier (BBB) and invade the brain. Methods C57BL/6 mice were infected intravenously with bioluminescent pneumococci, and treated with ceftriaxone (1 hour postinfection) and anti-pIgR and PECAM-1 antibodies (1 or 5 hours postinfection), then monitored for 5 and 10 days. Bacterial brain invasion was analyzed using IVIS imaging and bacterial counts. Results Ceftriaxone, given early after pneumococcal challenge, cleared pneumococci from the blood but not from the brain. After combining ceftriaxone with receptor blockade, using anti-pIgR and PECAM-1 antibodies, we found 100% survival after 5 and 10 days of infection, in contrast to 60% for ceftriaxone alone. Combined antibiotic and antibody treatment resulted in no or few viable bacteria in the brain and no microglia activation. Antibodies remained bound to the receptors during the study period. Receptor blockade did not interfere with antibiotic permeability through the BBB. Conclusions We suggest that adjunct treatment with pIgR and PECAM-1 antibodies to antibiotics may prevent pneumococcal meningitis development and associated brain damages. However, further evaluations are required. antibody treatment, PECAM-1, pIgR, pneumococcal meningitis, Streptococcus pneumoniae Streptococcus pneumoniae (the pneumococcus) is a major cause of bacterial meningitis globally, and approximately 100 000 cases are reported annually [1–3]. Pneumococcal meningitis is mainly caused by bacteria that penetrate the blood-brain barrier (BBB) and invade the brain [4, 5]. Despite access to antimicrobial agents and introduction of conjugated pneumococcal vaccines (PCVs), mortality remains high at 10%–40% [1, 3]. Pneumococcal meningitis is treated with antibiotics, preferably β-lactams. In the pre-antibiotic era, mortality rates were close to 100% [6]. Introduction of PCVs has decreased the incidence of pneumococcal meningitis caused by vaccine-type strains, but the incidence of invasive pneumococcal disease caused by nonvaccine-types has increased [7–9]. Meningitis symptoms, such as rash, severe headache, and impaired consciousness, usually develop late when the disease is already at an advanced stage [10, 11]. Among survivors of childhood meningitis, approximately 50% develop chronic sequelae such as behavioral and/or intellectual disorders, hearing loss, or other neurological deficiencies [12]. Hence, early interventions that minimize bacterial entry into the brain are crucial to enhance survival of the patients and to minimize brain damage of survivors. Pneumococci penetrate the BBB by binding to the polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1) expressed on the brain endothelium [5, 13]. Using a bacteremia-derived meningitis model, we recently reported that blocking pIgR, and/or PECAM-1, using knockout mice and/or antibodies to the 2 receptors, led to a significant reduction of bacteria in the brain compared with untreated wild-type mice after 14 hours of systemic infection [13]. In this study, we investigated the therapeutic effect of one-time treatment with antibodies against the 2 receptors, in a long-term pneumococcal infection for up to 10 days. METHODS Pneumococcal Strains and Growth Conditions The bioluminescent S pneumoniae strain TIGR4 Xenogen 35 (PerkinElmer) was used in the in vivo mouse experiments. Pneumococci were grown in Todd-Hewitt broth with 0.5% yeast extract at 37°C, as previously described [13, 14]. Mouse Experiments Animal experiments were approved by the local ethical committee (Stockholms Norra djurförsöksetiska nämnd). The bacteremia-derived meningitis model was performed as previously described [13, 14]. We used C57BL/6 mice, 6–7 weeks old (obtained from Charles River, Sulzfeld, Germany), for all experiments. A total of 200 µL 5 × 107 colony-forming units (CFU) pneumococci was injected intravenously into the tail vein, and survival of mice was monitored for either 5 or 10 days according to the local approved ethical regulations. After sacrifice, unattached bacteria in the blood stream were removed by perfusion with sterile phosphate-buffered saline (PBS) [13, 14]. Bioluminescent signals from the brain of mice were then imaged with the IVIS Spectrum Imaging System. For antibody treatment in mice, 20 µg/mL antibodies (anti-pIgR and PECAM-1 antibodies were obtained from Abcam, catalog numbers ab170321 and ab28364, respectively) were administered intravenously, in a volume of 200 µL, either 1 hour or 5 hours after pneumococcal challenge. Ceftriaxone ([Sigma-Aldrich] 100 mg/kg) was administered intravenously, either alone or in combination with antibodies, 1 hour after pneumococcal challenge. Antibodies and ceftriaxone were diluted in sterile PBS as previously described [13]. To test the possible interference of anti-pIgR and PECAM-1 antibodies in antibiotic penetration of the BBB, C57BL/6 mice, 6–7 weeks old, were either systemically treated with ceftriaxone only or ceftriaxone combined with anti-pIgR and PECAM-1 antibodies (same dose of antibiotic and antibody concentrations as stated above). Clinical symptoms were monitored for 12 hours after treatment (either with ceftriaxone or with ceftriaxone combined with antibodies) according to the local approved ethical regulations. Quantification of IVIS-Bioluminescent and Iba-1-Fluorescent Signals Bioluminescence signals from IVIS imaging analysis and fluorescent signal of the microglial marker Iba-1 were quantified using Image J as previously described [13]. In brief, bioluminescent signals were selected using the function Image-Adjust-Color Threshold. For the bioluminescence signal of the IVIS imaging, RGB Profile Plot was generated for each image taken with the IVIS Spectrum System, and intensities of blue/green/red colors, mild/severe/very severe infection, respectively, were measured separately. For the fluorescence signal of Iba-1, RGB Profile Plot was generated for each image taken in each group and in each time-point postinfection, and intensities of the red colors were measured separately. Antibodies, Lectin, and Isotype Controls Immunofluorescence detection was performed using antibodies diluted in sterile PBS with 5% fetal calf serum (Biochrom). For detection of the BBB, endothelium DyLight 594-labeled LEL lectin (Vector Laboratories, catalog number DL-1177) diluted 1:200 was used [13–15]. For detection of mouse PECAM-1 and pIgR, a rabbit antimouse PECAM-1 antibody (Abcam, catalog number ab28364) and a rat antimouse pIgR antibody (Abcam, catalog number ab170321) both diluted 1:50 were used. For the detection of microglia, a goat anti-Iba-1 antibody (Abcam, catalog number ab5076) diluted 1:100 was used. The primary antibodies to detect pIgR and PECAM-1 were used in combination with the secondary antibodies Alexa Fluor 350 Goat anti-Rabbit (for the detection of PECAM-1) and Alexa Fluor 488 Goat anti-Rat (for the detection of pIgR) both diluted 1:500 (Thermo Fisher Scientific, catalog numbers A-11046 and A-11006, respectively). The primary antibody to detect Iba-1 was used in combination with the secondary antibody Alexa Fluor 488 Donkey anti-Goat (Thermo Fisher Scientific, catalog number A-11055). As isotype controls, rabbit IgG (Innovative Research), rat IgG (Sigma-Aldrich), and goat IgG (Santa Cruz Biotechnology, catalog number sc-2028) were used at the same dilution as those for the primary antibodies, no fluorescent signal was detected. Immunofluorescence Stainings and High-Resolution Microscopy Imaging Slides with 5-µm brain cryosections were fixed with acetone for 10 minutes and dried. Incubations with the antibodies and lectin were performed for 1 hour, in the dark in case of fluorophore-labeled antibodies. Slides were washed in PBS twice for 5 minutes between each incubation step. At the end of the staining, a 5 µL drop of Vectashild solution (Vector Laboratories) was added to each section and a coverslip was applied. For Iba-1 staining of microglia (results shown in Figure 3A and C), 10 µm brain cryosections were used. Microscopic analysis was performed using a Delta Vision Elite high-resolution microscope (Applied Precision). For the imaging analysis shown in Figure 3A, a 100× objective was used to gain the highest magnification combined with the highest resolution possible. For the imaging analysis shown in Figures 3C and 4A, a 20× objective was used to gain a general comprehensive overview of microglia and receptor signals. For each microscopy analysis, brain sections were imaged using the same imaging setup (percentage laser intensity and exposure time). The z-stacks images were acquired using a scientific complementary metal-oxide-semiconductor camera and processed with Softworx imaging program (Applied Precision). Quantification of Receptor Signal Intensity From Immunofluorescence Images Signal intensity from immunofluorescence images was performed as previously described [14]. In brief, using the Threshold function of Image J, we measured the area covered by the DyLight 594-labeled LEL lectin, brain endothelium marker, and the area of either pIgR or PECAM-1 receptors to determine the area occupied by the 488 nm (receptors) signal and 594 nm (brain vasculature) signals, respectively. We calculated the receptor to brain endothelium ratio by dividing the surface of the 488 nm signal by the total area of the brain endothelium of the 59 nm signal. Tissue sections from 3 mice were analyzed for each group, and we used 6 brain sections per mice. Ten images were taken from each brain section. The averages of each group were calculated for the final quantification and statistical analysis. Antibiotic-Susceptibility Testing of Brain Homogenates and Minimum Inhibitory Concentration Analysis for Ceftriaxone Streptococcus pneumoniae strain TIGR4 Xenogen 35 was streaked onto blood-agar plates. Whatman paper discs (Sigma-Aldrich) were applied on the agar. Brain homogenates from mice treated either with ceftriaxone or ceftriaxone combined with anti-pIgR and PECAM-1 antibodies were pipetted on the paper discs, and, as positive control, 2 mL ceftriaxone solution (100 mg/kg, the same concentration as that used in the mouse experiments [see the “Mouse Experiments” section]) was used; PBS was used as negative control. Plates were incubated overnight at 37°C with 5% CO2, and zones of bacterial growth inhibition were analyzed. For minimum inhibitory concentration (MIC) analysis using E-test, a bacterial suspension in PBS made by harvesting fresh colonies of S pneumoniae Xenogen 35 was streaked on a blood-agar plate (14 cm diameter), and an E-strip of the antibiotic ceftriaxone was placed on the agar surface. The plate was incubated overnight at 37°C with 5% CO2 and then analyzed. Statistical Analysis For multiple comparisons, the nonparametric analysis of variance test was used to assess differences between groups, then the Dunn’s test was used to make pairwise comparisons. For 2 group comparisons, the non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as the Mann-Whitney test) was used (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001). RESULTS Receptor Blockade Confers 100% Protection Towards Systemic Pneumococcal Infection and Prevents Bacterial Invasion of the Brain To study the therapeutic effect of antibody treatment after pneumococcal challenge, we first used our bacteremia-derived meningitis model [13, 14] with an infection lasting for 5 days, using the pneumococcal strain of serotype 4, TIGR4 Xenogen 35, and gave antibodies to pIgR and PECAM-1 one time, 1 hour post pneumococcal challenge (Figure 1A). Bacterial invasion of the brain was monitored by calculating the number of CFU in the brain tissue and detection and quantification of the bioluminescent signal generated by luminescent bacteria in the brain (Figure 2A and B). Mice treated with antibodies alone showed a longer survival than mice without treatment, but they reached high bacteremia levels at the second day of infection and succumbed (Figure 1A and Supplementary Figure S1A). At the time of sacrifice, mice that only received antibodies had a 10-fold lower number of bacteria (CFU) in the brain compared with untreated mice (Figure 2A) and lower bioluminescent signals (Figure 2B). Figure 1. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies combined with ceftriaxone treatment of mice infected with pneumococci intravenously led to 100% survival. Survival of mice infected with pneumococci intravenously and treated with anti-pIgR and PECAM-1 antibodies 1 hour postinfection after (A) 5 days and (B) 10 days of infection. Ten mice in each experimental group were used in 2 experiments with 5 mice/group. Figure 1. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies combined with ceftriaxone treatment of mice infected with pneumococci intravenously led to 100% survival. Survival of mice infected with pneumococci intravenously and treated with anti-pIgR and PECAM-1 antibodies 1 hour postinfection after (A) 5 days and (B) 10 days of infection. Ten mice in each experimental group were used in 2 experiments with 5 mice/group. Figure 2. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies prevent pneumococcal invasion of the brain. Measurements of pneumococcal invasion of the brain tissue after intravenous pneumococcal challenge (survival experiment in Figure 1) after (A–C) 5 days and (D–F) 10 days of infection. (A and C) Amount of pneumococci (colony-forming units [CFU]/mL) in the brain of mice at time of sacrifice, (A) 5 days postinfection, and (C) 10 days after infection, in red mice that did not survive and in green mice that survived. The non-parametric analysis of variance test was used to assess differences between the groups, and the Dunn’s test was used to make pairwise comparisons (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001). (B and D) IVIS bioluminescent signals of pneumococci in the brain of mice at the time of sacrifice and relative quantification (B) 5 days and (D) 10 days postinfection. Within red borders mice that did not survive, and within green borders mice that survived. Abbreviation: n. d., not detected. Figure 2. View largeDownload slide Anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies prevent pneumococcal invasion of the brain. Measurements of pneumococcal invasion of the brain tissue after intravenous pneumococcal challenge (survival experiment in Figure 1) after (A–C) 5 days and (D–F) 10 days of infection. (A and C) Amount of pneumococci (colony-forming units [CFU]/mL) in the brain of mice at time of sacrifice, (A) 5 days postinfection, and (C) 10 days after infection, in red mice that did not survive and in green mice that survived. The non-parametric analysis of variance test was used to assess differences between the groups, and the Dunn’s test was used to make pairwise comparisons (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001). (B and D) IVIS bioluminescent signals of pneumococci in the brain of mice at the time of sacrifice and relative quantification (B) 5 days and (D) 10 days postinfection. Within red borders mice that did not survive, and within green borders mice that survived. Abbreviation: n. d., not detected. Ceftriaxone is a third-generation cephalosporin that has been used previously in mice models to treat meningitis [13, 16, 17]. We next studied the effect of ceftriaxone treatment using the pneumococcal strain TIGR4, which is susceptible to ceftriaxone and has an MIC value of 0.032 µg/mL. A single ceftriaxone dose of 100 mg/kg, the same concentration as used previously in experimental meningitis mouse models [13, 17], given 1 hour after intravenous challenge with TIGR4, cleared pneumococci from the blood in 6 of 10 animals that survived after 5 days. However, at sacrifice, these mice all had viable bacteria in the brain (5 × 103 to 104 CFU/ mL), and a luminescence signal from the brain was detected (Figure 2A and B). Four of the antibiotic only treated mice succumbed before 5 days of infection with high bacteremia levels (Supplementary Figure S1A), and high numbers of bacteria were found in the brain (1–5 × 105 CFU/mL) (Figure 2A and B). We then administered anti-pIgR and PECAM-1 antibodies together with ceftriaxone and observed that all 10 mice survived (Figure 1A). Six of the mice showed no signs of pneumococcal invasion of the brain (Figure 2A and B). The remaining 4 animals had bacteria in the brain but in lower numbers compared with the surviving mice treated with only ceftriaxone (Figure 2A). Notably, when anti-pIgR and PECAM-1 antibodies were administered 5 hours postinfection (4 hours post ceftriaxone), all mice survived, but the numbers of bacteria in the brain were slightly higher compared with when the antibodies were given together with ceftriaxone 1 hour postinfection (Figure 2A and B). The result suggests that the time of administration will influence the amount of bacteria invading the brain and the effect of antibody treatment. Furthermore, we investigated the effect of antibody treatment in a longer term infection of 10 days. Untreated mice and mice that received only antibodies showed similar survival rates to that observed after the 5-day infection (Figure 1B). Four of the 10 ceftriaxone only treated mice succumbed between day 3 and 4 with high bacteremia levels (Figure 1B and Supplementary Figure S1B), and high numbers of pneumococci were found in the brain (3–9 × 105 CFU/mL) (Figure 2C and D). The 6 ceftriaxone-treated mice that survived also showed viable bacteria in the brain (5 × 102 to 5 × 103 CFU/mL) at sacrifice, and a high luminescence signal from the brain was detected (Figure 2C and D). All 10 mice that received ceftriaxone combined with anti-PECAM-1 and pIgR antibodies survived (Figure 1B). Eight mice had no viable bacteria, and 2 mice showed a very low number of bacteria in the brain (1–3 × 101 CFU/ mL) (Figure 2C), which were not detected using the IVIS Imaging System (Figure 2D). Receptor Blockade Reduces Neuroinflammation We then asked whether blockade of the 2 endothelial receptors prevents neuroinflammation. Microglia, the resident macrophages of the brain, quickly react towards invading pathogens undergoing activation [18]. Activated microglia change their morphology in comparison to resting microglia; their central soma becomes rounder and bigger, and the long branches become thicker and shorter [14]. Using high-resolution and high-magnification immunofluorescence microscopy, we found that at 14 hours postinfection, the brain of untreated mice showed severe neuroinflammation, and all microglia detected displayed a round soma with all cellular branches completely retracted (Figure 3A). At the same time point, brains of mice treated with antibodies showed some activated microglia but also microglia that displayed long and thin cell processes, indicating a less pronounced neuroinflammation compared with untreated mice (Figure 3A). Forty percent of the mice (4 out of 10) that were treated with ceftriaxone only, and that succumbed on day 3–4, showed severe neuroinflammation (Figure 3B). Moreover, at the same time point, the brains of mice treated with ceftriaxone combined with anti-pIgR and PECAM-1 antibodies showed normal microglia, indicating no major signs of neuroinflammation (Figure 3A). After the 10-day infection, we found among survivors some activated microglia in the brains of mice that were treated only with ceftriaxone, whereas mice treated with ceftriaxone combined with anti-pIgR and PECAM-1 antibodies showed normal microglia with no signs of activation (Figure 3A). Figure 3. View largeDownload slide Receptor blockade reduces microglia activation and neuroinflammation. Brain sections were collected after intravenous challenge of mice with pneumococci and used for high-magnification (100× objective) microscopy imaging to analyze the morphological changing during microglial activation. (A) The panel “14 hours” shows brain sections of untreated mice and of mice treated with antibodies to polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1, 14 hours postinfection. Untreated mice displayed activated microglia with round soma and complete retraction of cellular branches (red arrows), whereas brains of mice treated with antibodies showed both activated (red arrows) and normal microglia (blue arrows). The panel “Day 3–4” shows brain tissue section of mice treated with ceftriaxone only (nonsurvivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 3–4 days postinfection. Mice treated with ceftriaxone displayed severe microgliosis (red arrows), whereas the brain of mice that received ceftriaxone combined with antibodies showed normal microglia with no major signs of neuroinflammation (blue arrows). The panel “Day 10” shows brain tissue section of mice treated with ceftriaxone only (survivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 10 days postinfection. Mice treated with ceftriaxone only showed some activated microglia (red arrows), whereas no signs of microgliosis (blue arrows) were observed in brains from mice treated with ceftriaxone combined with antibodies. The black scale bar represents 5 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (B) Brain sections collected after intravenous challenge of mice with pneumococci at the same time points as in A and used for low magnification (20× objective) microscopy imaging to analyze Iba-1 signal intensity as marker of microglial activation. Low magnification imaging was performed to gain a comprehensive overview of microglial activation of the mice. The black scale bar represents 25 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (C) Quantification of Iba-1 signal intensity in the brain of the mice in the groups and time points shown in B. Each column and bar in the graph represent the average and standard deviation values calculated using all Iba-1 signal intensities measured in each image taken for each group and each time point postinfection, respectively. For 2-group comparison, the non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used (*, P < .05; **, P < .01). Abbreviation: n.s., nonsignificant. Figure 3. View largeDownload slide Receptor blockade reduces microglia activation and neuroinflammation. Brain sections were collected after intravenous challenge of mice with pneumococci and used for high-magnification (100× objective) microscopy imaging to analyze the morphological changing during microglial activation. (A) The panel “14 hours” shows brain sections of untreated mice and of mice treated with antibodies to polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1, 14 hours postinfection. Untreated mice displayed activated microglia with round soma and complete retraction of cellular branches (red arrows), whereas brains of mice treated with antibodies showed both activated (red arrows) and normal microglia (blue arrows). The panel “Day 3–4” shows brain tissue section of mice treated with ceftriaxone only (nonsurvivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 3–4 days postinfection. Mice treated with ceftriaxone displayed severe microgliosis (red arrows), whereas the brain of mice that received ceftriaxone combined with antibodies showed normal microglia with no major signs of neuroinflammation (blue arrows). The panel “Day 10” shows brain tissue section of mice treated with ceftriaxone only (survivors) and of mice treated with ceftriaxone combined with antibodies to pIgR and PECAM-1, 10 days postinfection. Mice treated with ceftriaxone only showed some activated microglia (red arrows), whereas no signs of microgliosis (blue arrows) were observed in brains from mice treated with ceftriaxone combined with antibodies. The black scale bar represents 5 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (B) Brain sections collected after intravenous challenge of mice with pneumococci at the same time points as in A and used for low magnification (20× objective) microscopy imaging to analyze Iba-1 signal intensity as marker of microglial activation. Low magnification imaging was performed to gain a comprehensive overview of microglial activation of the mice. The black scale bar represents 25 µm. Nine brain sections from 5 mice in each group were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken for each group and each time point postinfection. (C) Quantification of Iba-1 signal intensity in the brain of the mice in the groups and time points shown in B. Each column and bar in the graph represent the average and standard deviation values calculated using all Iba-1 signal intensities measured in each image taken for each group and each time point postinfection, respectively. For 2-group comparison, the non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used (*, P < .05; **, P < .01). Abbreviation: n.s., nonsignificant. To quantify microglia activation, we performed low-magnification imaging of microglia using brain sections of mice from the same groups and time points postinfection, as shown in Figure 3A. As previously described, activation of microglia is also characterized by upregulation of the microglial marker Iba-1 [14, 19]. Detection of Iba-1 (Figure 3B) and quantification of the intensity of the Iba-1 signal (Figure 3C) confirmed the results obtained using high-magnification imaging (Figure 3A). Fourteen hours postinfection, antibody-treated mice showed a lower Iba-1 signal intensity than untreated mice (Figure 3B and C). Mice that did not survive after ceftriaxone treatment 3–4 days postinfection showed a very high Iba-1 signal intensity compared with mice treated with ceftriaxone combined with antibodies (Figure 3B and C), and all of these mice survived. The Blockade of the Receptors In Vivo Is Long Lasting The low number of bacteria in the brain after 1 single dose of antibody treatment, despite high numbers of bacteria in the blood stream, suggests that the receptor blockade is long lasting. To investigate whether both anti-pIgR and PECAM-1 antibodies remained bound to the brain endothelium at sacrifice, we performed immunofluorescence staining on brain sections from the mice used in the 5-day infection experiment. Brain sections from mice that had been treated with antibodies were stained with only the secondary antibodies to detect bound PECAM-1 and pIgR antibodies, respectively. As positive control, brain sections from untreated mice were stained with primary antibodies to detect pIgR and PECAM-1, followed by the respective secondary antibodies. Finally, negative control brain sections from untreated mice were stained with only secondary antibodies. Staining with secondary antibodies showed similar pIgR and PECAM-1 signals with only a minor decrease of the signal intensity to the level that was observed for both receptors in the positive control sections (Figure 4A and B), indicating that both receptors were still blocked after 5 days treatment with only 1 single dose of antibodies. As expected, staining with the secondary antibodies of brain sections from untreated mice resulted in no fluorescent signal (Figure 4A and B). Figure 4. View largeDownload slide Blockade of the 2 endothelial receptors with antibodies is long lasting. To study whether anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies remained bound to the brain endothelium at sacrifice, brain sections from uninfected mice (positive control) and from mice treated with antibodies were stained only with secondary antibodies, supposing that the primary antibodies were already bound to PECAM-1 and pIgR. (A) Signals of PECAM-1 (blue) and pIgR (green) were detected on the blood-brain barrier endothelium (red) 5 days after antibody treatment (antibody treatment + secondary antibodies). Mice that did not receive antibodies showed no fluorescent signal (negative control). The black scale bar represents 25 µm. Nine brain sections from 5 mice that received ceftriaxone combined with antibodies and 9 brain sections from 5 untreated mice were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken. (B) Quantification of the intensity of the receptor signal shows that the signal was comparable to the one observed for both receptors in the positive control. Each column and bar in the graph represent the average and standard deviation values calculated using the intensity signal measured in each image taken (among all brain tissue sections analyzed for each group), respectively. Only a minor decrease was detected, which was not statistically significant (n. s.).The non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used. Figure 4. View largeDownload slide Blockade of the 2 endothelial receptors with antibodies is long lasting. To study whether anti-polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM)-1 antibodies remained bound to the brain endothelium at sacrifice, brain sections from uninfected mice (positive control) and from mice treated with antibodies were stained only with secondary antibodies, supposing that the primary antibodies were already bound to PECAM-1 and pIgR. (A) Signals of PECAM-1 (blue) and pIgR (green) were detected on the blood-brain barrier endothelium (red) 5 days after antibody treatment (antibody treatment + secondary antibodies). Mice that did not receive antibodies showed no fluorescent signal (negative control). The black scale bar represents 25 µm. Nine brain sections from 5 mice that received ceftriaxone combined with antibodies and 9 brain sections from 5 untreated mice were analyzed. Ten images were taken from each brain section. Images shown are representative of all images taken. (B) Quantification of the intensity of the receptor signal shows that the signal was comparable to the one observed for both receptors in the positive control. Each column and bar in the graph represent the average and standard deviation values calculated using the intensity signal measured in each image taken (among all brain tissue sections analyzed for each group), respectively. Only a minor decrease was detected, which was not statistically significant (n. s.).The non-parametric, 2-tailed, Wilcoxon’s rank-sum test (also known as Mann-Whitney test) was used. Receptor Blockade Does Not Interfere With Antibiotic Penetration Across the Blood-Brain Barrier Furthermore, we assessed whether administration of anti-pIgR and PECAM-1 antibodies interferes with the penetration of antibiotics across the BBB. Uninfected mice were either treated with ceftriaxone only or ceftriaxone combined with anti-pIgR and PECAM-1 antibodies and sacrificed after 12 hours, and brain homogenates were used in an antibiotic-sensitivity test. Brain homogenates from mice that received only ceftriaxone were able to inhibit pneumococcal growth on blood-agar plates (Supplementary Figure S2A). Brain homogenates from mice that received ceftriaxone combined with antibodies resulted in similar-sized inhibition zones, suggesting that receptor blockade does not interfere with the ceftriaxone permeability of the BBB (Supplementary Figure S2B and S2C). DISCUSSION Our previous findings suggest that pneumococcal entry from the blood to the brain is a relatively slow process, because only a fraction of circulating small single cocci in the blood stream seems capable of invading the BBB [14]. In this study, we found that ceftriaxone treatment combined with antibodies led to a lower number of bacteria in the brain in the 10-day infection than in the 5-day infection. This lower number could partly be explained by the poor nutrient environment in the brain that reduced bacterial growth rates, but it could also be explained by the action of microglia, which, similar to macrophages, kill invading pathogens. Ceftriaxone, similar to other β-lactam antibiotics, only act on growing bacteria [20, 21]. This may explain why we obtained clearance of bacteria from the blood with only 1 single dose of ceftriaxone, without eradicating those that had entered into the brain. Our data show that a single-dose treatment with anti-pIgR and PECAM-1 antibodies in combination with ceftriaxone led to 100% survival and abolished bacterial invasion of the brain and development of lethal meningitis, which potentially could reduce the risk for future chronic sequelae. It is interesting to note that the same dose of ceftriaxone was more successful in reducing the bacteremia levels when administered in combination with anti-pIgR and PECAM-1 antibodies (Supplementary Figure S1A and B). Pneumococcal trafficking across the BBB leads to inflammatory responses in the brain [22]. Inflammation in the central nervous system leads the recruitment of immune cells (such as leukocytes) to the sites of infection [22]. We hypothesize that when the pIgR and PECAM-1 receptors are not blocked, pneumococci in the blood stream may invade the brain and other organs together with recruited immune cells, thereby potentially reducing the number of immune cells in the circulation and therefore leading to less pneumococcal clearance from the blood. In contrast, we speculate that when the receptors are blocked, pneumococcal brain invasion is reduced, and the immune cells remain in the blood, thereby potentially clearing the infection. CONCLUSIONS In a clinical perspective, receptor blockade may be an approach to reduce bacterial invasion into the brain, because the antibody blockade appears to be long lasting. Early antibody blockade may protect the brain while providing a longer time window for proper choice of antibiotic and antibiotic eradication of bacteria from the blood stream, and thereby increase survival and potentially reduce the risk of neurological sequelae. However, because interventions were administered rapidly after infection in this study, implications for preventing meningitis progression and sequelae will require further evaluation in animal models using antibody administration repeatedly and at later time points. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We thank professor Staffan Normark for scientific discussions. We also thank Kenth Andersson for technical assistance during the mouse experiments and Anna Granlund (veterinary) for medical consult concerning the use of ceftriaxone in the mouse experiments. Finanical support. This work was funded by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Swedish Foundation for Strategic Research, and Stockholm County Council. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. 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The Journal of Infectious DiseasesOxford University Press

Published: Apr 26, 2018

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