Bacteria growth and inoculum preparation
PA was selected for this study because it is known to be the most common cause of nosocomial pneumonia (14). PA (PAO1 strain) (ATTCC; #27853) was cultured following manufacturer’s instructions. Prior to infection, freshly cultured bacteria from frozen aliquots (-80 °C) were grown overnight at 37 °C in tryptic soy broth (Remel; #R455052) with vigorous agitation (180 rpm) and harvested in log phase to an optical density at 600-nm (OD600) of 0.3 (only for survival studies, mice were inoculated with OD600 = 0.6). Bacteria were centrifuged at 3,000 rpm for 10 min, rinsed and resuspended in sterile phosphate-buffered saline (PBS, pH 7.4). Serial dilutions and colony count of the inoculum were performed to obtain the number of colony-forming units (CFUs) per mL (CFU/mL). For histology experiments and multiphoton imaging, PA labelled with a green fluorescent protein gene (PA-GFP; ATCC; #10145GFP) was used. PA-GFP was cultured in nutrient agar (BD Difco™; #213000) or broth (BD Difco™; #234000) supplemented with 300 µg ampicillin following manufacturer’s instructions as described above and then inoculated into mice.
Animal Model Of Pneumonia
Male and female C57BL/6 mice between 8 and 16 weeks of age were used in all experiments. All mice were housed in a temperature and humidity-controlled housing with a 12-hour light:dark diurnal cycle. Littermates of the same age were allocated to experimental groups. All experimental animal protocols were approved by the University of South Florida Institutional Animal Care and Use Committee (IACUC; protocol 8421R) and were conducted in accordance with the Guide for Care and Use of Laboratory Animals.
Under anesthesia, forty-microliters of bacteria reconstituted in sterile PBS [OD600 = 0.3; 2x107 (CFUs/mL)] were directly injected into the mouse lungs by intratracheal instillation using a 22-G cannula via the oropharynx using a mouse intubation kit (Hallowell EMC, MA) and an otoscope. Control group received the same volume of sterile PBS (uninfected group). After inoculation, mice were kept in a supine position for a few breaths or at least 30 seconds. Once awake, mice were returned to their cages, monitored regularly and allowed access to food and water. For clinical relevance, antibiotics (imipenem + cilastatin formulated in 0.9% saline solution) were administrated subcutaneously at a dose of 5 mg/Kg starting at 6 hours post-infection. The general conditions of each mouse were monitored over a period of 7 days. All procedures were performed in a bio-safety cabinet in a designated bio-safety level-II room according to the Biosafety protocols approved by the University of South Florida.
Measurement of microvascular permeability in vivo
The leakage of circulating solutes across microvessels was evaluated by measuring tissue (brain and lung) accumulation of tracer molecules of different sizes using the ratiometric assay and near-infrared imaging (NIR) as we previously described (15). Time-course of vascular permeability changes was assessed in anesthetized mice intravenously injected through the retro-orbital venous sinus with sodium fluorescein (NaFl; 5 µL/g of a 100 mg/mL NaFl solution; 376 Da; Sigma-Aldrich; #6377) at 24 hours, 7 days and 1-month post-infection. Then, mice were anesthetized with urethane (1.75 mg/Kg; intraperitoneally) and transcardially perfused with ice-cold 0.1 M PBS. After dissection, brains and lung lobes were harvested, weighed and homogenized. Tissue homogenates were centrifuged at 12,000 × g for 20 min at 4°C. The fluorescence of a 100 µL aliquot of the brain and lung samples along with 100 µL of blank or standards was measured on a fluorescence microplate reader (SpectraMax M3; Molecular Devices, Sunnyvale, CA). The concentrations of the samples were within the linear range of the standard curve. Amount of dye (ng) was normalized per pg or µg protein in the extract.
In a different set of experiments, permeability to Alexa Fluor 70-kDa (5 µg/g of a 1 mg/mL solution; Sigma-Aldrich; #R9379) was assessed in the lungs of controls and infected animals at 24 hours post-infection. For NIR of lungs (left lobe), animals received albumin (Albumin-CF®680 dye; 〜70-kDa; 5 µg/g of a 1 mg/mL solution; Biotum; #20292). Tracer was allowed to distribute in freely moving mice for 4 hours and then animals were cardially perfused with PBS. For NIR imaging of brains, anesthetized animals received a combined injection of 10-kDa dextran (10 µg/g of a 1 mg/mL solution; CFTM790-10-kDa dextran; #80121; Stellar Scientific) and albumin (Albumin-CF®680 dye) intravenously. Organs (left lung lobe and whole brain) were imaged at 700 and 800 nm with a near-infrared imaging system (Odyssey CLx; LI-COR Biosciences, Lincoln, NE).
Measurements Of Bacteria Load In Tissues And Csf
Mouse lungs and spleen were harvested at different time points after infection, weighted and then homogenized in tryptic soy broth under sterile conditions. One hundred µL of the homogenate were plated on tryptic soy agar plates for quantification of colonies. CSF samples were obtained under anesthesia through the cisterna magna using a custom-made glass cannula. Ten-fold serial dilutions of the homogenates and CSF were prepared and spread onto agar plates. Next day, CFUs were counted. For the identification of bacteremia (qualitatively), 100 µL of blood were obtained under sterile conditions and cultured in culture vials (BD Bactec™ peds plus culture vials; #442020) for 4 days. Then, 100 µL of the culture vials were streaked onto a 5% sheep blood in tryptic soy agar plates (Hardy diagnostics; #A10) for colony counts.
Proinflammatory Mediators In Plasma And Brain Tissue
Plasma samples obtained from blood collected by cardiac puncture in heparin tubes (BD Microtainer® blood collection tubes; BD Biosciences; #365965) were analyzed for IL-6, IL-1β and TNF-α using indirect sandwich enzyme-linked immunosorbent assay (ELISA) (Sigma-Aldrich; #RAB0308 and #RAB0477) according to the manufacturer’s instructions.
Total RNA was extracted from brain cortices and hippocampi from PA-infected and control animals using TRIzol® Reagent (Invitrogen; #15596) and RNeasy Plus kit (Qiagen). RNA concentration was measured using NanoDrop One Spectrophotometer (Thermo scientific). Reverse transcription (RT)-PCR of RNA was performed using High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific; #4387406). cDNA was amplified for real-time detection with TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific; #4369016) plus individual primers for IL-1β, IL-6, TNF-α, ICAM-1, VCAM-1, CXCL1 and CXCL2 (FAM tagged; Thermo Fisher Scientific) on a Biorad CFX Connect™ machine. Expression data were normalized to Gapdh mRNA levels. The data were calculated as 2(Ct(Gapdh–gene of interest)) to compare infected group to controls. MIQE guidelines were followed for all the PCR experiments and analysis (16).
Flow Cytometry
Mice were euthanized and perfused with sterile PBS through the left ventricle of the heart. The brains (400–500 mg; without the cerebellum) were harvested, cut along the coronal plane with a razor into three pieces and digested into cell suspensions using a Multi Tissue Dissociation kit 1 (Miltenyi Biotec, Germany; #130-110-201) and a gentleMACS™ OctoDissociator with heaters (Miltenyi Biotec; #130-096-427) following manufacturer’s instructions. During the final steps, myelin and erythrocytes were removed using a neural debris removal kit (Miltenyi Biotec; 130-109-398) and red cell lysis solution (Biolegend; #420301), respectively. Total cell count in each brain was quantified using a Luna-II automated cell counter. Cells were then subjected to antibody staining for flow cytometry analysis. The cell suspension was stained with a panel of antibodies against CD45-Alexa 700 (1:1200; Biolegend; #147716) and CD11b-PE dazzle (1:800; Biolegend; #101256). A corresponding isotype IgG antibody was used as negative staining control. Dead cells were excluded using the live/dead stain Zombie NIR fixable viability dye (1:2000; Biolegend; #423105). The samples were run on a Cytek® Northern Lights™ cytometer (Cytek biosciences) and data were analyzed using FlowJo (FlowJo LLC, Ashland, OR). Cell populations were calculated based on relative abundance obtained by flow cytometry (number = total cell count x % of positive cells) (17). Cells were gated using FSCA and FSCH to identify singlets, then gated for live cells by taking a population that was negative for live/dead stain. Antibodies concentrations were chosen based on previous antibody titrations to determine the dilution that provides the best separation of cell populations. Each “n” refers to a single brain obtained from a single mouse, so that each n represents a different mouse. Brains were not pooled together for these experiments. See Suppl. Figure 1 for gating strategy used.
Endothelial Cell Culture
Primary mouse brain microvascular endothelial cells (Cell Biologics, Chicago, IL; #C57-6023) were grown in the recommended culture medium (Cell Biologics; #M1168) supplemented with 10% (v/v) fetal calf serum at 37°C and 5% CO2, split at a ratio of 1:2 and used at passage two. Endothelial cell monolayers were used for barrier integrity and immunofluorescence experiments.
Assessment Of Barrier Function By Electric Cell-substrate Impedance Sensing (Ecis) Assay
Barrier integrity of brain microvascular endothelial cells was performed using a protocol previously described by our group (15, 18). Cell monolayers were grown to reach confluence in order to obtain a polarized endothelium as evidenced by high and stable transendothelial electrical resistance (TER, ohm) values (15). Mouse brain endothelial cell barrier function was determined by measuring the cell-cell adhesion barrier resistance to electric current using an ECIS system (Model ZƟ; Applied BioPhysics Inc., Troy, NY). Briefly, 250 µL of cell suspension (5 Å~ 105 cells per mL) were seeded to each well of two ECIS culture arrays consisting of 8-wells with 10-gold microelectrodes (8W1E+) following manufacturer’s instructions. Resistance was recorded in real time with the following settings: alternating current (1-volt) and 4-kHz frequency at 7-second intervals. Data were normalized to baseline measurements just prior to the onset of treatment (t = 0) and expressed as resistance change (ohms). Both data acquisition and processing were performed using the ECIS Z-theta Analysis Software supplied by Applied Biophysics to determine the resistance (ohm @ 4kHz) values reflecting cell monolayer integrity and overall response to cytokines. In a different set of experiments, cells were exposed to IL-1β (Miltenyi Biotec; #130-094-053) and IL-1β + TNF-α (Biolegend; #575204). Cytokines were dissolved in PBS + 0.1% BSA at 2, 20 and 200 ng/mL. Vehicle control was diluent alone at the same volume as treatment. Data were normalized to baseline, and comparisons were made between cytokine treatment and vehicle control. Data show mean ± S.E.M. (n = 3).
Immunocytochemistry
Brain endothelial cell monolayers were plated on Lab-Tek® chamber slides (Thermofisher Scientific; #1777399PK) and exposed to cytokines (IL-1β, 20 ng/mL), washed in PBS containing 2 mM CaCl2 and 2 mM MgCl2 and then fixed in 2% paraformaldehyde (PFA) in PBS for 5 min followed by 4% PFA for 15 min. Cells were washed in PBS twice, permeabilized for 10 min (in 0.1% Triton-X/PBS) and blocked (10% normal donkey serum in 3% BSA/PBS) for 1 h at room temperature. Slides were incubated with ZO-1 primary antibody (1:100; Thermofisher; #40-2200) overnight at 4°C. Next day, slide chambers were washed seven times and incubated with appropriate secondary Alexa Fluor® antibodies (diluted at 1:500). Chambers were carefully removed, and slides were then cover slipped using ProLong™ diamond antifade mountant with DAPI (Life Technologies; #P36966).
Tissue Processing And Histology
Tissues (brain and lung) harvested from animals 24 hours after infection with GFP-labelled PA were subjected to multiphoton imaging. Mice were anesthetized and injected intravenously with 200 µL of Evans blue solution containing 3% Evans blue fluorescent dye (Sigma-Aldrich; #E2129) in 5% BSA/PBS to visualize vasculature prior to organ extraction and ex-vivo multiphoton imaging.
For visualizing PA distribution in tissue by confocal microscopy, mice infected with GFP-labelled PA and controls were anesthetized and intracardially perfused through the left ventricle with PBS and 4% PFA thereafter. Organs (lungs, spleen and brains) were post-fixed in 4% PFA overnight, rinsed and preserved in 10% sucrose solution in PBS for one day or until the tissue sank to the bottom. Mouse tissues were collected and embedded in Tissue Plus® O.C.T. compound (Fisher HealthCare; #4585) in disposable base molds (Fisher HealthCare; #22363553). Blocks were then frozen in liquid nitrogen and stored in − 80°C until sectioned in a cryostat. Frozen blocks were cut into sections (15 µm thick) on a Leica CM1950 cryostat and slides were stored at − 20°C until use. On the day of the experiment, slides were thawed in the hood overnight and stained with Dylight 488-conjugated Lycopersicum esculentum lectin (1:100; Vector laboratories; #DL-1174) to visualize brain microvessels. Slides were mounted in ProLongTM diamond antifade mountant with DAPI.
Brain microvessels from control and PA-infected mice were freshly isolated from brain cortices as previously described (19). Briefly, pial vessels were carefully removed, and the remaining tissue was homogenized with a loose-fitting Dounce homogenizer in MCDB 131 medium. Homogenates were then centrifuged for 5 min at 2,000g at 4 ºC. Pellets were resuspended in 15% (wt/vol) dextran-PBS and centrifuged again at 10,000g for 15 min at 4 ºC. Myelin was carefully removed from the tube and the pellet was removed with 1 mL DPBS and passed through a 40 µm strainer. The strainer was reversed and washed with 10 mL MCDB 131 media to retrieve the microvessels. Microvessels were fixed in 2% PFA and cytospinned onto microscope slides.
For immunohistochemistry, slides were permeabilized and blocked for 1 hour at RT in 10% normal donkey serum diluted with PBS/3% BSA and 0.3% Triton-X. Slides were incubated overnight with the primary antibodies against neutrophils (1:100; Cederlane; #CL8993AP). Lectin was used to label blood vessels. All antibody dilutions used were optimized by titration.
For visualizing lung structure, hematoxylin and eosin (H&E) staining was performed. Animals were transcardially perfused via the left ventricle with PBS first and then 4% PFA. After one day of post-fixation in the same fixative, lungs were embedded in paraffin using an automatic tissue processor (Thermo Scientific) and then sectioned (4 µm) using a microtome. Slides were deparaffinated and then stained with H&E following standard procedures. All slides were imaged at the same time and using the same settings with a brightfield Olympus VS120 slide scanning imaging system (x20 objective).
Microscopy And Imaging
Confocal microscopy was performed using two different lenses ×20 HC PL APO (0.75NA [numerical aperture], WD [working distance] 0.62 mm) and a ×63 HC PL APO (Oil immersion, 1.4NA, WD 0.14 mm). Images were obtained as z-stacks using Leica STED SP8 Laser Confocal Microscope and image software (LAS X). For confocal microscopy, GFP-labelled PA was excited with the 488-nm laser.
Two-photon imaging was performed using an Olympus FV1000 MPE microscope equipped with 4 spectral detectors, a x25 XL Plan N 1.05 N.A. water immersion objective and a Coherent Chameleon Ultra II IR laser. For multiphoton imaging, GFP-labelled PA was excited with 924-nm laser pulses, emission was detected at 500–520 nm. Evans blue was excited at 945-nm and detected at 620–750-nm. The raw 3D images were reconstructed from z-stacks using Imaris software 9.7 (RRID:SCR_007370).
Open Field Behavioral Test
Spontaneous locomotor activity and anxiety-like behavior were measured by open field test assessed at 1-week after PA infection (20). Mice were placed in an open field chamber and were allowed free exploration for 15 min. The distance traveled was recorded and analyzed using ANY-maze® video tracking system (Stoelting Co, IL, USA). Increased overall distance travelled in the chamber and time spent in the center were considered an index of greater locomotor activity and anxiety-like behavior.
Hematology And Biochemistry
White blood cell counts, cell differentials (neutrophils and monocytes) and total protein were measured in mouse blood collected in heparin tubes using a ProCyte Dx hematology analyzer (IDEXX, Westbrook, Maine). Blood pH was measured in fresh blood using an iSTAT device (Abbott, Hightstown, New Jersey).
Statistical analysis
Data were analyzed using GraphPad Prism (version 9.4.0; GraphPad Software Inc., San Diego, CA). Reported values are expressed as mean ± standard error of the mean (SEM). For animal experiments, female and male mice were randomly selected and used as biological replicates. Normality of the data was tested using the Shapiro–Wilk test. For data that were not normally distributed (nonparametric data), the Mann–Whitney test (two groups, one variable) or Kruskal–Wallis test followed by Dunn’s correction (> 2 variables) was used. Data with normal distribution were analyzed by a two-tailed unpaired Student’s t–test (one variable) or one–way ANOVA with Dunnett’s or Tukey’s correction (> 2 variables). Statistical test used to analyze the data is described in the figure legends. In all tests a 95% confidence interval was used. Figures show exact P values.