Animals
We used CRISPR-Cas9 technology 19,20 to create a humanized murine model of G6PD deficiency containing the G6PD A- variant (rs1050828, Val68Met). Valine 68 was substituted to Methionine using an oligouncleotide template for homology-directed repair (HDR) containing the homologous human DNA sequence. Founder mice and N1 offspring were confirmed through sequence analysis and a stable colony was obtained through strategic back-crossing to wild-type mice. We previously reported that erythrocyte G6PD activity from hemizygous male mice was approximately 5% of WT-G6PD activity21.
For in vivo pneumonia studies, a sample size of 8 animals per group was determined a priori to achieve a power of 0.83 for a 2-tailed test using an α of 0.05. A skilled technician who was blinded to the biological hypothesis performed the inoculations, monitoring, and harvest. Only male mice were used in this study as G6PD deficiency is X-linked. Male mice aged 12 – 14 weeks were used for experiments with WT littermates used as controls. Briefly, mice were inoculated via direct intratracheal instillation with K. pneumoniae at 103 CFU inoculum. Animals were sacrificed and tissues were harvested after 24- and 48- hours in independent experiments.
Euthanasia and Ethical Approval
Animals were euthanized at time of experimental procedures using overdose isoflurane followed by exsanguination via inferior vena cava. The mice were monitored carefully and euthanized if they met predefined criteria for euthanasia. All procedures were performed with approval of the Institutional Animal Care and Use Committee at the University of Pittsburgh (protocol numbers 18052522 and 18063096). All procedures are in accordance with relevant guidelines and regulations including ARRIVE guidelines.
Klebsiella pneumoniae strains
K. pneumoniae strain: The strain used in this study was K. pneumoniae strain 43816 serotype 2 (American Type Culture Collection, Manassas, VA).
K. pneumoniae inoculation
K. pneumoniae strains were grown overnight in tryptic soy broth (TSB) for 18 hours shaking at 250 rpm at 37°C. A 1:100 dilution of bacteria in TSB was then incubated at 37°C for 1.5 hours. The bacterial inoculum was prepared in PBS to OD600nm = 0.2. Mice were instilled with 103 CFU inoculum by the intratracheal route. We have previously described a detailed method of intratracheal administration of bacteria by direct visualization 22,23.
Bronchoalveolar Lavage
A detailed method of bronchoalveolar lavage fluid (BALF) collection has been previously described 24,25. Briefly, at the specified times, mice were anesthetized with isofluorane. The thoracic cavity was opened by midline incision. The trachea was exposed and cannulated with a 20-gauge catheter, which was secured with a 2-0 silk suture. The left main stem bronchus was identified and divided at the hilum, and the entire left lung was removed. BAL was performed on the right lung with PBS containing 0.6 mM EDTA instilled in one aliquot of 0.6 mL, followed by three aliquots of 0.5 mL.
Determination of bacterial burden
The left lung, spleen, and right medial lobe of the liver were removed, and placed in 1 ml of sterile water. Tissues were immediately homogenized on ice with a tissue homogenizer. Blood was drawn in citrate-phosphate-dextrose treated 23-gauge syringes from the inferior vena cava of each anesthetized mouse. Serial 1:10 dilutions of isolated whole blood and tissue homogenates in sterile PBS were plated on tryptic soy agar. Plates were incubated for 18 h at 37˚C after which colonies were counted.
Isolation and culture of bone marrow-derived macrophages (BMDMs)
Isolation and differentiation of bone marrow-derived myeloid cells to non-activated macrophages has been previously described 26. Briefly, bone marrow cells from WT and G6PD A- mice were allowed to adhere and cultured in differentiation media (DMEM, 20% neonatal calf serum, 30% L929 conditioned medium, 1% penicillin-streptomycin) at a density of 1 x 106 cells/ml in 100 x 20mm sterile petri dishes for 7 days. Medium was replaced at day 5. After 7 days, differentiated BMDMs were cultured in 24-well plates at a density of 2.5 x 105 cells/ well in a final volume of 500 µL 24-hours prior to subsequent use in in vitro pHrodo phagocytosis assay.
Isolation of bone marrow neutrophils
Extraction of bone marrow cells was performed as described previously 27, and neutrophils were isolated from cell suspensions via negative selection (Miltenyi Neutrophil Isolation Kit, #130-097-658) according to the manufacturer’s instructions. Isolated cells were diluted in serum-free OptiMEM to a final concentration of 4x104 cells per 30 µL media. The final cell suspension was >95% polymorphonuclear cells as determined by cytospin preparations. These cells were immediately used in the modified Amplex Red assay as described below. Unused cell suspensions were centrifuged at 300 x g for 10 minutes, lysed, and pellets stored at -80°C.
pHrodo™ in vitro Phagocytosis Assay
K. pneumoniae strain 43816 serotype 2 was grown to OD600nm = 0.2 then heat-killed at 60°C for one hour. Heat-killed bacteria were centrifuged at 12,000 rpm for 2 min, resuspended in 100 mM sodium bicarbonate, centrifuged at 12,000 rpm for 2 min, then resuspended in 200 µL sodium bicarbonate. pHrodo™ iFL Green STP Ester dye (ThermoFisher #P36012) at 10 mg/mL was diluted in the bacterial suspension to 1 mM, then incubated for one hour in the dark. Unconjugated dye was removed through successive washing steps with PBS and 100% methanol. BMDMs at a density of 2.5x105 cells/well were infected with heat-killed, labeled-bacteria at MOI=10 for 90 min. Plates were centrifuged at 1500 rpm for 5 min at 4°C to synchronize phagocytosis. As a negative control, cytochalasin D [10 µM] was applied to selected wells one hour prior to infection to inhibit phagocytic uptake. Adherent cells were isolated by vigorous washing with ice cold PBS. pHrodo™-positive macrophages were examined by flow cytometry analysis. The phagocytosis of pHrodo labeled KP2 in BMDMs was detected as green fluorescence positive events by BD FACScalibur. Four wells of WT and four wells of G6PD A- BMDMs were obtained from one mouse each as technical duplicates.
In vivo Phagocytosis Assays
For measuring in vivo phagocytosis, pHrodo labeled K. pneumoniae was instilled intratracheally into mice at a concentration of ~5 x 107 CFU in 0.1 mL total volume. After one hour, mice were euthanized and BAL was performed using 1-mL of 0.6mM EDTA in PBS followed by three sequential lavages of 0.9-mL. BAL cells were labeled with fluorochrome-conjugated antibodies against Ly6G PE and F4/80 APC. The flow cytometry data were acquired on BD FACSCalibur. The positive events were back-calculated as absolute BAL cell counts based upon total cell counts.
Enzyme-Linked Immunosorbent Assays (ELISAs)
ELISA Quantikine-sets for murine interleukin-1β (IL-1β), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from R&D Systems and the levels of cytokines in BALF were measured according to the manufacturer’s instructions (Minneapolis, Minnesota).
Measurement of Extracellular H2O2 Production
The measurement of extracellular H2O2 production was performed using BMDMs and bone marrow neutrophils using a modified Amplex red assay. Briefly, H2O2 production was quantified in whole cells by Amplex® Red as previously described28–30. Either bone marrow derived macrophages or bone marrow derived neutrophils were prepared as described above, resuspended in OptiMEM and then plated (4 × 104 cells/well) into 96‐well plate in assay buffer (25 mM Hepes, pH 7.4, containing 0.12 M NaCl, 3 mM KCl, 1 mM MgCl2) supplemented with 0.1 mM Amplex® Red, and 0.32 U/ml of horse radish peroxidase (HRP). The reaction was started by addition of 5µM phorbol 12-myristate 13-acetate (PMA). Fluorescence measurements were made using a Biotek Synergy 4 Hybrid Multi-Mode Microplate Reader with a 530/25 excitation and a 590/35 emission filter. The reaction was monitored at 37 oC for 1 h. A standard curve of known H2O2 concentrations was included on each plate. NADPH oxidase (NOX) activity was obtained by calculating the rate of H2O2 production as RFU/min/40,000 cells after subtracting the equivalent value given by cells treated with 300 U/ml of catalase. Data are expressed as fold change of WT vehicle control.
Phagocytosis Marker Assay
BMDMs were stimulated with LPS at 100 ng/mL (ULTRA PURE LPS from Escherichia coli O111:B4, #421, List Labs) or saline vehicle for 4 hours. Flow cytometry analysis was performed using antibodies against the following markers: CD16 (BioLegend, #158010), CD18 (BioLegend, #101405), CD32 (BioLegend, #156406), CD21/35 (BioLegend, #123418), CD36 (BD Biosciences, #562477), CD64 (BD Biosciences, #740622), CD204 (BD Biosciences, #748091), CD206 (BioLegend, #141719), CD11c (BioLegend, #117348), CD11b (BD BioSciences #553311), MARCO (R&D, #FAB2956N).
Statistical Analysis
A Student t test was used to compare differences between two groups. A 2-tailed Mann-Whitney U test was used for data that was not normally distributed. Differences were considered significant for p-values < 0.05, and significance was Bonferroni-adjusted for multiple comparisons where appropriate. All statistical analysis was performed using GraphPad Prism 9.5.0.