Özet

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Published: September 28, 2022
doi:

Özet

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Abstract

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease.

This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors.

Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

Introduction

Streptococcus pneumoniae (pneumococcus) are Gram-positive bacteria that asymptomatically reside in the nasopharynx of most healthy individuals1,2. Promoted by factors that are not completely defined, pneumococci can transition from benign colonizers of the nasopharynx to pathogens that spread to other organs resulting in serious infections, including otitis media, pneumonia, and bacteremia3. Pneumococcal disease presentation is, in part, dependent on strain-specific differences, including the serotype, which is based on the composition of capsular polysaccharides. There have been over 100 serotypes characterized so far, and some are associated with more invasive infections4,5. Several other factors increase the risk of pneumococcal disease. One such factor is viral infection, where the risk of pneumococcal pneumonia is increased 100-fold by IAV6,7. Historically, S. pneumoniae is one of the most common causes of secondary bacterial pneumonia following influenza and is associated with worse outcomes8. Another major risk factor is advanced age. In fact, S. pneumoniae is the leading cause of community-acquired bacterial pneumonia in elderly individuals above 65 years old9,10. Elderly individuals account for the majority (>75%) of deaths due to pneumonia and influenza, indicating that the two risk factors-aging and IAV infection-synergistically worsen disease susceptibility11,12,13,14. However, the mechanisms by which viral infection prompts the transition of pneumococci from asymptomatic colonizer to invasive pathogen and how this is shaped by host factors remain poorly defined. This is largely due to the absence of a small animal model that recapitulates the transition from asymptomatic pneumococcal colonization to critical clinical disease.

Co-infection studies have classically been modeled in mice inoculated with pneumococci directly into the lungs 7 days following influenza infection15,16. This reproduces the susceptibility to secondary bacterial pneumonia and is ideal for studying how antiviral immune responses impair antibacterial defenses17. However, longitudinal studies in humans have demonstrated that pneumococcal carriage in the nasopharynx, where the bacteria can form asymptomatic biofilms18, is uniformly associated with invasive diseases19,20. Bacterial isolates from infections of the middle ear, lung, and blood are genetically identical to those found in the nasopharynx20. Thus, to study the transition from asymptomatic carriage to invasive disease following IAV infection, a model was established in which mice were intranasally administered biofilm-grown pneumococci followed by IAV infection of the nasopharynx21,22. Viral infection of the upper airway led to changes in the host environment that led to the dispersal of pneumococci from biofilms and their spread to the lower airways21. These dispersed bacteria had upregulated expression of virulence factors important for infection, converting them from colonizers to pathogens21. These observations highlight the complex interaction between the virus, host, and bacteria and demonstrate that the changes to the host triggered by viral infection have a direct impact on the pneumococcal behavior, which, in turn, alters the course of bacterial infection. However, this model fails to recapitulate the severe signs of illness observed in humans, likely because the virus is limited to the nasal cavity, and the systemic effects of viral infection on host immunity and lung damage are not recapitulated.

We recently established a model that incorporates the complex interaction between the host and pathogens but also more closely mimics the disease severity observed in humans23. In this model, mice are first infected intranasally with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This resulted in bacterial dissemination to the lungs, pulmonary inflammation, and illness that progressed to lethality in a fraction of young mice23. This previous study demonstrated that both viral and bacterial infection altered host defense: viral infection promoted bacterial dissemination, and prior bacterial colonization impaired the ability of the host to control pulmonary IAV levels23. Examining the immune response revealed that IAV infection diminished the antibacterial activity of neutrophils, while bacterial colonization blunted the type I interferon response critical to antiviral defense23. Importantly, this model reproduced the susceptibility of aging. Compared to young mice, old mice displayed signs of disease earlier, showed more severe clinical illness, and succumbed to infection more frequently23. The work presented in this manuscript shows that the degree of disease is also dependent on the bacterial strain, because invasive pneumococcal strains display more efficient dissemination upon IAV infection, show more overt signs of pulmonary inflammation, and result in accelerated rates of disease compared to non-invasive strains. Thus, this S. pneumoniae/IAV co-infection model permits the detailed examination of both pathogen and host factors and is well-suited for studying immune responses to polymicrobial infections at the different phases of disease progression.

Protocol

All animal studies were performed in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the University at Buffalo Institutional Animal Care and Use Committee.

1. Preparing chemically defined media (CDM)

  1. Prepare the stocks as follows:
    1. Dissolve the mix I compounds listed in Table 1 in 100 mL of ultrapure water while stirring. Store in 200 µL aliquots at −20 °C.
    2. Dissolve the mix II compounds listed in Table 1 in 20 mL of 0.1 M NaOH while stirring. Store in 100 µL aliquots at −20 °C.
    3. Dissolve the mix III compounds listed in Table 1 in 1 mL of ultrapure water while stirring. Store in 10 µL aliquots at 4 °C.
    4. Dissolve the mix IV compound listed in Table 1 in 1 mL of ultrapure water while stirring. Store in 10 µL aliquots at −20 °C.
    5. Dissolve the compounds listed in Table 2 initially in 15 mL of ultrapure water while stirring. Adjust the pH to 7.0 with a few drops of 0.1 M NaOH and adjust the final volume to 20 mL using ultrapure water. Store in 1 mL aliquots at −20 °C.
    6. Dissolve the compounds listed in Table 3 in 90 mL of ultrapure water on a hot plate at 50 °C while stirring. Adjust the pH to 7.0 with 0.1 M NaOH, and then adjust the final volume to 100 mL using ultrapure water. Store in 5 mL aliquots at −20 °C.
  2. Make the starter stock freshly every time by dissolving the compounds in Table 4 in 70 mL of ultrapure water while stirring.
  3. To the fresh starter stock, add the following mix stocks in order: 200 µL of Mix I stock (Table 1), 80 µL of Mix II stock (Table 1), 10 µL of Mix III stock (Table 1), 10 µL of Mix IV stock (Table 2), 1 mL of Vitamin stock (Table 3), and 5 mL of Amino Acid stock (Table 4).
  4. Once the stocks have been added, adjust the final volume to 100 mL by adding 30 mL of ultrapure water to the beaker.
  5. Supplement the CDM with compounds from Table 4. Once mixed thoroughly, filter-sterilize and store at 4 °C for a maximum of 2 weeks.

2. Growing the S. pneumoniae biofilm

  1. Prepare RP-10 medium by mixing 445 mL of RPMI 1640 with 50 mL of heat-inactivated fetal bovine serum (FBS) and 5 mL of penicillin/streptomycin at 10,000 U/mL and 10,000 µg/mL, respectively.
  2. Grow the NCI-H292 (H292) mucoepidermoid carcinoma cell line. Add the cells from one purchased vial to 5 mL of RP-10 medium in a T-25 tissue culture-treated flask. Incubate at 37 °C/5% CO2 for 3-5 days to reach 100% confluence.
  3. Check the cells under a light microscope using 10x magnification to assess confluency.
    NOTE: When all the cells are in contact with other cells and there are no gaps in between, then the desired 100% confluency is reached.
  4. Wash the cells 2x in 5 mL of room-temperature PBS. Ensure the buffer is calcium free to avoid chelating the EDTA in the following step.
  5. Add 1 mL of trypsin-EDTA to the flask and incubate at 37 °C/5% CO2 for 5-10 min until the cells detach. Neutralize with 4 mL of RP-10 medium. Gently mix by pipetting up and down and transfer to a 50 mL conical tube.
  6. Add 500 µL of the cell suspension per well to a tissue culture-treated 24-well plate. From a confluent T-25 flask, expect 2 × 106-4 × 106 cells/mL.
  7. On the following day, check the cells under a light microscope to make sure that they are confluent, as in step 2.3. If they are not, then incubate for longer.
  8. Once the H292 cells are 100% confluent in the 24-well plate, gently wash the cells 3x with 1 mL of room-temperature PBS to ensure that no medium containing antibiotics or debris remains.
  9. After washing the cells, add 250 µL/well of 4% paraformaldehyde to fix the cells. Incubate for either 1 h on ice or overnight at 4 °C.
  10. The night prior to cell fixation, streak the S. pneumoniae strain of interest on blood agar plates and incubate overnight at 37 °C/5% CO2.
    NOTE: The data presented here are with the following S. pneumoniae strains obtained via collaborative exchange: serotype 19F otitis media isolate EF303024, classical serotype 2 Avery strain D3925, and serotype 4 bacteremia isolate TIGR426. The strains are also available from public collections referenced in the Table of Materials.
  11. Prepare CDM plus oxyrase (0.15 U/mL) by adding 100 µL of oxyrase (30 U/mL) to 20 mL of CDM.
    NOTE: Oxyrase is used to eliminate oxygen to allow the efficient growth of S. pneumoniae in liquid culture27.
  12. Inoculate the bacteria from the plate into fresh CDM + oxyrase by washing the bacteria off the plate by adding 1 mL of the CDM + oxyrase and gently lifting the bacterial colonies using the side of a 1 mL pipette tip, being careful to not scrape the agar. Alternatively, use an inoculating loop to lift the bacteria and inoculate them into a tube containing 1 mL of the CDM + oxyrase.
  13. Dilute the bacteria in CDM + oxyrase to a starting OD600 of 0.05.
  14. Grow the bacteria in a loosely capped 50 mL conical tube standing at 37 °C/5% CO2 until an OD600 of 0.2 is reached (this will take anywhere between 2-5 h). Check the OD600 every hour to ensure the OD does not exceed 0.2.
  15. Once the OD has reached 0.2, vortex the bacterial culture tube. Seed 0.5 mL of the bacteria on the fixed H292 cells and add another 0.5 mL of CDM + oxyrase medium per well. Add 1 mL of CDM + oxyrase to the control wells with no bacteria. Incubate the plate for 48 h at 34 °C/5% CO2.
    NOTE: Growth at 34 °C is used to more closely mimic the lower temperature in the nasopharynx21.
  16. Every 12 h following the initial seeding, gently remove 0.5 mL of the medium and replenish with 0.5 mL of fresh CDM + oxyrase. Be careful not to disrupt the forming biofilm. Check the bottom of the plate for biofilm and look for increasing cloudiness as time goes on due to the growth of the biofilm. To control for contamination, check the wells without bacteria to ensure that the control wells remain clear.
  17. At 48 h post inoculation, remove the supernatant and wash 2x very gently with 1 mL of PBS. Resuspend in 1 mL of fresh CDM and pipette up and down vigorously to lift the biofilm. For each bacterial strain, pool the bacteria from all the wells into a 50 mL conical tube. Mix well by gently tilting the tightly capped tube up and down several times.
  18. To the 50 mL conical tube, add 40% glycerol in CDM at equal volumes to achieve a bacterial suspension with a final concentration of 20% glycerol. Aliquot 1 mL into microcentrifuge tubes, flash-freeze on dry ice, and save at −80 °C.
  19. Prior to use, enumerate the bacteria by thawing one aliquot on ice, spinning the tube at 1,700 × g for 5 min, removing the supernatant, resuspending the pellet in 1 mL of PBS, and plating serial dilutions on blood agar plates28.
  20. Grow the agar plates overnight at 37 °C/5% CO2 and count the colonies at relevant dilutions to obtain the bacterial concentration in colony-forming units (CFU)/mL.
    ​NOTE: It is recommended to enumerate the bacteria in the stocks at least a day after freezing or later, as there is a drop in bacterial viability within the first 24 h. The stored frozen aliquots can be used for subsequent infection of mice for a maximum of 2 months.

3. Intranasal inoculation of mice with biofilm-grown S. pneumoniae

  1. Purchase mice and use at the desired age.
    NOTE: Mice aged 3-4 months old are preferred to model young hosts, and mice aged 21-24 months old can be used to model elderly individuals >65 years of age29. The data presented here are with C57BL/6 male mice.
  2. Thaw the biofilm-grown bacterial aliquots on ice and spin at 1,700 × g for 5 min. Carefully remove and discard the supernatant without disrupting the pellet, wash the bacteria by resuspending the pellet in 1 mL of PBS, and spin again at 1,700 × g for 5 min. Remove the supernatant and resuspend the pellet in the volume needed to reach the desired concentration (aim for 5 × 106 CFU/10 µL for intranasal inoculation). Confirm the amounts of bacteria administered by plating the prepared inoculum on blood agar plates as in step 2.19.
  3. Inoculate the mice intranasally with 5 × 106 CFU by pipetting 5 µL of the diluted inoculum into each naris. Make sure to hold the mice firmly, stabilizing the head, until the volume is inhaled (typically within seconds of pipetting the volume into the nares). Perform this step in the absence of anesthesia to prevent pulmonary aspiration of the inoculum.

4. Viral infection with influenza A virus (IAV)

  1. At 48 h following intranasal inoculation with S. pneumoniae, thaw the IAV strain of interest on ice.
    NOTE: The data presented here are with a mouse-adapted strain of influenza A virus A/PR/8/34 H1N1 that was obtained via collaborative exchange30.
  2. Once the virus has thawed, dilute the virus in PBS to the desired concentration; aim for 20 plaque-forming units (PFU)/50 µL for intratracheal infection and 200 PFU/10 µL for intranasal infection. For mock-infected and bacteria-only groups, use PBS to inoculate the mice.
  3. Place ophthalmic lubricant on the eyes of the mice prior to anesthesia. Anesthetize the mice using 5% isoflurane and confirm anesthesia by a firm toe pinch.
  4. Once the animal is anesthetized, remove it from the isoflurane chamber and immediately infect the anesthetized mice with 50 µL (20 PFU) of IAV intratracheally using blunt tweezers to pull the tongue out of the mouth and pipetting the volume of liquid down the trachea.
  5. Place the mice in a separate cage and monitor until complete recovery (they are able to maintain sternal recumbency [able to lay upright on the chest]).
  6. Following recovery, immediately intranasally inoculate the mice with 10 µL (200 PFU) of IAV using the inoculation method in step 3.3.
  7. House mice that have undergone single or dual bacterial and viral infection with the same infection group and separate them from the other groups.

5. Monitoring the mice for disease symptoms

  1. Monitor the mice daily for at least 10 days and blindly score for signs of sickness as follows:
    1. Score as follows for weight loss: 0 = 5% or less; 1 = 5%-10%; 2 = 10%-15%; 3 = 20% or more. Euthanize the mice using CO2 inhalation when the weight loss score is at 3.
    2. Score as follows for activity: 0 = normal/active; 1 = moving but slightly diminished; 2 = diminished; 3 = severely diminished/lethargic (only moves if touched), 4 = coma/immobile. Euthanize the mice when the activity score is at 3.
    3. Score as follows for posture: 0 = no hunch (normal); 1 = slightly hunched posture; 2 = severe hunch. Euthanize the mice when the posture score is at 2.
    4. Score as follows for the eyes: 0 = normal; 1 = protruding; 1 = sunken; 1 = closed; 1 = discharge. It can be a combination. Add the totals for the final eye score.
    5. Score as follows for breathing: 0 = Normal breathing; 1 = irregular or altered (higher/lower rate); 2 = labored (exaggerated effort or gasping). Euthanize the mice when the breathing score is at 2.
  2. Based on the above criteria, add the individual scores for a total clinical score of healthy (0) to extremely sick (15). Consider any mouse displaying a total score above 2 to be sick. Humanely euthanize any mice displaying a total score above 9 or the indicated scores for each criterion and mark them on the survival curve.

6. Processing of infected tissues for bacterial enumeration

  1. At 48 h following IAV infection, euthanize the mice.
  2. Place the mouse in a supine position. Using 70% ethanol, spray the chest and abdomen of the mouse to clean the fur. Using forceps, pinch the fur and skin in the middle of the mouse and cut the fur with 4.5 in dissection scissors to expose the area from the liver up to the chest.
  3. Blood collection
    1. Using dissection scissors, gently cut into the peritoneal cavity to expose the liver. Using forceps, expose the hepatic portal vein at the top of the liver near the diaphragm. Cut the hepatic portal vein using the dissection scissors. Once the blood starts to pool in the peritoneal cavity, collect 10 µL of blood using a micropipette and place into 90 µL of anticoagulant solution (50 mM EDTA solution in PBS) in a microcentrifuge tube for plating for bacterial burden.
    2. Use a P-1000 micropipette to collect the rest of the blood, place it in a blood collection tube, and centrifuge at 7,600 × g for 2 min to collect the serum. Save the sera in microcentrifuge tubes at −80 °C for subsequent analysis of any desired cytokine or metabolite.
  4. Lung collection
    1. Using dissection scissors, make a cut up the sides of the exposed rib cage and gently pull the ribs up toward the head of the mouse to expose the heart. Insert a 25 G needle attached to a 10 mL syringe prefilled with PBS into the right ventricle and begin slowly perfusing. Look for bleaching of the lungs as an indicator of successful perfusion. Flush slowly to avoid breaking the pulmonary tissue.
    2. Lift the heart with the forceps and make a cut to separate the lungs and heart. Once separated, pick up all lobes of the lung with the forceps and rinse in a dish with sterile PBS to remove any residual blood. In a Petri dish, mince the lungs into small pieces and mix well. Remove half of the lung mix for determination of the bacterial CFU or viral PFU and place it in a round bottom 15 mL tube prefilled with 0.5 mL of PBS for homogenization.
      NOTE: It is important not to take different lobes of the same lung for the various assessments. Instead, all the lobes should be minced, mixed well together, and parsed out equally for the different assessments.
    3. Remove the other half of the lung for flow cytometry (section 7 below) and place it in a non-tissue culture-treated 24-well plate with each well prefilled with 0.5 mL of RP-10. Leave at room temperature until processing.
  5. Nasopharynx collection
    1. At the neck, use the dissection scissors to cut away the fur, and then cut away the muscle and expose the trachea.
      NOTE: The trachea is a tube-like structure located under the muscle.
    2. Place small forceps under the trachea at a distance of 1 cm from the mouse's jaw to stabilize it. Using dissection scissors, gently make a 0.1 cm slit on the anterior portion of the trachea, avoiding cutting the trachea completely.
    3. Prepare a 1 mL syringe filled with 0.5 mL of PBS with 0.58 mm tubing attached to a 25 G needle. Collect the nasal wash by inserting the tubing into the trachea going upward toward the nasopharynx. Once resistance is felt entering the nasal cavity, place a microcentrifuge tube at the nose and slowly flush the PBS through the trachea to collect the nasal lavage.
    4. Place the mouse in a prone position. Spray the head of the mouse with ethanol. Use dissection scissors to cut the fur and mystacial pad to expose the head bone of the mouse.
    5. Using the dissection scissors, make a 1 cm cut down the sides of the mandible and between the eyes. Using forceps, slowly pull the facial bones away from the body to expose the nasal cavity.
    6. Use forceps to gently remove the nasal tissue and place it into a round bottom tube prefilled with 0.5 mL of PBS for homogenization.
  6. To homogenize the collected tissue, first clean the homogenizer probe by putting it in 70% ethanol and turning on the homogenizer at 60% power for 30 s. Repeat the step in sterile water for 10 s. Homogenize each tissue for 1 min. Clean the homogenizer probe in sterile water between each sample and in a fresh tube of 70% ethanol between each organ and sample group.
  7. Enumeration of bacterial numbers
    1. Once all the organs have been harvested and homogenized, plate serial dilutions on blood agar plates. To calculate the total CFU, use 10 µL to plate and note down the final volume in mL for each sample. Plate the nasopharynx samples on blood agar plates supplemented with 3 µg/mL gentamicin to select for the growth of S. pneumoniae while inhibiting the growth of other microorganisms that colonize that tissue. Incubate overnight at 37 °C/5% CO2.
    2. To enumerate the bacterial CFU for the lung and nasopharynx, first count the colonies on the blood agar plates. Then, use equation (1) and equation (2) to calculate the amount per mL and total number.
      Amount per mL = number of colonies × dilution factor × 100   (1)
      Total number = amount per mL × total volume per sample   (2)
      NOTE: In equation (1), 100 is used to multiply since 10 µL is plated, which is a 100-fold dilution of 1 mL. The total volume per sample in equation (2) is from step 6.7.1, which results in the limit of detection of 100 per organ.
    3. To enumerate the bacterial CFU For bacteremia, first count the colonies on the blood agar plates. Then, use equation (3) to determine the amount per mL of blood.
      Amount per mL of blood = number of colonies × dilution factor × 100 × 10   (3)
      NOTE: In equation (3), 100 is used as 10 µL is plated, which is a 100-fold dilution of 1 mL, and 10 indicates a 1:10 dilution of the blood in anticoagulant. This results in the limit of detection of 1,000/mL.

7. Processing of the lung samples for flow cytometry

  1. Prepare the required media as follows:
    1. Prepare RP-10 as described in step 2.1.
    2. Prepare digestion buffer by mixing RP-10 with 2 mg/mL collagenase and 30 µL/mL DNase I.
    3. Prepare lysis buffer by dissolving 8.29 g of NH4Cl, 1 g of NaHCO3, and 0.038 g of EDTA in 1 L of H2O.
    4. Prepare 10x FACS buffer by mixing 450 mL of HBSS with 50 mL of heat-inactivated FBS and 5 g of sodium azide.
    5. Prepare 1x FACS buffer by diluting 50 mL of 10x FACS buffer in 450 mL of HBSS.
  2. Take the lung samples from step 6.4.3 and place in a 24-well plate. Add 500 µL of digestion buffer to each well. Incubate for 45 min up to 1 h at 37 °C/5% CO2.
  3. Prefill 50 mL conical tubes for each sample with 5 mL of RP-10. When the incubation is over, place a 100 µm filter at the top of the 50 mL conical tube and wet it with 1 mL of RP-10.
  4. Using a P-1000 micropipette, move the digested lungs and place them on the filter. Use the plunger of a 3 mL syringe to mash the organ. Rinse 2x with 1 mL of RP-10 each time.
  5. Spin the samples at 4 °C and 327 × g for 5 min. Aspirate the supernatant and resuspend the pellet in 1 mL of lysis buffer. Leave for 3 min to allow lysis of the red blood cells. Neutralize with 5 mL of RP-10.
  6. Spin the samples at 4 °C and 327 × g for 5 min. Aspirate the supernatant, resuspend the pellet in 1 mL of RP-10, and take 10 µL for counting the samples.
  7. Spin the samples at 4 °C and 327 × g for 5 min. Aspirate the supernatant and resuspend the pellet in RP-10 at 2 × 106-4 × 106 cells/mL. Add 60 µL of each sample into a 96-well plate to stain for the desired cell types23 listed in step 7.9, Table 5, and Table 6.
  8. Spin the plate at 4 °C and 327 × g for 5 min.
  9. Meanwhile, prepare the antibody master mixes, florescent minus one (FMOs), and single-stain controls with the desired antibodies. To stain for polymorphonuclear leukocytes (PMNs), macrophages, monocytes, dendritic cells, and T cells, use the antibodies and final dilutions listed in Table 5 and Table 6. Use a total volume of 100 µL/well of the antibody mix. Follow the dilutions listed in the tables for determining the appropriate volume of the master mix and the individual antibodies required.
  10. When the spin is done (step 7.8), decant the supernatant, resuspend the pellets in 100 µL of the antibody mixes, FMOs, or single-stain controls, and incubate on ice for 30 min in the dark.
  11. Wash the cells 2x by adding 150 µL of FACS buffer to the wells and spinning the plate at 4 °C and 327 × g for 5 min.
  12. When the spin is done, decant the supernatant, resuspend the pellets in 100 µL of fixation buffer, and incubate on ice for 20 min.
  13. Wash the cells 2x by adding 150 µL of FACS buffer to the wells and spinning the plate at 4 °C and 327 × g for 5 min.
  14. Prepare labeled FACS tubes with 200 µL of FACS buffer. Resuspend the pellets in 150 µL of FACS buffer. Individually filter each sample into their corresponding FACS tube using a 100 µm filter. Keep on ice or at 4 °C and protected from the light until ready to analyze.
  15. Analyze the cells using a flow cytometer.

8. Plaque assay for enumerating IAV

  1. Prepare the required media as follows:
    1. Prepare infection medium by dissolving 2.5 g of bovine serum albumin (BSA) into 40 mL of DMEM while stirring at 37 °C for 10-20 min until dissolved. Filter-sterilize into 460 mL of DMEM.
    2. Prepare 2.4% microcrystalline cellulose by dissolving 1.2 mg of microcrystalline cellulose into 50 mL of H2O. Autoclave on the liquid setting and store at room temperature.
    3. Prepare 5% of BSA DMEM by dissolving 2.5 g of BSA into 40 mL of DMEM while stirring at 37°C for 10-20 min. Add the remaining 10 mL of DMEM for a final volume of 50 mL. Filter-sterilize and store at 4 °C.
    4. Prepare 2x MEM/0.5% BSA by mixing 1 mL of 5% BSA DMEM with 9 mL of 2x MEM.
    5. Prepare low-viscosity overlay medium by mixing a 1:1 ratio of 2.4% microcrystalline cellulose and 2x MEM/0.5% BSA with 1 mg/mL TPCK (inhibitor of chymotrypsin) trypsin.
    6. Prepare EMEM/10% FBS by mixing 450 mL of Eagle's Minimum Essential Medium (EMEM) with 50 mL of heat-inactivated FBS.
  2. Grow the Madin-Darby canine kidney (MDCK) cell line. Add the cells from one purchased vial to 5 mL of EMEM/10% FBS in a T-25 tissue culture-treated flask. Incubate for 3-5 days at 37 °C/5% CO2 until the cells reach 100% confluence. Check for confluency as in step 2.3.
  3. Remove and discard the culture medium, and rinse 2x with 5 mL of room-temperature PBS. Add 1 mL of trypsin-EDTA to the flask and incubate at 37 °C/ 5% CO2 for 10-15 min until the cells detach. Once lifted, neutralize with 4mL of EMEM/10% FBS to obtain a cell suspension at 2 × 105 cells/mL.
  4. Seed the MDCK cells in a 12-well tissue culture-treated plate by adding 1 mL of resuspended cells per well (at 2 × 105 cells/well) 1 day before beginning the plaque assay.
    NOTE: Make sure the cells reach 100% confluency prior to use and incubate for longer if needed to reach confluency.
  5. For use as standards, make 10-fold serial dilutions (106-101) of IAV stock (of a known titer) in the infection medium listed in step 8.1.1. Make 1.2 mL of each dilution to test in triplicate.
  6. Thaw the organ homogenates on ice. Spin down on a tabletop centrifuge at 2,000 × g and collect the clear supernatant.
  7. Repeat step 8.5 but with the supernatant from the samples in step 8.6.
  8. Aspirate the medium from the cells and wash 2x with 1 mL of PBS to remove all the FBS.
  9. Add 300 µL of each standard dilution or serially diluted sample gently along the side of each well, beginning at the highest dilution to the lowest, and do this in triplicate.
  10. Place the plates in the incubator at 37 °C/5% CO2, shaking the plate every 10 min for a total of 50 min. Make sure to place them flat in the incubator and do not stack them.
  11. After the 50 min, wash the cells 2x with 1 mL of PBS.
  12. Add 2 mL of the low-viscosity overlay medium into each well except the lowest-dilution and no-virus wells; to those, add infection medium and trypsin.
  13. Place the plate back into the incubator at 37 °C/5% CO2 for 2-4 days to achieve plaques that can be visualized by the naked eye.
  14. Wash the plates by adding 2 mL of PBS into each well fast from the side and gently shake to suspend the settled low-viscosity overlay medium.
  15. Discard the whole liquid volume in the well by gently pipetting the medium off.
  16. Repeat the wash one more time with 2 mL of PBS in each well, and then discard the whole liquid volume by gentle pipetting.
  17. To fix the plaques, add 500 µL of 4% paraformaldehyde into each well, shake, and let sit for 30 min.
  18. Wash slowly down the side with 1 mL of PBS; then, gently discard the liquid.
  19. Add 500 µL of 1% crystal violet (diluted in water) to each well to cover the cell monolayer. Incubate for 5 min.
  20. Wash with 1 mL of tap water. Make sure to discard all the liquid in the well by gentle pipetting. Place the plate upside down on a diaper pad to dry overnight.
  21. Count the plaques visually and save the images on any available imager.

Representative Results

Biofilm-grown S. pneumoniae (Figure 1A) were used to infect mice (Figure 1B) using a small 10 µL inoculum delivered intranasally to unanesthetized mice. This small-volume inoculum results in consistent pneumococcal carriage restricted to the nasopharynx (Figure 2A, +sp groups) while avoiding systemic spread (Figure 2B,C, +sp groups). Two days following intranasal inoculation, the mice were infected with a murine-adapted H1N1 influenza A virus A/PR/8/34 (IAV)22,30 delivered both intranasally and intratracheally to achieve consistent delivery of specific amounts to the nasopharynx and the lungs23.

Here, the model was used to compare the course of disease following viral infection in mice intranasally challenged with different strains of S. pneumoniae, including TIGR4 and D39, which are invasive strains that result in pneumonia that progresses to bacteremia, and EF3030, which is an otitis media strain21,24,25,26,31. The disease presentation in S. pneumoniae/IAV co-infected mice was dependent on the bacterial strain (Figure 2). While there was no significant difference in bacterial numbers of the nasopharynx (Figure 2A) among any of the strains, S. pneumoniae TIGR4 and D39, but not EF3030, disseminated to the lungs by 48 h post IAV infection (Figure 2B). Forty percent of the mice intranasally infected with S. pneumoniae TIGR4 displayed bacterial dissemination to the lungs, and of those, half of them became bacteremic (Figure 2C), consistent with prior findings23.

Mice intranasally infected with S. pneumoniae D39 showed more efficient dissemination, because spread to the lungs was observed in 100% of the co-infected mice (Figure 2B). Similar to S. pneumoniae TIGR4, half of those experienced bacteremia (Figure 2C). In tracking the overall survival, regardless of the bacterial strain, the rate of survival of co-infected mice was significantly lower than the mice singly challenged with S. pneumoniae alone for all the strains tested (Figure 2D). Compared to the control mice challenged with IAV alone, the mice intranasally infected with S. pneumoniae TIGR4 and D39, but not EF3030, displayed accelerated rates of disease. By day 2 post IAV infection, 30% (D39) and 20% (TIGR4) of mice had succumbed, while the IAV-only control groups did not start to succumb until day 5 post challenge (Figure 2D). The mice co-infected with S. pneumoniae EF3030 and IAV had delayed symptoms, more similar to the IAV-only controls (Figure 2D). These findings demonstrate that the co-infection model results in disease in young healthy mice that is bacterial strain-dependent, which makes it ideal for exploring the bacterial factors required at each step of disease progression.

This model was used to assess the presence of various immune cells in the lungs (cell types and gating strategy in Figure 3) following IAV infection in mice intranasally inoculated with different strains of S. pneumoniae. The bacterial strains D39 and TIGR4, which dispersed into the lungs following IAV infection, elicited a significant increase above baseline (uninfected) in the influx of inflammatory immune cells from the circulation, such as neutrophils (PMNs) and monocytes, while EF3030 did not (Figure 4AC). IAV infection alone elicited a significant increase above baseline in the influx of immune cells important for host defense against viral infection, such as NK cells and gamma-delta T cells (Figure 4AC). These antiviral responses were significantly blunted in mice intranasally infected with S. pneumoniae prior to viral challenge (Figure 4AC). This is consistent with prior studies assessing cytokine responses that found that S. pneumoniae carriage blunted the production of type I interferons and impaired the ability of the host to control IAV loads in the lungs23. These findings demonstrate that the co-infection model can be used to study how immune responses change in mono versus polymicrobial infections.

This model was also used to assess the effect of aging on the course of disease following IAV infection in mice intranasally infected with S. pneumoniae TIGR4. In singly infected mice, the viral titers did not vary between the young and aged cohorts (Figure 5A)23. As in prior studies23, old mice displayed earlier and significantly more severe signs of disease compared to their young counterparts, as demonstrated by the higher clinical scores (Figure 5B). Consistent with the disease symptoms, old mice inoculated with S. pneumoniae started dying faster within 24 h post IAV infection, and all of them succumbed to the disease, whereas the young controls survived the infection at a significantly higher (33%) rate (Figure 5C). These findings demonstrate that the co-infection model can be used to detect more severe disease in vulnerable hosts, making it ideal for exploring host factors that confer resistance or susceptibility to co-infection.

Figure 1
Figure 1: Timeline of co-infection and organ processing for the assessment of immune cell influx and pathogen burden. (A) Streptococcus pneumoniae are grown in biofilms. (B) Mice are inoculated intranasally with 5 × 106 CFU of the indicated biofilm-grown S. pneumoniae strain to establish nasopharyngeal carriage or left untreated. Forty-eight hours later, the mice are either mock treated with PBS or receive 200 PFU of influenza A virus PR8 intranasally and 20 PFU intratracheally. Mice are monitored over time for clinical disease scores and survival. (C) At 48 h post IAV infection, bacterial CFU or viral PFU in the different organs or immune cell influx in the lungs are assessed. Abbreviations: CFU = colony-forming units; PFU = plaque-forming units; IAV = influenza A virus PR8; IT = intratracheally; NP = nasopharyngeally. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Dual intranasal/intratracheal IAV infection of S. pneumoniae-inoculated mice leads to bacterial spread and disease that is dependent on the bacterial strain. Young (10-12 weeks old) male C57BL/6 (B6) mice were infected as in Figure 1. Bacterial numbers in the (A) nasopharynx, (B) lungs, and (C) blood were all determined at 48 h post IAV infection. (B,C) Percentages denote the fraction of mice that exhibited spread. (D) Survival was monitored for 10 days post IAV infection. Pooled data from (A,B) n = 5, (C) n = 11, and (D) n = 6 mice per group are shown. Each circle corresponds to one mouse, and the dashed lines indicate the limit of detection. (AC) *, indicates a significant difference (p < 0.05) between the indicated groups as determined by the Kruskal-Wallis test. (D) *, indicates a significant difference (p < 0.05) between +sp and Co-inf mice per bacterial strain as determined by the log-rank (Mantel-Cox) test. Abbreviations: +sp = mice infected intranasally with bacteria only using the indicated strain; Co-inf = bacterial-infected mice that were infected with IAV; IAV = mice that received the influenza A virus; CFU = colony-forming units. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Immune cell gating strategy. The lungs were harvested, and the immune cell influx was determined by flow cytometry. The representative gating strategy of the different cell types is shown. (A) CD45+, live single cells were gated on and the percentages of (B) PMNs (Ly6G+, CD11b+), macrophages (Ly6G, Ly6C, F480+), and monocytes (Ly6G, Ly6C+), (C) DCs (Ly6G, CD11c+) and NK cells (NK1.1+, CD3), (D) TCR γΔ and CD8 (CD8+, TCRβ+) and CD4 (CD4+, TCRβ+) T cells were determined. Abbreviations: SSC-A = side scatter-peak area; FSC-A = forward scatter-peak area; FSC-H = forward scatter-peak height; SSC-W = side scatter-peak width; L/D = live/dead; FMO = fluorescent minus one; NK = natural killer; PMN = polymorphonuclear leukocyte; DC = dendritic cell; TCR = T cell receptor. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Pulmonary immune responses are bacterial strain-dependent. Young (10-12 weeks old) C57BL/6 male mice were either uninfected, singly inoculated with the indicated Streptococcus pneumoniae strain (+sp), singly challenged with IAV (IAV), or co-infected with S. pneumoniae and IAV (Co-inf). Forty-eight hours following IAV infection (see the experimental design in Figure 1), the lungs were harvested, and the immune cell influx was determined by flow cytometry following the gating strategy in Figure 3. (A) The average percentages of each indicated cell type within the CD45 gate are displayed for all the treatment groups on the heat map. (B) Representative dot plots of cell types that displayed significant differences between treatments are shown for each mouse group. (C) The percentages of the indicated immune cell types are shown. Each circle corresponds to one mouse. (A,C) Pooled data from n = 5 mice per group are shown. *, indicates a significant difference (p < 0.05) between Co-inf and uninfected; $, indicates a significant between IAV and uninfected; #, indicates a significant difference between Co-inf and IAV alone. Significant differences between the challenge groups for each cell type were determined by ANOVA followed by the Tukey's test. Abbreviations: NK = natural killer; PMN = polymorphonuclear leukocyte; DC = dendritic cell; TCR = T cell receptor; IAV = influenza A virus. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Aging and increased host susceptibility to IAV/Streptococcus pneumoniae co-infection. Young (10-12 weeks) and aged (21-22 months) C57BL/6 male mice were co-infected with S. pneumoniae TIGR4 i.n. and IAV i.n. and i.t. (as in Figure 1) or singly challenged with IAV alone. (A) Viral titers were determined 48 h later. Asterisks indicate statistical significance (p < 0.05) as determined by the Student's t-test. Data are pooled from n = 4 mice per group. (B) Clinical score and (C) survival were monitored over time. (B) The mean ± SEM pooled from n = 6 mice per group are shown. Asterisks indicate statistical significance (p < 0.05) between the young versus old mice at the indicated timepoint as determined by the Mann-Whitney test. (C) Data are pooled from n = 6 mice per group. Asterisks indicate statistical significance (p < 0.05) between the young versus old mice as determined by the log-rank (Mantel-Cox) test. Abbreviations: IAV = influenza A virus; i.n. = intranasally; i.t. = intratracheally; SEM = standard error of the mean. Figure 5A is reprinted with permission from Joma et al.23. Please click here to view a larger version of this figure.

Mix I stock for CDM
Adenine 0.1 g
D-Alanine 0.25 g
CaCl2 Anhydrous 0.025 g
Manganese Sulfate 0.03 g
Cyanocobalamin 100 µL of 10 mg/mL stock
Para-Aminobenzoic Acid 400 µL of 5 mg/mL stock
Pyridoxamine 2HCl 100 µL of 10 mg/mL stock
Mix II stock for CDM
Guanine 0.05 g
Uracil 0.05 g
Mix III stock for CDM
Ferric Nitrate 9H2O 50 mg/mL
Ferric Sulfate 7H2O 10 mg/mL
Mix IV stock for CDM
Beta-Nicotinamide adenine dinucleotide 25 mg/mL

Table 1: Mix I, II, III, and IV stocks for CDM. Abbreviation: CDM = chemically defined media.

Vitamin Mix Stock for CDM
Pyridoxal Hydrochloride 0.8 g
Thiamine Cl2 0.4 g
Riboflavin 0.4 g
Ca-pantothenate 0.4 g
Biotin 0.04 g
Folic Acid 0.4 g
Niacinamide 0.4 g

Table 2: Vitamin Mix Stock for CDM. Abbreviation: CDM = chemically defined media.

Amino Acid Stock for CDM
L-Alanine 0.480 g
L-Arginine 0.250 g
L-Asparagine 0.700 g
L-Aspartic Acid 0.600 g
L-Cysteine 1.000 g
L-Cystine 0.100 g
L-Glutamic Acid 0.200 g
L-Glutamine 0.780 g
L-Glycine 0.350 g
L-Histidine 0.300 g
L-Isoleucine 0.430 g
L-Leucine 0.950 g
L-Lysine 0.880 g
L-Methionine 0.250 g
L-Phenylalanine 0.550 g
L-Proline 1.350 g
L-Serine 0.680 g
L-Threonine 0.450 g
L-Tryptophan 0.100 g
L-Valine 0.650 g

Table 3: Amino Acid Stock for CDM. Abbreviation: CDM = chemically defined media.

Starter Stock for CDM
Dextrose 1.0 g
Magnesium Sulfate-7-Hydrate 0.070 g
Potassium Phosphate Dibasic 0.02 g
Potassium Phosphate Monobasic 0.1 g
Sodium Acetate Anhydrous 0.45 g
Sodium Bicarbonate 0.25 g
Sodium Phosphate Dibasic 0.735 g
Sodium Phosphate Monobasic 0.32 g
Final Supplements for CDM
Choline Chloride 0.1 g
L-Cysteine HCl 0.075 g
Sodium Bicarbonate 0.25 g

Table 4: Starter stock and final supplements for CDM. Abbreviation: CDM = chemically defined media.

Antibody/Fluorophore Clone Dilution Factor
L/D for UV excitation N/A 0.38888889
Ly6G AF 488 1A8 0.25
CD11b APC M1/70 0.25
CD11c PE N418 0.18055556
Mouse Fc Block 2.4G2 0.11111111
F4/80 PE Cy7 BM8 0.18055556
Ly6C BV605 AL-21 0.25
CD103 BV 421 M290 0.18055556
CD45 APC-eF-780 30-F11 0.18055556

Table 5: Antibody panel 1.

Antibody/Fluorophore Clone Dilution Factor
L/D for UV excitation N/A 0.388888889
TCR-β APC Cy7 H57-597 0.180555556
CD4 V450 (Pacific Blue) RM4-5 0.25
CD8 BV650 53-6.7 0.180555556
Mouse Fc Block 2.4G2 0.111111111
CD45 PE 30-F11 0.180555556
CD3 AF488 145-2C11 0.180555556
TCR- γΔ APC GL-3 0.180555556
NK1.1 AF 700 PK136 0.180555556

Table 6: Antibody panel 2.

Discussion

Most of the existing S. pneumoniae/IAV co-infection experimental studies rely on bacterial delivery into the lungs of mice pre-infected with IAV. These models have helped identify changes in the pulmonary environment and systemic immune response that render the host susceptible to secondary bacterial infection15,16,17,32,33,34,35,36,37. However, these models have failed to mimic the transition of S. pneumoniae from an asymptomatic colonizer to a pathogen capable of causing serious lung and systemic infections. Further, these models are not suitable for studying the host factors and host-pathogen interactions in the upper respiratory tract that contribute to susceptibility to infection. A prior model for the movement of pneumococci from the nasopharynx to the lung after IAV infection relied on bacterial infection of the nasopharynx followed by viral infection. However, it failed to reproduce the severe signs of disease observed in human patients21. The modified murine infection model described here recapitulates the transition of S. pneumoniae from asymptomatic carriage to a pathogen that causes severe clinical disease.

A critical step of this model is establishing S. pneumoniae infection in the nasopharynx. Streptococcus pneumoniae form biofilms and colonize the nasopharynx at different efficiencies21,38. To establish consistent infection, at least 5 × 106 CFU of the biofilm-grown bacterial strains tested so far are required23. It is recommended that any new bacterial strain be tested for stable infection of the nasopharynx prior to viral infection. For viral co-infection, previous studies have found that intranasal infection with IAV is required for the dispersion of the bacteria from the nasopharynx21,22,23. In those prior studies, 500 PFU of IAV for intranasal delivery were used, while in this study, 200 PFU were sufficient to increase bacterial numbers in the nasopharynx. IAV infection is not limited to the upper airways and can spread to the lungs39,40, which is key for rendering the pulmonary environment more permissive for bacterial infection15,16,41. The delivery of IAV to the lungs can be achieved by either intranasal delivery or intratracheal installation of anesthetized mice. Prior work with BALB/cByJ mice found that intranasal delivery results in viral pneumonia21; however, access of the inoculum to the lungs following intranasal inoculation is more restricted in C57BL/6 mice. In C57BL/6 mice, intratracheal installation is required for consistent delivery of the virus23. In this model, prior bacterial colonization accelerates the presentation of disease symptoms after viral infection23. As viral infection can itself cause disease symptoms with potential variation in kinetics, it is recommended to first test a range of doses for any new viral strain tested and choose a dose that reveals accelerated kinetics in co-infected hosts.

The lungs provide another critical readout for disease evaluation in this model. For the assessment of pathogen burden and immune cell influx, a lung from the same mouse can be used. However, as infection and inflammation severity can differ between lobes, it is recommended to not take different lobes of the same lung for the various assessments. Rather, all the lobes can be minced into small pieces, mixed well together, and then parsed out equally for the different assessments. Similarly, the nasopharynx can be used for the enumeration of bacterial CFU or viral PFU and immune response. However, the number of cells obtained from the washes and tissue is too low to perform flow cytometry without pooling the samples from mice within the same group. Alternatively, inflammation in the nasopharynx can be assessed histologically23.

A critical feature of this model is that it recapitulates the clinical disease seen in patients. In humans, secondary pneumococcal pneumonia following IAV infection often results in obvious signs of disease, including cough, dyspnea, fever, and muscle aches that can lead to hospitalizations, respiratory failure, and even death8,15,42,43. This model recapitulates the severe signs of clinical disease observed in humans in terms of difficulty in breathing (reflected in the breathing score) and overall malaise (reflected in posture and movement scores) displayed by the mice, as well as death in some of the healthy young controls. The exacerbated disease symptoms in co-infected mice are likely a result of both bacterial dissemination to the lungs and impaired viral clearance in mice with pneumococcal carriage23. A limitation of the model is that the incidence of clinical disease and bacterial dissemination from the nasopharynx varies between mice and is influenced by bacterial strain, host age, and genotype21,22,23. Reflecting this, for invasive strains, the progression from localized infection (with no detectable bacteremia) to death can occur within 24 h. Therefore, for a true assessment of systemic spread, bacteremia should be followed over shorter intervals (every 6-12 h). Similarly, the disease score can change rapidly, particularly in the first 72 h following co-infection. Therefore, to closely track the disease symptoms, it is advisable to monitor mice three times per day for days 1-3 post IAV infection.

In summary, this model replicates the movement of S. pneumoniae from an asymptomatic colonizer of the nasopharynx to a pathogen capable of causing pulmonary and systemic disease upon IAV infection. In this model, IAV triggers the transition of S. pneumoniae via modifying the bacterial behavior in the nasopharynx, increasing bacterial spread to the lung, and altering antibacterial immunity23. Similarly, bacterial carriage blunts the antiviral immune responses and impairs IAV clearance from the lungs23. This renders this model ideal for parsing out changes in immune responses in single versus polymicrobial infections. Additionally, the course of disease following co-infection is, in part, dependent on the strain of pneumococci present in the nasopharynx. Therefore, the model is suited to dissecting the bacterial factors required for asymptomatic colonization versus pathogenic transition of S. pneumoniae. Lastly, this model reproduces the susceptibility of aging to co-infections, and although this was not tested here, it can be easily used to assess the impact of host background on the disease course. In conclusion, separating carriage and disease into distinct steps provides the opportunity to analyze the genetic variants of both the pathogens and the host, allowing the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. Moving forward, this model can be used to tailor treatment options for vulnerable hosts.

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

We would like to thank Nick Lenhard for the critical reading and editing of this manuscript. We would also like to thank Andrew Camilli and Anthony Campagnari for the bacterial strains and Bruce Davidson for the viral strains. This work was supported by the National Institute of Health Grant (R21AG071268-01) to J.L. and the National Institute of Health Grants (R21AI145370-01A1), (R01AG068568-01A1), (R21AG071268-01) to E.N.B.G.

Materials

4-Aminobenzoic acid  Fisher AAA1267318 Mix I stock
96-well round bottom plates Greiner Bio-One 650101
100 µm Filters Fisher 07-201-432
Adenine Fisher AC147440250 Mix I stock
Avicel Fisher 501785325 Microcyrstalline cellulose 
BD Cytofix Fixation Buffer  Fisher BDB554655 Fixation Buffer
BD Fortessa Flow cytometer 
BD Intramedic Polyethylene Tubing  Fisher 427410 Tubing for nasal lavage
BD Disposable Syringes with Luer-Lok Tips (1 mL) Fisher 14-823-30
BD Microtainer Capillary Blood Collector and BD Microgard Closure Fisher 02-675-185 Blood collection tubes
Beta-Nicotinamide adenine dinucleotide Fisher AAJ6233703 Mix IV stock
Biotin Fisher AC230090010 Vitamin stock
C57BL/6J mice The Jackson Laboratory #000644 Mice used in this study
Calcium Chloride Anhydrous Fisher Chemical C77-500 Mix I stock
CD103 BV 421 BD Bioscience BDB562771 Clone: M290 DF 1:200
CD11b APC Invitrogen 50-112-9622 Clone: M1/70, DF 1:300
CD11c PE BD Bioscience BDB565592 Clone: N418 DF 1:200
CD3 AF 488 BD Bioscience OB153030 Clone: 145-2C11 DF 1:200
CD4 V450 BD Horizon BDB560470 Clone: RM4.5 DF 1:300
CD45 APC eF-780 BD Bioscience 50-112-9642 Clone: 30-F11 DF 1:200
CD45 PE Invitrogen 50-103-70 Clone: 30-F11 DF 1:200
CD8α BV 650 BD Horizon BDB563234 Clone: 53-6.7 DF 1:200
Choline chloride Fisher AC110290500 Final supplement to CDM
Corning Disposable Vacuum Filter/Storage Systems Fisher 09-761-107 Filter sterilzation apparatus 
Corning Tissue Culture Treated T-25 Flasks Fisher 10-126-9
Corning Costar Clear Multiple Well Plates Fisher 07-201-590
Corning DMEM With L-Glutamine and 4.5 g/L Glucose; Without Sodium Pyruvate Fisher MT10017CM
Cyanocobalamin Fisher AC405925000 Mix I stock
D39 National Collection of Type Culture (NCTC) NCTC 7466 Streptococcus pneumoniae strain
D-Alanine Fisher AAA1023114 Mix I stock
D-Calcium pantothenate Fisher AC243301000 Vitamin stock
Dextrose Fisher Chemical D16-500 Starter stock
Dnase  Worthington Biochemical  LS002147
Eagles Minimum Essential Medium ATCC 30-2003
EDTA VWR BDH4616-500G
EF3030 Center for Disease Control and Prevention  Available via the isolate bank request Streptococcus pneumoniae strain, request using strain name
F480 PE Cy7 BD Bioscience 50-112-9713 Clone: BMB DF 1:200
Falcon 50 mL Conical Centrifuge Tubes Fisher 14-432-22 50 mL round bottom tube
Falcon Round-Bottom Polypropylene Test Tubes With Cap Fisher 14-959-11B 15 mL round bottom tube
Falcon Round-Bottom Polystyrene Test Tubes (5 mL) Fisher 14-959-5 FACS tubes 
FBS Thermofisher 10437-028
Ferric Nitrate Nonahydrate Fisher I110-100 Mix III stock
Fisherbrand Delicate Dissecting Scissors Fisher 08-951-5 Instruments used for harvest
Fisherbrand Disposable Inoculating Loops  Fisher 22-363-602 Inoculating loops 
Fisherbrand Dissecting Tissue Forceps Fisher 13-812-38 Forceps for harvest
Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL Fisher 05-408-137 Micocentrifuge tubes
Fisherbrand Sterile Syringes for Single Use (10 mL) Fisher 14-955-459
Folic Acid Fisher AC216630500 Vitamin stock
Gibco RPMI 1640 (ATCC) Fisher A1049101
Gibco DPBS, no calcium, no magnesium Fisher 14190250
Gibco HBSS, calcium, magnesium, no phenol red Fisher 14025134
Gibco MEM (Temin's modification) (2x), no phenol red Fisher 11-935-046
Gibco Penicillin-Streptomycin (10,000 U/mL) Fisher 15-140-122
Gibco Trypan Blue Solution, 0.4% Fisher 15-250-061
Gibco Trypsin-EDTA (0.25%), phenol red Fisher 25-200-056
Glycerol (Certified ACS) Fisher G33-4
Glycine Fisher AA3643530 Amino acid stock
Guanine Fisher AAA1202414 Mix II stock
Invitrogen UltraComp eBeads Compensation Beads Fisher 50-112-9040
Iron (II) sulfate heptahydrate Fisher AAA1517836 Mix III stock
L-Alanine Fisher AAJ6027918 Amino acid stock
L-Arginine Fisher AAA1573814 Amino acid stock
L-Asparagine Fisher AAB2147322 Amino acid stock
L-Aspartic acid  Fisher AAA1352022 Amino acid stock
L-Cysteine Fisher AAA1043518 Amino acid stock
L-Cysteine hydrochloride monohydrate Fisher AAA1038914 Final supplement to CDM
L-Cystine Fisher AAA1376218 Amino acid stock
L-Glutamic acid  Fisher AC156211000 Amino acid stock
L-Glutamine Fisher O2956-100 Amino acid stock
L-Histidine Fisher AC166150250 Amino acid stock
LIFE TECHNOLOGIES LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation Invitrogen 50-112-1524 Clone: N/A DF 1:500
L-Isoleucine Fisher AC166170250 Amino acid stock
L-Leucine Fisher BP385-100 Amino acid stock
L-Lysine Fisher AAJ6222514 Amino acid stock
L-Methionine Fisher AAA1031822 Amino acid stock
Low endotoxin BSA  Sigma Aldrich A1470-10G
L-Phenylalanine Fisher AAA1323814 Amino acid stock
L-Proline Fisher AAA1019922 Amino acid stock
L-Serine Fisher AC132660250 Amino acid stock
L-Threonine Fisher AC138930250 Amino acid stock
L-Tryptophan Fisher AAA1023014 Amino acid stock
L-Valine Fisher AAA1272014 Amino acid stock
Ly6C BV 605 BD Bioscience BDB563011 Clone: AL-21 DF 1:300
Ly6G AF 488  Biolegend NC1102120 Clone: IA8, DF 1:300
Madin-Darby Canine Kidney (MDCK) cells  American Type Culture Collection (ATCC) CCL-34 MDCK cell line for PFU analuysis 
Magnesium Sulfate 7-Hydrate Fisher 60-019-68 CDM starter stock
Manganese Sulfate Fisher M113-500 Mix I stock
MilQ water Ultra-pure water
Mouse Fc Block BD Bioscience BDB553142 Clone: 2.4G2 DF 1:100
MWI VETERINARY PURALUBE VET OINTMENT Fisher NC1886507 Eye lubricant for infection
NCI-H292 mucoepidermoid carcinoma cell line  ATCC CRL-1848 H292 lung epithelial cell line for biofilm growth
Niacinamide Fisher 18-604-792 Vitamin stock
NK 1.1 AF 700 BD Bioscience 50-112-4692 Clone: PK136 DF 1:200
Oxyrase For Broth 50Ml Bottle 1/Pk Fisher 50-200-5299 To remove oxygen from liquid cultures
Paraformaldehyde 4% in PBS Thermoscientific J19932-K2
Pivetal Isoflurane  Patterson Veterinary 07-893-8440 Isoflurane for anesthesia during infection 
Potassium Phosphate Dibasic Fisher Chemical P288-500 Starter stock
Potassium Phosphate Monobasic Fisher Chemical P285-500 Starter stock
Pyridoxal hydrochloride  Fisher AC352710250 Vitamin stock
Pyridoxamine dihydrochloride Fisher AAJ6267906 Mix I stock
Riboflavin Fisher AC132350250 Vitamin stock
Sodium Acetate VWR 0530-500G Starter stock
Sodium Azide  Fisher Bioreagents BP922I-500 For FACS buffer
Sodium Bicarbonate Fisher Chemical S233-500 Starter stock and final supplement to CDM
Sodium Phosphate Dibasic Fisher Chemical S374-500 Starter stock
Sodium Phosphate Monobasic Fisher Chemical S369-500 Starter stock
TCR APC  BD Bioscience 50-112-8889 Clone: GL-3 DF 1:200
TCRβ APC-Cy7 BD Pharmigen BDB560656 Clone: H57-597 DF 1:200
Thermo Scientific Blood Agar with Gentamicin Fisher R01227 Blood agar plates with the antibiotic gentamicin 
Thermo Scientific Trypsin, TPCK Treated Fisher PI20233
Thiamine hydrochloride Fisher AC148991000 Vitamin stock
TIGR4 ATCC BAA-334 Streptococcus pneumoniae strain
Uracil Fisher AC157300250 Mix II stock
Worthington Biochemical Corporation Collagenase, Type 2, 1 g Fisher NC9693955

Referanslar

  1. Kadioglu, A., Weiser, J. N., Paton, J. C., Andrew, P. W. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nature Reviews Microbiology. 6 (4), 288-301 (2008).
  2. Obaro, S., Adegbola, R. The pneumococcus: Carriage, disease and conjugate vaccines. Journal of Medical Microbiology. 51 (2), 98-104 (2002).
  3. Chong, C. P., Street, P. R. Pneumonia in the elderly: A review of the epidemiology, pathogenesis, microbiology, and clinical features. Southern Medical Journal. 101 (11), 1141-1145 (2008).
  4. Kadioglu, A., Andrew, P. W. Susceptibility and resistance to pneumococcal disease in mice. Briefings in Functional Genomics and Proteomics. 4 (3), 241-247 (2005).
  5. Ganie, F., et al. Structural, genetic, and serological elucidation of Streptococcus pneumoniae serogroup 24 serotypes: Discovery of a new serotype, 24C, with a variable capsule structure. Journal of Clinical Microbiology. 59 (7), 0054021 (2021).
  6. Centers for Disease Control and Prevention. Estimates of deaths associated with seasonal influenza — United States. MMWR. Morbidity and Mortality Weekly Report. 59 (33), 1057-1062 (2010).
  7. Shrestha, S., et al. Identifying the interaction between influenza and pneumococcal pneumonia using incidence data. Science Translational Medicine. 5 (191), (2013).
  8. McCullers, J. A. Insights into the interaction between influenza virus and pneumococcus. Clinical Microbiology Reviews. 19 (3), 571-582 (2006).
  9. Pneumococcal Disease Global Pneumococcal Disease and Vaccine. Centers for Disease Control and Prevention Available from: https://www.cdc.gov/pneumococcal/global.html (2018)
  10. Grudzinska, F. S., et al. Neutrophils in community-acquired pneumonia: Parallels in dysfunction at the extremes of age. Thorax. 75 (2), 164-171 (2020).
  11. Boe, D. M., Boule, L. A., Kovacs, E. J. Innate immune responses in the ageing lung. Clinical and Experimental Immunology. 187 (1), 16-25 (2017).
  12. Krone, C. L., van de Groep, K., Trzcinski, K., Sanders, E. A., Bogaert, D. Immunosenescence and pneumococcal disease: An imbalance in host-pathogen interactions. The Lancet Respiratory Medicine. 2 (2), 141-153 (2014).
  13. Cho, S. J., et al. Decreased NLRP3 inflammasome expression in aged lung may contribute to increased susceptibility to secondary Streptococcus pneumoniae infection. Experimental Gerontology. 105, 40-46 (2018).
  14. Disease Burden of Influenza. Centers for Disease Control and Prevention Available from: https://www.cdc.gov/flu/about/burden/index.html (2018)
  15. McCullers, J. A. The co-pathogenesis of influenza viruses with bacteria in the lung. Nature Reviews Microbiology. 12 (4), 252-262 (2014).
  16. McCullers, J. A., Rehg, J. E. Lethal synergism between influenza virus and Streptococcus pneumoniae: Characterization of a mouse model and the role of platelet-activating factor receptor. The Journal of Infectious Diseases. 186 (3), 341-350 (2002).
  17. Metzger, D. W., Sun, K. Immune dysfunction and bacterial coinfections following influenza. Journal of Immunology. 191 (5), 2047-2052 (2013).
  18. Chao, Y., Marks, L. R., Pettigrew, M. M., Hakansson, A. P. Streptococcus pneumoniae biofilm formation and dispersion during colonization and disease. Frontiers in Cellular and Infection Microbiology. 4, 194 (2014).
  19. Bogaert, D., De Groot, R., Hermans, P. W. Streptococcus pneumoniae colonisation: The key to pneumococcal disease. The Lancet Infectious Diseases. 4 (3), 144-154 (2004).
  20. Simell, B., et al. The fundamental link between pneumococcal carriage and disease. Expert Review of Vaccines. 11 (7), 841-855 (2012).
  21. Marks, L. R., Davidson, B. A., Knight, P. R., Hakansson, A. P. Interkingdom signaling induces Streptococcus pneumoniae biofilm dispersion and transition from asymptomatic colonization to disease. mBio. 4 (4), 00438 (2013).
  22. Reddinger, R. M., Luke-Marshall, N. R., Sauberan, S. L., Hakansson, A. P., Campagnari, A. A. Streptococcus pneumoniae modulates Staphylococcus aureus biofilm dispersion and the transition from colonization to invasive disease. mBio. 9 (1), 02089 (2018).
  23. Joma, B. H., et al. A murine model for enhancement of Streptococcus pneumoniae pathogenicity upon viral infection and advanced age. Infection and Immunity. 89 (8), 0047120 (2021).
  24. Andersson, B., et al. Identification of an active disaccharide unit of a glycoconjugate receptor for pneumococci attaching to human pharyngeal epithelial cells. Journal of Experimental Medicine. 158 (2), 559-570 (1983).
  25. Avery, O. T., Macleod, C. M., McCarty, M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. The Journal of Experimental Medicine. 79 (2), 137-158 (1944).
  26. Tettelin, H., et al. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science. 293 (5529), 498-506 (2001).
  27. Tothpal, A., Desobry, K., Joshi, S. S., Wyllie, A. L., Weinberger, D. M. Variation of growth characteristics of pneumococcus with environmental conditions. BMC Microbiology. 19 (1), 304 (2019).
  28. Bou Ghanem, E. N., et al. Extracellular adenosine protects against Streptococcus pneumoniae lung infection by regulating pulmonary neutrophil recruitment. PLoS Pathogens. 11 (8), 1005126 (2015).
  29. Bou Ghanem, E. N., et al. The alpha-tocopherol form of vitamin E boosts elastase activity of human PMNs and their ability to kill Streptococcus pneumoniae. Frontiers in Cellular and Infection Microbiology. 7, 161 (2017).
  30. Tait, A. R., Davidson, B. A., Johnson, K. J., Remick, D. G., Knight, P. R. Halothane inhibits the intraalveolar recruitment of neutrophils, lymphocytes, and macrophages in response to influenza virus infection in mice. Anesthesia & Analgesia. 76 (5), 1106-1113 (1993).
  31. Aaberge, I. S., Eng, J., Lermark, G., Lovik, M. Virulence of Streptococcus pneumoniae in mice: A standardized method for preparation and frozen storage of the experimental bacterial inoculum. Microbial Pathogenesis. 18 (2), 141-152 (1995).
  32. McCullers, J. A., Bartmess, K. C. Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae. The Journal of Infectious Diseases. 187 (6), 1000-1009 (2003).
  33. Smith, A. M., McCullers, J. A. Secondary bacterial infections in influenza virus infection pathogenesis. Current Topics in Microbiology and Immunology. 385, 327-356 (2014).
  34. Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., Tuomanen, E. I. Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature. 377 (6548), 435-438 (1995).
  35. Ballinger, M. N., Standiford, T. J. Postinfluenza bacterial pneumonia: Host defenses gone awry. Journal of Interferon & Cytokine Research. 30 (9), 643-652 (2010).
  36. Sun, K., Metzger, D. W. Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection. Nature Medicine. 14 (5), 558-564 (2008).
  37. Nakamura, S., Davis, K. M., Weiser, J. N. Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice. Journal of Clinical Investigation. 121 (9), 3657-3665 (2011).
  38. Blanchette-Cain, K., et al. Streptococcus pneumoniae biofilm formation is strain dependent, multifactorial, and associated with reduced invasiveness and immunoreactivity during colonization. mBio. 4 (5), 00745 (2013).
  39. Rello, J., Pop-Vicas, A. Clinical review: Primary influenza viral pneumonia. Critical Care. 13 (6), 235 (2009).
  40. Torres, A., Loeches, I. M., Sligl, W., Lee, N. Severe flu management: A point of view. Intensive Care Medicine. 46 (2), 153-162 (2020).
  41. Bakaletz, L. O. Viral-bacterial co-infections in the respiratory tract. Current Opinion in Microbiology. 35, 30-35 (2017).
  42. Palacios, G., et al. Streptococcus pneumoniae coinfection is correlated with the severity of H1N1 pandemic influenza. PLoS One. 4 (12), 8540 (2009).
  43. Dhanoa, A., Fang, N. C., Hassan, S. S., Kaniappan, P., Rajasekaram, G. Epidemiology and clinical characteristics of hospitalized patients with pandemic influenza A (H1N1) 2009 infections: The effects of bacterial coinfection. Virology Journal. 8, 501 (2011).

Play Video

Bu Makaleden Alıntı Yapın
Lenhard, A., Joma, B. H., Siwapornchai, N., Hakansson, A. P., Leong, J. M., Bou Ghanem, E. N. A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness. J. Vis. Exp. (187), e64419, doi:10.3791/64419 (2022).

View Video