Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Podsumowanie

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Streszczenie

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen¹. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types². In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway³, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens⁴, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit⁵. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear⁶. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Wprowadzenie

Animal models, especially mice, have been extensively used to explore immune responses against pathogens. Although innate and acquired immunity differ between rodents and humans⁷, the ease in breeding and the development of knockouts for numerous genes, make mice an excellent model to study immune responses⁸. The immune response is complex and results from the interaction of a pathogen, the resident microbial flora and several immune (lymphocytes, neutrophils, macrophages) and non-immune (epithelial cells, endothelial cells) cellular types². In vitro studies do not allow observing these complex interactions and mainly focus on unique cell-pathogen interactions. While animal models must be used with caution and limited to very specific and relevant questions, mouse models provide a good insight into the mammal immune response in vivo and may address parts of important clinical questions⁷.

In the airways, the microbial community is complex associating a large number of different microorganisms⁶. While what constitutes a "normal" airway microbiome remains to be determined, resident communities are frequently polymicrobial, and originate from diverse ecological sources. Patients with suppurative chronic lung disease (cystic fibrosis, bronchectasis) or mechanically ventilated patients exhibit a particular flora due to colonization of the airways by environmentally-acquired microorganisms⁹. Pseudomonas aeruginosa and Candida albicans are both problem pathogens⁵, frequently isolated together from tracheobronchial samples, and responsible of severe opportunistic infection in these patients, especially in the intensive care unit (ICU)⁴.

Isolation of these microorganisms during acute pneumonia in ICU results in anti-microbial treatment against P. aeruginosa but yeast are usually not considered pathogenic at this site⁵. In vitro interactions between P. aeruginosa and C. albicans have been widely reported and showed that these microorganisms can affect the growth and the survival of each other but studies could not conclude if the presence of C. albicans is detrimental or beneficial for the host¹⁰. Mouse models were developed to address this relevance of P. aeruginosa and C. albicans in vivo, but the interaction between microorganisms was not the key point. Indeed, the model was established to evaluate the involvement of C. albicans in host immune response, and outcome.

A previous model established by Roux et al already used an initial colonization with C. albicans followed by an acute lung infection induced by P. aeruginosa. Using their model, the authors found a deleterious role of prior C. albicans colonization¹¹. However Roux et al used a high load of C. albicans in their model with 2 x 10⁶ CFU/mouse during 3 consecutive days. We established a 4-day model of C. albicans airway colonization, or at least persistence without lung injury, In this model C. albicans was retrieved up to 4 days after a single instillation of 10⁵ CFU per mouse (Figure 2B) ^12,13. After 4 days, no evidence of inflammatory cell recruitment, inflammatory cytokine production nor epithelial damage was observed. At 24 - 48 hr, at the peak presence of C. albicans, even though a cellular and cytokine innate immune response was observed, there was no evidence of lung injury. Surprisingly, mice thus colonized with C. albicans 48 hr prior to intranasal instillation of P. aeruginosa had attenuated infection compared to mice with P. aeruginosa infection alone. Indeed, mice exhibited lesser lung injury and decreased bacterial burden^12,13.

Several hypotheses could explain this beneficial effect of prior colonization with C. albicans on P. aeruginosa-mediated acute lung infection. First, an interspecies cross-talk involving each microorganisms quorum-sensing systems, the homoserinelactone-based P. aeruginosa system and the farnesol-based C. albicans system, were evaluated. Second, C. albicans acting as a "decoy" target for P. aeruginosa diverting the pathogen from lung epithelial cells was studied. Both hypotheses were invalidated (unpublished data). The third hypothesis was that of a "priming" of the innate immune system by C. albicans responsible for an enhanced subsequent innate response against P. aeruginosa. This last hypothesis was confirmed. Indeed C. albicans colonization led to a priming of innate immunity through IL-22, mainly secreted by innate lymphoid cells, resulting in increased bacterial clearance and reduced lung injury¹².

In conclusion, the host is a central actor in the interaction between microorganisms modulating the innate immune response and involving different inflammatory cell types. While these complex immune interactions can be dissected in vitro the initial hypotheses can only be provided by appropriate in vivo models. The following protocol provides an example of in vivo study of host-mediated pathogen interaction that may be adapted to others microorganisms.

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Protokół

The regional ethics regional committee for animal experiments has approved this method, in accordance with national and international animal care and use in investigational research guidelines.

1. Sample Collection

1. Sample storage
   1. Collect all samples and immediately store at - 20 °C or on ice until freezer storage to avoid deterioration. Place sterile phosphate buffered saline (PBS) on ice to improve broncho-alveolar lavages (BAL) performance.
2. Surgery
   1. Sterilize all surgical equipment using an autoclave.
      NOTE: If possible, it is recommended to use two different sets of instruments for abdominal and thoracic steps to avoid cross-contamination. Required dissection equipment is detailed in Figure 4A

2. Mice, Bacterial and Yeast Strains

1. House mice in compliance with local use of animals in research committee guidelines in a ventilated rack without exceeding 5 mice per cage, with food and water ad lib, in a biosafety level 2 housing facility due to the use of biosafety level 2 micro-organisms : P. aeruginosa and C. albicans.
2. Keep the bacterial strains at -80 °C in 40% glycerol medium.
   1. Add bacteria directly from frozen stock into culture tube containing 3 ml of sterile Luria-Bertani broth using a 10 µl inoculation loop. Leave O/N at 37 °C with orbital shaking (400 rpm) the day before instillation.
   2. Harvest bacteria by centrifugation at 2,000 x g for 5 min.
   3. Aspirate the supernatant into an appropriate closed biohazard waste disposal. Observe white adherent pellet at the bottom of culture tube.
   4. Wash and suspend the pellet using 5 ml of PBS.
   5. Repeat steps 2.2.2 to 2.2.3 a second time to perform a second wash.
   6. Resuspend the pelleted bacteria using 1 ml of PBS and brief vortexing.
   7. Determine the inoculum density using optical densitometer at 600 nm using an Optical Density Meter. A density of 0.9 corresponds to 10⁹ CFU/ml for PAO1, dilute accordingly.
      NOTE: This result has to be obtained for each used strain by determining density of successive dilutions of a calibrated inoculum.
   8. Verify the inoculum by serial logarithmic dilutions and plate 100 µl of each dilution on bromocresol purple agar (BCP) plates and O/N culture. Administer each mouse intranasally 50 µl of the solution containing 1 x 10⁸ to 2 x10⁸ CFU per ml (5 x 10⁶ to 1 x10⁷ CFU per mouse).
3. Use C. albicans SC5314 as a reference strain. Conserve the strain in 40% glycerol medium at -80°C.
   1. Supplement Yeast-Peptone-Dextrose broth with 0.015% amikacin to avoid bacterial contamination and facilitate further count.
   2. Add yeast using 10 µl inoculation loop into prepared YPD-broth supplemented with amikacin O/N at 37°C.
   3. Harvest yeast by centrifugation at 2,000 x g for 5 min.
   4. Remove supernatant into an appropriate bioharzard waste disposal. White adherent pellet should be observed in the bottom of culture tube.
   5. Wash and suspend the pelleted bacteria using 5 ml of PBS and brief vortexing.
   6. Repeat steps 2.2.2 to 2.2.3 a second time to perform a second wash.
   7. Resuspend the pellet using 1 ml of PBS and brief vortexing.
   8. Determine the size of the inoculum by counting on a Mallassez hematocytometer using a standard microscope at 40x magnification.
      NOTE: Concentration (in CFU/ml) is obtained using the following formula: (number of yeast x 10⁵)/(number of counted grid rectangles on the Mallassez hematocytometer).
   9. Verify by serial logarithmic dilution to 10^-5 and 10^-6 to confirm that solution contains 2 x 10⁶ CFU/ml.
   10. Plate on YPD agar plates supplemented with 0.015% amikacin.

3. Airways Colonization by C. albicans

NOTE: After environmental adaptation, mice are weighed twice a day.

1. Under a fume hood, deposit 500 µl of Sevoflurane onto a 4 cm x 4 cm gauze (open-drop technique)¹⁴.
   1. Immediately place the gauze on the floor of an approximately 750 ml induction chamber. Immediately place a mesh-raised platform above the gauze to avoid direct contact between the animal and the gauze.
   2. Close airtight lid and wait 1 to 2 min to allow diffusion of Sevoflurane in the chamber.
   3. Transfer the mouse from cage to mesh platform and close lid. Light anesthesia with conserved spontaneous breathing should be achieved in 30 to 45 sec.
   4. Monitor for hypotonia by observing loss of righting reflex at which point the mouse can be removed from the box and instilled.
2. Intranasal instillation (Can be performed in 10 sec by a trained operator).
   1. Hold the mouse one-handed nestled on its back held upright (Figure 4B).
   2. Using index finger, support the head and use the thumb to keep the jaw closed to avoid expectoration (Figure 4C).
   3. As described at step 2.3.8, ensure that the prepared C. albicans solution contains 2 x10⁶ CFU per ml for an 50 µl instilled volume.
      NOTE: The second critical point is the instilled volume. A volume lower than 50 µl could result in an insufficient colonization or inhomogeneous instillation of airway, a larger volume may induce drowning/suffocation and death.
   4. Instil the mouse intra-nasally by approaching the pipette to the nostrils.
   5. Pipette a 50 µl drop forming a bubble containing the solution on the nostrils, subsequently inhaled by the spontaneously breathing mouse.
   6. Place mouse in a recovery area (e.g., a large well-aerated bare cage with an overhead heating lamp). Mice must be monitored until complete awakening. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency and righting reflex. At this point, the mouse can be returned to a normal housing cage.

4. P. aeruginosa-induced Acute Lung Infection

NOTE: Mice are weighed during the four following days. Normally, mice gain weight during C. albicans-mediated airway colonization (Figure 2A).

1. Prepare the suspension containing P. aeruginosa the day of the instillation after O/N growth (section 2.2).
2. Anesthetize briefly using inhaled Sevoflurane as described above (section 3.1).
   NOTE: To perform acute lung infection, recommended bacterial burden are suggested in Table 1.
3. Instil the mouse as described above (section 3.2) with particular attention to post-instillation recovery.

5. Measure of Lung Injury Index

1. Prepare a solution containing FITC-labeled albumin. Inject this solution 2 hr before animal euthanasia.
   1. Weigh 0.2 mg of albumin-FITC with the appropriate equipment.
   2. Add 0.2 mg into 1 ml PBS. Briefly vortex. If not used immediately, place the solution in foil to avoid exposure to ambient light.
   3. Inject intra-peritoneally 200 µl of FITC-labeled albumin solution to each mouse.
2. Euthanasia
   1. Weigh the mice for the last weight data.
   2. Euthanize a single mouse in accordance with local use of animals in research committee guidelines using one intra-peritoneal injection of a lethal overdose of pentobarbital : 300 µl of 5.47% pentobarbital.
   3. Remove mouse from the cage and receives lethal injection by operator.
   4. Following injection, transfer the mouse alone to another cage, hidden from any other animals. Observe the mouse until absence of movement. Confirm death is by absence of movement, particularly respiratory movement, lack of pulse.
   5. Perform surgical collection of samples on dead animals, therefore without anesthetics nor analgesics.
3. Surgical sample collection: Thoracic stage.
   NOTE: To maintain sterile conditions, all surgery is performed using sterile equipment in a biosafety level 2 environment.
   1. Apply ethanol to the skin. Perform a midline skin incision from sternum to mid abdomen with scissors. From midline incise along the ribcage on either side. Fold back the skin on either side of the thorax to visualize the rib cage.
   2. Perform a vertical incision of the ribcage on either side going up towards the clavicles in order to be able to recline the entire anterior chest wall with the sternum allowing the perfect visualization of heart and lungs (Figure 5A, 5B).
   3. Collect blood using a pre-heparined syringe by puncturing the heart next to the interventricular artery. Withdraw a minimum of 500 µl to obtain at least 100 µl of plasma. Place the blood sample on ice.
   4. Perform a midline cervical incision to visualize the trachea (Figure 5B and 5C). Carefully dissect the fascia around the trachea. Place a suture behind the trachea (Figure 5C and 5D). Subsequently the suture will be closed around the cannulating needle to ensure proper lavage.
   5. Catheterize the trachea using the 20-G modified gavage needle (Figure 5D and 4A). Tie a surgical knot around the cannulated trachea with the previously placed suture.
   6. To perform bronchoalveolar lavages (BAL), gently and progressively inject and draw 500 µl of ice-cold PBS into/from the lung. Place the sample on ice to avoid cellular lysis.
   7. Repeat step 5.3.6, 3 times to obtain a total of 1,500 µl BAL fluid and pool lavage samples into a 2 ml centrifuge tube (Figure 5E).
   8. Remove the lungs from the chest. Place a lung segment (size should correspond to the half of one lung or a lobe) into 1.5 ml centrifuge tube and store rapidly at - 80°C.
   9. Place a lung segment in a pre-weighed hemolysis tube containing PBS to determine bacterial burden and place it on ice.
4. Surgical sample collection: abdominal stage.
   1. Perform another incision on the left-side of the abdomen. Observe the spleen through the peritoneum.
   2. Remove the spleen and place into a second hemolysis tube containing 1 ml PBS and place on ice.
5. Lung injury index
   NOTE: Alveolar-capillary membrane permeability is assessed by measuring FITC-labeled-albumin leakage from the vascular compartment to the alveolar-interstitial compartment.
   1. Centrifuge blood sample and BAL fluid for 10 min at 1,500 x g. Collect the supernatants into new centrifuge tubes. The pellets correspond to recruited plasma or BAL cells and should be placed on ice.
   2. Add 100 µl of each blood supernatant (plasma, yellow) or BAL supernatant to a 96-well transparent plate (300 µl wells). Place a foil on the plate if not used immediately.
   3. Measure fluorescence levels in plasma and BAL supernatants using a fluorescence microplate reader (excitation, 487 nm; emission, 520 nm).
   4. Determine the lung injury index by calculating the fluorescence ratio [(BAL supernatant/blood supernatant) x 100].
6. Bronchoalveolar lavage (BAL) differential cell count.
   NOTE: Use the cell pellet obtained from centrifugation of BAL fluid at step 5.5.1.
   1. If needed, use red-cell lysis buffer. Add 500 µl of red-cell lysis buffer into the centrifuge tube containing the cell pellet. Briefly vortex and leave 10 min on ice. Add 500 µl PBS to stop red-cell lysis.
   2. Harvest cells by centrifugation for 10 min at 1,500 x g. Remove supernatant and suspend the cell pellet into 1 ml of sterile PBS. Enumerate cells on a Mallassez hematocytometer. Using a Hemacytometer to Count Cells. Concentrate cells on a slide with a cytospin.
   3. Stain cells using coloration kit allowing cell identification and count (macrophages, lymphocytes, neutrophils).
7. Lung bacterial burden and bacterial dissemination
   NOTE: To assess lung bacterial burden and bacterial dissemination, lungs and spleen were respectively collected and stored into pre-weighed hemolysis tubes containing 1 ml of PBS (step 5.3.9).
   1. Weigh hemolysis tubes containing 1 ml PBS and either lung or spleen. Homogenize the samples with a tissue homogenizer to obtain lung homogenates and spleen homogenates.
   2. Deposit 100 µl of tissue homogenates into centrifuge tubes containing 900 µl of sterile PBS to obtain serial logarithmic dilutions.
   3. Plate the two last appropriate diluted samples (10^-3 and 10^-2) on either BCP-agar for P. aeruginosa or YPD-amikacin-supplemented agar for C. albicans lung and spleen burden determination.
   4. Incubate the plates O/N at 37 °C. The following day, enumerate the colonies on plates.
   5. Index the result to lung weight to obtain a CFU per gram of lung. Lung sample sizes are not the same, the results should be expressed in CFU per gram of lung.
      Formula for the index is: [CFU] x [weight of hemolysis tube and lung] - [weight of hemolysis tube].

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Wyniki

As seen previously during the protocol description, the experiment needs 5 day to complete (Figure 1: experiment timeline). One operator is solicited during the entire run of the experiment and can handle the processes up to a maximum of 10 mice. If more animals are required, two persons are needed particularly for surgical sample collection. Indeed all samples must be collected in under 2 hr to avoid an increased passive alveolar-capillary leakage of FITC-labeled albumin in the last mice.

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Dyskusje

Animal models, particularly mammals, are useful to elucidate complex mechanisms of host-pathogen interaction in the fields of immunity. Of course, the need for information obtainable only from animal models must be essential; otherwise, use of animals must be replaced by in vitro models. This animal model illustrates the insight that can only be provided by an animal model since the interaction between pathogens is mediated by a multi-component host response. Mice currently used to study this host-pathogen inter...

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Materiały

Name	Company	Catalog Number	Comments
Sevorane, Sevoflurane	Abott	05458-02	250 ml plastic bottle
Fluorescence Reader Mithras  LB940	Berthold Technologies	reference in first column	no comment
Bromo-cresol purple agar	Biomerieux	43021	20x per unit
Pentobarbital sodique 5.47%	CEVA	6742145	100 ml plastic bottle
2-headed valve	Distrimed	92831	no comment
Sterile inoculation loop 10 µl	Dutscher	10175	x1,000 conditioning
Insuline syringes 1 ml	Dutscher	30003	per 100 conditioning
2 positions Culture tube 8 ml	Dutscher	64300	no comment
Ultrospec 10	General Electric life sciences	80-2116-30	no comment
Hemolysis tubes 13 x 75 mm	Gosselin	W1773X	per 100
PBS - Phosphate-Buffered Saline	Life technologies	10010023	packaged in 500 ml
amikacin 1 g	Mylan	62516778	per 10
Heparin 10,000 UI in 2 ml	Pan pharma	9128701	10x per unit
RAL 555 coloration kit	RAL Diagnostics	361550	3 flacons of 100 mL
1.5 ml microcentrifuge tube	Sarstedt	55.526.006	x 1000
Transparent 300 µl 96-well plate	Sarstedt	82 1581500	no comment
Yest-peptone-Dextrose Broth	Sigma	95763	in powder
FITC-albumin	Sigma	A9771	in powder
Luria Bertani Broth	Sigma	L3022	in powder
25-gauge needle	Terumo or unisharp	A231	x 100 conditioning
Cytocentrifuge	Thermo Scientific	A78300003	no comment