Murine Models of Eosinophil Function in Fungal and Viral Infections

Abstract

Eosinophils are granulocytes that were historically considered to be terminally differentiated at the time of bone marrow egress. However, more recent evidence provides a new outlook on these cells as complex immunomodulators that are involved in host defense and homeostasis. Our work established a role for eosinophils as mediators of antiviral immune responses during influenza in hosts that were sensitized and challenged with fungal allergens. Herein, we describe methods for working with murine eosinophils in the context of influenza A virus.

1. Introduction

Eosinophils are terminally differentiated granulocytes that have been ascribed numerous and varied roles in allergy, remodeling/repair, immune and metabolic homeostasis, helminth infection, and anti-pathogen defense (reviewed in [1,2]). In a healthy individual, there are very few eosinophils circulating in the blood and residing in the lungs [3,4]. However, during diseases such as allergic bronchopulmonary aspergillosis and allergic asthma, a distinct cytokine panel comprising of interleukin (IL)-3 and −5 and granulocyte macrophage colony stimulating factor (GM-CSF) drive eosinophil production and release from the bone marrow to the bloodstream after which they migrate to and accumulate in the lungs (reviewed in [5]).

The airways are susceptible to bacterial, viral and fungal infections that bring pathogens into intimate contact with eosinophils in the respiratory system of hosts with diseases such as asthma in which eosinophils have accumulated in the lungs. Eosinophils have anti-bacterial and -viral properties that are beneficial during infection by providing both innate and adaptive protection against invading pathogens (reviewed in [6]). Eosinophil granules contain antimicrobial components such as cationic proteins and a litany of cytokines, which can be selectively released by piecemeal degranulation to kill pathogens or augment the immune response against them [7]. The release of extracellular DNA traps associated with granule proteins such as MBP and ECP immobilizes bacteria and bring them in direct contact with antimicrobial components within the extracellular DNA traps [8,9]. Furthermore, eosinophils are able to present peptides associated with MHCI and MHCII to T-cells, leading to the expansion of pathogen-specific lymphocytes that contribute to the resolution of infection ([10,11] and reviewed in [12]). We have shown that preceding antigen presentation, influenza A virus (IAV) triggers eosinophils to up-regulate MHCI and CD86 – critical components of antigen presentation to CD8⁺ T-cells [10]. In mice with influenza, adoptively transferred eosinophils exposed to homologous IAV migrate to the T-cell zones of mediastinal lymph nodes, which can then interact with resident CD8⁺ T-cells [10].

Investigating the interactions between eosinophils and respiratory pathogens requires a repertoire of models that successfully induce eosinophilia and virus infection in the lungs and can recapitulate the scenario in vitro. Herein, we describe protocols we have used to examine the response of eosinophils to IAV in vivo and in vitro to demonstrate a protective role for eosinophils during influenza in mice, and to further delineate the effect of IAV infection on eosinophil transcription and surface antigen expression [13,10,14].

2. Materials

All reagents to be used in mice should be sterile.

2.1: Development and characterization of a model of asthma and influenza that protects the host from severe influenza

Madin-Darby canine kidney (MDCK.2) cells (ATCC), MEM-BSA media for infection as detailed by Eisfeld et al. [15], N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin, influenza virus strain A/CA/04/2009, 1× calcium and magnesium-free phosphate buffered saline (PBS) at pH 7.2, 10× PBS, 0.5% solution of fresh chicken red blood cells, Aspergillus fumigatus antigen (Greer Laboratories) and culture (ATCC, NIH 5233), Alum, ketamine, xylazine, C57BL/6 mice, isoflurane and anesthesia apparatus.

2.2: Determination of the in vivo relevance of eosinophils during influenza pathogenesis

70% ethanol, RPMI 1640, C-tubes and GentleMACS^™ and mouse lung dissociation kit (Miltenyi Biotec, Germany), Hanks’ balanced salt solution (HBSS) with 2 mM EDTA, 40 μm cell straining sieves, 1.084 g/mL Ficoll-Paque® (FP, GE Healthcare), trypan blue, cytospin centrifuge (ThermoFisher Scientific) HemaDiff staining kit, fetal bovine serum (FBS), 1× PBS at pH 7.2, buprenorphine, ketamine, xylazine, topical antibiotic, surgical instruments, stainless steel surgical wound clips and applicator.

2.3: Dynamics of eosinophil responses to IAV

Eosinophil growth media as described by Dyer et al. [16], recombinant mouse (rm) IL-5 and rmGM-CSF, influenza virus strain A/PR/08/1934 or A/CA/04/2009, TPCK-treated trypsin, GolgiPlug (BD Biosciences), RNaseZap® (Invitrogen), RNeasy Mini® extraction kit (Qiagen, Germany), β-mercaptoethanol, RNA bioanalyzer.

3. Methods

All work should be performed under aseptic conditions unless otherwise indicated.

3.1: Development and characterization of a model of asthma and influenza that protects the host from severe influenza

1. To prepare the virus stock, culture the selected strain of IAV in MDCK cells by inoculating a confluent monolayer grown in a 24 well plate with 0.1 multiplicity of infection (MOI, see Note 1) in 0.1 mL of IAV in MEM-BSA media for 1 hour at 37°C/5% CO₂. Aspirate the inoculum, wash monolayer twice with 1×PBS and add 1 mL of MEM-BSA media supplemented with 1 μg/mL TPCK-treated trypsin. Incubate plate for 72 hours at 37°C/5% CO₂. Observe cells for cytopathic effect (CPE) daily and harvest the supernatant at 72 hours into a pre-chilled 50 mL polypropylene tube. Centrifuge at 700 ×g for 10 minutes at 4°C to remove cell debris. Transfer the supernatant that contains the virus into a pre-chilled glass bottle on ice and swirl to mix prior to aliquoting into cryovials at 100–200 μL volumes. Store stock virus at −80°C until use. To measure the virus titer, quick thaw a virus stock tube and prepare 1:10 serial dilutions in MEM-BSA media and inoculate confluent monolayers of MDCK cells grown in a 96 well plate with 0.1 mL of each dilution in quadruplicate. Incubate the plate for 1 hour at 37°C/5% CO₂. Wash the plate twice with 1×PBS and add 0.2 mL of MEM-BSA media supplemented with 1 μg/mL TPCK-treated trypsin and incubate plate for 72 hours at 37°C/5% CO₂. Perform a hemagglutination assay by mixing 50 μL of cell culture supernanant with 50 μL of with 0.5% chicken red blood cells and incubating at RT for 30 minutes. Calculate the stock titer as tissue culture infectious dose-50 (TCID₅₀)/mL using the Reed-Muench method.

2. To induce allergic asthma in mice, follow methods previously described in great detail by Hoselton and Schuh [17]. Briefly, prepare 0.2 mL of antigen [10 μg of Aspergillus fumigatus antigen adsorbed in 100 μL Alum and 100 μL of 1×PBS for 30 minutes] for each mouse. Inject 100 μL subcutaneously and 100 μL intraperitoneally into the mouse. Rest animal for one week and administer 20 μg of A. fumigatus antigen in 20 μL of 1×PBS intranasally, once weekly for three weeks. Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine cocktail to deliver 75mg/kg ketamine and 25 mg/kg xylazine and wait until respiration rate stabilizes to a slow rhythmic breathing patters and the mouse is unresponsive to a toe pinch. Expose the anesthetized mouse to airborne conidia from an 8-day old culture of A. fumigatus for 10 minutes, rest the mouse for 2 weeks and repeat the inhalation challenge [18,19].

3. Calculate the volume of stock required for the preparation of 20,000 TCID₅₀/mL of virus (see Note 2) according to the formula below to administer 1000 TCID₅₀ of the virus when inoculated with 50 μL intranasally.

   $$C_1{V}_1=C_2{V}_2$$

   C₁ is the stock concentration of virus, V₁ is volume of stock required to make working concentration virus, C₂ is the working concentration of virus and V₂ is the volume of working concentration of virus. Add V₂-V₁ mL of 1× PBS into a sterile glass culture tube. Take out the virus stock vial from −80°C and thaw for 1 min in water bath at room temperature (RT). Vortex vial for 30 seconds and add V₁ mL of virus stock solution to the glass culture tube. Close the lid and vortex the working solution of the virus and maintain on ice until use (see Note 3).

4. Place a clean sterilized mouse cage on a heating pad at 37°C. Record the weight of each mouse prior to virus infection. Categorize animals according to the groups; naïve, acute asthma-only (AA; allergic animal), Flu-only (infected with IAV) and acute asthma and influenza group (AA+Flu; allergic mice infected with IAV) (see Note 4). Place the induction chamber within a class II biosafety cabinet and connect to anesthesia apparatus. Fill the vaporizer in the anesthesia apparatus with isoflurane and switch on the O₂ tank regulator. Place the mice inside the anesthesia chamber and close the lid. Turn on the vaporizer knob and adjust the flow rate to 2.5%. When mouse movement is minimal and the respiration rate stabilizes (about 4–5 minutes) (see Note 5), inoculation may proceed.

5. Mix the working stock of virus prior to each inoculation. Pipet 50 μL of the prepared virus inocula into the nares of the anesthetized mouse while holding the mouse vertically as shown in Figure 1. Hold the mouse in the vertical position for a few seconds to allow the inoculum to travel to the lungs. Place the infected mouse supine into the clean cage placed on a heating pad and record the time of infection. Observe the mouse until it fully regains consciousness and moves around normally. Remove the cage from the heating pad and ensure the animal has access to food and water ad libitum.

6. Weigh and record body weight of each infected mouse every 24 h until the termination of the experiment noting any severe signs of influenza such as dull fur, hunching, non-responsiveness, dehydration etc. (see Note 6).

7. Euthanize naïve and AA group of mice at D0 time point by placing them in CO₂ chamber for 3–5 mins. Check mouse for the faded eye color and lack of respiration. Remove mouse from the chamber and perform cervical dislocation as a secondary measure of euthanasia. Euthanize ‘AA+Flu’ and ‘Flu-only’ group of mice on days 1, 3, 5, 7, and 9 (or at desired timepoints) post virus infection. Collect tissue as needed for the study (see Note 7).

Figure 1. Representation of mouse infection with influenza A virus inoculum.

The induction chamber is used to anesthetize the mouse with isoflurane and 50 μL of prepared working stock of influenza virus (kept in a glass vial on ice) is used to inoculate the mouse dropwise into each nostril gently holding the chin in place to prevent any mouth breathing that may occur under anesthesia. [The glass sash in the biosafety cabinet was raised above the secure mark for the purpose of taking this photograph. The virus inoculations should be performed under aseptic technique ensuring all safety measures are taken to prevent exposure to this biohazardous agent.]

3.2: Determination of the in vivo relevance of eosinophils during influenza pathogenesis

1. Subject mouse to the fungal asthma model as described in step 2 in section 3.1 above. Euthanize the mouse at day 7 after the second allergen exposure as in step 7 in section 3.1. After cervical dislocation, wet the mouse thoroughly with 70% ethanol and transfer into a class II biosafety cabinet. Using sterilized instruments, harvest the lungs and place into a 2 mL Eppendorf tube containing 1 mL of RPMI and place the tube on ice.

2. Transfer the lungs into a C-tube containing mouse lung dissociation reagent cocktail according to the manufacturer’s guidelines. Dissociate the lungs using the Miltenyi Biotech GentleMACS^™ at 37°C (see Note 8), then add 4 mL of HBSS/EDTA buffer to the lung homogenate and strain through a 40 μm sieve into a sterile polypropylene tube. Centrifuge sample at 600 ×g for 10 minutes at RT. Aspirate the supernatant and resuspend the pellet in 4 mL of HBSS/EDTA buffer.

3. Bring 1.084 g/mL FP solution to RT in the dark. Invert FP several times to mix and aliquot 3 mL of FP into 15 mL polypropylene tube. Layer the single cell suspension from step 2 gently on top of FP and centrifuge at 960 ×g for 30 mins at 20°C. Remove layers above (see Note 9) and resuspend the cell pellet (which contains the majority of eosinophils and red blood cells) in 9 mL of ice-cold water. Gently pulse vortex for 1 minute and add 1 mL of 10× PBS to restore isotonic coniditions. Strain sample through a 40 μm sieve into a sterile polypropylene tube and centrifuge at 300 ×g for 10 minutes at RT. Re-suspend pellet in 15 mL of 1×PBS/5% FBS and transfer into a T75 flask. Place the flask flat in an incubator at 37°C/5% CO₂ for 1 hour to promote the attachment of any contaminating adherent cells. Collect the non-adherent cells in suspension and centrifuge at 300 ×g for 10 min at RT. Re-suspend the pellet in 1 mL HBSS and remove 20 μL for enumeration and cytospin preparation while saving the remaining cells in ice on the dark.

4. Enumerate cell yield by trypan blue dye exclusion test (see Note 10). Cytospin cells onto glass microscope slides at 500 rpm for 5 mins and stain with HemaDiff staining kit (fix in methanol for 30 sec, xanthene for 30 sec and thiazine for 30 sec). Wash slide with distilled water and observe under microscope. Confirm isolation of eosinophils (see Note 11) after differential staining through morphology of the nucleus and the presence of punctate red staining in the cytoplasm (Figure 2A).

5. Wash the cell suspension in 1×PBS and resuspend cells at the desired concentration (see Note 10) in 1×PBS in a sterile 5 mL polystyrene tube placed in ice. Prepare IAV-infected recipient mice (Section 3.1 Step 5) for adoptive transfer of eosinophils.

6. Administer buprenorphine subcutaneously to recipient mouse at 0.1 mg/kg. Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine cocktail to deliver 75 mg/kg ketamine and 25 mg/kg xylazine. Load a 1 mL syringe with the cell suspension and affix a 30-gauge needle. Once the mouse is non-responsive to toe pinch and has a slow rhythmic breathing pattern, place it on an inclined surgical board and hook its upper incisors to horizontally affixed dental floss to provide easy access to its trachea. Rub down fur on the throat with 70% ethanol for 1 minute and make a small incision using sterilized instruments to expose the trachea and inject 50 μL of purified eosinophils (see Note 12) into the trachea. Gently pull the skin back together and apply a stainless steel wound clip (see Note 13) and a topical antibiotic. Place mouse supine in a clean cage placed on a heating pad (set at 37°C) and observe until it fully recovers from anesthesia and begins moving around the cage. Ensure the mouse has full access to food and water. Administer a dose 0.1 mg/kg buprenorphine subcutaneously 24 hours later. Observe the animal daily for signs of infection at the surgical site and apply topical antibiotic as needed. Daily weight should be recorded as detailed in Section 3.1 step 6. Recipient mice can be euthanized at predetermined timepoints in the study to collect the desired tissues. Eosinophils may be purified from the lungs of recipient animals to determine whether granularity was maintained post-transfer (Figure 2B).

Figure 2. Morphology of mouse eosinophils.

(a) Light micrograph of an eosinophil purified from the lungs of a mouse that was sensitized and challenged with A. fumigatus. Scale bar = 10 μm (b) Transmission electron micrograph of an eosinophil purified from the lungs of a virus-infected mouse that was a recipient of adoptive transfer. Scale bar = 1μm (c) Light micrograph of a mouse bone marrow-derived eosinophil. Scale bar = 10 μm

3.3: Dynamics of eosinophil responses to IAV

1. Differentiate bone marrow-derived eosinophils (BMdEos) from mouse tibia and femur bone marrow for 14 days by supplementing the media with growth factors and cytokines as described in detail elsewhere [16], or use purified peripheral mouse eosinophils as described in Section 3.2 above. Morphology of a 14-day old BMdEos culture is shown in Figure 2C. Centrifuge eosinophils at 300 ×g for 10 min at 15°C and resuspend cells at 1 million cells/mL in eosinophil growth media supplemented with 10 ng/mL rmIL-5 and seed a 96-well plate at 5×10⁵ eosinophils per well in 50 μL. Prepare working stock of IAV at the desired MOI as described in section 3.1. Add the calculated volume of IAV to wells allocated for infection and equal volume of media to wells allocated to be un-infected controls (see Note 14). Mix by pipetting and incubate for 1 h at 37°C/5% CO₂ (see Note 15). Wash cells and resuspend in eosinophil growth media with 10 ng/mL rmIL-5, 5 ng/mL rmGM-CSF, and 1μg/mL of TPCK-treated trypsin. Incubate 96 well plate for desired timepoints.

2. These infected eosinophils can then be used in assays to determine downstream effects of their activation under various other conditions such as when co-cultured with T cells as shown in Figure 3.

3. If cells are to be used for transcriptome analysis, seed eosinophils in growth media with rmIL-5 and rmGM-CSF in a 12 well plate at a concentration of 1 million cells/mL and infect at a low MOI (see Note 16). At 6 hours prior to harvest, add 1 μL GolgiPlug (Brefeldin A) per 1 mL media to the eosinophils to prevent the release of endogenous RNases. Gently rock the plate to mix before placing back at 37°C/5% CO₂ for the remaining 6 hours. At harvest, place plate containing eosinophils on ice for 1 min and then transfer eosinophils to 15 mL polypropylene tubes. Centrifuge eosinophils at 200 ×g for 10 min in a centrifuge pre-cooled to 4°C and aspirate supernatant (see Note 17). However, if components in the supernatant are of interest, remove supernatant being careful not to disturb the cell pellet. Wipe work bench, pipets and gloved hands with RNaseZap®. Perform RNA extraction using Qiagen RNeasy Mini® kit, with 10 μL β-mercaptoethanol added per 1 mL RLT buffer as per the manufacturer’s recommendation. Elute RNA in nuclease-free water. Determine yield, and 260/280 and 260/230 ratios for the RNA sample. If necessary, treat with DNase to remove any residual DNA. Determine RNA quality using a RNA bioanalyzer such as the Agilent 2100 Bioanalyzer (see Note 17). Store RNA at −80°C until use in microarray assay.

Figure 3. Example of flow cytometric gating for eosinophil antigen presentation markers in influenza A virus exposed eosinophils co-cultured with T cells.

Aspergillus fumigatus allergen-pulsed eosinophils were exposed influenza A virus at a multiplicity of infection of 0.1 for one day in the presence of 5 ng/mL GM-CSF. CD4⁺ and CD8⁺ T cells isolated from a naïve mouse spleen was cultured overnight in the presence of 2.5 μg/mL concanavalin A in the presence of 10 ng/mL interleukin 2. Eosinophils and T cells were co-cultured at a 1:1 ratio for one hour prior to staining for flow cytometric analysis. (a) Scatter properties of culture conditions show canonical placement of eosinophils and T cells. (b) Eosinophils were gated based on Siglec-F expression and (c) their expression of major histocompatibility (MHC) molecules and CD86 co-stimulatory molecule were quantified.

4. Notes

1. Virus inoculum should be 0.1 MOI in relation to the cell number in the confluent well. We seed 2.5 × 10⁵ MDCK cells per well in a 24 well plate. We then add 2.5 × 10⁴ TCID₅₀ IAV per well. If the starting virus concentration is not known, add 0.1 mL of the stock virus and observe the CPE daily. Harvest the supernatant when approximately 75% of the MDCK cells in the monolayer are rounded or at 72 hours (whichever comes first).

2. The infectious dose of IAV depends on the needs of the investigator. Since we wanted to use a clinical isolate of IAV, and because we aimed to investigate the immune parameters of non-lethal influenza, we performed a dose curve with our virus strain and arrived at 1000 TCID₅₀ per mouse to be optimum. However, it should be noted that even with 20× the dose of A/CA/04/2009 virus, allergic mice still lost <10% of body weight compared to Flu-only mice (Figure 4).

3. If V₁ is < 20 μL, first prepare a 1:10 serial dilution of the stock virus to ensure proper inoculum preparation through precise pipetting.

4. We infect allergic mice with IAV at 7 days after the second allergen inhalation in our AA+Flu model. By extending the time between the fungal exposure and the IAV infection to 28 days, we have also developed a chronic asthma and influenza (CA+Flu) model which can be used to determine the impact of IAV in remodeled airways [19].

5. Anesthesia must be optimal, characterized by rhythmic slow breathing, to ensure proper lung inoculation. Over-anesthetized mice will have the inoculum accumulate in the larynx and swallow the inoculum immediately as the anesthesia wears off. Under-anesthetized mice will breathe out during intranasal inoculation. In both situations, less than the desired dose of virus will reach the lungs, and mice will not develop influenza marked by minimal reduction in weight and low viral titers.

6. With the infectious dose of IAV that we give (1000 TCID₅₀/mouse) we expect about 12–15% weight loss in the Flu-only control wild type C57BL/6 strain mice. Greater than 20% weight loss is unexpected with this dose of IAV and if an animal loses >20% of its starting body weight, it has severe influenza (viral pneumonia) and should be euthanized immediately. Our experience with the AA+Flu mice (wild type C57BL/6 strain) has been that they do not lose weight in response to 1000 TCID₅₀ A/CA/04/2009 IAV or A/PR/8/1934 [19].

7. We routinely collect the bronchoalveolar lavage fluid, blood, lung lobes (cardiac lobe and bottom of proximal lobe for RNA, top of the proximal lobe and distal lobe for protein, and the left lobe for histology), and the mediastinal lymph nodes and spleens for the measurement of varying immune parameters. We collect whole lungs from infected animals to determine the viral burden by TCID₅₀ method as described in Section 3.1 step 1. Eosinophils in the bronchoalveolar lavage and/or lungs can be identified by flow cytometry. In general, we gate the stained cell population from the bronchoalveolar lavage (or lungs) to exclude very small events and gate to exclude doublets. Following this, we identify eosinophils as events that express CCR3 and Siglec-F as shown in Figure 5.

8. In the event that a GentleMACS^™ machine is unavailable, transfer the lungs to a sterile Petri dish and chop into very small pieces using sterile scissors and forceps. Wash the Petri dish with 1 mg/mL collagenase containing HBSS solution and transfer contents into a polypropylene tube. Place the tube in a shaking dry incubator at 37°C for 30 minutes.

9. We have noted that some eosinophils are found in the buffy coat. Cytospins can be prepared from a small sample in each layer to verify the presence of eosinophils in each layer. In order to save as many eosinophils as possible from the mouse lung sample, we process the buffy coat separately and combine cells together at the end.

10. On average, using this method of eosinophil isolation, we obtain about 6 million eosinophils from the lungs of an allergic mouse. Because we have noted that about 425,000 eosinophils are located in the airways of an allergic mouse at the time of IAV infection, we have intratracheally transferred 700,000 eosinophils into recipient mice in our work expecting some not to reach the airways and for some to die.

11. Major contaminants using this method are cells that morphologically resemble macrophages and lymphocytes. However, anywhere from 90 – 97 % purity can be achieved and the variations are dependent on investigator that runs the asthma model and conducts the purification.

12. We deliver 50 μL of 1×PBS as a surgical control and 50 μL containing the same number of splenocytes from a naïve mouse as a non-eosinophil control. Mouse BMdEos may substitute mouse lung-purified eosinophils.

13. We generally do not remove the surgical clip as we have observed that the clips fall off naturally as the wound heals. When the clip has naturally fallen off, be sure to remove it from the cage bedding to ensure the safety of the laboratory mouse.

14. UV-inactivated IAV can be used as a ‘mock-infection’ instead of the media control. Our data indicate no difference in eosinophil responses when exposed to media or UV-inactivated IAV [10].

15. It is expected that approximately 15–20 % eosinophils die during this 1 hour incubation with IAV. Infected eosinophils that survive this initial exposure survive for 5–7 days afterwards when cultured in the presence of rmIL-5.

16. In our experience, we routinely recovered poor quality RNA from eosinophils that were infected with high MOI of IAV. Since we have shown that IAV causes piecemeal degranulation in eosinophils [10], it is possible that RNA integrity is compromised by exposure to RNases that are released by the activated eosinophil. Therefore, while we infect eosinophils with higher MOI for RNA analysis by quantitative PCR, we recommend low MOI for microarray analysis which requires high integrity RNA.

17. At harvest, if the supernatant looks turbid after this centrifugation step, centrifuge the cells again at 300 ×g for 10 min at 4°C.

18. RNA yield from eosinophils is expected to be low, due to low transcriptional activity of eosinophils. High RIN values are sometimes elusive using eosinophils, in particular following infection with IAV. While RIN values of 8 are desirable, we have used RIN values of 6 or greater successfully in microarray assays.

Figure 4. Example of body weight curve as a marker of influenza in mice.

Mice subjected to the fungal asthma model and age-matched controls were infected with 20,000 TCID₅₀ A/CA/04/2009 influenza virus and the change in body weight in reference to their starting body weight immediately before the virus infection was quantified. **P<0.01, ***P<0.001 by two-way ANOVA with Sidak’s multiple comparison test.

Figure 5. Example of flow cytometric gating strategy for eosinophils in bronchoalveolar lavage (BAL) samples from in vivo model of asthma and influenza comorbidity.

(a) Scatter plot of BAL sample shows the canonical placement of general cell populations within the gate drawn to eliminate small events. (b) Cells are then gated to capture singlets prior to population separation to (c) identify events that are positive for both CCR3 and Siglec-F which are then denoted as eosinophils. Inset is a light micrograph of an eosinophil within the BAL cell population. (a) and (b) are representative of a sample from an allergic mouse while (c) are representative of samples acquired equivalent to day 7 after influenza A virus (IAV) infection.