The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.
Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.
A biofilm is a community of surface-associated microbes or non-attached aggregates encased in an extracellular polymeric substance (EPS)1,2. These communities protect the encased microorganisms from environmental stressors, including tolerance to antimicrobial agents and the immune system3. Several pathogenic microbial species form biofilms that have been associated with chronic infections4. The development of biofilms is an intricate process involving attachment to surfaces, EPS production, cell proliferation, biofilm structuring, and cell detachment5. Once cells disperse to form a biofilm, they remain planktonic or translocate to a new substratum and re-initiate biofilm development6.
Staphylococcus aureus, an opportunistic pathogen, follows a general scheme of biofilm development, including attachment, proliferation, maturation, and dispersal7. The attachment process in S. aureus biofilms is dictated by hydrophobic interactions, teichoic acids, and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)8,9. As the proliferation of S. aureus begins, EPS, which primarily consists of polysaccharides, proteins, extracellular DNA, and teichoic acids, is produced5. As EPS components are produced, various exoenzymes and small molecules are also produced, contributing to the biofilm 3-dimensional structure and aiding in detachment5. S. aureus takes advantage of this highly coordinated lifestyle to establish various chronic infections, including infections due to the indwelling of medical devices10.
Methicillin-resistant S. aureus (MRSA) is one of the leading causes of infections related to indwelling medical devices, such as central venous and urinary catheters, prosthetic joints, pacemakers, mechanical heart valves, and intrauterine devices11. During such infections, neutrophils are the first host immune cells recruited to the infection site to combat pathogens via multiple strategies12. These include phagocytosis, degranulation, reactive oxygen and nitrogen species (ROS/RNS) production, or release of neutrophil extracellular traps (NETs) to eliminate pathogens13.
Generation of ROS upon phagocytosis of microbes is one of the key antimicrobial responses exhibited by neutrophils14. Phagocytosis is enhanced if microbes are coated in opsonins, particularly immunoglobulins and complement components found in serum15. The opsonized microbes are then recognized by cell surface receptors on neutrophils and engulfed, forming a compartment called the phagosome15. Neutrophils generate and release ROS in the phagosome via the membrane-associated NADPH-oxidase16. This multi-component enzyme complex generates superoxide anions by transferring electrons to molecular oxygen16. Additionally, neutrophils also generate RNS through the expression of inducible nitric oxide synthase (iNOS)17. These high superoxide and nitric oxide radicals within the phagosome have broad antimicrobial activities. They can interact with metal centers in enzymes and damage nucleic acids, proteins, and cell membranes of the pathogen18,19,20,21. Numerous microbes adopt a biofilm lifestyle and employ different strategies to evade killing by ROS22,23. Thus, standardized assays that couple biofilms with neutrophils to quantify ROS are beneficial for consistent results.
While assays, such as quantifying neutrophil ROS production, provide information about the responses of neutrophils to biofilms, the ability to visualize the interactions of neutrophils within a biofilm can also serve as a powerful tool. The use of fluorescent dyes for microscopy often requires optimization to obtain high-quality images that can be used for microscopy imaging analysis. The flexibility to optimize some conditions is limited as neutrophils can undergo cell death post-isolation. Furthermore, biofilms are typically washed to remove the planktonic population from the experimental set-up before the addition of neutrophils. While washing, variability between replicate biofilms may arise due to loss of partial biomass if biofilms are loosely adhered to the surface.
Broadly, current methods in the field to analyze interactions between neutrophils and biofilms mainly include microscopy, flow cytometry, and colony-forming units (CFU) enumeration24,25,26,27. Microscopy involves the use of dyes that either directly stain the neutrophils and biofilms, or target various neutrophil responses against microbes such as NET formation, degranulation, and cell death25,28. A subset of these responses, such as neutrophil cell death and degranulation, can also be analyzed via flow cytometry, but requires neutrophils to be preferentially unassociated with large aggregates of microbes in a biofilm28,29. Flow cytometry can also quantify some biofilm parameters, such as cell viability27. These processes, however, require disruption of the biofilm biomass and would not be useful to visualize other important interactions such as the spatial distribution of neutrophils and their components within a biofilm27,29,30.
The present protocol focuses on adapting some of the traditionally used methods to study neutrophil-biofilm interactions on biofilms that have been optimized to provide minimal variability during handling. This protocol thus provides standardized methods to grow and quantify biofilms, isolate primary human neutrophils from peripheral blood, quantify ROS production, and visualize biofilm-neutrophil interactions via microscopy. This protocol can be adapted to different systems to understand biofilm-neutrophil interactions while considering the heterogeneity among donor pools.
All procedures were approved by the Ohio State University Institutional Review Board (IRB) (2014H0154). Informed written consent was obtained from all the donors for collecting peripheral blood to isolate primary human neutrophils. Staphylococcus aureus (USA300 LAC)31 was used as the model organism for performing the experiments. The experiments were performed with proper personal protective equipment (PPE) due to potential exposure to a bloodborne pathogen.
1. Preparation of in vitro biofilm
2. Quantification of biofilm biomass
3. Neutrophil isolation
NOTE: Neutrophils were isolated following a previously published method with minor changes36. This isolation protocol combines density gradient centrifugation first, followed by 3% dextran sedimentation. This section only covers the overall neutrophil isolation protocol, focusing on the changes made to the published protocol. Furthermore, the protocol outlined below is one of the many methods that can isolate neutrophils, and can be substituted as needed. Other methods for isolating neutrophils include the use of cell separation media or magnetic antibody cell separation37.
4. Measurement of ROS produced by neutrophils
5. Imaging biofilm-neutrophil interactions
The media used to grow bacterial biofilms influence the survival of neutrophils. Different media were tested to reduce the effect of media alone on the viability of neutrophils for studying neutrophil-biofilm interactions (Figure 1). Bacterial growth media such as Tryptic Soy Broth minimizes the viability of neutrophils, such that ~60% of neutrophils are alive after a 30 min incubation period at 37 °C with 5% CO2. Mammalian cell culture media, such as MEMα, does not affect the viability of neutrophils and supports the growth of S. aureus biofilms. In fact, minimal media promotes robust growth of biofilms in other bacteria46,47.
To assess the effect of media on biofilm growth and variability in biofilm biomass quantification after washing the biomass to eliminate planktonic cells, an 18 h S. aureus biofilm was grown in a 96-well plate, with wells treated or untreated with poly-L-Lysine. A nutrient-rich (Tryptic Soy Broth (TSB)) and minimal (MEMα) media were used as-is or supplemented with 2% glucose. The biofilm biomass stained with CV revealed that S. aureus biofilm grown in MEMα supplemented with 2% glucose produced the most robust biofilm among all tested media (Figure 2A). Furthermore, biofilms grown in PLL pretreated wells containing MEMα + 2% glucose showed less variability than biofilms in PLL-untreated wells containing MEMα + 2% glucose. These biofilms showed less variability in quantification via CV assay35 and the CFU/mL when plated after precisely handling biofilms for biomass quantification. These biofilms contained, on average, 1 x 108 CFU/mL, as demonstrated by plating the biofilms in 3 separate days (Figure 2B). This number is useful in determining the number of neutrophils to add to the biofilms for neutrophil functionality assays.
To measure ROS production by neutrophils in response to biofilms, S. aureus biofilms were grown statically for 18-20 h in a 96-well plate. Biofilms were then opsonized, and neutrophils were added. ROS production was then measured for 60 min (Figure 3A). The area under the curve is calculated from the kinetic curve to quantify total ROS production by neutrophils. Neutrophils treated with an agonist, such as PMA, used as a control, show an increased ROS production. In the absence of biofilms, neutrophils treated with PMA showed robust ROS production. In the presence of S. aureus biofilm, the overall ROS production by neutrophils treated with PMA decreased. In the absence of PMA, neutrophils solely rely on their interaction with the biofilm, which further reduces the amount of ROS produced (Figure 3B).
To visualize the neutrophil-biofilm interactions using fluorescence microscopy, a GFP-expressing strain of S. aureus, Blue CMAC dye, and ethidium homodimer-1, that stains the cytoplasm of live cells and DNA of dead cells, respectively, were used. S. aureus biofilm was grown for 18 h in a 6 µ-channel slide. Blue CMAC dye-labeled neutrophils were added along with ethidium homodimer-1 to the washed biofilms and incubated for 30 min at 37 °C with 5% CO2 prior to imaging. Wide-field fluorescent microscopy revealed that many neutrophils were localized to the surface of S. aureus biofilms, while a few are within the biofilm (Figure 4A). The interaction between S. aureus cells within neutrophils was also apparent (Figure 4C). Most of the S. aureus cells interacting with neutrophils (cyan) were dead (magenta), while a few remained alive (yellow) as determined by live-dead staining (Figure 4C). For comparison, GFP-expressing S. aureus biofilms were stained with ethidium homodimer-1, which revealed a fraction of the dead S. aureus population within the biofilm (Figure 4B). Non-viable neutrophils that were positive for ethidium homodimer-1 were quantified using analysis software (see Table of Materials) after incubation with S. aureus biofilms. Approximately 48% of neutrophils were already dead within 30 min of incubation with S. aureus biofilm. During optimization of the microscopy protocol, the effect of washing the biofilm and neutrophils after 30 min of incubation to remove non-adhered neutrophils was also assessed, revealing around 33% of dead neutrophils still attached to the biofilm (Figure 4D).
Figure 1: LIVE-DEAD assay compares neutrophil survival between bacterial and mammalian growth media. Neutrophils were isolated and incubated in HBSS, MEMα, TSB, or 0.1% SDS for 30 min. LIVE-DEAD staining was performed using Calcein AM (live) and ethidium homodimer-1 (dead). Percent of live neutrophils was determined, where HBSS-incubated neutrophils were treated as 100% live neutrophils. Results represent an average of two independent experiments performed in triplicate, with neutrophils obtained from two different donors. Data are presented as mean ± SD (*p < 0.05, ****p < 0.0001. One-way ANOVA). Please click here to view a larger version of this figure.
Figure 2: Quantification of biofilm biomass in different conditions and bacterial viability count of biofilms grown in the optimized conditions. (A) S. aureus was seeded in a 96-well plate either coated or uncoated with poly-L-Lysine (PLL). Biofilms were grown in TSB, MEMα, or either of the media supplemented with 2% glucose under static conditions for 18 h. Crystal violet (CV) staining was performed to stain biofilm biomass. The eluted CV stain was diluted at 1:10 and read in a microplate reader. Results represent an average of three independent experiments performed in triplicate. Data are presented as mean ± SD. The SD for each group is shown at the bottom to demonstrate different biofilm growth conditions variability. (B) Bacterial CFU counts were obtained from biofilms grown in an optimized medium (MEMα + 2% glucose). The 18 h static biofilms were subjected to the same number of washes followed by a 10 min sonication to loosen the biofilm biomass and passed through a 22G needle to disrupt the aggregates prior to plating. Results represent three replicates performed in triplicate. Data are presented as mean ± SD (ns = not significant. One-way ANOVA). Please click here to view a larger version of this figure.
Figure 3: Quantification of ROS production by neutrophils via chemiluminescence assay. (A) Neutrophils (PMN) were incubated with HBSS-washed S. aureus biofilms (BF) either in the presence (closed gray triangle) or absence (open gray inverted triangle) of PMA to measure ROS production by neutrophils. Luminol was used to detect ROS every 3 min for 60 min in a microplate reader. While neutrophils treated with PMA in the absence of a biofilm (closed black circle) served as a positive control, neutrophil only (open black circle) and biofilm only (open gray triangle) groups served as negative controls. Data represent an average of two independent experiments performed in triplicate with neutrophils obtained from two different donors. Data are presented as mean ± SD. (B) The area under the curve from (A) was calculated to quantify the total ROS generated by the neutrophils. The data are represented as mean ± SD. (***p < 0.0001. One-way ANOVA). Please click here to view a larger version of this figure.
Figure 4: Visualization of the interaction between S. aureus biofilm and neutrophils using wide-field fluorescence microscopy. Blue CMAC dye-labeled neutrophils (cyan) were supplemented with ethidium homodimer-1 (magenta; dead) prior to incubating with an 18 h S. aureus biofilm (yellow). Biofilm-neutrophil interactions were imaged using wide-field fluorescent microscopy and images processed using an image analysis software. Experiments were performed with three different donors. Representative images are presented as (A) 3D view of S. aureus biofilm with live (cyan) and dead (magenta; a few indicated with white arrows) neutrophils, (B) 3D view of an S. aureus biofilm in the absence of neutrophils with either live S. aureus expressing GFP (yellow) or dead S. aureus stained with ethidium homodimer-1 (magenta), (C) an orthogonal view of S. aureus and neutrophil interaction as depicted by the xy, yz, and xz planes, and (D) quantification of neutrophil viability in the presence of S. aureus biofilm after 30 min either immediately (unwashed) or after three rounds of washes with HBSS to remove non-adhered neutrophils (washed). Neutrophil cell death is presented as mean ± SD (Student's t-test). Scale bar indicates 50 µm in (A) and (B) and 10 µm in (C). Please click here to view a larger version of this figure.
There have been numerous efforts to grow robust and reproducible S. aureus biofilms for downstream experiments in vitro48,49,50. A standardized protocol is outlined that takes advantage of the cationic nature of PLL, as well as supplementing the media with glucose for the growth of robust in vitro S. aureus biofilms. The addition of PLL allows for better attachment of the negatively charged bacterial cell to the positively charged PLL coated surfaces. It is important to note that PLL at a 10 µg/mL concentration has antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, and S. aureus when incubated for 24 h51. The same concentration is used to coat surfaces; however, excess PLL is aspirated, making the concentration of PLL lower than 10 µg/mL when seeding for biofilm growth.
It is important to note that PLL has worked only in specific growth media such as MEMα with 2% glucose, where it was observed that S. aureus produced robust biofilms with minimal variability (Figure 2A). PLL concentration to be used in conjunction with other media types would require further optimization, such as using an increased concentration of PLL to coat the wells. Additionally, these conditions have been optimized for a monospecies S. aureus biofilm. While chronic wound biofilms are often polymicrobial, standardizing assays to study monospecies biofilm and its interactions with neutrophils and other immune cells is key in understanding their contribution to pathogenesis52. These standardized protocols can be optimized further to sustain and study polymicrobial biofilms and their interactions with neutrophils.
It was also observed that rich bacterial culture media, such as TSB, led to a loss of neutrophil viability (Figure 1). Therefore, growth conditions of S. aureus biofilms in MEMα, used for mammalian cell cultures, were optimized. For studies involving neutrophils, this media supports neutrophil viability and promotes S. aureus growth. While it was observed that media affects the viability of neutrophils, it is also important to consider that neutrophils isolated from peripheral human blood undergo apoptosis ex vivo with approximately 70% apoptotic neutrophils by 20 h53. This necessitates proper handling, such as storing the neutrophils on ice when preparing for experiments, using endotoxin-free reagents, and preventing activation of neutrophils by avoiding vortexing of samples with neutrophils.
The assessment of oxidative burst in neutrophils is routinely performed to determine the killing effect of neutrophils on the pathogen14,54,55. These studies are frequently performed with planktonic bacteria where neutrophils are added, and the oxidative burst response is quantified using luminol-amplified chemiluminescence that detects superoxide anions produced by neutrophils. The present protocol is modified by replacing planktonic bacteria with statically grown 18 h S. aureus biofilm. As such, neutrophils can be directly added to the biofilm to assess their activation. On the other hand, bacteria in biofilms produce enzymes, such as catalase and superoxide dismutase to detoxify ROS23,56. Staphylococcus epidermidis biofilms produce higher catalase than its planktonic counterpart under stress57. The total chemiluminescence of PMA-stimulated neutrophils in an S. aureus biofilm is significantly lower than the PMA-stimulated neutrophils where biofilm is absent (Figure 2). This may be due to the activity of these detoxifying enzymes. Furthermore, S. aureus biofilms produce several pore-forming toxins called leukocidins that kill neutrophils58. The reduced burst response is also likely due to the reduced viability of neutrophils in the presence of S. aureus biofilm. While this study uses luminol that detects the total ROS produced both inside and outside of the cells, other reagents, such as CM-H2DCFDA (5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate) or isoluminol, need to be considered if the goal of the work is to study intracellular or extracellular ROS production14,53,54 specifically.
The ability to visualize neutrophil-biofilm interactions via microscopy can be informative about the behavior of neutrophils and biofilms in the presence of each other. The excitation and emission spectra of the fluorescent dyes and proteins represent a snapshot of the interaction between an 18 h S. aureus biofilm and neutrophils after a 30 min incubation. To effectively capture signals from stained cells, it is important to limit exposure of the samples to light sources while setting up the samples for microscopy. While imaging, rapid photobleaching of the samples was avoided by lowering the intensity of the light source when adjusting all the parameters such as Z-stack height and exposure time for different channels.
These simple practices allowed for proper microscopy imaging where it was observed that few neutrophils are localized within the biofilm (Figure 4A). This may be due to spaces present within the biofilm as 18 h S. aureus biofilm grown in MEMα with 2% glucose does not uniformly cover the surface (Figure 4B). However, other studies' use of rich media has shown a uniform lawn of S. aureus biofilm growth and leukocytes penetrating through the biofilm30,58. Furthermore, it is also observed that there was neutrophil cell death after 30 min of incubation with S. aureus biofilms due to S. aureus biofilm-produced leukocidins that lyse neutrophils58 (Figure 4A,D). Addition of a wash step to remove non-adhered neutrophils after incubating them with biofilm for 30 min removed ~15% of dead neutrophils from the system compared to the unwashed group, in which microscopy was performed immediately after 30 min of incubation (Figure 4D). Neutrophils interacting with S. aureus were also observed (Figure 4C). Further experiments are required to assess whether S. aureus is engulfed by neutrophils or attached to the cell surface of neutrophils54. Imaging neutrophils and biofilms is the first step to evaluate several neutrophil functionalities downstream, such as phagocytosis and NETosis54,59. The effect of neutrophils on biofilms can also be assessed by quantifying the biofilm biomass, structural changes of the biofilm, and biofilm viability, among many others, using image analysis tools listed in step 5.6. Lastly, donor-to-donor variability exists in neutrophils; thus, it is recommended that at least three different donors be used for studies involving neutrophils.
Overall, standardized in vitro assays were combined to assess interactions between neutrophils and biofilms. Though these assays utilize S. aureus, the protocols described can easily be adapted to study other pathogens. While there are various in vivo models to study host-pathogen interactions, they can be expensive and labor-intensive, especially if the conditions are not optimized. Working with standardized in vitro assays allows one to optimize experimental conditions and confirm observations prior to moving to an in vivo system. Finally, various animal infection models have been used to study biofilm-neutrophil interactions in vivo. However, it is important to consider immunological differences between humans and animal models60,61,62,63. This necessitates using neutrophils derived from humans to study these complex host-pathogen interactions.
The authors have nothing to disclose.
This work was funded by the National Institute of Allergy and Infectious Diseases (R01AI077628) to DJW and an American Heart Association Career Development Award (19CDA34630005) to ESG. We thank Dr. Paul Stoodley for providing us with USA 300 LAC GFP strain. Furthermore, we acknowledge resources from the Campus Microscopy and Imaging Facility (CMIF) and the OSU Comprehensive Cancer Center (OSUCCC) Microscopy Shared Resource (MSR), The Ohio State University. We also thank Amelia Staats, Peter Burback, and Lisa Coleman from the Stoodley lab for performing blood draws.
0.9% sodium chloride irrigation, USP | Baxter | 2F7124 | Endotoxin-free; Used for isolation of neutrophils |
150 mL rapid-flow filter unit | Thermo Scientific | 565-0020 | |
200 proof ethanol | VWR | 89125-188 | |
3 mL syringe | BD | 309657 | Used for blood draw |
50 mL conical centrifuge tubes | Thermo Scientific | 339652 | |
60 mL syringe | BD | 309653 | Used for blood draw |
Agar | Fisher Bioreagents | BP1423-2 | |
Alcohol swab | BD | Used for blood draw | |
Band-aids | Used for blood draw | ||
BD Bacto Tryptic Soy Broth | BD | DF0370-07-5 | Combine with 1.5% agar to make Tryptic Soy Agar |
Cell counter | Bal Saupply | 202C | |
CellTracker blue CMCH | Invitrogen | C2111 | Blue CMAC Dye (BCD) |
Clear bottom 96-well flat bottom polystyrene plates | Costar | 3370 | |
Cotton gauze | Fisherbrand | 13-761-52 | Used for blood draw |
Crystal violet | Acros Organic | 40583-0250 | |
Culture tubes | Fisherbrand | 14-961-27 | Borosilicate Glass 13 x 100 mm |
D-(+)-glucose | Sigma | G-8270 | |
Dextran from Leuconostoc spp. | Sigma | 31392-250G | Used for isolation of neutrophils |
Dulbecco's phosphate buffered saline (DPBS) 1x | Gibco | 14190-144 | |
Ethidium homodimer-1 | Invitrogen | L3224 B | |
Ficoll-Paque plus | Cytiva | 17144003 | Used for isolation of neutrophils (density gradient medium) |
Hanks' balanced salt solution (HBSS) 1x | Corning cellgro | 21-022-CV | without calcium, magnesium, and phenol red |
Hemacytometer | Bright Line | ||
Heparin | Novaplus | NDC 63323-540-57 | 1000 USP units/mL, Used for blood draw |
IMARIS 9.8 | Oxford Instruments | Microscopy image analysis software | |
Luminol | Sigma | A8511-5G | |
Minimal essential media (MEM) Alpha 1x | Gibco | 41061-029 | |
Needle (23 G1) | BD | 305145 | Used for blood draw |
Nikon Eclipse Ti2 | Nikon | ||
NIS-Elements | Nikon | Quantification of dead neutrophils | |
Normal human serum | Complement Technology | NHS | |
Petri Dish (100 x 15 mm) | VWR | 25384-342 | |
Phorbol 12-myristate 13-acetate | |||
Poly-L-lysine solution | Sigma | P4707-50ML | |
Sodium chloride | Fisher Bioreagents | BP358-10 | Used for neutrophil isolation |
SoftMax Pro Software | Molecular Devices | Microplate reader software used for data acquisition | |
SpectraMax i3x | Molecular Devices | Microplate reader | |
Sterile water for irrigation, USP | Baxter | 2F7114 | Endotoxin-free; Used for neutrophil isolation |
Surflo winged infusion set | Terumo | SC*19BLK | 19 G x 3/4", used for blood draw |
Trypan blue stain (0.4%) | Gibco | 15250-061 | |
Turnicate | Used for blood draw | ||
UltraPure distilled water | Invitrogen | 10977015 | |
White opaque 96-well plates | Falcon | 353296 | Tissue culture treated and flat bottom plate |
μ-Slide VI 0.4 | Ibidi | 80601 | μ-channel slide |