Therapeutic stem cell‐derived alveolar‐like macrophages display bactericidal effects and resolve Pseudomonas aeruginosa‐induced lung injury

Abstract Bacterial lung infections lead to greater than 4 million deaths per year with antibiotic treatments driving an increase in antibiotic resistance and a need to establish new therapeutic approaches. Recently, we have generated mouse and rat stem cell‐derived alveolar‐like macrophages (ALMs), which like primary alveolar macrophages (1'AMs), phagocytose bacteria and promote airway repair. Our aim was to further characterize ALMs and determine their bactericidal capabilities. The characterization of ALMs showed that they share known 1'AM cell surface markers, but unlike 1'AMs are highly proliferative in vitro. ALMs effectively phagocytose and kill laboratory strains of P. aeruginosa (P.A.), E. coli (E.C.) and S. aureus, and clinical strains of P.A. In vivo, ALMs remain viable, adapt additional features of native 1'AMs, but proliferation is reduced. Mouse ALMs phagocytose P.A. and E.C. and rat ALMs phagocytose and kill P.A. within the lung 24 h post‐instillation. In a pre‐clinical model of P.A.‐induced lung injury, rat ALM administration mitigated weight loss and resolved lung injury observed seven days post‐instillation. Collectively, ALMs attenuate pulmonary bacterial infections and promote airway repair. ALMs could be utilized as an alternative or adjuvant therapy where current treatments are ineffective against antibiotic‐resistant bacteria or to enhance routine antibiotic delivery.

susceptible to bacterial lung infections. [2][3][4][5] Furthermore, in light of the SARS-CoV-2 pandemic, in developed countries ~8% of critically ill SARS-CoV-2 patients present with bacterial co-infections and antimicrobial therapies are routinely administered as a prophylactic measure, 6,7 potentially leading to a further increase in antibiotic resistance in our rapidly evolving healthcare landscape. 8,9 Recently, the World Health Organization published a list of 12 multi-antibioticresistant priority pathogens that pose the greatest threat to global human health; four of these directly colonize the lungs, namely Pseudomonas aeruginosa (P.A.), methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumonia, and Haemophilus influenzae. 10,11 Current trends suggest that traditional antibiotic therapy is becoming less effective in clearing infections, resulting in longer hospital visits, higher medical costs and increased mortality. 8 Thus, innovative approaches for new therapies need to be developed to combat antibiotic resistance. Cell-based therapies hold promise as a revolutionary therapeutic to treat respiratory infections and halt global antibiotic resistance.
Primary alveolar macrophages (1'AMs) are tissue-resident macrophages of the pulmonary innate immune system that maintain healthy airways by orchestrating the clearance pathogens and initiating an adaptive immune response. 12,13 Unlike bone marrowderived Myb + macrophages, which arise from adult hematopoietic stem cells, Myb − 1'AMs are from embryonic or foetal origin and self-replicate, making them an ideal candidate for cell-based therapies. [14][15][16] In recent years, we have developed novel alveolarlike macrophages (ALMs) that are derived from pluripotent stem cells (PSCs) and retain key embryonic signatures of Myb − 1'AMs. 17 Previously, we have demonstrated that ALMs reduce lung injury in mouse models of acute and chronic lung diseases, can internalize dead Staphylococcus aureus (S.A.) 17 and kill respiratory syncytial virus (RSV) and prevent RSV-induced lung injury. 18 Here, we further characterized ALMs for their efficacy to treat bacterial lung infections. We evaluated the non-pharmacological antimicrobial functions of ALMs, specifically for bacteria known to colonize the lungs of immune-compromised individuals, such as P.A. and S.A. in CF patients, and Escherichia coli (E.C.) that is nosocomially acquired in many pneumonia patients. Lastly, we also illustrate ALMs ability to resolve P.A.-induced lung injury in a preclinical model.

| Maintenance, expansion and culturing of alveolar-like macrophages
ALMs were differentiated from mouse (G4 DsRed-MST) and rat (DAc8) PSC lines and cultured in macrophage media as previously described. 17 For bacterial experiments, penicillin-streptomycin was removed 48 h before. ALMs were passaged every 5-7 days at a ratio of 1:10 using ReLeSR. Details of all reagents are reported in Table S1.

| Collection of mouse and rat primary alveolar macrophages
Animal experiments were completed in compliance with the Animal Care Committee at The Hospital for Sick Children (AUP #46059 and #59356). Adult male C57BL6/J mice and Sprague-Dawley rats were sacrificed with pentobarbitol. The thoracic cavity was opened and a tracheostomy was performed, then the lungs were instilled with Dulbecco's phosphate-buffered saline (DPBS), and bronchoalveolar lavage fluid (BALF) was collected. For mice, lungs were lavaged with a 24-gauge angiocath with 3 × 1 ml DPBS. For rats, lungs were lavaged with a 20-gauge angiocath with 3 × 10 ml DPBS. Each aliquot was used to lavage the lungs twice. BALF collected was centrifuged at 400g for 20 min. The pellet was resuspended in macrophage media, transferred to tissue culture plates and incubated. Primary

| In vitro proliferation of primary alveolar macrophages and alveolar-like macrophages
1'AMs and ALMs were cultured at the same density in macrophage media. Cells were imaged using the same x/y coordinates at ×100 magnification on Days 0-7 using a light microscope (Leica DMI300B) and Volocity software (Perkin Elmer Inc, Version 6.3).
The number of cells per field of view was manually counted.

| Flow cytometry characterization of primary alveolar macrophages and alveolar-like macrophages
Mouse ALMs were previously characterized for known 1'AM markers. 17 Here, we further characterized mouse ALMs and 1'AMs for their expression of toll-like receptor (TLR) 2, 4 and 5. Rat ALMs and 1'AMs were characterized using known 1'AM cell surface markers; CD45, 19 SIRPα, 19 CD11b/c 20 and mature macrophage marker, 21 along with TLR2, 4 and 5. Briefly, 1'AMs and ALMs were collected and the FcϒII receptor was blocked with anti-CD32 for 5 min. All antibodies were added for 30 min. Further details are outlined in Table S2 or previously described. 17 A Gallios 10/3 flow cytometer (Beckman-Coulter) was used for acquisition and data were analysed using Kaluza software (Beckman-Coulter). Inc), using Metamorph software (Molecular Devices). Image processing, including iterative restoration (confidence limit = 99%, 10 iteration limit = 10) was completed using Volocity. Live cell imaging of mouse ALMs cultured with E.C. and rat ALMs cultured E.C. and P.A., with was completed using a Leica CTRMIC 6000 confocal microscope as previously described. 17

| In vitro bactericidal capacity of rodent alveolar-like macrophages
To perform a gentamicin protection assay (GPA), ALMs were sus-

| In vivo proliferation and viability of mouse and rat alveolar-like macrophages
Mice and rats were anaesthetized with a loading dose of ketamine (mice 20 mg/kg, rat 75 mg/kg) and xylazine (mice 10 mg/kg, rat 25 mg/kg), followed by a 2 min exposure to 2% isofluorane. Four million mouse ALMs or 1 × 10 7 rat ALMs were instilled intratracheally into the lungs via either a 24-or 20-gauge angiocath. Mice ALMs expressed DsRed for tracking. 17 Rat ALMs were prestained with CellTrace™ for tracking. Following 24 h, BALF was collected and the viability of ALMs was determined by flow cytometry using cell viability VivaFix assay. Proliferation of ALMs was determined by flow cytometry using an anti-Ki67 antibody. Further antibody details are in Table S2. Flow cytometry results were gated using the DsRed and CellTrace™ dye fluorescence to distinguish the administered ALMs from primary BALF cells.

| In vivo characterization of mouse and rat alveolar-like macrophages and primary BALF cells
Mice and rats were anaesthetized and 4 × 10 6 DsRed-expressing ALMs or 10 × 10 6 CellTrace™-expressing ALMs, respectively, were instilled. Following 72 h, BALF was collected as described above.  Table S2.

| In vivo internalization and killing of E. coli and P. aeruginosa by rat and mouse alveolar-like macrophages
Live GFP-expressing E.C. and P.A., mouse DsRed ALMs and rat CellTrace™ ALMs were prepared as described above. For mice, 1 × 10 7 bacteria were instilled while rats were instilled with 1 × 10 8 bacteria. ALMs were instilled 30 min after the initial bacterial instillation to achieve a MOI of 10:1 for bacteria:ALMs (mice: 1 × 10 6 ALMs, rats: 1 × 10 7 ALMs).

2.11
| Internalization of E. coli and P. aeruginosa by rat and mouse alveolar-like macrophages BALF was collected 3 h post-instillation. Flow cytometry was performed on BALF, gating for GFP (bacteria) and DsRed or CellTrace™.
Mouse GFP + DsRed + and rat GFP + CellTrace™ + cells were considered ALMs that had internalized bacteria. Internalization was confirmed by creating cytospots and ensuring co-localization of GFP and DsRed or CellTrace™ staining. For cytospots, 1 × 10 5 ALMs were adhered to slides at 300g and fixed with 4% (v/v) PFA for 20 min.
Mouse GFP + DsRed + ALMs were imaged on a Leica DM 6000B microscope connected to a Leica TCS SP5 system using Leica Application Suite Advanced Fluorescence software (version 1.6.0) (Leica Microsystems Inc). GFP + CellTrace™ + ALMs were imaged using confocal microscopy and analysed as outlined above. Figure S1 provides a schematic experimental workflow.

| Killing capacity and planktonic bacterial load in P. aeruginosa-induced lung injury model in rats
To assess rat ALMs ability to kill P.A. from the lysis step onwards as described above. Bacterial load (BALF supernatant) and survival (ALMs and primary BALF cells) were calculated as described above using the T0 and T24 time points. Figure S2 provides a schematic experimental workflow. This experiment was not performed in mice as a significant number of cells need to be recovered for FACS, therefore requiring a large number of mice to be used that could not ethically be justified.

| Administration of alveolar-like macrophages in P. aeruginosa-induced lung injury model in rats
To determine the role of ALMs in P.A.-induced lung injury, 1 × 10 8 PA01 was intratracheally instilled into rats, with controls receiving DPBS. Six hours later, both groups were instilled with either 1 × 10 7 ALMs or a DPBS control. Rats were weighed daily for seven days. At Day 7 the right lung was collected to determine the wet/ dry weight ratio and the left lung was pressure fixed at 20 cm H 2 O with 4% (v/v) PFA and the tissue was processed through a number of ethanol, xylazine and wax baths before being embedded. Three 5 μm sections were cut 100 μm apart and stained with haematoxylin and eosin. From each section, 20 images were randomly taken in a grid-like fashion with a ×200 magnification on a light microscope and Volocity as described above. An injury score was assigned to each image based on the presence and severity of epithelial thickening, epithelial sloughing, oedema and infiltrate. Each parameter was scored out of two with a final score out of eight. Three researchers independently scored 60 images per rat in a doubleblinded fashion, and an average score was taken per rat from all three researchers. Figure S3 provides a schematic experimental workflow.

| Statistical analysis
Data are presented as mean ± SEM. Comparisons between groups were made using paired Student's t-test using Welch's correction where appropriate. Where multiple comparisons were required, either a one-way, two-way or repeated-measures ANOVA was used with either a Sidak or Tukey post hoc test. Statistical significance was set at p < 0.05. Statistical analysis and graphing were performed using GraphPad Prism 6.

| Alveolar-like macrophages proliferate in vitro
The proliferative capabilities of ALMs and 1'AMs were directly compared. The doubling rate of rat and mice ALMs was 34.20 ± 1.25 and 46.40 ± 1.73 h, respectively, while 1'AMs did not divide, and the number of 1'AMs even decreased over seven days ( Figure 1A and B).
The experiment was terminated at seven days as the ALMs overgrow the culture dish at this point, but after passaging they continue to proliferate and can be continuously cultured in vitro for at least two years without a reduction in growth rate. 17

| Alveolar-like macrophages express known primary alveolar macrophage cell surface markers
Rat ALMs and 1'AMs were compared for their surface marker expression ( Figure S4A, n = 5). The rat ALM population highly expressed the 1'AM markers SIRPα, CD11b/c, mature macrophage marker, however, did not express the pan myeloid marker CD45, and CD86. Rat 1'AMs highly expressed CD45 and SIRPα, however, did not express the mature macrophage marker, CD11b/c or CD86.

| Alveolar-like macrophages internalize live E. coli and P. aeruginosa
Scanning electron micrographs demonstrated P.A. associating with mouse ALMs (Figure 2A). Pseudopodia extend from the surface of the ALM and wrap around a P.A., followed by formation in the phagocytic cup before beginning to envelope a P.A. Confocal imaging confirmed that mouse ALMs ( Figure 2B) and rat ALMs ( Figure 2C) internalize live E.C. and P.A and retain them in vacuolar structures resembling phagosomes.  Figure 3B). Reinterpretation of this data illustrated that mouse and rat ALMs display similar bactericidal efficiencies to P.A. (Figure S5A).
Re-interpretation of this data illustrated that mouse and rat ALMs display similar bactericidal efficiencies to both laboratory and clinical strains of P.A. (Figure S5C).

| Alveolar-like macrophages adapt to the lung microenvironment in vivo
In mice and rats, three days post-ALM administration the expres-

| Alveolar-like macrophages internalize and kill bacteria in vivo without altering bacterial load in BALF
Firstly, we established that mouse ALMs could internalize E.C. and P.A. in vivo over 3 h; 15.88 ± 4.48% and 31.83 ± 3.54% (n = 3) of mouse ALMs internalize E.C. and P.A., respectively ( Figure 5A and B).
Next, we assessed the ability of rat ALMs and primary BALF cells

| Rat alveolar macrophages resolve P. aeruginosa-induced lung injury in vivo
Body weight was measured daily for seven days post-instillation.

F I G U R E 3
Bactericidal capacity of alveolar-like macrophages to kill P. aeruginosa, E. coli, S. aureus and clinical respiratory strains of P. aeruginosa in vitro. Bacterial percentage killing for mouse (A) and rat (B) ALMs targeting live laboratory strains of E.C., P.A. and S.A. were determined using a GPA. Bacterial per cent killing for mouse (C) and rat (D) ALMs targeting various clinical P.A. strains that are either tobramycin sensitive and are eradicated by antibiotics (ER) or are resistant to tobramycin and persist with antibiotic treatment (PR) were determined using a GPA. Data in all panels are presented as mean ± SEM, n = 3-5 separate experiments each with technical triplicates. Statistical comparisons were made against the T0 time point (data not shown)

| DISCUSS ION
Recently, the need to establish more effective therapeutic approaches towards lung diseases and infections has driven the development of cell-based therapies. 24,25 This rapidly evolving area led to the development of ALMs, 17  Surface expression of known 1'AM markers 20,21,26 was confirmed on rat ALMs establishing their similarity to 1'AMs. Rat ALMs similarly express CD86 and SIRPα; however, CD11b/c and the mature macrophage marker are highly expressed in rat ALMs compared to 1'AMs. 17 This differential expression could be due to variable activation states of in vitro ALMs and in vivo 1'AMs, which is known to alter marker expression. 20 CD45 is expressed by virtually all hematopoietic cells; however, some primitive macrophage subsets, such as microglia and those derived from yolk-sac haematopoiesis express low or no CD45. 27 Interestingly, mouse ALMs express CD45 but rat ALMs do not, suggesting that the expression of CD45 on ALMs is non-essential to their characteristics and function in general and re-affirming that ALMs retain a primitive signature. 17,28 Importantly, mouse and rat ALMs express TLRs 2, 4 and 5, known to be responsive to the pathogen-associated molecular patterns of LPS and flagellin 29,30 ; and ALMs proliferate more sustainably and rapidly than 1'AMs and primary bone marrow-derived macrophages. 17 Mouse 1'AMs have been cultured to 20 passages in vitro, 15,31 and human iPSC-derived macrophages have been harvested for up to three months post-differentiation in a bioreactor system 32 ; however, the rate of proliferation of our F I G U R E 4 Alveolar-like macrophages are viable but have reduced proliferative capacity in vivo. Mouse DsRed + ALMs (A) and rat CellTrace™ + ALMs (B) are viable and proliferative, when cultured to 50% confluency in vitro prior to instillation. 24 h post in vivo instillation ALMs were collected via bronchoalveolar lavage fluid (BALF). The mouse DsRed + ALM or rat CellTrace™ + ALM population in the BALF was separated from endogenous populations by flow cytometry. Mouse and rat ALMs retain their viability, but their proliferative capacity is diminished. Data in both panels are presented as mean ± SEM. Black histograms; unstained control cells, white histograms; stained cells. ESC-derived ALMs is significantly greater and they remain proliferative over longer periods of time. 17 ALMs are therefore easily scalable to therapeutic numbers, which is key to producing a cost and time effective cell-based therapy.
One of the major effector functions of 1'AMs is their ability to phagocytose and kill pathogens. 33 Thus, we determined the bactericidal effects of ALMs. After visualizing bacterial internalization, we confirmed that both rat and mouse ALMs effectively kill laboratory strains of E.C., P.A. and S.A. with greater than 70% killing efficiency. Moreover, ALMs efficiently kill clinical strains of P.A. It is important to recognize that ALMs effectively killed both eradicated and persistent P.A. clinical strains with the same efficiency as F I G U R E 5 Alveolar-like macrophages display bactericidal effects in vivo by internalizing P. aeruginosa and E. coli. Adult mice and rats were infected intratracheally with either live GFPexpressing E.C. and/or P.A. Thirty minutes post-infection, mice and rats were instilled intratracheally with mouse DsRed + ALMs or rat CellTrace™ + ALMs, respectively. Three hours following the initial infection mice and rats were sacrificed and BALF cells were collected. In the present study, we also characterized ALMs in vivo and demonstrated that ALMs remain viable (~95%) but drop their proliferation rate by ~50%, suggesting ALMs adapt to the lung niche and present as 1'AMs, which exhibit slow proliferation rates in vivo. 34 This reduction in proliferation could be beneficial, as it suggests ALMs are less likely to become tumorigenic, an important distinction as ALMs can be retained in the airways for at least 4 weeks. 17 In mice, ALM polarization towards a M1 or M2 phenotype was not observed; however, we observed that ALMs adapt to the lung niche by reducing their expression of CD86 and CD206, and increasing their expression of Siglec-F to adopt a phenotype more analogous to 1'AMs. We also observed a marginal increase in the CD11b + /GR-1 + neutrophil population in the BALF following ALM administration in mice. This increase from ~2% to ~8% is unlikely to indicate an acute inflammatory response as >90% neutrophils are observed in the BALF of inflammatory LPS models. 35,36 Unlike mice, rat ALMs exhibit a distinctly M2 phenotype, both in vitro and three days postinstillation in rat lungs. After transplantation, rat ALMs also adopt a phenotype similar to 1'AMs, as seen with a reduction in CD206 expression to 1'AM levels. it is plausible that the bacterial load may be reduced or resolved at a later time point, especially as the amelioration of weight loss was observed from Day 4 onwards. However, another mechanism for the ALM-driven resolution of lung injury could be rat ALMs maintaining a predominant M2 phenotype within the lung microenvironment. It is F I G U R E 7 Alveolar-like macrophages resolve P. aeruginosa-induced lung injury in vivo. Adult rats were infected intratracheally with P.A. or DPBS. Six hours post-instillation, rats were instilled intratracheally with either ALMs or DPBS. Rats were weighed daily, and histological analysis was performed at Day 7 postinstillation. (A) Rats in the PA01 + DPBS group was lighter than the DPBS + DPBS and DPBS + ALM groups by Day 4 and all three experimental groups by Day 5 onwards. Data are mean ± SEM, n = 3-4 rats per experimental group. Statistically significant difference; * denotes p < 0.05 between experimental groups in a Tukey's post hoc test. Data were expressed as a percentage gain in body weight to normalize for the variable starting weights (pre-instillation). (B) Lung histological analysis demonstrated that the injury score was significantly greater in the PA01 + DPBS group compared to the other three experimental groups, with no differences observed between the other groups. Data are mean ± SEM, n = 3-4 rats per experimental group. Experimental groups with different letters (above the bar) are significantly different from each other. (C-F) Representative histological images of DPBS + DPBS, DPBS + ALM, PA01 + DPBS and PA01 + ALM groups, respectively. Images were taken at ×200 magnification. Scale bars are 50 μm. Images are representative of 3-4 rats per experimental group probable that M2 ALMs can downregulate the magnitude and longevity of neutrophil influx and aid in efferocytosis, a known M2 macrophage effector function, 40 resulting in the mitigation of P.A.-induced structural lung injury and weight loss by Day 7. Future studies will be more expansive, including determining the bacterial load and cytokine profiles at different time points and whether greater ALM dosages can reduce the bacterial load even after 24 h.
Together, these data are the first to show ALM-driven resolution of P.A.-induced lung injury, and further supports the concept of ALMs promoting airway repair in disease models. 17 Recently, it has been reported that murine hematopoietic stem cell transplantation 39 or delivery of human iPSC-derived macrophages 32 rescued mice from P.A. airway infection; however, the critical distinction is that these graft-derived or instilled macrophages were of bone marrow or non-primitive origins, unlike ALMs that are engineered to reside in the lung for long periods of time. Moreover, in these studies the graft-derived macrophages already resided in the lung prior to infection 39 while human iPSC-derived macrophages were instilled at the time of infection. 32 In contrast, in our study ALMs were delivered post-infection, which more realistically mimics a clinical time-course.

| CON CLUS IONS
Collectively, the use of ALMs as a translational cell-based bactericidal therapy holds great promise in future. ALMs are a robust proliferative cell with the potential to be utilized as an alternative or adjuvant therapy where current strategies are ineffective against antibiotic-resistant bacteria or to enhance routine antibiotic delivery. Importantly, ALMs not only resolve lung injury but also are effective antibacterial cells that attenuate pulmonary bacterial infection and promote repair. Moreover, ALMs scalability and immunological functionality make them versatile cells that are permissible to transfection and thus could be altered to further enhance their bactericidal and immunoregulatory functions and be used as a platform to treat a multitude of lung diseases.