Ethical approval was obtained to recruit ventilated sepsis patients with and without ARDS (REC 16/WA/0169) and for the use of lung tissue samples from patients undergoing routine thoracic surgery (REC 17/WM/0272). For patients who lacked capacity, permission to enroll was sought from a personal legal representative following the UK Mental Capacity Act (2005). For patients with capacity, written informed consent was obtained from the patient.

Invasively ventilated adult patients with ARDS and sepsis were recruited from the intensive care unit of the Queen Elizabeth Hospital, Birmingham, UK, from December 2016 to February 2019, and BAL was collected as previously described (9). Demographic and physiological details of the patients can also be found in this prior publication. BAL fluid was rendered acellular by centrifuging at 500 g for 5 min. Acellular supernatant BAL was then pooled and stored at −80°C before use in this study.

Adult patients who underwent lung lobectomy as part of their clinical treatment plan for malignancy at Birmingham Heartlands Hospital from September 2017 to July 2019 were also recruited. Recruited patients were never-smokers or long-term ex-smokers (quit > 5 years), with normal spirometry and without airways disease. No patient received chemotherapy before surgery. Following lobectomy, lung tissue resection samples surplus to histopathological requirements were collected.

Macroscopically normal lung tissue samples were perfused with 0.15 M saline via pressure bag by inserting a needle (21-gauge) in bronchioles. When saturated, the tissue was gently massaged to facilitate emptying lavage fluid from the tissue, ready for the next instillation. This process was repeated until the lavage fluid contained <1 × 10⁴ cells/ml (23).

Cells were pelleted from the lavage fluid by centrifugation at 500 g for 5 min. Mononuclear cells were then separated by gradient centrifugation using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada), according to the instructions of the manufacturer. Mononuclear cells were then cultured in RPMI-1640 media supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (Sigma-Aldrich, Darmstadt, Germany) at 37°C and 5% CO₂ for 24 h to allow adherence. After 24-h culture, the wells were washed and media changed, thereby removing non-adherent mononuclear cells (24, 25). AMs were assessed for purity by cytospin (23); AM purity was consistently >95% across all samples.

The efferocytosis assay was modified from published protocols (26–29). Neutrophils were isolated from the blood of healthy volunteers using Percoll density centrifugation (30) as previously described by our group (31). Neutrophil purity was >96% as assessed by cytospin and viability >97% as assessed by trypan blue exclusion. Neutrophils were suspended in a 5 μM solution of CellTracker™ Deep Red fluorescent dye (ThermoFisher, Waltham, MA, USA) in 10% FCS/RPMI at 4 × 10⁶/ml, then incubated for 30 min at 37°C. Stained neutrophils were centrifuged at 1,500 g for 5 min then resuspended at 2 × 10⁶/ml in serum-free RPMI and incubated at 37°C and 5% CO₂ for 24 h to allow apoptosis. Flow cytometric assessment of neutrophil apoptosis was performed using a fluorescein isothiocyanate (FITC)-conjugated Annexin V and 7-aminoactinomycin D (7-AAD) apoptosis detection kit (BioLegend, San Diego, CA, USA): mean neutrophil apoptosis of 93% with necrosis of <2% was observed.

Alveolar macrophages were cultured at 2.5 × 10⁵/well in 24-well plates. As a negative control, 5 μg/ml Cytochalasin D (CytoD, Sigma-Aldrich, Darmstadt, Germany) was added for 30 min to inhibit actin filament polymerization required for efferocytosis. Stained apoptotic neutrophils (ANs) were added to AMs at a 4:1 ratio before incubation for 2 h at 37°C. The optimal assay duration of 2 h had previously been determined by time-course experiments. Media was removed and wells washed two times with ice-cold phosphate-buffered saline (PBS) to remove non-adherent/engulfed neutrophils. Cells were harvested using a 5-min TrypLE™ express (ThermoFisher, Waltham, MA, USA) incubation at 37°C, before acquisition using an Accuri C6 flow cytometer and software (BD Biosciences, Franklin Lakes, NJ, USA). AMs and ANs alone were used to set gates for their respective populations on forward and side-scatter plots. ANs alone were used to set a positive gate on the allophycocyanin (APC) plot, which was subsequently used to identify AMs which had engulfed ANs. Minimum 5,000 events gated as AMs were counted for each experimental condition and the percentage of APC⁺ AMs calculated. CytoD-treated AMs (negative control) determined the background fluorescence present due to ANs adhering to the surface of AMs but not being engulfed. This background fluorescence was subtracted from the percentage of APC⁺ AMs in other experimental conditions to give a corrected net efferocytosis index representative of neutrophil engulfment (Supplementary Figure 1). Steps were taken to avoid bias, including drawing gates based on single-cell populations (ANs and AMs) before assessing efferocytosis.

Alveolar macrophage phagocytosis assays were performed using pHrodo™ red Escherichia coli and Staphylococcus aureus BioParticle® conjugates (ThermoFisher, Waltham, MA, USA) in a 96-well plate according to the instruction of the manufacturer and as previously described (23). The pHrodo™ beads were prepared according to the instructions of the manufacturer at a final concentration of 1 mg/ml. AMs were seeded at 50,000 cells per well in black well, clear bottomed 96-well plates, and cultured overnight. For negative control wells, 5 μg/ml CytoD (Sigma-Aldrich, Darmstadt, Germany) was added for 30 min. A total of 50 μl of pHrodo bead suspension was added per well and incubated for 6 h at 37°C. After 6 h, cells were washed three times with PBS before adding 100 μl fresh PBS. Fluorescence was measured using a microplate reader (Synergy 2, Bio-Tek, Winooski, VT, USA) set at the excitation/emission spectra of pHrodo™ red dye: 560/585 nm. The negative control (cytochalasin D treated) AMs were used to determine the background fluorescence present due to stained pHrodo™ red BioParticles® adhering to the outside of macrophages but not being engulfed. This background fluorescence value was subtracted from fluorescence values of other experimental conditions, to give the corrected net fluorescence value the representative of phagocytosis. Phagocytosis results were expressed as fold change in relative fluorescence unit from untreated AMs.

Bronchoalveolar lavage from 14 recruited patients with sepsis-related ARDS was rendered acellular by centrifugation and then pooled. The acellular pooled BAL was mixed in a 1:1 ratio with 10% FCS/RPMI. To elicit functional changes associated with ARDS, AMs were treated with this 50% ARDS BAL mixture. AMs were also treated with a 1:1 mixture of 0.9% saline and 10% FCS/RPMI, as vehicle control (VC). Other treatments given in conjunction with 50% ARDS BAL or saline included 200 nM Y-27632 dihydrochloride (Rho-associated protein kinase inhibitor, Apexbio, Houston, TX, USA), 2 μM SF1670 (phosphatase and tensin homolog inhibitor, Selleckchem, Houston, TX, USA), and dimethyl sulfoxide (VC for Y-27632 and SF1670, Sigma-Aldrich, Darmstadt, Germany) at a 1:50,000 dilution. ROCK and PTEN inhibitor treatment doses were determined by dose response on untreated AM efferocytosis (Supplementary Figure 2). Other treatments not combined with 50% ARDS BAL or saline included 50 ng/ml interferon-γ (IFN-γ, Peprotech, UK), 1 μg/ml ultrapure lipopolysaccharide (LPS, Invitrogen, Waltham, MA, USA), 40 ng/ml interleukin-4 (IL-4, Peprotech, UK), and 40 ng/ml IL-13, (Peprotech, UK). Previous studies have shown that macrophage treatment with IFN-γ and LPS can induce a proinflammatory phenotype, whereas IL-4 and IL-13 treatment can induce a proresolving phenotype (17). Cytokine concentrations were based on published methods (32). The 1 μg/ml dose of LPS was based on the lowest dose required to elicit tumor necrosis factor-α (TNFα) production from AMs (Supplementary Figure 3). Efferocytosis, phagocytosis, apoptosis/viability, and RNA extraction for gene expression were performed 24 h after treatment with 50% ARDS BAL. Phenotyping was performed 48 h after treatment. AM apoptosis and viability were assessed using a flow cytometric apoptosis detection kit (BioLegend, San Diego, CA, USA).

Alveolar macrophages were labeled with the following antihuman antibodies or their isotype controls: CD206-APC, CD80-PE, CD163-FITC, Mer-APC, and SIRPα-FITC (see Supplementary Table 1). Surface marker expression was assessed by an Accuri C6 flow cytometer and software (BD Biosciences, Franklin Lakes, NJ, USA). AM population was gated on forward and side-scatter plot. The median fluorescence intensity (MFI) in relevant channels from isotype control AMs was subtracted from the MFIs of stained AMs, to give the net MFI for each antibody fluorophore. Results presented as fold change-corrected MFI, as a measure of change in cell surface expression, compared to VC (50% saline).

RNA was isolated from AMs using Nucleospin RNA kits (Machery-Nagel, Düren, Germany) as per the instructions of the manufacturer. RNA quantity was assessed with the NanoDrop 2000 UV-Vis Spectrophotometer (ThermoFisher, Waltham, MA, USA). One-Step Quantifast Probe RT-PCR Kits (Qiagen, Hilden, Germany) were used to assess gene expression with a CFX384 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA). Taqman® gene expression assays (ThermoFisher, Waltham, MA, USA) were purchased for 18S on VIC-MGB (ref 4318839) and RAC1 on FAM-MGB (Hs01025984_m1). PCR conditions were used as per the recommendation of the manufacturer. Triplicate data were analyzed using CFX Maestro software (BioRad, Hercules, CA, USA). Relative quantification of target gene mRNA was calculated relative to expression of 18s endogenous control gene.

Inflammatory cytokine (IL-6, IL-8, TNF-α, IL-1β, macrophage chemoattractant protein-1, IL-10, IL-1ra, and vascular endothelial growth factor) content of pooled patient BAL fluid was measured by a commercially available custom Magnetic Luminex® Performance Assay (R&D Systems, UK) as per the instructions of the manufacturer. Protein concentration in pooled patient BAL fluid was measured using the Pierce™ BCA (Bicinchoninic Acid) Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) as per the instructions of the manufacturer.

Data were analyzed using Prism 8 software (GraphPad, San Diego, CA, USA). Parametric data are shown as mean and SD. Non-parametric data are shown as the median and interquartile range (IQR). Differences between continuously distributed data were assessed using Welch's t-tests for parametric data or Mann–Whitney tests for non-parametric data. Differences between non-parametric paired data were assessed using Wilcoxon matched-pairs signed-rank test. Differences between three or more unpaired parametric data sets were assessed using ANOVA followed by Dunn's multiple comparison tests. Differences between three or more paired parametric data sets assessed using the repeated measures ANOVA followed by Tukey's multiple comparison tests. Two-tailed p-values of ≤ 0.05 were considered significant.

Samples of BAL from the first 14 patients with sepsis-related ARDS recruited to a previous study (9) were pooled and used to treat AMs isolated from lung resections. This pooled ARDS patient BAL was characterized with regards to inflammatory cytokine and LPS content (Table 1). AMs were isolated from the lung tissue of 16 patients who underwent lobectomy (mean yield of 9 million AMs per patient). The mean age of lobectomy patients was 70 years (SD = 6.9 years). The male:female split for lobectomy patients was 9:7.

Alveolar macrophage efferocytosis was impaired in sepsis patients with ARDS compared to lobectomy patients (Figure 1A, mean 7.6% [SD = 5.1] vs. 32.2% [SD = 9.4], p < 0.0001). We established an in vitro model of ARDS, by treating lung resection tissue AMs with pooled ARDS BAL to induce AM dysfunction. ARDS BAL or saline VC treatment did not affect AM apoptosis or viability compared to standard culture (Supplementary Figure 4). Treatment of AMs with ARDS BAL reduced efferocytosis compared to VC treatment (Figure 1B, mean 11.7% [SD = 6.4] vs. 24.7% [SD = 7.6], p = 0.0006). Treatment of AMs with ARDS BAL reduced Rac1 mRNA expression compared to VC treatment (Figure 1C, median of differences 0.48, p = 0.016), which supports our finding that ARDS BAL inhibits AM efferocytosis.

Treatment of AMs with VC reduced phagocytosis of both S. aureus and E. coli pHrodo® bioparticles compared to untreated controls (Figures 2A,B, p = 0.031). Treatment of AMs with ARDS BAL increased phagocytosis of S. aureus pHrodo® bioparticles compared to VC treatment (Figure 2A, median of differences 0.32, p = 0.031). There was no difference in AM phagocytosis of E. coli pHrodo® bioparticles following ARDS BAL treatment compared to VC treatment (Figure 3B, p = 0.063). Thus, ARDS BAL treatment had divergent effects on AM efferocytosis and phagocytosis.

Since ARDS BAL treatment of AMs had divergent effects on efferocytosis and phagocytosis, we used this in vitro model to investigate the association between AM phenotype and function. Treatment of AMs with ARDS BAL increased expression of CD206 (Figure 3A, mean fold change 0.39, p = 0.006) and MerTK (Figure 3B, mean fold change 0.3, p = 0.028) compared to VC treatment. Treatment of AMs with ARDS BAL decreased SIRPα expression compared to VC treatment (Figure 3C, mean fold change −0.26, p = 0.006). Treatment of AMs with ARDS BAL did not change the expression of CD163 or CD80 compared to VC treatment (Figures 3D,E, p > 0.05 for both). These changes in AM surface-receptor expression were incongruent with the observed defect in AM efferocytosis following ARDS BAL treatment.

Treatment of AMs with proinflammatory mediators IFN-γ and LPS also impaired efferocytosis (Figure 4A, mean difference 16.5%, p = 0.008). However, in contrast to ARDS BAL, proinflammatory mediator treatment decreased expression of both MerTK (Figure 4B, mean fold change −0.58, p = 0.015) and CD163 (Figure 4C, mean fold change −0.58, mean fold change −0.55, p = 0.005) while increasing expression of SIRPα (Figure 4D, mean fold change 2.48, p = 0.036). Proinflammatory mediator treatment had no significant effect on the expression of AM surface markers CD206 or CD80 (Figures 4E,F). Treatment with proresolving mediators IL-4 and IL-13 did not affect AM efferocytosis; however, their effect on AM surface-receptor expression was also assessed: expression of MerTK, CD163, and SIRPα was decreased while expression of CD206 was increased (Figures 4A–E). These findings indicate that the effect of ARDS BAL treatment on AMs cannot solely be explained by changes in surface-receptor expression.

Since this model effectively replicated in vitro the efferocytosis defect in ARDS AMs evident in vivo, we next used this model to explore the mechanism driving this defect. Rac1 intracellular signaling pathways are summarized in Figure 5. From this pathway, we identified ROCK and PTEN as potential targets to modify activity. The addition of ROCK-inhibitor to ARDS BAL treatment increased AM efferocytosis compared to treatment with ARDS BAL plus VC (Figure 6A, mean fold change 0.17, p = 0.009). The addition of PTEN inhibitor to ARDS BAL treatment did not affect efferocytosis compared to treatment with ARDS BAL plus VC (Figure 6B). ROCK inhibition did not affect AM phagocytosis of E. coli or S. aureus bioparticles (Supplementary Figure 5). ROCK inhibition also did not affect AM surface marker expression of CD206, CD163, CD80, SIRPα, and MerTK (Supplementary Figure 6).