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Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease

  • Camilla Margaroli
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Hamed Horati
    Affiliations
    Department of Pediatrics, Div. of Respiratory Medicine and Allergology, I-BALL program, Erasmus MC-Sophia Children's Hospital, University Hospital Rotterdam, Rotterdam, The Netherlands
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  • Luke W. Garratt
    Affiliations
    AREST-CF Program, Telethon Kids Institute, University of Western Australia, Perth, Australia
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  • Vincent D. Giacalone
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Craig Schofield
    Affiliations
    AREST-CF Program, Telethon Kids Institute, University of Western Australia, Perth, Australia
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  • A. Susanne Dittrich
    Affiliations
    Department of Translational Pulmonology, Translational Lung Research Center (TLRC), German Center for Lung Research (DZL) and Department of Pulmonology, and Critical Care Medicine, Thoraxklinik at the University of Heidelberg, Heidelberg, Germany
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  • Tim Rosenow
    Affiliations
    AREST-CF Program, Telethon Kids Institute, University of Western Australia, Perth, Australia
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  • Brian S. Dobosh
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Hong S. Lim
    Affiliations
    Department of Biomedical engineering, The Georgia Institute of Technology and Emory University, Atlanta, GA, United States of America
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  • Dario L. Frey
    Affiliations
    Department of Translational Pulmonology, Translational Lung Research Center (TLRC), German Center for Lung Research (DZL) and Department of Pulmonology, and Critical Care Medicine, Thoraxklinik at the University of Heidelberg, Heidelberg, Germany
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  • Mieke Veltman
    Affiliations
    Department of Pediatrics, Div. of Respiratory Medicine and Allergology, I-BALL program, Erasmus MC-Sophia Children's Hospital, University Hospital Rotterdam, Rotterdam, The Netherlands
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  • George L. Silva
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Milton R. Brown
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Carsten Schultz
    Affiliations
    Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR, United States of America
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  • Harm A.W.M. Tiddens
    Affiliations
    Department of Pediatrics, Div. of Respiratory Medicine and Allergology, I-BALL program, Erasmus MC-Sophia Children's Hospital, University Hospital Rotterdam, Rotterdam, The Netherlands
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  • Sarath Ranganathan
    Affiliations
    Department of Pediatrics, University of Melbourne, Melbourne, Australia

    Murdoch Children's Research Institute, and Department of Respiratory and Sleep Medicine, Royal Children's Hospital, Parkville, Australia
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  • Joshua D. Chandler
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Peng Qiu
    Affiliations
    Department of Biomedical engineering, The Georgia Institute of Technology and Emory University, Atlanta, GA, United States of America
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  • Limin Peng
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America

    Department of Biostatistics, Emory University School of Public Health, Atlanta, GA, United States of America
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  • Bob J. Scholte
    Affiliations
    Department of Pediatrics, Div. of Respiratory Medicine and Allergology, I-BALL program, Erasmus MC-Sophia Children's Hospital, University Hospital Rotterdam, Rotterdam, The Netherlands
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  • Marcus A. Mall
    Affiliations
    Department of Translational Pulmonology, Translational Lung Research Center (TLRC), German Center for Lung Research (DZL) and Department of Pulmonology, and Critical Care Medicine, Thoraxklinik at the University of Heidelberg, Heidelberg, Germany

    Department of Pediatric Pulmonology, Immunology and Intensive Care Medicine, Charité – Universitätsmedizin Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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  • Anthony Kicic
    Affiliations
    AREST-CF Program, Telethon Kids Institute, University of Western Australia, Perth, Australia

    Department of Respiratory and Sleep Medicine, Perth Children's Hospital and Faculty of Medicine, University of Western Australia, Perth, Western Australia, Australia

    School of Public Heath, Curtin University, Perth, Western Australia, Australia
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  • Lokesh Guglani
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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  • Stephen M. Stick
    Affiliations
    AREST-CF Program, Telethon Kids Institute, University of Western Australia, Perth, Australia

    Department of Respiratory and Sleep Medicine, Perth Children's Hospital and Faculty of Medicine, University of Western Australia, Perth, Western Australia, Australia
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  • Hettie M. Janssens
    Affiliations
    Department of Pediatrics, Div. of Respiratory Medicine and Allergology, I-BALL program, Erasmus MC-Sophia Children's Hospital, University Hospital Rotterdam, Rotterdam, The Netherlands
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  • Rabindra Tirouvanziam
    Correspondence
    Corresponding author.
    Affiliations
    Department of Pediatrics, Center for CF & Airways Disease Research, Children's Healthcare of Atlanta, IMPEDE-CF Program, Emory University School of Medicine, Atlanta, GA, United States of America
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Open AccessPublished:June 19, 2022DOI:https://doi.org/10.1016/j.jcf.2022.06.001

      Highlights

      • Macrophages are gradually replaced by neutrophils in early CF lung disease
      • Signaling through the PD-1 pathway is known to inhibit immune cell activation
      • Macrophage PD-1 expression associates with neutrophilia, infection and lung damage
      • Neutrophil-derived factors induce PD-1 expression in airway-like monocytes
      • Blocking PD-1 on patient airway leukocytes improves bacteria killing

      Abstract

      Background

      Macrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF.

      Methods

      Blood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays.

      Results

      Airway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance.

      Conclusion

      Taken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

      Keywords

      1. Introduction

      Resident macrophages play a key role in tissue homeostasis and responses to infection and injury [
      • Okabe Y
      • Medzhitov R.
      Tissue biology perspective on macrophages.
      ]. In the airways, macrophages quickly adapt to variations in oxygen pressure, inflammation, and the presence of pathogens [
      • Schaible B
      • Schaffer K
      • Taylor CT.
      Hypoxia, innate immunity and infection in the lung.
      ]. Exposure of airway macrophages at birth and thereafter to host- and microbe-derived factors shapes long-term responses and the likelihood to mount an aberrant response to later insults [
      • Torow N
      • Marsland BJ
      • Hornef MW
      • Gollwitzer ES.
      Neonatal mucosal immunology.
      ]. In cystic fibrosis (CF), airway epithelial cells and macrophages display intrinsic defects due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In particular, mucin hyperconcentration and dehydration can alter airway macrophages [
      • Trojanek JB
      • Cobos-Correa A
      • Diemer S
      • Kormann M
      • Schubert SC
      • Zhou-Suckow Z
      • et al.
      Airway mucus obstruction triggers macrophage activation and matrix metalloproteinase 12-dependent emphysema.
      ], which may contribute to abnormal immune regulation, and subsequent recruitment and activation of neutrophils, with ensuing lung damage [
      • Alexis NE
      • Muhlebach MS
      • Peden DB
      • Noah TL
      Attenuation of host defense function of lung phagocytes in young cystic fibrosis patients.
      ,
      • Deriy LV
      • Gomez EA
      • Zhang G
      • Beacham DW
      • Hopson JA
      • Gallan AJ
      • et al.
      Disease-causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages.
      -
      • Leveque M
      • Le Trionnaire S
      • Del Porto P
      • Martin-Chouly C
      The impact of impaired macrophage functions in cystic fibrosis disease progression.
      ]. In itself, CFTR dysfunction in CF airway macrophages may result in impaired phagocytosis and pathogen clearance [
      • Di A
      • Brown ME
      • Deriy LV
      • Li C
      • Szeto FL
      • Chen Y
      • et al.
      CFTR regulates phagosome acidification in macrophages and alters bactericidal activity.
      ,
      • Bruscia EM
      • Bonfield TL.
      Cystic fibrosis lung immunity: the role of the macrophage.
      -
      • Del Porto P
      • Cifani N
      • Guarnieri S
      • Di Domenico EG
      • Mariggio MA
      • Spadaro F
      • et al.
      Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa.
      ]. While the impact of intrinsic CFTR-linked defects on CF airway macrophage responses has been well studied, little is known about their regulation by extrinsic immunomodulatory pathways.
      Programmed cell death protein-1 (PD-1) is an immune checkpoint protein that regulates cell behavior during immune responses, with best known functions related to T-cell control in cancer and viral infection [
      • Keir ME
      • Butte MJ
      • Freeman GJ
      • Sharpe AH.
      PD-1 and its ligands in tolerance and immunity.
      ]. Comparatively, the role of PD-1 in macrophages is less well described, however there is increasing evidence of its role in tumor-infiltrating myeloid cells [
      • Rudd CE.
      A new perspective in cancer immunotherapy: PD-1 on myeloid cells takes center stage in orchestrating immune checkpoint blockade.
      ]. In blood monocytes during sepsis and in tumor-associated macrophages, PD-1 expression is associated with reduced uptake of bacteria and cell debris, respectively, suggesting a role for this pathway in shaping macrophage function [
      • Gordon SR
      • Maute RL
      • Dulken BW
      • Hutter G
      • George BM
      • McCracken MN
      • et al.
      PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity.
      ,
      • Bally AP
      • Lu P
      • Tang Y
      • Austin JW
      • Scharer CD
      • Ahmed R
      • et al.
      NF-kappaB regulates PD-1 expression in macrophages.
      -
      • Huang X
      • Venet F
      • Wang YL
      • Lepape A
      • Yuan Z
      • Chen Y
      • et al.
      PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis.
      ]. Recently, PD-1 signaling was also found to mediate extrinsic control of resident macrophages by immunosuppressive leukocytes recruited in a model of airway infection [
      • Lei GS
      • Zhang C
      • Lee CH.
      Myeloid-derived suppressor cells impair alveolar macrophages through PD-1 receptor ligation during Pneumocystis pneumonia.
      ]. We previously showed that neutrophils recruited to CF airways undergo reprogramming, leading to complex functional changes [
      • Dittrich AS
      • Kuhbandner I
      • Gehrig S
      • Rickert-Zacharias V
      • Twigg M
      • Wege S
      • et al.
      Elastase activity on sputum neutrophils correlates with severity of lung disease in cystic fibrosis.
      ,
      • Forrest OA
      • Ingersoll SA
      • Preininger MK
      • Laval J
      • Limoli DH
      • Brown MR
      • et al.
      Frontline Science: pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.
      ,
      • Laval J
      • Touhami J
      • Herzenberg LA
      • Conrad C
      • Taylor N
      • Battini JL
      • et al.
      Metabolic adaptation of neutrophils in cystic fibrosis airways involves distinct shifts in nutrient transporter expression.
      ,
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      -
      • Tirouvanziam R
      • Gernez Y
      • Conrad CK
      • Moss RB
      • Schrijver I
      • Dunn CE
      • et al.
      Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways.
      ]. Among those is the acquisition of immunosuppressive functions [
      • Makam M
      • Diaz D
      • Laval J
      • Gernez Y
      • Conrad CK
      • Dunn CE
      • et al.
      Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs.
      ], including expression of the PD-1 ligand, PD-L1, observed in adult patients with CF [
      • Ingersoll SA
      • Laval J
      • Forrest OA
      • Preininger M
      • Brown MR
      • Arafat D
      • et al.
      Mature cystic fibrosis airway neutrophils suppress T cell function: evidence for a role of arginase 1 but not programmed death-ligand 1.
      ].
      The early and massive recruitment of neutrophils to the CF airways [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ] suggests a change in the poise of resident airway macrophages, which have an essential role in repairing tissue damage and preventing untimely neutrophil recruitment to sites of injury [
      • Uderhardt S
      • Martins AJ
      • Tsang JS
      • Lammermann T
      • Germain RN.
      Resident macrophages cloak tissue microlesions to prevent neutrophil-driven inflammatory damage.
      ]. Inhibitory signaling in airway macrophages may reduce their capacity for blocking neutrophil recruitment in the early stages of inflammation in CF airways [
      • Montgomery ST
      • Mall MA
      • Kicic A
      • Stick SM.
      Hypoxia and sterile inflammation in cystic fibrosis airways: mechanisms and potential therapies.
      ], while simultaneously inhibiting their ability to clear bacteria. Since PD-1 signaling has been shown to downregulate macrophage poise in multiple inflammatory diseases [
      • Gordon SR
      • Maute RL
      • Dulken BW
      • Hutter G
      • George BM
      • McCracken MN
      • et al.
      PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity.
      ,
      • Huang X
      • Venet F
      • Wang YL
      • Lepape A
      • Yuan Z
      • Chen Y
      • et al.
      PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis.
      ,
      • Lei GS
      • Zhang C
      • Lee CH.
      Myeloid-derived suppressor cells impair alveolar macrophages through PD-1 receptor ligation during Pneumocystis pneumonia.
      ], we hypothesized that expression of PD-1 on airway macrophages in young children with CF would associate with progressive airway disease, and that blocking this signaling cascade could enhance bacteria killing. Therefore, we investigated the presence of the PD-1 pathway in airway macrophages and its relationship with intrinsic and extrinsic inflammatory factors critical for the early stages of CF lung disease.

      2. Results

      2.1 CF airway macrophages express high levels of surface PD-1

      To determine the presence of the PD-1 signaling pathway in early CF airway inflammation, immune cells from blood and bronchoalveolar lavage fluid (BALF) from 45 children with CF, aged 3 to 62 months, were analyzed by flow cytometry (Fig. S1). The cohort included equal numbers of male and female subjects, with a mean age of 30 months, and 11/45 subjects were diagnosed with proinflammatory pathogen infections. Twenty subjects were homozygous for F508del, twenty had compound heterozygous mutations with F508del, and five had other mutations (Table S1). These subjects were primarily White with pancreatic insufficiency, and none were prescribed CFTR modulators at the time of sample collection. For Perth cohort subjects, demographic data on genotype, sex and birth date were obtained from clinical records. Data on ethnicity, pancreatic sufficiency status and current treatment were recorded by parent questionnaires, with “data unavailable” indicating this was not answered on the questionnaire (Table S2).
      PD-1 expression on airway macrophages was higher than on airway neutrophils and T cells, by 10- and 17-fold respectively (Fig. 1A). Clustering analysis of CF airway macrophages with the spanning tree progression analysis of density normalized events (SPADE) algorithm [
      • Qiu P.
      Toward deterministic and semiautomated SPADE analysis.
      ] revealed a distinct pattern of PD-1 expression in different airway macrophage clusters, highlighting the intrinsic diversity of this immune cell population with regards to the PD-1 pathway (Fig. 1B). Airway macrophages and T cells showed significant expression of the PD-1 ligands, PD-L1 and PD-L2 (Fig. 1C and 1D), suggesting potential for activation of PD-1 signaling. While we observed differences in expression of PD-L1 and PD-L2 between neutrophils, macrophages and T cells, these may be partially caused by outliers. Indeed, there were 5-6 outlier points for expression of PD-L1 and PD-L2 on macrophages and T cells, and there were four subjects common to these groups of outliers. However, these subjects did not differ from the rest of the cohort based on demographic or inflammatory outcomes.
      Fig. 1
      Fig. 1PD-1 and its ligands, PD-L1 and PD-L2, are expressed in CF airway leukocytes. Flow cytometry analysis of BALF leukocytes from CF children shows higher PD-1 expression on airway macrophages (M) compared to neutrophils (N), and T cells (T) (A). SPADE analysis of airway macrophage PD-1 expression in different clusters (B). PD-L1 (C) and PD-L2 (D) expression in airway macrophages (M), neutrophils (N), and T cells (T) measured by flow cytometry shown as median fluorescence intensity (MFI). Data are shown as median and interquartile range, and analyzed by the Wilcoxon rank sum test, with significance levels indicated as * for p<0.05, ** for p< 0.01, *** for p< 0.001, and **** for p<0.0001.
      Given the pattern of expression of PD-1 and its ligands on airway immune cells, we investigated their relationship with clinical variables of early CF lung disease. No significant relationships were observed between the clinical outcomes and the levels of expression of PD-L1 or PD-L2. However, in a subset of 24 children with CF, for whom flow cytometry data and date-matched CT scans were acquired, PD-1 expression on airway macrophages correlated with both the total score of structural lung damage (%Dis), ranging between 0 and 5% (Rho = 0.51, p = 0.01, Fig. 2A), and the score for bronchiectasis (%Bx), ranging from 0 and 2.1% (Rho = 0.47, p = 0.01, Fig. 2B). Furthermore, while PD-1 expression on airway macrophages did not correlate with absolute macrophage count (Rho = -0.03, p = n.s., Fig. 2C), it correlated negatively with airway macrophage percentage (Rho = -0.57, p = 0.003, Fig. 2D), and positively with airway neutrophil absolute count (Rho = 0.50, p = 0.01) and percentage (Rho = 0.51, p = 0.009). These findings suggest a potential link between neutrophilic inflammation, airway macrophage PD-1 expression, and early structural lung damage in CF.
      Fig. 2
      Fig. 2PD-1 expression on airway macrophages correlates with lung disease in children with CF. PD-1 expression on CF airway macrophages is associated with PRAGMA-CF total disease score measured by CT (%Dis) (A) and with the bronchiectasis score (%Bx) (B). PD-1 expression on airway macrophages did not correlate with airway macrophage absolute count (C), but correlated negatively with airway macrophage percentage (D). Correlations were performed using the non-parametric Spearman's rank order correlation test and are shown as coefficient strength of Spearman Rho (left panel) and significance levels of p-values.

      2.2 CFTR genotype and sex do not influence PD-1 expression in CF airway macrophages

      Next, we investigated which intrinsic or extrinsic factors could contribute to PD-1 expression in airway CF macrophages. No significant difference was observed in the level of PD-1 expression based on sex (Fig. S2A) or between patients bearing F508Del homozygous vs compound heterozygous genotypes (Fig. S2B). Likewise, while uptake of oxidized lipids and the subsequent inability to efficiently metabolize them (lipidation index) correlates with a dysfunctional state in macrophages [
      • Feng B
      • Yao PM
      • Li Y
      • Devlin CM
      • Zhang D
      • Harding HP
      • et al.
      The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages.
      ], as described in atherosclerosis [
      • Moore KJ
      • Sheedy FJ
      • Fisher EA.
      Macrophages in atherosclerosis: a dynamic balance.
      ] and several lung conditions including CF [
      • Kazachkov MY
      • Muhlebach MS
      • Livasy CA
      • Noah TL.
      Lipid-laden macrophage index and inflammation in bronchoalveolar lavage fluids in children.
      ,
      • Knauer-Fischer S
      • Ratjen F.
      Lipid-laden macrophages in bronchoalveolar lavage fluid as a marker for pulmonary aspiration.
      ], in the subset of 22 children with CF for which Oil Red O staining was performed, no relationship was found between airway macrophage PD-1 expression and the lipidation index [
      • Colombo JL
      • Hallberg TK.
      Recurrent aspiration in children: lipid-laden alveolar macrophage quantitation.
      ] (Fig. S2C).
      Since lipidation, sex, and CFTR genotypes were not related to PD-1 expression on CF airway macrophages, we measured soluble and cell-bound inflammatory mediators in BALF and combined them with clinical variables in a bootstrap forest partitioning analysis (Table S3, N = 42 patients included). The main predictors for PD-1 expression on airway macrophages were patient age, neutrophil mediators, and infection with pro-inflammatory pathogens, defined by the presence of at least one of four pro-inflammatory pathogens, namely P. aeruginosa, S. aureus, H. influenzae, and Aspergillus spp. [
      • Gangell C
      • Gard S
      • Douglas T
      • Park J
      • de Klerk N
      • Keil T
      • et al.
      Inflammatory responses to individual microorganisms in the lungs of children with cystic fibrosis.
      ], at the time of BALF collection. While these factors may be related to each other, we recently showed that the presence of neutrophils in the airways of children with CF and their active exocytosis of NE-rich granules precedes colonization by pro-inflammatory pathogens, and that the extent of NE exocytosis did not differ between infected and non-infected groups at the single cell level [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ]. However, LPS is an inducer of PD-1 expression on macrophages [
      • Bally AP
      • Lu P
      • Tang Y
      • Austin JW
      • Scharer CD
      • Ahmed R
      • et al.
      NF-kappaB regulates PD-1 expression in macrophages.
      ] and upon infection with pro-inflammatory pathogens, neutrophils are increasingly recruited to the airways, resulting in the extracellular accumulation of neutrophil-derived mediators. Therefore, to untangle the contribution of neutrophil and bacterial mediators in inducing PD-1 expression, we performed subsequent analyses discriminating between patients that presented with pro-inflammatory pathogens and those that were classified as not infected.

      2.3 Neutrophil activation modulates PD-1 expression by CF airway macrophages

      Considerable overlap was observed between infected and non-infected groups in inflammatory mediator levels, suggesting that a high state of immune activation is not exclusive to patients with airway infections (Fig. S3). We then assessed factors correlated with PD-1 expression on airway macrophages in the absence of infection (Fig. 3A). Significant correlations were observed for extracellular NE (Rho = 0.62, p = 0.02) and IL-8 (Rho = 0.48, p = 0.04), suggesting a role for neutrophil-derived soluble factors in the upregulation of PD-1 in airway macrophages.
      Fig. 3
      Fig. 3PD-1 expression on CF airway macrophages is associated with neutrophilic inflammation. PD-1 expression on CF airway macrophages correlates with neutrophil-derived mediators in young children with CF with negative airway cultures. Correlations are shown as coefficient strength of Spearman Rho (left panel) and corresponding significance levels of p-values (right panel, with grey indicating p > 0.05) (A). In vitro transmigration of monocytes into neutrophil conditioned media (illustrated in Fig. S4) show modulation of PD-1 expression in monocytes exposed to the CF-like neutrophil conditioned media (B). Data are shown as median and interquartile range, and analyzed by the Wilcoxon rank sum test, with significance levels indicated as * for p< 0.05.
      To determine whether neutrophil-released mediators could directly modulate airway monocyte/macrophage surface PD-1 expression, we exploited an in vitro transmigration model previously used to recreate the CF airway neutrophil phenotype observed in vivo [
      • Forrest OA
      • Ingersoll SA
      • Preininger MK
      • Laval J
      • Limoli DH
      • Brown MR
      • et al.
      Frontline Science: pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.
      ], which includes increased lifespan, active release of pro-inflammatory cytokines and exocytosis of granule enzymes such as NE and myeloperoxidase. Upon in vitro generation of CF airway neutrophils, media was conditioned with these cells and then subsequently applied to a blood monocyte transmigration model [
      • Ford BD
      • Moncada Giraldo D
      • Margaroli C
      • Giacalone VD
      • Brown MR
      • Peng L
      • et al.
      Functional and transcriptional adaptations of blood monocytes recruited to the cystic fibrosis airway microenvironment in vitro.
      ] (Fig. S4). Interestingly, monocytes that transmigrated into the conditioned media from CF airway-like neutrophils showed increased expression of surface PD-1 compared to both their blood counterpart and also to those monocytes that transmigrated into the transmigration control supernatant (conditioned media from neutrophils transmigrated towards the leukotriene B4 - LTB4) (Fig. 3B). Our experimental model suggests soluble mediators from neutrophils recruited to CF airways could be directly modulating airway monocyte/macrophage PD-1 expression.

      2.4 PD-1 increases uniformly on CF airway macrophages upon infection

      Since the likelihood of infection increases with age and correlates with enhanced neutrophil recruitment to CF airways [
      • Pittman JE
      • Calloway EH
      • Kiser M
      • Yeatts J
      • Davis SD
      • Drumm ML
      • et al.
      Age of Pseudomonas aeruginosa acquisition and subsequent severity of cystic fibrosis lung disease.
      ,
      • Coburn B
      • Wang PW
      • Diaz Caballero J
      • Clark ST
      • Brahma V
      • Donaldson S
      • et al.
      Lung microbiota across age and disease stage in cystic fibrosis.
      ], we next fitted a logistic regression model for infection given age, and then a multivariate linear regression model for PD-1 expression on airway macrophages adjusted for age and infection. Unsurprisingly, we found that older children were more likely to harbor infections (p = 0.038) and that age was also a significant predictor of PD-1 expression (p<0.001). However, even after adjusting for age, the effect of infection on PD-1 expression remained significant (p<0.001). These findings support the notion that while age increases the risk of infection in CF, both age and infection can independently impact PD-1 expression on airway macrophages in children with CF.
      Next, we investigated whether the increase of PD-1 expression on airway macrophages upon infection was confined to CF or if it could be applicable to age-matched controls. Therefore, we collected blood and BAL from 10 age-matched non-CF disease control children with aerodigestive diagnosis, prone to aspiration-induced inflammation, and analyzed the cells by flow cytometry. Similar to children with CF, airway macrophages showed higher PD-1 expression compared to airway neutrophils and T cells by 10- and 18-fold, respectively (Fig. S5A). Furthermore, we found that the two cohorts did not differ in their lipidation index (Fig. S5B), neutrophil percentage (Fig. S5C), or macrophage percentage (Fig. S5D). However, macrophage count was elevated in CF compared to non-CF disease control BALF, consistent with previous studies [
      • Brennan S
      • Sly PD
      • Gangell CL
      • Sturges N
      • Winfield K
      • Wikstrom M
      • et al.
      Alveolar macrophages and CC chemokines are increased in children with cystic fibrosis.
      ]. CF patients with the highest absolute macrophage counts tended to be younger (approximately 0.25-2 years) (Fig. S5E).
      The significant increase in PD-1 expression on CF airway macrophages in the presence of infection compared to non-CF aerodigestive controls (Fig. 4A) suggests differential regulation of this pathway in CF airway disease. Clustering analysis showed that PD-1 expression was upregulated uniformly when comparing infected and non-infected subgroups (Fig. 4B), suggesting that infection with pro-inflammatory pathogens does not correlate with the modulation of PD-1 expression on any particular SPADE-defined subset of airway macrophages. Interestingly, among all surface markers included in our cytometry panel, PD-1 emerged as the most significant feature in the airway macrophage population discriminating between infected and non-infected children with CF (p<0.0001, paired sample t-test). None of the other surface markers listed as significant discriminators between the two groups reproduced the uniform pattern observed with PD-1. Furthermore, differences in airway macrophage cell frequencies between the infected and not infected groups were observed (Fig. 4C). Out of the 100 clusters in the SPADE tree, 52 clusters, matching the lower PD-1 expression originally shown in Fig. 1B, were more prevalent in the non-infected group. Taken together, these results show that while certain airway macrophage subsets become more prevalent during infection, all airway macrophages upregulate surface PD-1 expression upon presence of pro-inflammatory pathogens.
      Fig. 4
      Fig. 4PD-1 expression and function in CF airway macrophages associates with airway infection. Children with CF showed higher PD-1 expression on airway macrophages upon infection compared to non-CF disease control children (N = 22 and N = 10, respectively). Data are shown as median and interquartile range, and analyzed by the Wilcoxon rank sum test, with significance levels indicated as ** for p< 0.01, *** for p<0.001 (A). SPADE analysis of CF airway macrophages shows that the difference in PD-1 expression levels measured between infected vs. non-infected CF children is due to uniform PD-1 modulation across all macrophage subsets (dark red) (B), and changes in frequency in defined cell clusters (C). Subset size reflects the number of macrophages with similar levels of marker expression. BALF leukocytes from eight young children with CF were treated ex vivo with anti-PD1 (antibody) combined with a SHP2 inhibitor (SHP099) while co-incubated with P. aeruginosa for 1 hour (D-F). Expression of PD-1 on macrophages and frequency of PD-1% macrophages was compared in samples with and without bacteria coincubation. Subject-matched data points are connected and analyzed by the Wilcoxon rank sum test (N = 5, no differences). Killing was determined by CFU counts (N = 8). Treatment effectiveness was calculated by subtracting the effect of therapy on bacteria alone and killing capacity of the cells without treatment. Subject-matched data points are connected, and analyzed by the Wilcoxon rank sum test with significance levels indicated as ** for p< 0.01.

      2.5 PD-1 blockade in CF airway cells improves bacterial clearance

      Next, we investigated whether presence of pro-inflammatory bacteria could modulate PD-1 expression on airway macrophages. To this end, we designed a short-term culture assay in which fresh BALF samples from CF children were collected (demographics in Table S3), minimally processed to remove endogenous bacteria and incubated with the pro-inflammatory pathogen P. aeruginosa. Coincubation with bacteria did not change overall level of PD-1 expression on macrophages or the frequency of cells positive for PD-1 compared to cells not exposed to bacteria in vitro (Fig. 4D-E). To determine whether surface PD-1 on airway immune cells was active and influencing cell behavior we leveraged the same short-term culture assay and assessed bacterial killing in the presence or absence of PD-1 signaling blockade using a combination of a PD-1 blocking antibody and treatment with SHP099 (inhibitor of the PD-1-associated phosphatase SHP2). In a subset of 8 primary BALF samples from children with CF with sufficient leukocyte yield to conduct this assay, bacterial killing significantly improved upon PD1 blockade (Fig. 4F), suggesting that surface PD-1 is indeed active in airway leukocytes in CF and that its activity reduces bacterial clearance.
      Finally, we investigated if PD-1 expression was dependent on CFTR function using our in vitro monocyte transmigration model (Fig. S4). Monocytes from healthy control (CFTR-competent) donors were incubated with CFTR inhibitor 172 and monocytes from CF (CFTR-defective) donors were incubated with elexacaftor/tezacaftor/ivacaftor, then transmigrated to LTB4 or CFASN with the same drugs present. Neither treatment significantly impacted expression of PD-1 or PD-L1 in either transmigration condition, though some donor variability was observed (Fig. S6). Overall, our findings are consistent with the notion that the CF airway microenvironment, rather than intrinsic differences in CFTR function in monocytes/macrophages, leads to a change in poise in part linked to PD-1 signaling. This change is concomitant with early neutrophilic inflammation and a decrease in bacterial killing capacity, opening the path for chronic CF airway disease (Fig. 5).
      Fig. 5
      Fig. 5Pathological model of PD-1 signaling in CF airway monocytes/macrophages. Increased PD-1 expression on CF monocytes/macrophages increases with age, and associates with chronic neutrophilic inflammation and hyperexocytosis and bacterial infection in the airways.

      3. Discussion

      Immune responses in CF airways depend on close interactions between the CF epithelium, which shows enhanced pro-inflammatory signaling [
      • Stolarczyk M
      • Scholte BJ.
      The EGFR-ADAM17 axis in chronic obstructive pulmonary disease and cystic fibrosis lung pathology.
      ,
      • Montgomery ST
      • Dittrich AS
      • Garratt LW
      • Turkovic L
      • Frey DL
      • Stick SM
      • et al.
      Interleukin-1 is associated with inflammation and structural lung disease in young children with cystic fibrosis.
      ], resident macrophages and recruited neutrophils, mucus, and colonizing pathogens, which evolve over the course of the disease [
      • Margaroli C
      • Tirouvanziam R.
      Neutrophil plasticity enables the development of pathological microenvironments: implications for cystic fibrosis airway disease.
      ,
      • Quinn RA
      • Adem S
      • Mills RH
      • Comstock W
      • DeRight Goldasich L
      • Humphrey G
      • et al.
      Neutrophilic proteolysis in the cystic fibrosis lung correlates with a pathogenic microbiome.
      ]. Macrophages and neutrophils in particular collaborate and modulate each other during responses [
      • Silva MT.
      When two is better than one: macrophages and neutrophils work in concert in innate immunity as complementary and cooperative partners of a myeloid phagocyte system.
      ,
      • Janardhan KS
      • Sandhu SK
      • Singh B.
      Neutrophil depletion inhibits early and late monocyte/macrophage increase in lung inflammation.
      ], by dynamically sharing released factors such as NE [
      • McGowan SE
      • Stone PJ
      • Calore JD
      • Snider GL
      • Franzblau C.
      The fate of neutrophil elastase incorporated by human alveolar macrophages.
      ], implementing bacterial clearance [
      • Soehnlein O
      • Kai-Larsen Y
      • Frithiof R
      • Sorensen OE
      • Kenne E
      • Scharffetter-Kochanek K
      • et al.
      Neutrophil primary granule proteins HBP and HNP1-3 boost bacterial phagocytosis by human and murine macrophages.
      ], and orchestrating gene expression [
      • Brook M
      • Tomlinson GH
      • Miles K
      • Smith RW
      • Rossi AG
      • Hiemstra PS
      • et al.
      Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation.
      ]. Taken together, data presented here are consistent with a model in which airway macrophages in children with CF undergo apparent exhaustion, marked by increased PD-1 expression, which associates with neutrophilic inflammation and infection with pro-inflammatory pathogens, resulting in significant lung damage. However, while at later stages of disease this phenomenon may be more widespread to the whole lung, in early CF lung disease, upregulation of PD-1 on airway macrophages likely occurs only in discrete diseased areas.
      We recently showed that presence of a pathogenic neutrophil subset in CF airways, which actively exocytoses NE-rich granules, is detectable prior to the sustained presence of pro-inflammatory pathogens, and whose prevalence is not influenced by a patient's infection status [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ]. Here, we showed that macrophage PD-1 expression was not directly modulated by presence of pro-inflammatory bacteria in vitro, but expression was elevated in children with CF who had detectable airway infection. Further, the differential expression of PD-1 observed between patients with CF and the control cohort could be due to epigenetic reprogramming induced by the local microenvironment [
      • Hey J
      • Paulsen M
      • Toth R
      • Weichenhan D
      • Butz S
      • Schatterny J
      • et al.
      Epigenetic reprogramming of airway macrophages promotes polarization and inflammation in muco-obstructive lung disease.
      ]. Macrophages have previously been proposed as potential therapeutic targets for CF airway disease [
      • Bonfield TL.
      Macrophage dysfunction in cystic fibrosis: a therapeutic target to enhance self-immunity.
      ]. However, studies addressing the impact of new CFTR modulators and correctors have yet to fully address their influence on intrinsic defects of CF airway macrophages [
      • Hisert KB
      • Schoenfelt KQ
      • Cooke G
      • Grogan B
      • Launspach JL
      • Gallagher CG
      • et al.
      Ivacaftor-induced proteomic changes suggest monocyte defects may contribute to the pathogenesis of cystic fibrosis.
      ]. In that context, there is a clear need for the development of new therapeutic agents aiming at the direct modulation of extrinsic regulatory pathways in macrophages, which may reinvigorate pathogen clearance and delay neutrophil takeover seen in the early phase of pro-inflammatory pathogen infection and lung damage in children with CF. Here, we showed preliminary evidence that PD-1 blockade in airway leukocytes improved bacterial clearance. Because PD-1 is known to play a key role in controlling macrophage responsiveness in several conditions, modulation of PD-1 signaling may be of interest in airway diseases other than CF which also feature chronic bacterial infections [
      • Gordon SR
      • Maute RL
      • Dulken BW
      • Hutter G
      • George BM
      • McCracken MN
      • et al.
      PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity.
      ,
      • Huang X
      • Venet F
      • Wang YL
      • Lepape A
      • Yuan Z
      • Chen Y
      • et al.
      PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis.
      ,
      • Lei GS
      • Zhang C
      • Lee CH.
      Myeloid-derived suppressor cells impair alveolar macrophages through PD-1 receptor ligation during Pneumocystis pneumonia.
      ].
      Further studies are needed to address downstream effects of PD-1 signaling in CF airway macrophages, although prior evidence linked PD-1 signaling to changes in autophagy [
      • Chatterjee P
      • Patsoukis N
      • Sari D
      • Boussiotis VA.
      PD-1 couples glucose starvation with autophagy and survival through AMPK-mediated phosphorylation of Ulk1.
      ], which controls pathogen clearance by macrophages [
      • Yuan K
      • Huang C
      • Fox J
      • Laturnus D
      • Carlson E
      • Zhang B
      • et al.
      Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages.
      ]. Moreover, while P. aeruginosa did not change PD-1 levels on macrophages, it may modulate macrophage responsiveness by altering expression of the PD-1 ligand PD-L1, which would increase the likelihood of PD-1 inhibitory signaling in these cells [
      • Avendano-Ortiz J
      • Llanos-Gonzalez E
      • Toledano V
      • Del Campo R
      • Cubillos-Zapata C
      • Lozano-Rodriguez R
      • et al.
      Pseudomonas aeruginosa colonization causes PD-L1 overexpression on monocytes, impairing the adaptive immune response in patients with cystic fibrosis.
      ]. While we did not observe a direct link between CFTR function and PD-1 expression on airway-conditioned monocytes in vitro, further studies are needed to determine if altered PD-1 signaling and/or intrinsic effects of CFTR deficiency in airway macrophages cause an inability to clear pathogens in CF children. While it has been previously shown that CFTR mutations can affect in vitro responses of monocyte-derived macrophages [
      • Tarique AA
      • Sly PD
      • Holt PG
      • Bosco A
      • Ware RS
      • Logan J
      • et al.
      CFTR-dependent defect in alternatively-activated macrophages in cystic fibrosis.
      ], less is known about CFTR function in resident human airway macrophages.

      3.1 Limitations of study

      In this study we consider the transition from macrophage dominance to neutrophil dominance in the lung, but we do not define specific subsets of macrophage-lineage cells, such as inflammatory monocytes, monocyte-derived macrophages, and alveolar macrophages in BAL samples. Future studies implementing new techniques such as CITE-Seq are needed to pursue deeper investigation of CF airway monocyte/macrophage subsets revealed in our initial flow cytometry and SPADE analysis, with a particular focus on changes in PD-1 expression upon infection and neutrophil recruitment. Also, while this study shows a novel immunomodulatory pathway present in CF airway disease, it does not delineate whether the enhanced PD-1 expression on airway macrophages observed in patients with detectable infection is a cause or a consequence of the progressive neutrophil takeover. Indeed, a limitation in both this and prior [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ] studies is that sensitive bacterial metagenomic analyses were not available, such that we cannot fully state that given patients were free of pro-inflammatory pathogens even if clinical microbiology cultures were negative. However, the in vitro modulation of PD-1 expression by neutrophil-derived factors did occur in the absence of pathogens or pathogen-derived molecules, suggesting a host-host interaction between CF airway neutrophils and monocytes/macrophages. Given limitations in BAL volume and cell number from pediatric subjects, we made a concerted effort to maximize use of each sample, but were not able to assess all possible targets of interest. One example is IFNα which has been reported to induce PD-1 expression in macrophages and should be assessed in future studies. Even though our in vitro data suggest little acute impact of CFTR modulator therapy on lung-recruited monocytes, it will also be interesting to assess lung macrophage PD-1 expression and poise in BAL from infants with CF as CFTR modulator therapy becomes available to toddlers and infants.

      4. Methods

      4.1 Experimental model and subject details

      4.1.1 Study visits

      Prospective study visits were conducted on 45 CF children aged 3 to 62 months of age enrolled in AREST-CF (Perth, Australia), I-BALL (Rotterdam, The Netherlands) and IMPEDE-CF (Atlanta, GA, USA) early disease surveillance programs, and 10 age-matched non-CF controls undergoing bronchoscopy for clinical indications at the Aerodigestive Clinic at Children's Healthcare of Atlanta (Atlanta, GA, USA). The study was approved by relevant Institutional Ethical Review Boards at each site (1762/EP for AREST-CF; NL49725.078.14 for I-BALL; IRB#00097352 for IMPEDE-CF).

      4.1.2 H441 cell line

      H441 cells (ATCC, Cat# HTB-174) were cultured in DMEM/F-12 media supplemented with 10% FBS (Corning), 2mM glutamine (Sigma), and 100 U/mL-0.1mg/mL penicillin/streptomycin (Sigma). 2.5 × 105 H441 were harvested at passages 2-3 and cultured at air-liquid interphase on the Alvetex scaffold (Reprocell) coated with Rat tail collagen (Sigma) as previously described [
      • Dobosh B
      • Giacalone VD
      • Margaroli C
      • Tirouvanziam R.
      Mass production of human airway-like neutrophils via transmigration in an organotypic model of human airways.
      ]. The basolateral medium DMEM/F-12 media supplemented with 2% Ultroser G (Crescent Chemical Co), 2mM glutamine (Sigma) and 100 U/mL-0.1mg/mL penicillin/streptomycin (Sigma) was replaced every two days. After 14 days the membranes were inversed to allow neutrophil loading on the basolateral side and apical migration as described below.

      4.2 Method details

      4.2.1 Human sample collection and processing

      Blood and BALF were collected from CF children (N = 45) and non-CF disease controls (N = 10). Demographic information for CF and non-CF disease control subjects is provided in Table S1. Blood was collected in K2-EDTA tubes by venipuncture, cells and plasma were separated by centrifugation and the cellular fraction was used for flow cytometry. BALF was collected under general anesthesia using sterile saline as per the standard clinical procedure previously described [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ]. BALF was then mechanically dissociated on ice in presence of 2.5 mM EDTA, cells were recovered after an 800 x g, 10-minute centrifugation at 4°C, then washed and used for downstream assays. BALF supernatant was further spun at 3,000 x g for 10 minutes at 4°C to remove debris and bacteria, and stored at -80°C until use. Differential cell counts and Oil Red O staining with lipid laden macrophage index [
      • Colombo JL
      • Hallberg TK.
      Recurrent aspiration in children: lipid-laden alveolar macrophage quantitation.
      ] were performed by the clinical pathology laboratories at the Erasmus MC/Sophia Children's Hospital and Emory University/Children's Healthcare of Atlanta. Infection status was determined by clinical microbiology using standard aerobic culture techniques at each respective institution, and classified for presence of the pro-inflammatory pathogens P. aeruginosa, S. aureus, H. influenzae, and Aspergillus spp. [
      • Gangell C
      • Gard S
      • Douglas T
      • Park J
      • de Klerk N
      • Keil T
      • et al.
      Inflammatory responses to individual microorganisms in the lungs of children with cystic fibrosis.
      ]. Due to the nature of the sample collection process, the age of the patients, and cell yield, it was not possible to perform all assays on all collected samples. A detailed tally describing the assays performed on each sample is provided in Table S2.

      4.2.2 Chest-computed tomography (CT) scans

      Patients underwent a chest CT-scan without anesthesia using a Siemens SOMATOM® Force ultra-fast scanner at the Erasmus MC/Sophia Children's Hospital or under general anesthesia at the Perth Children's Hospital. The CT-images were scored for structural lung damage using the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis (PRAGMA-CF) scoring method [
      • Rosenow T
      • Oudraad MC
      • Murray CP
      • Turkovic L
      • Kuo W
      • de Bruijne M
      • et al.
      PRAGMA-CF. A quantitative structural lung disease computed tomography outcome in young children with cystic fibrosis.
      ]. Repeatability of PRAGMA-CF score has been previously described. Disease score (%Dis) was the total lung volume proportion of bronchiectasis, mucus plugging, and bronchial wall thickening.

      4.2.3 Flow cytometry and SPADE analysis

      Multiparametric flow cytometry analysis of whole blood and BALF cells was standardized across study sites by integrating two stringent calibration steps prior and post- sample acquisition using a fluorescent bead-based method (Biolegend), as previously illustrated [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ] which provide constant and robust output from the flow cytometers, and used premixes of the antibodies listed above to stain blood and BAL cells. Samples were pre-stained for 10 minutes on ice in the dark with the Human TruStain FcX Fc blocking solution and the Zombie Aqua reagent (Biolegend). In addition, we also check for specificity of antibody staining with fluorescence-minus one controls, as previously explained [
      • Tirouvanziam R
      • Diaz D
      • Gernez Y
      • Laval J
      • Crubezy M
      • Makam M.
      An integrative approach for immune monitoring of human health and disease by advanced flow cytometry methods.
      ]. The following staining was subsequently added on ice in the dark for 30 minutes: anti-human CD3 (pan-T cells), CD45 (pan-leukocytes), CD63, PD-1, PD-L1, PD-L2, CD66b, CD33, CD41a (all from Biolegend), CD66a (Novus Biologicals) and Siglec-8 (R&D Systems). Cells were washed, fixed in Lyse/Fix Phosflow (BD Biosciences) and acquired on a FACS Fortessa or LSRII (BD Biosciences). Analysis and compensation were performed in FlowJo V9.9.5 (FlowJo, LLC). Macrophages, neutrophils, and T cells were gated from blood and airway samples as detailed in Fig. S1. While gating of blood leukocyte populations is straightforward, gating of airway leukocytes is more challenging. Our gating strategy was devised to identify all monocyte/macrophage-lineage cells but was not sufficient to identify subsets. CD45 is a pan-leukocyte marker and was used to exclude epithelial cells and debris. CD3 was used to separate T cells from myeloid cells, which were separated into monocyte/macrophage and neutrophil populations using CD33 [
      • Hoyer JD
      • Grogg KL
      • Hanson CA
      • Gamez JD
      • Dogan A.
      CD33 detection by immunohistochemistry in paraffin-embedded tissues: a new antibody shows excellent specificity and sensitivity for cells of myelomonocytic lineage.
      ]. Clustering analysis and group comparisons were performed in SPADE V4.0 [
      • Qiu P.
      Toward deterministic and semiautomated SPADE analysis.
      ] using the following parameters: arcsinh transformation cofactor (150), neighborhood size (5), local density approximation factor (1.5), maximum allowable cells in pooled downsampled data (50,000), target density with fixed number of cells remain (20,000), K-means algorithm with 100 clusters.

      4.2.4 Assays for soluble mediators in BALF

      BALF cytokines were quantified using a U-PLEX multiplex chemiluminescent ELISA assay (Meso Scale Diagnostics), per manufacturer's protocol. In vitro enzymatic activity assays for NE and MMP12 were performed using Förster resonance energy transfer probes NEmo-1 and LaRee-5 respectively (Sirius Fine Chemicals SiChem GmbH), as described [
      • Gehrig S
      • Mall MA
      • Schultz C.
      Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters.
      ].

      4.2.5 In vitro transmigration

      Blood neutrophils and monocytes were isolated from healthy donors using Polymorphprep (Accurate Chemical) and RosetteSep (STEMCELL technologies) with Ficoll (GE Healthcare) kits, respectively, per manufacturers’ protocols. Isolated blood neutrophils were resuspended in RPMI (Corning), loaded on the wells and transmigrated towards the chemoattractant control leukotriene B4 (LTB4, 100 nM, Sigma) or CF airway supernatant (CFASN, devoid of cells and bacteria), as we have detailed previously [
      • Forrest OA
      • Ingersoll SA
      • Preininger MK
      • Laval J
      • Limoli DH
      • Brown MR
      • et al.
      Frontline Science: pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.
      ]. Neutrophils transmigrated through the epithelium to either LTB4 (transmigration control) or CFASN (CF airway-like condition) were collected after 4 hours, washed three times, and cultured in fresh RPMI for 8 hours at 37°C to generate conditioned media. These conditioned media were further purified by sequential centrifugations at 800 x g for 10 minutes, followed by 3,000 x g for 10 minutes. Isolated blood monocytes were then transmigrated in turn through the epithelium into the neutrophil-derived conditioned media in new transmigration chambers for 5 hours and analyzed by flow cytometry as detailed above, with the replacement of anti-human CD33 by anti-human CD115 (Biolegend). All transmigrations were performed in an incubator with 5% CO2 at 37°C. Monocytes from CF donors were isolated using the same procedure, with subject demographics summarized in Table S4.

      4.2.6 Ex vivo bacterial killing assay

      Overnight cultures of the pro-inflammatory bacteria Pseudomonas aeruginosa (strain PAO1) were subaliquoted and grown to reach the exponential growth phase. The bacteria were then incubated in RPMI, supplemented with 10% FBS (Corning), on an end-over-end rotating wheel for 30 minutes at 37°C. Fresh BALF leukocytes were resuspended in RPMI, 10% FBS and incubated at 37°C, 5% CO2 for 15 minutes. Co-incubation of bacteria and immune cells, in presence or absence of PD-1 blockade, was performed at a multiplicity of infection of 1, in RPMI, 10% FBS, on an end-over-end rotating wheel for 1 hour at 37°C, 5% CO2. PD-1 blockade was accomplished using 1 µg/mL of Ultra-LEAF purified anti-human PD-1 blocking antibody (Biolegend) combined with 80 nM of SHP099 (Cayman Chemical), a small molecule SHP2 inhibitor [
      • Chen YN
      • LaMarche MJ
      • Chan HM
      • Fekkes P
      • Garcia-Fortanet J
      • Acker MG
      • et al.
      Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.
      ]. Total bacterial killing was assessed by gently lysing the cells in 0.1% Triton-X and plating of bacteria on an agar plate overnight at 37°C. Bacterial killing capacity was calculated using colony forming units (CFU), with the bacteria, RPMI, 10% FBS condition set as 100% survival. Treatment efficacy was calculated by subtracting the effect of PD-1 blockade on bacteria alone and the killing capacity of immune cells without treatment from the condition where bacteria, immune cells, and PD-1 blockade were all present. Demographic information for CF subjects used for the bacteria killing assays is summarized in Table S4.

      4.3 Quantification and statistical analysis

      4.3.1 Statistical analysis

      Data were compiled in Excel (Microsoft) and transferred to JMP13 (SAS Institute) and Prism v7 (GraphPad) for statistical analysis and graphing, respectively. The effect of study site on measured variables was corrected, while adjusting for age using a non-parametric empirical Bayesian method as previously described [
      • Margaroli C
      • Garratt LW
      • Horati H
      • Dittrich AS
      • Rosenow T
      • Montgomery ST
      • et al.
      Elastase exocytosis by airway neutrophils Is associated with early lung damage in children with cystic fibrosis.
      ]. Predictor screening analysis was performed using bootstrap forest partitioning in JMP13, while simple linear fitting modelling was performed in R. Correlations were calculated using Spearman's rank-order correlation, a non-parametric rank-based calculation that is relatively insensitive to outlier effect.

      Funding

      National Institutes of Health (NIH, USA) R01HL126603 (to H.H., M.R.B, B.J.S., H.M.J., L.P., and R.T.); I-BALL monitoring program in Rotterdam and research coordinators are partly funded by Dutch CF foundation (NCFS-HIT-CF); [email protected] Research Development Program Fellowship (to C.M.) as funded by the US Cystic Fibrosis Foundation (MCCART15R0); UAB Research Development Program Fellowship (to C.M.) funded by the US CFF (ROWE19R0); CFF Postdoc-to-Faculty Award to C.M. (MARGAR21F5); National Health and Medical Research Council (NHMRC; Australia) 1142505 (to L.W.G, R.T., A.K., and S.M.S.), and Peter Doherty Fellowship 1141479 (to L.W.G.); Western Australian Department of Health Merit Award (to L.W.G.); National Science Foundation (NSF, USA) CCF1552784 (to P.Q.); ISAC Marylou Ingram Scholarship (to P.Q.); the German Ministry for Education and Research (FKZ 82DZL00401 and FKZ 82DZL004A1 to M.A.M.), the German Research Foundation (SFB-TR84TP B08 to M.A.M), the Einstein Foundation Berlin (EP-2017-393 to M.A.M), the German Cystic Fibrosis Association Mukoviszidose e. V. (Project number 1605 to A.S.D.), and the Heidelberg Research Center for Molecular Medicine Career Development Fellowship (to A.S.D.). AREST CF is supported by several sources, including the NHMRC, NIH, US Cystic Fibrosis Foundation Therapeutics, and Cystic Fibrosis Australia. IMPEDE-CF is supported by the [email protected] Research Development Program Pilot Fund (to R.T. and L.G.) as funded by the US Cystic Fibrosis Foundation (MCCART15R0).

      Author Contributions

      All authors read, reviewed and agreed to submit the manuscript. Study conception: S.M.S., R.T., H.M.J.; Study implementation: C.M., H.H., L.W.G., V.D.G., C.S., L.G.; Supervision of statistical analyses: L.P; Unrestricted access to all data: H.H., L.G., R.T., C.M. H.M.J.; Critical technical and/or logistical support: A.S.D., T.R., B.S.D., H.S.L., D.L.F, M.V., G.L.S., M.R.B., C.S., H.A.W.M.T., S.R., J.D.C., P.Q., L.P, B.J.S., M.A.M., A.K., L.G.; Manuscript writing: C.M., R.T., V.D.G.; Manuscript review: H.H., L.W.G., T.R., B.J.S., J.D.C., B.J.S., M.A.M., A.K., L.G.,S.M.S, H.M.J.

      Declaration of Competing Interest

      None.

      Acknowledgements

      We thank all children and parents willing to enroll in the study cohorts in Rotterdam (The Netherlands), Perth (Australia), and Atlanta (USA). For the Erasmus Medical Center we would like to thank the Department of Cell Biology for flow cytometry core services, pediatric pulmonologists for performing bronchoscopies, research coordinators E. van der Wiel and B. Manai for patient inclusion and study management, and the Department of Pathology for providing lipidation slides and data. For the Telethon Kids Institute we would like to thank the flow cytometry core, and for Children's Healthcare of Atlanta and Emory University the pediatric flow cytometry core. We also thank Drs. D. Weiss and D. Hufnagel for assistance with the bacterial killing assay.

      Appendix. Supplementary materials

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