Macrophage dysfunction in cystic fibrosis: Nature or nurture?

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect the homeostasis of chloride flux by epithelial cells. This has deleterious consequences, especially in respiratory epithelia, where the defect results in mucus accumulation distinctive of cystic fibrosis. CFTR is, however, also expressed in phagocytic cells, like macrophages. Immune cells are highly sensitive to conditioning by their environment; thus, CFTR dysfunction in epithelia influences macrophages by affecting the lung milieu, but the mutations also appear to be directly consequential for intrinsic macrophage functions. Particular mutations can alter CFTR's folding, traffic of the protein to the membrane and function. As such, understanding the intrinsic effects of CFTR mutation requires distinguishing the secondary effects of misfolded CFTR on cell stress pathways from the primary defect of CFTR dysfunction/absence. Investigations into CFTR's role in macrophages have exploited various models, each with their own advantages and limitations. This review summarizes these methodologic approaches, discussing their physiological correspondence and highlighting key findings. The controversy surrounding CFTR‐dependent acidification is used as a case study to highlight difficulties in commensurability across model systems. Recent work in macrophage biology, including polarization and host–pathogen interaction studies, brought into the context of CFTR research, offers potential explanations for observed discrepancies between studies. Moreover, the rapid advancement of novel gene editing technologies and new macrophage model systems makes this assessment of the field's models and methodologies timely.

several CFTR mutations that can lead to CF phenotypes, which may have differential effects on macrophage function. This review seeks to answer the following questions: why is studying CFTR's role in macrophages relevant? What are the challenges in understanding the effects of CFTR mutations in macrophages? Why is modeling CF macrophage function difficult?
Studying the role of CFTR in macrophages is complicated by the specificities of Cftr gene mutations and the commensurability of model systems used. The consequences of CFTR mutations can be extrinsic or intrinsic. Extrinsic effects refer to the consequences of epithelial dysfunction on the lung milieu because this milieu dictates the differentiation and modality of the immune cells including macrophages.
Intrinsic effects include the loss of CFTR function (primary effect), as well as secondary consequences, like increased cell stress due to CFTR misfolding and intracellular protein aggregation. Parsing these 3 effect types, extrinsic, primary, and secondary, is further complicated by the inherent limitations of how CF can be modeled. The multiplicity of models and methodologies used to study CF macrophages exposes the inherent difficulty of working with this heterogenous cell type.
This review expands on a 2016 review by Bruscia and Bonfield,9 where the authors delineate intrinsic and acquired factors accounting for macrophage alteration in CF. We argue that "intrinsic" should be parsed in terms of the effects of CFTR's absence or dysfunction (primary), but also in terms of the influence of CFTR protein misfolding on cellular stress pathways (secondary). With this framework in mind, we critically assess methodologies used for evaluating CFTR's role in macrophages, with a focus on the models themselves.

The CF lung milieu conditions macrophages
The perturbed equilibrium of the pulmonary environment in PWCF predisposes the lung toward proinflammatory cytokine production and phagocyte recruitment. 10,11 The lung-resident alveolar and interstitial macrophages are yolk sac derived, and seeded during embryonic hematopoiesis. 12 Alveolar macrophages act as sentinels of the lung, maintaining homeostasis and, upon activation, secreting proinflammatory cytokines to recruit neutrophils, whereas interstitial macrophages play a regulatory role in lung tissue and contribute to the adaptive immune response. 13 Pathogens encounter alveolar macrophages as part of the first-line of defense in the airway lumen but in CF, mucus accumulation compromises host-pathogen interaction. 14 In CF, infiltrating neutrophils release reactive oxygen species (ROS) and elastase, and continued response without resolution of inflammation leads to airway epithelium damage and promotes fibrosis (reviewed in ref. 10).
This tissue damage, with compromised mucociliary clearance, exacerbates the risk of infection. Elastases also cleave receptors necessary for host-pathogen recognition, making phagocytosis less efficient (reviewed in ref. 9).
Pseudomonas aeruginosa and Burkholderia cepacia complex are among several opportunistic bacterial pathogens often encountered

Key Questions
What are the extrinsic and intrinsic (direct and indirect) con-

CFTR is expressed in macrophages
The influence of CFTR mutations in macrophages requires elucidation because CF is characterized by opportunistic bacterial infections, and it is likely that immune cell dysfunction contributes to the chronicity of infection. CFTR is expressed in human macrophages and neutrophils as demonstrated by the isolation of Cftr mRNA as early as 1991 6 ; hence, mutated CFTR in these cells may contribute to immunodysregulated phenotypes. CFTR is a large, glycosylated transmembrane protein, which can complicate efficient detection by immunoblotting. However, due in part to the multitude of available antibodies (including mutation-and phospho-sensitive ones 20,21 ), CFTR has been detected in macrophages both at the surface and intracellularly by immunoblotting, 22 flow cytometry, 23 and immunofluorescence microscopy. 24 Human peripheral blood MDMs and monocytes express CFTR, albeit with variation in abundance between blood donors. 5,25,26 CFTR expression is reduced on the surface of monocytes from PWCF. 26 Investigating CFTR's contributions to macrophage function has become more practicable with advances in gene editing tools 27 and macrophage models. 28 CFTR's chloride efflux functionality has been investigated in CF and non-CF macrophages from humans and other species using whole-cell patch-clamp assays and fluorescence-based chloride ion flux measurement. 26,29,30 Confirming CFTR expression in tissue-resident alveolar macrophages has been challenging. Di

The CFTR mutations in lung epithelia influence phagocytes
CFTR mutations in epithelial cells can extrinsically influence macrophage function by contributing to lung dysfunction because macrophages' inflammatory profile depends on cues from the lung environment (for a detailed review, see ref. 9). The CF lung milieu differs starkly from the healthy lung, with altered inflammatory cytokines, neutrophil effectors, and hydrostatic pressure. 33 CF modifier genes also influence the lung environment. 34 Mucopurulent material harvested from CF airways is sufficient to recapitulate the CF inflammatory phenotype in non-CF alveolar macrophages. 31 However, both pulmonary macrophages and MDMs with the CFTR mutation have an exaggerated cytokine response to bacterial lipopolysaccharide, 18 and gene expression profiles are altered in monocytes and MDMs from PWCF. 25,35 Since MDMs are not exposed to the airways, pulmonary environmental conditioning is not the only factor in inflammation. Systemic cytokine levels are altered in PWCF, which could influence MDM phenotype. 36 The cytokine profiles of the CF lung are conducive to both proinflammatory and reparatory types of polarization. 9 Both proinflammatory "M1"-and reparatory "M2"-polarized alveolar macrophages increased in ΔF508-CFTR mice, complicating the immune response phenotype. 37 The evolving understanding of macrophage activation from "M1"/"M2" polarization to a spectrum model of intermediate phenotypes 38 could inform interpretations of previous work, spotlighting the influence of CF environmental cues in macrophage polarization.

Pleiotropic dysfunction of CFTR mutations in macrophages
Mutations in CFTR have consequences for the folding, trafficking, and degradation of the protein. 39 Over 2000 mutations in the Cftr gene have been recorded, and these are classified according to their effects: absence or reduction of the protein, defective anion transport, or membrane localization (reviewed in ref. 40). Accordingly, parsing the contributions of misfolding or CFTR dysfunction to pathology is complicated.
The most common Cftr gene mutation (class II) observed in PWCF results in the loss of a phenylalanine residue at position 508 (ΔF508), and occurs in over two-thirds of PWCF worldwide. 3 Misfolded CFTR is targeted for endoplasmic reticulum (ER)-associated degradation.
Although most of ΔF508-CFTR is degraded in this way, misfolded CFTR trafficked to the plasma membrane can be removed by a peripheral protein quality control system. 41 This means that the ΔF508 mutation has primary consequences, affecting CFTR's chloride channel activity, but also secondary consequences for endocytic recycling, ER stress, and the unfolded protein response in cells, as misfolded CFTR is targeted for ubiquitination and proteasome degradation. 39,42-44 A recent study using peripheral blood monocytes from PWCF suggested that mutated CFTR did not inherently contribute to inflammasome activation in these cells, 45 but rather that inflammasome activation was a secondary consequence of cell stress induced by the burden of processing misfolded protein.
CFTR mutation in phagocytes is associated with alterations in cytokine profiles, 18 bactericidal activity, 15 lysosome maturation, 46 adhesion, 47 and dysfunction of other ion channels. 40 CFTR inh -172 treatment of "M1"-and "M2"-polarized non-CF human MDMs decreased ability to phagocytose Escherichia coli bioparticles, recapitulating the phenotypes observed in CF MDMs. 35 MDMs isolated from PWCF are less effective at killing intracellular P. aeruginosa than non-CF macrophages, 5 but CFTR deficiency does not compromise ROS-mediated killing. 48 These findings suggest that whatever leads to compromised bacterial killing in CF macrophages is independent from the oxidative burst. However, PWCF have defective Ca 2+ -dependent PKC activation of NADPH oxidase in response to Burkholderia cenocepacia and compromised bacterial killing. 49 Differences in the way bacterial species interface with macrophages may account for these discrepancies.
Even at potentially low levels, CFTR (dys)function seems to be physiologically relevant for macrophage defense activities, albeit by undefined mechanisms. Defective CFTR appears to enhance bacterial survival in the macrophage phagolysosome, as demonstrated in CFTR-defective alveolar macrophages infected with P. aeruginosa. 8 Burkholderia delay phagosome maturation in RAW 264.7 cells (a murine macrophage line): this effect was exaggerated with the CFTR inhibitor, suggesting that CF macrophages may have compounded difficulties clearing the intracellular infection. 46 This may be related to compromised autophagy because defective autophagy has been observed in ΔF508-CFTR epithelia, 50,51 and also in MDMs from PWCF. 15 Autophagy is cytoprotective, and CFTR dysfunction led to protein aggregation in these epithelial cells. Functional autophagy is necessary for the clearance of B. cenocepacia in human CF MDMs, 52 as inducing autophagy by rapamycin enhanced bacterial clearance in CF macrophage models. 17 Although CFTR-containing protein aggregates have not been directly shown in macrophages, Abdulrahman et al. 53 have observed that Beclin 1, a critical component of the early stages of autophagosome formation sequestered in mutant CFTR aggresomes, forms aggregates in ΔF508-CFTR murine macrophages.
Together, these findings cumulatively suggest that a secondary effect of CFTR mutation could be dysfunctional autophagy and the creation of a bacterial niche in CF macrophages. 54 Additionally, the cophenomena observed in immune cells with dysfunctional CFTR include up-regulated proinflammatory cytokine production, altered TLR4 activity, and elevated MMP12 activity, suggesting CFTR's interactions with other proteins could augment inflammation. 40 Chloride ion is an important signaling effector in cells (reviewed in ref. 55). Thus, altered chloride ion flux due to CFTR dysfunction has implications for signaling pathways in addition to contributing to airway surface liquid dehydration. Intracellular chloride levels influence Ca 2+ signaling 56 and can promote inflammation in airway epithelium. 57 CFTR inhibition enhances Ca 2+ efflux in macrophages. 30 Treatment with CFTR inh -172 blocked forskolin-induced Cl − efflux in healthy murine macrophages, but neither forskolin nor CFTR inh -172 treatment influenced CF macrophages' Cl − efflux, 58 suggesting CFTR is a functional ion channel in non-CF macrophages.

Mouse macrophages
Rosen et al. 64  The first mouse models were generated from the C57BL/6 strain in 1992, and initial work was epithelium focused. Dorin et al. 66,67 knocked out exon 10 of the Cftr gene, creating mice with low, but not absent, expression of CFTR, due to alternative splicing. Alveolar macrophages from these Cftr MHH mice have higher levels of ceramide. 24 Snouwaert et al. 68 generated a mouse model with a disrupted Cftr gene by adding a premature stop codon in exon 10 after Ser489 (S489X). Alveolar macrophages from S489X-CFTR mice have been isolated from bronchoalveolar lavage (BAL) and macrophages have been derived from bone marrow (differentiated using M-CSF). 18 TLR4 internalization is abnormal in murine BM-derived S489X-CFTR macrophages and the rate of protein degradation in lysosomes is also slower. 58 The ΔF508-CFTR mouse (129/Ola X FVB/N background) reproduces the phenylalanine deletion observed in human CF. 69 Levels of alveolar macrophages and CCL2 were elevated in these mice, with isolated cells showing enhanced LPS-induced proinflammatory mediator production. 37 Moreover, monocytes from these mice exhibited compromised adhesion to ICAM-1, with reduced trafficking in vivo. 47 72 Interestingly, genetic complementation of Cftr in the airway epithelium was sufficient to correct inflammatory abnormalities in mice with the G551D-Cftr mutation. 73 This finding suggests that the effects of epithelial dysfunction induced by this mutation were more consequential for immune cells than intrinsic Cftr mutation, at least in the mouse model.

Human macrophages
Human alveolar macrophages can be obtained through BAL, or peripheral blood monocytes can be induced to differentiate into macrophages through treatment with M-CSF or GM-CSF. 74 The genetic heterogeneity of CF and non-CF controls, as well as differences in ontogeny, may account for discrepancies in cytokine profiles reported for CF and non-CF macrophages. 65

Cell line-based models as tools for recapitulating CF phenotype
CF cell lines were initially developed mostly from epithelial cells but also in fibroblasts. 81 Mechanistic work regarding folding, trafficking, and stability of CFTR and its mutants has made use of heterologous expression in Hela and Cos7 models, in addition to human bronchial epithelial cell lines. 41 88 Concerns about CFTR inh -172 ′ s off-target effects notwithstanding, many of these studies support the theory that intrinsic CFTR defects have consequences for leukocyte function.
Moreover, simply inhibiting CFTR does not holistically recapitulate the ΔF508-CFTR phenotype, as exemplified in a macrophage Listeria monocytogenes infection model. 89 In epithelial cells, CFTR also acts as a signaling hub through its interaction with numerous kinases and adaptor proteins (reviewed in ref. 90). This regulatory network includes the actin cytoskeleton, which is known to be dysregulated in cells with mutated CFTR (reviewed in ref. 91). CFTR may similarly contribute to signaling coordination in immune cells. For this reason, the mutated CFTR phenotype cannot be simply recapitulated by inhibiting CFTR's channel function.
THP-1 treated with CFTR inh -172 has been used to represent the human CF phenotype. 46

The contested role of CFTR in phagosome acidification: a case study to highlight interdisciplinary contribution
CFTR's contribution to lysosomal acidification through chloride channel activity has been controversial. There have been several contradictory studies in both macrophage and epithelial models. 8,59,88,[94][95][96][97] Experiments have used human and murine cells; epithelia and leukocytes; primary cells differentiated in vitro and cell lines (along with stark differences in measurement protocols and fluorescent dyes), making meaningful commensurability challenging, as many of the authors acknowledge. The methodology and findings of these studies are reviewed elsewhere 95,98 with the authors concluding that lysosome acidification appears to be CFTR independent. Quantification of acidification was based on ratiometric imaging using pH-sensitive and pH-insensitive dyes. Although the chloride ion flux data have been interpreted to support a role for ClC7 (a Cl − /H + antiporter) in reacidification, 99 by and large the consensus from these studies is that cation counterion flux is key to lysosome acidification, and CFTR does not play a direct role. This is also supported by a more recent study using surface-enhanced Raman spectroscopy-based nanosensors showing that phagosomal acidification was the same for CF and non-CF MDMs, 100  The hypothetic mechanism was differential activity of ceramide sphingomyelinase at different pH values, but whether CFTR directly contributed was not established. Setting aside the question of bacterial effectors, differential phagolysosomal acidification occurs in other contexts. Canton et al. 105 have shown that there are differences in acidification kinetics in differentially polarized human monocyte-derived macrophages, using a protocol based on ratiometric imaging of FITC fluorescence (Since FITC's pK a of 6.3 limits its dynamic range, these data are not readily commensurable with the Oregon Green-based phagolysosomal pH data in CFTR-deficient cells.). These data suggest that "M1-" and "M2"-polarized macrophages have different phagosome maturation (and acidification), with "M1" phagosomes being near neutral, whereas "M2"'s rapidly dropped to below pH 5. The authors hypothesize that proton consumption through ROS generation contributes to more alkaline conditions in "M1", along with "M1"'s delayed fusion with  37 Tarique et al. 35 have shown that dysfunctional CFTR affects cytokine responsiveness in "M2" but not "M1" polarization in human MDMs, but how polarization is coordinated in the infected CF lung requires further elucidation. CFTR activity is compromised in acidic environments, 59 so if CFTR influences acidification in any way, its role is likely to be indirect.

CONCLUDING REMARKS
CFTR mutations can extrinsically, indirectly or directly, influence alveolar macrophage function but there is no ideal model system for parsing these consequences. The consequences are further complicated by the type of macrophage (tissue or monocyte-derived, mouse or human) and its activation state. Furthermore, live engulfed bacteria can affect phagocytosis, which has implications for studies assessing the influence of CFTR mutation on phagosome dynamics. This complexity could partially account for observed discrepancies in findings. We have also highlighted how differences in models and methods could account for differences between studies. Contextualizing these studies within more recent work on the in-kind contribution.