The Impact of Highly Effective Modulator Therapy on Cystic Fibrosis Microbiology and Inflammation

INTRODUCTION: CYSTIC FIBROSIS AND CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MODULATORS

Cystic fibrosis (CF), a multiorgan, life-limiting, autosomal-recessive genetic disease, develops when individuals possess disease-causing mutations in both copies of the gene encoding the CF transmembrane conductance regulator (CFTR).¹ CFTR functions as an anion channel,^2,3 and insufficient CFTR activity leads to decreased cellular transport of chloride and bicarbonate (and likely other anions), abnormal composition of secreted luminal fluids and other cellular impairments, and subsequent organ dysfunction.^4–6 primary morbidity and mortality in CF result from a vicious cycle of impaired mucociliary clearance and chronic airway infection and inflammation, which causes progressive lung function decline and respiratory failure.¹ The prognosis for people with CF (PwCF) has changed markedly over the past several decades, largely because of developments in understanding of the molecular mechanisms that underlie CF.⁷

The biggest advance in the past decade was the identification of CFTR modulators, compounds that target the underlying molecular defect causing disease by increasing the abundance of functional CFTR protein in the cell.⁶ The thousands of disease-causing mutations in the CFTR gene fall into 5 categories: (1) null mutations, (2) folding mutations, (3) gating mutations, (4) conductance mutations, and (5) insufficient protein production.^6,8 The first CFTR modulator, ivacaftor, was initially approved to treat G551D mutations, a class 3 mutation in which the CFTR channel folds and localizes correctly in the cell but does not open appropriately. Ivacaftor potentiates anion flux by increasing the open state of the CFTR channel.⁹ In the approximately 5% of PwCF with G551D mutations, ivacaftor produces significant increases in lung function and weight gain and decreases in pulmonary exacerbation frequency.^10,11 Ivacaftor also markedly reduces sweat chloride,¹⁰ the gold standard diagnostic test for CF, and the most direct measurement of CFTR anion transport activity.¹² Ivacaftor-induced changes thus became the benchmark for which subsequent modulators would be judged as highly effective CFTR modulator therapy (HEMT).⁶

Development then focused on discovery of modulators for the most common CFTR mutation, delta F508 (F508del). The F508del mutation is present in at least 1 copy in approximately 90% of PwCF, and approximately 50% are homozygous for this mutation.¹³ Identifying molecules to correct the F508del mutation proved more challenging, as this mutation causes misfolding of the CFTR protein in the endoplasmic reticulum (ER), with subsequent destruction of most of the protein before it can reach the target location in the cell.¹⁴ Initial combinations of modulators (ie, lumacaftor/ivacaftor and tezacaftor/ivacaftor) demonstrated efficacy in restoring CFTR activity in vitro^15–17 but induced only modest clinical and symptomatic changes in most PwCF^18–20 (and thus did not qualify as HEMT). The combination of 3 compounds, elexacaftor/tezacaftor/ivacaftor (ETI), was a breakthrough for enhancing CFTR channel activity in vivo in people with either 1 or 2 F508del mutations,^21,22 and ETI is now also considered HEMT.⁶

HEMT has been life changing for many PwCF, leading to significant decreases in respiratory symptoms and pulmonary exacerbations, and improvements in lung function and quality of life.^{10,21,23–26} Although the dramatic changes in respiratory symptoms suggest that HEMT is altering the CF lung environment, the exact mechanisms by which CFTR modulators alter the CF airway are less clear. HEMT has been shown to increase mucociliary clearance.²⁷ Decreases in sweat chloride caused by HEMT correlate with increases in airway surface liquid (ASL) pH.²⁸ These findings are consistent with HEMT restoring chloride and bicarbonate secretion by epithelial cells in the airway, thus removing one major contributor to progressive airway damage in PwCF (Fig. 1A). However, the influence of HEMT on the complex interactions between ASL composition, mucus obstruction, infection, and inflammation is only beginning to be elucidated.

Fig. 1.

Pulmonary damage in CF is associated with abnormal anion secretion, inflammation, and infection. (A) In contrast with healthy individuals, the CF lung exhibits many physiologic dysregulations, including abnormal secretion of ions and impaired mucociliary clearance, nonresolving inflammation, airway infection, and progressive bronchiectasis. These processes amplify each other and contribute to the long-term respiratory morbidity. (B) Impact of CFTR dysfunction (eg, F508del) on the many networks that promote airway injury. Lack of CFTR function in epithelial cells compromises airway anion balance favoring accumulation of an abnormal mucus layer that traps inhaled or aspirated bacteria. Certain CF pathogens with specific genetic and metabolic properties outcompete other members of the airway microbiome in the CF lung, which is detrimental for lung homeostasis. CFTR function deficiency in both epithelial and myeloid cells stimulates release of inflammatory mediators, which contribute to airway damage, release of damage-associated molecular patterns (DAMPs), and infection. These processes culminate in progressive pulmonary function decline and clinical deterioration.

This article reviews the data regarding how HEMT impacts CF airway infection and inflammation, and describes the evolving understanding of the molecular and cellular mechanism by which HEMT induces these changes.

PULMONARY DAMAGE IN CF IS DUE TO INTERACTIONS BETWEEN AIRWAY INFLAMMATION, INFECTION, ABNORMAL ANION SECRETION, AND IMPAIRED MUCOCILIARY CLEARANCE

At baseline health, PwCF experience nonresolving low-grade inflammation in their airways, a process that is initiated by impaired anion secretion and impaired mucociliary clearance and then stoked by chronic airway infections (Fig. 1B). The intricate relationship between these processes becomes more complex as patients age and lung damage accumulates.

Before addressing how HEMT changes CF airway pathophysiology, one must first delineate the intersections of infection, inflammation, impaired ion transport, and impaired mucociliary clearance in the CF lung.

Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction and Impaired Mucociliary Clearance Induces Inflammation Independent of Infection

Initial models of CF airway inflammation postulated that impaired mucociliary clearance led to bacterial airway infection, which stimulated the inflammatory response. Recent studies in human infants and newer animal models have suggested that lack of CFTR activity produces abnormal inflammation even in the absence of airway infection.²⁹ In young children with CF, levels of bronchoalveolar lavage (BAL) fluid inflammatory markers (eg, neutrophils, interleukin [IL]-8, extracellular DNA) correlate highly with the concentration of BAL mucins. These findings were observed in children without culture or molecular evidence of infection, and without radiologic (ie, computed tomography [CT] scan) evidence of airway damage, suggesting that mucous abnormalities and inflammation precede and may incite airway structural damage and infection.³⁰ Animal models also indicate that CF airway inflammation arises independent of infection.³¹ When CF ferrets and pigs are administered sufficient antibiotics from birth to prevent airway infection, these animals still develop airway structural damage, mucus impaction, and neutrophilic airway inflammation.^32,33 These studies suggest that lack of CFTR activity on lung cells and resulting abnormalities in airway mucus and electrolytes promote inflammatory responses in the absence of infection.

Several mechanisms could account for these observations. Mucus obstruction in the airway and the associated hypoxia cause cellular stress, necrosis, and release of damage-associated molecular patterns (DAMPs), which trigger airway epithelial cell inflammatory responses^34,35 (see Fig. 1B). Abnormal composition of ASL has also been linked to aberrant immune cell responses. Scnn1b-Tg mice, which overexpress the epithelial sodium channel ENaC and develop dehydrated ASL, develop increased numbers of IL-17 secreting T cells, similar to cells seen in airways of PwCF; however, there is no increase in numbers of these cells in Cftr^−/− mice, which do not develop spontaneous lung disease.³⁶ Additionally, the structural cells of the airway and lung vasculature may promote inflammation when they lack sufficient CFTR activity. Endothelial cells lacking functional CFTR express IL-8 constitutively,³⁷ and epithelial cells lacking functional CFTR demonstrate enhanced and prolonged production of inflammatory cytokines IL-6 and IL-8 at baseline and after stimulation.^38–40 CF has thus been described as a disease of mucosal immune dysregulation.⁴¹

Chronic Infections Induce an Inflammatory Response

Bacterial airway infection occurs as early as infancy in CF, and CF airway microbiota measured by culture-independent analyses of BAL samples diverge from those of children with other airway diseases within the first few years of life.^42,43 Patterns in age-related pathogen prevalence in CF are well known and include predominance of Staphylococcus aureus and Haemophilus influenzae in early childhood, followed by increasing prevalence rates of CF pathogens (eg, Pseudomonas aeruginosa, methicillin-resistant S aureus, nontuberculous mycobacteria, Achromobacter, and Burkholderia) through adolescence and early adulthood.¹³ Chronic airway infection with CF pathogens is associated with adverse clinical outcomes, including increased pulmonary exacerbations and accelerated lung function decline.^44–46 The relationship between airway infection and inflammation in CF begins in the first years of life, with obligate and facultative anaerobes detectable in BAL fluid and associated with increases in total bacterial load and inflammation. Subsequent infection with CF pathogens in early childhood is associated with further increases in inflammation.⁴⁷ The relative abundance of CF pathogens then increases throughout adolescence and adulthood in parallel with decreases in bacterial community diversity, progressive bronchiectasis, declining lung function, and increasing inflammation.^48–50 In this context, inflammation is largely a response to airway damage and chronic infections. In addition, studies that evaluate inflammatory cells and mediators before and following antibiotic regimens highlight how airway bacterial burden and composition strongly influence inflammatory responses in the CF airway.^51–55

Immune Dysfunction as a Primary Consequence of Impaired Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction on Immune Cells Exaggerates Inflammation

Although CFTR expression by epithelial cells accounts for much of the pathology manifest in PwCF, CFTR is also expressed in immune cells.⁵⁶ Immune abnormalities observed in cells recovered from PwCF (compared with healthy donor cells) may be caused by lack of CFTR function on the immune cells or secondary effects of immune cells maturing within the unique airway and circulatory milieu generated by CF.^57–61 Studies in the past 2 decades, largely using animal models and in vitro cell systems that are free of the confounding presence of secondary effects of CF disease, have revealed how lack of functional CFTR alters responses of immune cells,^56,62 including neutrophils,^63,64 macrophages,⁶⁵ T cells,^36,66 B cells,^67,68 dendritic cells,⁶⁹ and platelets.⁷⁰ In CF animal models of airway infection or acute lung injury, inflammation is generally exaggerated compared with wild type animals; thus, restoration of CFTR activity by HEMT could potentially eliminate at least 1 contributor to deleterious inflammation.^32,71–73

Multiple mechanisms have been proposed for how CFTR molecules participate in intracellular processes that regulate immune functions, including the contribution of CFTR ion transport to the phagolysosomal milieu,^74,75 CFTR’s role as a scaffold to promote interactions of other proteins at the membrane,^76–78 and the requirement of CFTR for normal cellular responses to production of potentially toxic reactive oxygen species (ROS).⁷⁹ A more recent body of work demonstrates that CFTR dysfunction induces proinflammatory cellular metabolism.^80–82 These bioenergetic alterations are key for tissue homeostasis, as the same pathways that regulate ATP synthesis control the release of inflammatory mediators.^83,84 These abnormalities in cellular metabolism may be most critical in myeloid cells, as more than 95% of cells in the lumen of the CF airway are neutrophils and macrophages.^85,86 Resting myeloid cells, such as lung-resident macrophages, generate energy through oxidative phosphorylation (OXPHOS), a process that occurs in mitochondria (Fig. 2A). However, upon sensing of inflammatory stimuli such as bacterial ligands, OXPHOS is impaired, and, instead of generating energy, mitochondria become major sources of ROS.⁸⁷ If not adequately regulated, excess ROS can damage the airway mucosa. Recent studies have shown that CF macrophages exhibit mitochondrial dysfunction and excessive ROS production, even before infection.^88,89 This detrimental ROS accumulates in the CF lung and promotes protein aggregation and airway damage, contributing to long-term respiratory disease. In CF cells harboring class 2 CFTR mutations (protein processing mutations), ROS release is facilitated by reduced levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which is a major metabolic checkpoint that requires CFTR to limit mitochondrial oxidative metabolism⁹⁰ (Fig. 2B). CFTR and PTEN form a metabolo-regulatory complex at the cell membrane, which is compromised in CF myeloid cells. The excessive ROS release by CF cells is worsened by infection, as P aeruginosa, a major CF pathogen, further reduces the function of PTEN in the cell.^41,88,90,91 Thus, pro-oxidant metabolic dysfunction in CF myeloid cells contributes to pulmonary injury.

Fig. 2.

CF myeloid cells exhibit proinflammatory metabolic dysregulation. (A) In healthy myeloid cells, WT CFTR interacts with many proteins at the cell membrane, including PTEN. PTEN not only restricts ROS generation, but also promotes OXPHOS and energy synthesis by mitochondria. Glycolysis is low, limiting the production of inflammatory mediators that rely on carbohydrate breakdown. (B) Myeloid cells harboring type II CFTR mutations accumulate CFTR in the endoplasmic reticulum (ER). This produces ER stress, promoting glycolysis and inflammatory signaling. Lack of surface-attached CFTR also compromises the ability of CF cells to regulate OXPHOS, prompting ROS release. One factor contributing to this OXPHOS dysregulation is reduced interaction of CFTR with PTEN. CFTR-PTEN complex dysfunction favors secretion of succinate and itaconate, which stimulate synthesis of extracellular polysaccharides (EPSs) and biofilm by S aureus and. P aeruginosa. (C) Myeloid cells harboring type III CFTR mutations secrete potassium, which activates inflammatory pathways that rely on glycolysis, such as the inflammasome. As a consequence of increased glycolysis, mitochondrial OXPHOS is jeopardized, contributing to ROS release and inflammatory damage.

Aerobic glycolysis (the Warburg effect) becomes the major mode of energy production in macrophages stimulated by bacteria, in which OXPHOS is impaired.⁸⁴ This metabolic pathway also contributes to inflammation.^83,92 Glycolysis promotes the synthesis of many inflammatory cytokines, including IL-1β and IL-6. These cytokines activate phagocytes to fight infection and can aggravate tissue damage if not adequately regulated.⁴¹ Both CF macrophages and neutrophils exhibit a distinctive glycolytic signature that predisposes them to oversecrete inflammatory cytokines. In myeloid cells harboring class II CFTR mutations, this enhanced carbohydrate break-down is facilitated by reduced function of the CFTR-PTEN complex. In response to different CF pathogens like P aeruginosa, Burkholderia cenocepacia, and S aureus, phagocytes harboring CFTR alterations that compromise PTEN function produce exaggerated levels of IL-1β, IL-6, and many oxidants that jeopardize pulmonary tissue integrity.^65,90,93,94

Enhanced glycolysis is not exclusively associated to class 2 CFTR mutations, as cells exhibiting class 3 CFTR mutations (gating mutations) also trigger inflammatory pathways that require glucose catabolism to function. In cells harboring class 3 CFTR mutations, lack of chloride secretion promotes extracellular potassium (K⁺) accumulation.⁹⁵ This K⁺ activates the inflammasome, which is a major proinflammatory cytoplasmic complex that synthetizes IL-1β in response to glycolysis^6,9,83,96 (Fig. 2C). Additionally, the presence of class 2 CFTR mutations (whether 1 or 2 copies) can stimulate cellular oxidative metabolism independent of PTEN and extracellular K⁺ accumulation (as illustrated in Fig. 2B for class 2 CFTR mutations). Accumulation of dysfunctional CFTR in the ER, as seen in cells harboring either F508del/F508del or G551D/F508del mutations, activates cell stress and the unfolded protein response (UPR).^96,97 In CF phagocytes, UPR triggers ROS generation, augmented glycolysis, and also synthesis of inflammatory cytokines that can damage the local mucosa, like IL-6 and tumor necrosis factor alpha (TNFα).^96,98 Thus, glycolytic signaling enhanced by CFTR dysfunction also worsens CF lung inflammatory disease.

Immune Dysfunction Secondary to Cystic Fibrosis Chronic Disease Influences Inflammation

Many immune cells involved in CF airway disease are cells recruited from the circulation.^86,99–102 In the blood stream, and while migrating into lung tissue, these cells are exposed to circulating products of chronic infection and inflammation and other sequelae of CF (eg, protein calorie malnutrition, abnormal glucose control, or medications). These factors influence immune cell responses and may counteract the abnormalities caused by primary lack of CFTR function on these cells. For example, whereas macrophages lacking CFTR activity (either from mice, or matured in vitro from human blood monocytes) exhibit exaggerated lipopolysaccharide (LPS) responses,^73,103 monocytes studied ex vivo from PwCF exhibit an LPS-tolerant phenotype with decreased LPS responses.^104,105 Transcriptional profiling of peripheral blood mononuclear cells (PBMCs) from PwCF compared with healthy controls show a general downregulation of inflammatory genes, and this phenotype could be conferred on healthy donor cells by exposing them to CF plasma.¹⁰⁶ Functionally, immune cells from whole blood of healthy controls mount stronger Toll-like receptor (TLR)-mediated innate immune inflammatory responses (TMIIR) than cells from PwCF, and the PwCF that demonstrated the most robust TMIIR also demonstrated better lung function.⁵⁸

One hypothesis for why circulating immune cells may have blunted inflammatory responses in PwCF is that chronic infection in the airways results in a low-level leak of inflammatory mediators and bacterial products, including LPS, into the circulation.¹⁰⁷ Continuous exposure to low levels of inflammatory stimuli can cause immune cells to develop a tolerant phenotype.¹⁰⁸ Thus, PBMCs from PwCF with milder lung disease (ie, people who presumably are not leaking tolerogenic amounts of LPS and other inflammatory stimuli into the blood) produce more robust inflammatory responses to subsequent stimulation with bacterial ligands.⁵⁸ Complicating this hypothesis is the finding that levels of circulating markers of endothelial function in CF plasma are skewed to suggest more intact endothelial barrier function in PwCF compared with healthy controls.¹⁰⁹ However, this skewed response might also be associated with elevated endothelial damage, probably by an unbalanced inflammatory response, forcing the cells to oversynthetize these markers to stimulate tissue repair. Further studies are needed to understand the mechanisms of secondary effects of CFTR deficiency on immune cells.

CHANGES IN INFLAMMATION AND INFECTION AFTER INITIATION OF HIGHLY EFFECTIVE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MODULATOR THERAPY

Three types of studies have shaped understanding of how HEMT alters cellular responses, inflammation, and composition of airway microbiome in PwCF: in vitro studies of cells treated with HEMT, CF animal models, and human studies of response to HEMT (Table 1).

Table 1.

Effects of highly effective cystic fibrosis transmembrane conductance regulator modulator therapy on infection, inflammation, and lung function in cystic fibrosis

Clinical Endpoints Known to Improve with HEMT	Pathways with Evidence of HEMT Modulation or Improvement	Active Areas of HEMT Impact Investigation
Infection
Drug: ivacaftor (HEMT) Clinical observations: either decreased prevalence rates or delayed incidence of certain CF pathogens, such as P aeruginosa, S aureus, S maltophilia, and Aspergillus	Drug: ivacaftor (HEMT) or lumacaftor In vitro: Increased phenotypic polarization, phagocytosis, and bacterial killing by CF, both monocytes and macrophages	Impact of short and long-term HEMT on pathogen load, survival, and lifestyle (eg, biofilm, planktonic) in the CF airway Effect of HEMT on lung microbiome diversity and its influence on CF lung function Effect of antibiotic treatment and its interaction with HEMT Evaluation of confounding parameters (eg, lung function, comorbidities) on the long-term impact of HEMT on CF pathobiology
Inflammation
Drug: ivacaftor (HEMT) Clinical observation: improved lung function and reduced airway accumulation of inflammation markers (eg, neutrophil elastase [NE], IL- 8, IL-6, IL-1β)	Drug: ivacaftor (HEMT) In vivo: reduced airway damage and abundance of inflammation markers in CF ferrets and rats Drug: ivacaftor (HEMT), elexacaftor/tezacaftor/ ivacaftor (ETI) (HEMT), tezacaftor/ivacaftor (HEMT) Ex vivo: increased ability of different CF myeloid cells to regulate inflammation: (eg, more neutrophil apoptosis, augmented monocyte adhesion and trafficking, and reduced monocytes/ PBMCs inflammasome activation) Variable expression of cell activation markers, including IFNγ and TLR signalling	Influence of HEMT on myeloid and epithelial cell pathways that promote CF lung inflammatory damage: (eg, NFkB, inflammasome, extracellular potassium accumulation, hypoxia, recruitment of myeloid cells to the airway and regulation of their effector function) Impact of HEMT on adaptive immune cells that influence CF lung inflammation: subtypes of effector CD4+ T cells (eg, TH1,TH2, TH17), regulatory T cells (Tregs)
Metabolism
	Drug: lumacaftor, lumacaftor/ ivacaftor Ex vivo: augmented PTEN levels in CF PBMCs	Effects of HEMT on oxidative metabolic pathways that contribute to CF lung damage (eg, ROS release, glycolysis, PTEN function, and synthesis of immunometabolites that fuel pathogen metabolism in the lung)
Lung function
Drug: ivacaftor (HEMT), elexacaftor/tezacaftor/ ivacaftor (ETI) (HEMT) Clinical observation: enhanced LCI, augmented FEV1, increased mucociliary clearance, improvements in chest CT scan and MRI		How HEMT modulates lung function in hosts exhibiting chronic infection Utilization of CF animal models (eg, pig, ferrets, rats) Developing and testing new and more potent HEMT in these models

Highly Effective Cystic Fibrosis Transmembrane Conductance Regulator Modulator Therapy and In Vitro Cell Culture

Culturing cells in vitro in the presence of CFTR modulators reverses some CF immune cell defects. Defects in adhesion and trafficking observed in monocytes from PwCF and the genotype F508del homozygous could be reversed by addition of the CFTR corrector lumacaftor.¹¹⁰ Similarly, impaired phagocytosis and killing of P aeruginosa by F508del homozygous human monocyte-derived macrophages were restored to levels seen in healthy control macrophages following culture with lumacaftor.¹¹¹ These studies confirm that deficient CFTR channel activity at the level of the immune cell is responsible for abnormal responses, and restoration of CFTR activity by CFTR modulators is sufficient to remedy the impairments.

Some aberrant inflammatory responses observed in CF epithelial cells are also corrected by modulators. Treatment of in vitro cultured CF epithelial cells with various inflammatory stimuli results in marked increases in activation of the transcription factor nuclear factor kappa-B (NFKB) and secretion of the neutrophil chemoattractant IL-8, as compared to cells with functional CFTR.^39,112 Exposure of CFTR F508del homozygous epithelial cells in vitro to lumacaftor/ivacaftor markedly dampens the intracellular signaling cascade in response to inflammatory stimuli and transcription of IL-8.¹¹³ Furthermore, if a CF epithelial cell monolayer is injured in vitro, treatment of the monolayer with lumacaftor/ivacaftor enhances repair.¹¹⁴ Thus, HEMT directly dampens aberrant inflammation and helps restore homeostasis in CF both epithelial and immune cells. Interestingly, 2 studies recently demonstrated that exposure of epithelial cells to inflammatory mediators enhances their responsiveness to HEMT in vitro, manifested by improved CFTR function.^115,116 This result underscores the complex interactions between CFTR function and inflammation, and highlights the importance of assessing the chronic effects of HEMT on inflammation using animal models, and in human subjects.

Highly Effective Cystic Fibrosis Transmembrane Conductance Regulator Modulator Therapy and Animal Models of Cystic Fibrosis

Although in vitro studies provide direct evidence that HEMT alters cellular inflammatory responses, in vivo studies are required to understand how these HEMT-induced changes impact CF systemic and airway inflammation. Animal models permit manipulation of different contributors to systemic and airways disease with clearer interpretation of endpoints than in human studies, which can be confounded by genetic variability, behavioral differences, and environmental factors. However, studying chronic CF airway infection and inflammation has been challenging, because disrupting CFTR in smaller mammals has not recapitulated human CF lung disease.^31,117 CF rats develop abnormal airway mucus, impaired mucociliary clearance, and exaggerated airway inflammation,^71,118 but do not become spontaneously infected. Only the CF pig and ferret develop airway pathology similar to CF in people, including spontaneous bacterial airway infections.^31,117 Pigs, rats, and ferrets have been generated that express G551D CFTR, which can be potentiated effectively by ivacaftor.^118–120 G551D-CFTR ferrets administered ivacaftor beginning in utero are spared airway pathology seen in CF ferrets not receiving modulators, but then develop airway inflammation when ivacaftor therapy is withdrawn.¹¹⁹ G551D-CFTR rats raised for 6 months in the absence of ivacaftor demonstrate increased markers of airway inflammation in BAL fluid compared with wild-type rats. Levels of some markers interferon (IFNγ0, IL-1α, and IL-1β) normalized following 1 week of ivacaftor, whereas others did not (IL-6, TNFα),⁷¹ indicating that restoration of CFTR activity in airways with established disease may not completely reverse inflammation, even in the absence of chronic infection. Experiments in these model systems in which chronic lung disease and associated infection are established followed by initiation of HEMT and characterization of how inflammation changes acutely and over time will provide important information to link cellular data generated in vitro with observations of PwCF on HEMT. In addition, pig and ferret models have the potential to better understand how chronic airway infections evolve in individuals with established lung disease and bronchiectasis who start treatment with HEMT.

EFFECTS OF HIGHLY EFFECTIVE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MODULATOR THERAPY ON INFLAMMATION AND INFECTION IN HUMAN SUBJECTS

Infection

Epidemiologic studies of culture-based data

The first study to prospectively evaluate PwCF starting HEMT was the GOAL (G551D ObservationAL) study, which followed individuals aged 6 and older with G551D CFTR mutations as they initiated treatment with ivacaftor.²⁷ Subjects in the GOAL study had decreased prevalence rates of P aeruginosa and Aspergillus 1 year following ivacaftor initiation, with certain subjects (primarily those with better lung function and history of intermittent, as opposed to chronic, P aeruginosa infection) clearing their P aeruginosa infection.¹²¹ Similarly, registry-based observational studies from the United States and United Kingdom of outcomes 3 years following ivacaftor approval identified decreased prevalence rates of most CF pathogens (S aureus, P aeruginosa, Staphylococcus maltophilia, Aspergillus), with NTM as the notable exception.¹²² Although data on CFTR modulators other than ivacaftor are limited, a single-center analysis of patients on either ivacaftor or ivacaftor/lumacaftor showed delays in incident CF pathogen infection with P aeruginosa or S aureus in those on modulators compared with matched controls.¹²³ These studies demonstrate the likely significant impact that HEMT will have on reducing CF pathogen infection over time, with the greatest impact on those who start HEMT at younger ages with preserved lung function.¹²⁴ In addition to improving inflammation and mucociliary clearance, other potential mechanisms through which HEMT are likely to impact infection include direct antimicrobial activity of HEMT, HEMT synergy with antibiotics, and niche modification impacting pathogen virulence.^125–128

Culture-independent studies

Culture-independent studies have been less consistent in showing impact of HEMT on CF airway infection, and overall have included variable changes in bacterial community diversity, bacterial load, and relative abundances of specific taxa (including CF pathogens and certain obligate and facultative anaerobes).^{124,129–131} For example, although ivacaftor significantly increased mucociliary clearance in the GOAL study, no significant changes were noted in bacterial load, bacterial community diversity, or in inflammatory biomarkers in the first 6 months of treatment. These results contrast with a similar, prospective study of an Irish cohort of 12 adults with CF and G551D mutations starting ivacaftor.¹³² This study observed a rapid (within the first week of therapy) and significant decrease in P aeruginosa burden across the first 6 months of treatment; however, P aeruginosa burden then increased by the end of the first year of treatment, and none of the subjects cleared their P aeruginosa infection, despite maintaining improvements in lung function, radiographic improvements, and sustained reduction in inflammatory markers. Subsequent studies have similarly shown no significant decreases in P aeruginosa burden or total bacterial load with ivacaftor treatment.¹²⁹ Several factors likely contribute to differences observed across studies, including differences in patient populations (eg, ages of subjects, CFTR mutations, severity of disease, types of airway infections, and use of CF therapies such as antibiotics) that may confound results or obscure signals. For example, ivacaftor-associated reductions in total bacterial load were observed in an Australian cohort, but only after controlling for changes in antibiotic exposure.¹³³ Future studies accounting for changes in antibiotic use and other potential confounders (eg, age, lung function, or comorbidities) across longer-term use of HEMT are needed to more fully understand the impact on HEMT on the many aspects of CF airway infection, including CF pathogens, bacterial load, anaerobes, and bacterial community structure.

Inflammation

Measurements of lung inflammation

Several modalities that assess overall lung health and function in patients with CF provide indirect measures of inflammation, including spirometry, lung clearance index (LCI), and radiologic imaging. HEMT improves airflow, as measured by forced expiratory volume in 1 second (FEV₁).^11,21,22 Multiple breath washout assesses ventilation heterogeneity in the lung, as reported by LCI,^134,135 and preliminary studies have demonstrated improvements in LCI with HEMT.^136,137 Likewise, radiology (eg, chest CT scan and MRI) can assess lung damage,^138–140 and studies of individuals before and after HEMT have demonstrated improvements in radiographic markers associated with inflammation.^130,132,141 However, these tests evaluate the complex endpoint of lung damage, with structural changes, mucus impaction, bronchoconstriction, and airway inflammation all potentially contributing.^142,143

Direct measurements of airway inflammation (and infection) can be obtained by evaluating airway secretions (eg, BAL or induced or spontaneously expectorated sputum).^85,144 In CF, sputum biomarkers for quantitating airway inflammation, both for making clinical decisions and as endpoints in clinical trials, remain elusive.¹⁴⁵ However, higher concentrations of some airway inflammatory markers, such as neutrophil elastase (NE), correlate with severity of CF lung disease,^146–148 and also decrease in sputum following antibiotic treatment of CF pulmonary exacerbations.⁵¹ In the GOAL study, no difference in sputum inflammatory cytokines and biomarkers was detected in specimens obtained 6 months after initiation of ivacaftor.^27,124 As with bacterial burden, this contrasted with the results of the Irish cohort study, in which initiation of ivacaftor was associated with a rapid and sustained (at >600 days) drop in sputum inflammatory biomarkers (NE, IL-8, and IL-1β).¹³² These disparate results may reflect the fact that the GOAL study did not detect changes in bacterial burden in the airway following ivacaftor, whereas the Irish study did.

Because HEMT has led to decreased sputum production in many PwCF, alternate modalities for sampling airway inflammation are needed. A recent study reported on use of nasal lavage to obtain epithelial lining fluid (ENL),¹⁴⁹ and noted decreased levels of ENL IL-1β and IL-6 in subjects starting ivacaftor. Prior studies have shown that levels of inflammatory biomarkers in CF nasal lavage correlate with degree of sinus disease.¹⁵⁰ Whether nasal/upper airway inflammation mirrors bronchial/lower airway inflammation in CF, and whether HEMT alters upper airway and lower airway inflammation in a similar manner, remain to be determined. However, assays to evaluate CF airway inflammation that do not require lower airway specimens will be essential for characterizing CF lung inflammation in the era of HEMT.

Ex vivo studies of immune cell response

Investigations of how HEMT changes circulating immune cells can help elucidate how HEMT may influence lung inflammation, as circulating cells traffic to inflamed CF airways.^86,99,100 Some impairments described in CF immune cells are mitigated by initiation of HEMT. HEMT restored phenotypic polarization and phagocytosis in monocyte-derived macrophages (MDMs) generated from PwCF receiving ivacaftor to levels similar to those exhibited by healthy donor MDMs.¹⁵¹ Delayed neutrophil apoptosis, thought to contribute to nonresolving CF airway inflammation, is corrected in individuals with CFTR-G551D mutations after initiation of ivacaftor.¹⁵² Dysregulated inflammasome activation is also thought to contribute to excessive inflammation in the CF airway.⁹¹ Monocytes from individuals treated with ETI demonstrated decreased expression of the P2×7 receptor, which promotes ATP-induced inflammasome activation, and were more resistant to inflammasome activation than monocytes from the same individuals before receiving ETI.¹⁵³ Similar changes in inflammasome activation were seen in a study of PBMCs from PwCF homozygous for F508del mutations before and after initiation of tezacaftor/ivacaftor.¹⁵⁴ Inflammation resulting from abnormal cellular metabolism in CF cells is also likely to be dampened by CFTR modulators, which enhance CFTR channel stability and function, and are thus expected to restore interactions with key metabolic checkpoints at the cell membrane, such as PTEN. Administration of lumacaftor/ivacaftor to PwCF homozygous for F508del mutations increased PTEN levels in peripheral blood mononuclear cells.⁹⁰ Studies performed in vitro have demonstrated that ectopic PTEN expression in either F508del homozygous or control cells reduces mitochondrial ROS and glucose breakdown,⁸⁸ suggesting that HEMT will shift cellular metabolism in vivo to a less inflammatory state.

Other studies have characterized how HEMT alters circulating immune cell inflammatory phenotypes, using expression of different protein surface markers and cellular transcriptomes. Monocytes and neutrophils isolated from subjects at 1 and 6 months after initiation of ivacaftor exhibited decreases in expression of proteins associated with activation.¹⁵⁵ In a proteomic analysis, monocytes recovered 1 week after subjects started ivacaftor displayed changes in plasma membrane proteins, suggesting that cells had improved adhesion and trafficking and suppressed responses to the inflammatory cytokine IFNγ.¹⁵⁶ Follow-up studies of ex vivo monocytes from a separate cohort initiating ivacaftor demonstrated a marked reduction in monocyte responses to IFNγ.¹⁵⁷ In vitro functional studies of CF monocytes confirmed that impaired CFTR activity results in a monocyte adhesion deficiency, and this impairment is reversed by CFTR modulators.¹¹⁰ Transcriptome analysis of PBMCs recovered from subjects in the GOAL cohort 1 month after starting ivacaftor revealed decreased transcription of genes involved in TLR signaling and other immune inflammatory pathways.¹⁵⁸ In contrast, transcriptomic analysis of monocytes isolated 1 week after initiation of ivacaftor identified an almost nonoverlapping set of differentially expressed genes (DEGs) compared with those identified in the GOAL cohort at 1 month after initiation of ivacaftor, and these DEGs suggested that ivacaftor caused an overall more activated state of CF blood monocytes.¹⁵⁹ These disparate results may reflect the multiple ways in which CFTR dysfunction affects inflammation. For example, immune cells may acutely become more activated in the days after initiation of HEMT if there is a reduction in the immune tolerance previously described for CF monocytes¹⁰⁵ and leukocytes.⁵⁸ However, as HEMT leads to improvements in both pulmonary and systemic health over weeks to months, circulating immune cells may gradually be restored to a more quiescent and less activated state.

Some clinical complications seen in PwCF have been linked to sequelae of CFTR deficiency and immune impairments, including bronchiectasis and P aeruginosa airway infection, and may not be significantly improved by HEMT. For example, PwCF tend to have T cell responses skewed toward T_H2 and T_H17 lineages, phenotypes that cause exaggerated inflammation with poor infection-fighting potential.^36,160,161 In a study comparing T cells from PwCF with and without modulator treatment, no differences in T cell populations or concentrations of serum T cell-related cytokines were detected.¹⁶² T cell populations from subjects with non-CF bronchiectasis were similar to those from subjects with CF, suggesting that bronchiectasis, and not CFTR dysfunction, drives skewing of T cell populations. These data highlight the multifactorial contributions to CF airway inflammation and indicate that some deleterious inflammation will likely persist despite HEMT in people with more advanced lung disease.

Cystic fibrosis macrophage oxidative metabolism contributes to bacterial adaptation to the cystic fibrosis transmembrane conductance regulator-mutant lung

Although recent studies have begun to shed light on the impact of HEMT on CF immune cell function, how these drugs influence the metabolism of CF phagocytes, and how changes in phagocyte metabolism will alter host-pathogen interaction in the CF lung, remain unknown. The pro-oxidative metabolic abnormalities of CF myeloid cells are associated with release of many metabolites, like succinate and itaconate, that participate in immune signaling but also trigger phenotypic changes in CF airway pathogens.¹⁶³ While succinate is a proinflammatory metabolite that supports synthesis of cytokines that can damage mucosal tissues, like IL-1β,¹⁶⁴ itaconate acts as a regulatory determinant rapidly synthetized in inflamed environments.¹⁶⁵ Itaconate not only limits the release of detrimental cytokines by effector phagocytes, but also maintains tissue homeostasis during inflammatory diseases. In contrast with healthy individuals, metabolomic analyses have shown that sputum and BAL fluid of PwCF accumulate succinate and itaconate.¹⁶⁶ Genetic and metabolic analyses of P aeruginosa isolates from chronic CF infections demonstrate metabolic adjustments consistent with exposure to abundant levels of succinate and itaconate.¹⁶⁶ In response to this metabolic pressure, P aeruginosa generates biofilms, which promote its ability to evade phagocytosis, antibiotics, complement and antibodies. CF host-adapted P aeruginosa strains upregulate specific genetic clusters that support the utilization of succinate and itaconate, indicating that CF airway immunometabolites nourish respiratory pathogens instead of promoting their eradication.¹⁶⁶ Similarly, S aureus also responds to CFTR-controlled immunometabolites. Longitudinal analysis of S aureus strains from PwCF demonstrates activation of specific genes in response to itaconate, including those involved in the generation of extracellular polysaccharides and biofilm.¹⁶⁷ These CF-adapted bacterial strains modify their own metabolism in response to itaconate, diverting their utilization of carbohydrates for the generation of biofilm in the lung.

These data strongly indicate that CF pathogens activate sophisticated mechanisms of pathogenesis in response to the oxidative metabolic environment set by CFTR-mutant lung cells and highlight the complex interactions between infection and inflammation in the CF airway.

Future studies in HEMT-treated PwCF should include characterization of immunometabolites in BAL and sputum, as well as how HEMT alters the presence and phenotypes of myeloid cells that participate in immunometabolite generation. It is likely that the metabolic effects of HEMT on the type of organisms recovered from CF airway will vary as a function of both the class of CFTR mutation studied and the metabolic preferences of the pathogen.

CONCLUDING REMARKS

HEMT promises to significantly improve lifespan and quality of life for most PwCF, largely because of HEMT’s impact on almost all aspects of CF airway pathophysiology: mucus obstruction, ASL composition, infection, and inflammation. Although the improvements in CF airway infection and inflammation seen with HEMT are promising, these studies also emphasize the difficulty in determining which aspects of infection and inflammation will be resolved with HEMT and which may persist. CF airway inflammation (like inflammation in other chronic diseases) is not 1 process, but rather multiple immune processes initiated to eliminate infectious threats, repair damaged tissue, and attempt to restore homeostasis.¹⁶⁸ Although some contributors to CF inflammation are reversed directly by HEMT, others may be indirectly affected by HEMT, and others may not be affected at all. Similarly, HEMT’s impact will likely differ across the CF population, with larger impact on infection and inflammation seen in individuals who start HEMT at a younger age, before the establishment of chronic infection and bronchiectasis. Finally, HEMT’s impact on extrapulmonary manifestations of CF (eg, nutrition, intestinal inflammation, sinus disease) and on CF therapies (eg, reducing use of mucolytics and antibiotics) are likely indirect routes through which HEMT will continue to impact airway infection and inflammation.¹⁶⁹ Future studies are needed to continue to understand the mechanisms of the post-HEMT state, including further development of CF animal models to parse the intricate interactions between HEMT, metabolism, inflammation, and infection to continue to develop novel approaches for personalized care and optimal treatments for PwCF.

KEY POINTS.

• Cystic fibrosis (CF) airway disease results from the complex interactions of 3 pathologic processes: (1) primary airway epithelial cell deficiency in CF transmembrane conductance regulator (CFTR) activity causing abnormal air surface liquid composition and impaired mucociliary clearance, (2) chronic airway infections, and (3) nonresolving inflammation.

• Highly effective CFTR modulator therapy (HEMT) acts to restore CFTR localization and function in the cell, enhancing mucociliary clearance and improving measures of pulmonary function; however, how HEMT changes airway infection and inflammation remains poorly understood.

• CF airway inflammation is a multifactorial process, and the impact of HEMT varies across the different contributors. Similarly, the impact of HEMT on inflammation and infection will differ across the different stages of CF lung disease.

• More research is needed to better understand how HEMT changes CF airway infection and inflammation to best predict how pulmonary disease will evolve in patients with CF receiving HEMT and to direct future treatment strategies

CLINICS CARE POINTS.

• Highly effective modulator therapy (HEMT) improves lung function and quality of life for most people with cystic fibrosis (CF) and eligible CFTR mutations.

• There are multiple contributors to inflammation and infection in the lungs of people with CF.

• Lung inflammation and infection are not fully resolved by HEMT, especially in those people with CF with established or advanced lung disease.

Abbreviations

CF

cystic fibrosis

HEMT

highly effective CFTR modulator therapy

PwCF

people with cystic fibrosis

ASL

airway surface liquid

CFTR

cystic fibrosis transmembrance conductance regulator

DAMPs

damage associated molecular patterns

ROS

reactive oxygen species

OXPHOS

oxidative phosphorylation

ER

endoplasmic reticulum

UPR

unfolded protein response

LCI

lung clearance index

ETI

elexacaftor / tezacaftor / ivacaftor

NE

neutrophil elastase

PTEN

phosphate and tensin homolog deleted on chromosome 10

LPS

lipopolysaccharide

PBMC

speripheral blood mononuclear cells

TLR

Toll-like receptor

TMIIR

Toll-like receptor-mediated innate immune inflammatory responses

FEV₁

forced expiratory volume in one second

BAL

bronchoalveolar lavage