# Moving the Dial on Airway Inflammation in Response to Trikafta® in Adolescents with Cystic Fibrosis

Agathe Lepissier, Anne Sophie Bonnel, Nathalie Wizla, Laurence Weiss, Marie Mittaine, Katia Bessaci, Eitan Kerem, Véronique Houdouin, Philippe Reix, Christophe Marguet, et al.

► To cite this version:

Agathe Lepissier, Anne Sophie Bonnel, Nathalie Wizla, Laurence Weiss, Marie Mittaine, et al. Moving the Dial on Airway Inflammation in Response to Trikafta® in Adolescents with Cystic Fibrosis. American Journal of Respiratory and Critical Care Medicine, 2023, Online ahead of print. 10.1164/rccm.202210-1938LE. inserm-03944455

HAL Id: inserm-03944455

https://inserm.hal.science/inserm-03944455v1

Submitted on 23 Jul 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
4. Carr TF, Granell R, Stern DA, Guerra S, Wright A, Halonen M, et al. High insulin in early childhood is associated with subsequent asthma risk independent of body mass index. *J Allergy Clin Immunol Pract* 2022;10:785–792.e5.

5. Taussig LM, Wright AL, Holberg CJ, Halonen M, Morgan WJ, Martinez FD. Tucson children's respiratory study: 1980 to present. *J Allergy Clin Immunol* 2003;111:661–675. [Quiz, p. 676.]

6. Ferreira SS, Nunes FPB, Casagrande FB, Martins JO. Insulin modulates cytokine release, collagen and mucus secretion in lung remodeling of allergic diabetic mice. *Front Immunol* 2017;8:633.

7. Proskocil BJ, Calco GN, Nie Z. Insulin acutely increases agonist-induced airway smooth muscle contraction in humans and rats. *Am J Physiol Lung Cell Mol Physiol* 2021;320:L545–L556.

8. Nie Z, Proskocil B, Jacoby D, Fryer A. Potentiation effect of insulin on M3 muscarinic receptor agonist induced airway smooth muscle contraction. *Eur Respir J* 2019;54:PA4207.

9. Hancox RJ, Landhuis CE. Correlation between measures of insulin resistance in fasting and non-fasting blood. *Diabetol Metab Syndr* 2011;3:23

Copyright © 2023 by the American Thoracic Society

<figure><img src="image_2.png" /></figure>

Check for updates

Moving the Dial on Airway Inflammation in Response
to Trikafta in Adolescents with Cystic Fibrosis

Agathe Lepissier¹, Anne Sophie Bonnel²,³, Nathalie Wizla⁴,
Laurence Weiss⁵, Marie Mittaine⁶, Katia Bessaci⁷, Eitan Kerem⁸,
Véronique Houdouin⁹,¹⁰, Philippe Reix¹¹,¹², Christophe Marguet¹³,¹⁴,
Isabelle Sermet-Gaudelus¹,²,¹⁰,¹⁵, and the MODUL-CF Pediatric
French Network for Cystic Fibrosis

¹INSERM U1151, Institut Necker Enfants Malades, Paris, France; ²Hôpital Necker Enfants Malades, Assistance Publique Hôpitaux de Paris, Paris, France; ³Hôpital André Mignot, Le Chesnay, France; ⁴Hôpital Jeanne de Flandre, Lille, France; ⁵Hôpital de Hautepierre, Strasbourg, France;

⁶Hôpital des Enfants-CHU de Toulouse, Toulouse, France; ⁷American Memorial Hospital, Reims, France; ⁸Hadassah Hebrew University Medical Center, Jerusalem, Israel; ⁹Hôpital Robert Debré, Paris, France;

¹⁰Université Paris Cité, Paris, France; ¹¹Hôpital Femme Mère Enfant, Hospices Civils de Lyon, Bron, France; ¹²UMR 5558 CNRS Equipe EMET Université Claude Bernard Lyon 1, Lyon, France; ¹³CHU de Rouen, Rouen, France; ¹⁴UMR 1311, Université de Normandie, Rouen, France;

¹⁵European Respiratory Network-Lung, Frankfurt, Germany

ªThis article is open access and distributed under the terms of the
Creative Commons Attribution Non-Commercial No Derivatives
License 4.0. For commercial usage and reprints, please e-mail
Diane Gern (dgern@thoracic.org).

*Co–last authors.

Supported by Association Vaincre La Mucoviscidose grant
RC20220503008 and a Hadassah-France Research Grant.

Author Contributions: A.L. performed the experiments and wrote the manuscript. A.S.B. coordinated the study. N.W., L.W., M.M., K.B., V.H., and P.R. enrolled the patients and reviewed the manuscript. E.K. and C.M. enrolled the patients, participated to the design of the study, and edited the manuscript. I.S.-G. designed the study, obtained the funding, coordinated the study and the experiments, and wrote the manuscript.

Originally Published in Press as DOI: 10.1164/rccm.202210-1938LE
on January 4, 2023

To the Editor:

Cystic fibrosis (CF) lung disease is characterized by excessive and sustained airway inflammation (1). The CFTR (cystic fibrosis transmembrane conductance regulator) highly effective modulator therapy (HEMT) elexacaftor, tezacaftor, and ivacaftor (ETI) demonstrated remarkable pulmonary improvement in patients with CF (2). Although the respiratory benefit reflects a better airway surface hydration and improved mucociliary clearance, data are missing regarding the effect of ETI on inflammation, specifically in patients with relatively mild lung disease. The aim of the current study is to assess the short-term effect of ETI on lung inflammation in adolescents with CF.

Methods

**Study design.** Patients with CF were enrolled in a multicenter real-life longitudinal study (NCT04301856). Data were collected at initiation (M0) and 1 month (M1). This included antibiotic treatment, pulmonary function tests, sweat tests, inflammatory markers in blood sampling (C-reactive protein [CRP], leukocytes, and polymorphonuclear neutrophil [PMN] counts), sputum cultures, and inflammatory biomarkers. The study was approved by the Centre Institutional Review Board. All parents gave informed consent.

**Sputum sample preparation.** Sputum was collected by sputum induction. Sputum samples were immediately placed on ice and frozen at -80°C. After thawing, 200 µl of sputum was processed as per laboratory protocol, and supernatant was frozen at -80°C until analysis (3, 4).

**Sputum inflammatory biomarkers.** Neutrophil elastase (NE) activity was measured using a fluorometric assay (Sigma-Aldrich). IL-1β, IL-4, IL-6, IL-8, IFN-γ, and TNF-α (tumor necrosis factor α) concentrations were measured using V-PLEX Viral Panel 2 Human Kit, and V-PLEX Human IL-8 Kit (Meso Scale Discovery, Inc.). All tests were performed in triplicate.

**Statistical analyses.** Data were expressed as median (interquartile range). Comparisons were analyzed using the Fisher test, paired Student's t test, ANOVA, or simple regression analysis. FEV₁ change at M1 was quantified by its relative change from baseline.

Results

**Patient population.** Seventy-six European White adolescents (33 girls, median [interquartile range] age, 15 [3] yr), were enrolled from seven centers across France. Forty-six patients were p.Phe508del homozygous and had received Orkambi (lumacaftor/ivacaftor) before enrollment; the 30 remaining patients were compound heterozygous for F508del and a minimal function mutation and had not been previously treated with CFTR modulators.

Most of the patients presented with mild lung disease, as assessed by a median predicted FEV₁ (ppFEV₁) of 76% (Table 1). Patients colonized with *Pseudomonas aeruginosa* (n = 18), *Stenotrophomonas maltophilia*, or *Achromobacter xylosoxidans* (n = 17) had a significantly decreased ppFEV₁ (71% [18%] vs. 80% [20%]; *P* = 0.04). Thirty-one patients received oral or intravenous (IV) antibiotic therapy at initiation of ETI because of bronchial exacerbation.

**Lung function and clinical parameters.** Patients presented a significant improvement after 1 month of ETI treatment relative to baseline in both weight (+1.1 [2.9] kg) and FEV₁ (+24% [31%]),
<table><caption>**Table 1.** Change in Clinical, Respiratory, and Bacteriological Endpoints and Inflammatory Biomarkers</caption><thead><tr><th>Parameters</th><th>M0</th><th>M1</th><th>P Value</th></tr></thead><tbody><tr><td>Clinical and microbiological endpoints</td><td></td><td></td><td></td></tr><tr><td>Weight, kg</td><td>50 (11.7)</td><td>51.9 (10.2)</td><td>&lt;0.0001</td></tr><tr><td>Sweat chloride, mEq/L</td><td>92.5 (29.5)</td><td>44.0 (30)</td><td>&lt;0.0001</td></tr><tr><td>FEV<sub>1</sub>, L</td><td>2.4 (0.9)</td><td>3.1 (1.1)</td><td>&lt;0.0001</td></tr><tr><td>FEV<sub>1</sub>, pp</td><td>76.0 (21)</td><td>96.0 (24)</td><td>&lt;0.0001</td></tr><tr><td>FVC, L</td><td>3.3 (1.1)</td><td>3.7 (1.2)</td><td>&lt;0.0001</td></tr><tr><td>FVC, pp</td><td>90.0 (16)</td><td>102.0 (18)</td><td>&lt;0.0001</td></tr><tr><td>FEF<sub>25-75</sub>, L</td><td>2.3 (1.4)</td><td>3.3 (1.7)</td><td>&lt;0.0001</td></tr><tr><td>FEF<sub>25-75</sub>, pp</td><td>58.0 (40.5)</td><td>86.5 (46.7)</td><td>&lt;0.0001</td></tr><tr><td>Bronchial colonization</td><td></td><td></td><td></td></tr><tr><td>Staphylococcus aureus</td><td>55</td><td>49</td><td>0.007</td></tr><tr><td>Pseudomonas aeruginosa</td><td>17</td><td>12</td><td>&lt;0.0001</td></tr><tr><td>Achromobacter xylosoxidans or Stenotrophomonas maltophilia</td><td>17</td><td>3</td><td>0.01</td></tr><tr><td>Blood inflammatory biomarkers</td><td></td><td></td><td></td></tr><tr><td>Leukocytes, nb/µl</td><td>7,465 (3,192)</td><td>6,400 (2,595)</td><td>&lt;0.0001</td></tr><tr><td>Polymorphonuclear neutrophil count, nb/µl</td><td>4,035 (2,810)</td><td>3,100 (1,846)</td><td>&lt;0.0001</td></tr><tr><td>CRP, mg/L</td><td>0.5 (3.4)</td><td>0.0 (0.2)</td><td>0.0002</td></tr><tr><td>Sputum inflammatory biomarkers</td><td></td><td></td><td></td></tr><tr><td>Neutrophil elastase, µg/ml</td><td>23.4 (100.8)</td><td>2.1 (4.6)</td><td>&lt;0.0001</td></tr><tr><td>TNF-α, pg/ml</td><td>12.4 (36.75)</td><td>2.6 (11.4)</td><td>0.15</td></tr><tr><td>IFNy, pg/ml</td><td>3.8 (9.1)</td><td>4.0 (8.4)</td><td>0.25</td></tr><tr><td>IL-1β, pg/ml</td><td>132.2 (482.1)</td><td>22.9 (76.5)</td><td>0.02</td></tr><tr><td>IL-4, pg/ml</td><td>0.3 (0.5)</td><td>0.2 (0.4)</td><td>0.75</td></tr><tr><td>IL-6, pg/ml</td><td>7.0 (28.1)</td><td>4.9 (17.4)</td><td>0.28</td></tr><tr><td>IL-8, pg/ml</td><td>25,682.2 (70,448.2)</td><td>10,969.9 (28,618.9)</td><td>0.006</td></tr></tbody></table>

Definition of abbreviations: CRP = C-reactive protein; FEF<sub>25-75</sub> = forced mid-expiratory flow; M0 = month 0; M1 = month 1; nb = number; pp = percentage predicted; TNF = tumor necrosis factor.

Data are given as median (interquartile range) or bronchial colonization refers to the chronic identification of bacteria in sputum.

and 52 patients demonstrated a decrease in sweat test below 60 mmol/L (Table 1). Five patients with chronic colonization for *P. aeruginosa* and 14 with *A. xylosoxidans* or *S. maltophilia* demonstrated negative cultures 1 month after ETI initiation. The change in ppFEV<sub>1</sub> was not significantly different in patients previously treated with Orkambi (+21% [32%] vs. +29% [23%] for patients naive to CFTR modulators) or those receiving antibiotics at ETI initiation (+21% [24%] vs. +28% [30%] in untreated patients). ppFEV<sub>1</sub> was significantly increased in patients with *P. aeruginosa*, *A. xylosoxidans*, or *S. maltophilia* in sputum at M0 (+32% [28%] vs. +20% [23%] in noncolonized patients; *P* = 0.03).

**Inflammation biomarkers.** At baseline, blood concentrations of CRP, leukocytes, and PMN count were within the normal range in contrast to the sputum inflammatory markers, which were elevated (5). ppFEV<sub>1</sub> was negatively correlated with sputum biomarkers, including NE (*P* = 0.0006) and IL-8 (*P* = 0.009), as well as with blood CRP (*P* = 0.00004), leukocytes (*P* = 0.00004), and PMN counts (*P* = 0.01).

All blood and sputum inflammatory biomarkers decreased at M1. The difference was significant for NE, IL-1β, and IL-8 in sputum and CRP, leukocytes, and PMN count in blood (Figure 1 and Table 1). FEV<sub>1</sub> change was significantly correlated with that of sputum NE (*P* = 0.02), blood CRP (*P* = 0.04), leukocytes (*P* = 0.0007), and PMN count (*P* = 0.0002), and nearly at the significant concentration for sputum IL-8 (*P* = 0.07). Interestingly, some patients did not experience respiratory improvement despite a significant change in inflammatory biomarkers. There was no correlation between changes in sweat test and blood or sputum inflammatory

biomarkers. Change in NE at 1 month was not significantly affected by previous treatment with Orkambi (−16 [89] µg/ml vs. −17 [98] µg/ml), antibiotic course at initiation (−16 [80] µg/ml vs. −19 [123] µg/ml). Although baseline NE was more elevated in patients with *P. aeruginosa*, *A. xylosoxidans*, or *S. maltophilia* (66 [111] µg/ml vs. 17 [83] µg/ml; *P* = 0.1), negativation of sputum culture for these bacteria at M1 did not significantly impact NE change (−52 [136] µg/ml vs. −81 [68] µg/ml; not significant). A similar profile was observed for the other biomarkers.

## Discussion

This study in adolescents with relatively mild lung CF disease shows a decrease in sputum and blood inflammatory markers at 1 month after ETI initiation.

There are contradictory reports regarding the effect of HEMT on inflammation (6–8). Our observation that NE, IL-8, and IL-1β decrease significantly in sputum in parallel to CRP and PMN count in blood provides some evidence that ETI blunts neutrophil-derived inflammation in the short term. Once in the CF lung, PMNs acquire a modified phenotype that promotes NE release either free in the airway or bound to PMNs (4). As previously reported for circulating PMNs (9), our results suggest that ETI may also reverse this dysregulation in the lung. Importantly, those changes occurred regardless of whether patients were chronically infected with *P. aeruginosa*, *A. xylosoxidans*, or *S. maltophilia* or had been treated with antibiotics at initiation, suggesting an antiinflammatory effect, independent of infection.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Concentration of sputum NE, IL-1β, IL-8, and blood CRP, PMN, and leukocyte counts at baseline (M0) and M1 treatment with elexacaftor, tezacaftor, and ivacaftor. \*P<0.05, \*\*P<0.01, \*\*\*P<0.001, and \*\*\*\*P<0.0001. CRP = C-reactive protein; M0 = month 0; M1 = month 1; NE = neutrophil elastase; PMN = polymorphonuclear neutrophil.</figcaption></figure>

Given the strong association between neutrophilic inflammation and progression of lung damage in early life, our results may suggest that HEMT may prevent or at least attenuate longer-term pulmonary decline (10). An important point is that, at the individual level, the improvement of ppFEV₁ was not directly associated with the variation of the inflammatory markers. This can be the result of irreversible damage. Conversely, patients with remaining significant amounts of NE may require adjunctive anti-elastase therapy. This illustrates the complexity of understanding the exact targets of HEMT on the inflammatory cascade in CF airways and how restoration of CFTR function may affect inflammation and vice versa. This also outlines the necessity of longitudinal studies in the CF population to assess whether this antiinflammatory effect is sustained. ■

**Author disclosures** are available with the text of this letter at www.atsjournals.org.

**Acknowledgment:** The authors thank Flora Zavala for fruitful discussion for the manuscript, Nathalie Servel for experimentation, Kate Hayes for

helpful manuscript editing, and Anthony Rignani, Fatiha Hassani, and Ania Acher for data collection.

Correspondence and requests for reprints should be addressed to Isabelle Sermet-Gaudelus, M.D., Ph.D., Paris, France. Email: isabelle.sermet@aphp.fr.

References

1. Cohen-Cymberknoh M, Kerem E, Ferkol T, Elizur A. Airway inflammation in cystic fibrosis: molecular mechanisms and clinical implications. *Thorax* 2013;68:1157–1162.

2. Middleton PG, Mall MA, Drevínek P, Lands LC, McKone EF, Polineni D, et al.; VX17-445-102 Study Group. Elexacaftor-tezacaftor-ivacaftor for cystic fibrosis with a single Phe508del allele. *N Engl J Med* 2019;381:1809–1819.

3. McElvaney OJ, Gunaratnam C, Reeves EP, McElvaney NG. A specialized method of sputum collection and processing for therapeutic interventions in cystic fibrosis. *J Cyst Fibros* 2019;18:203–211.

4. 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. *Am J Respir Crit Care Med* 2019;199:873-881.

5. Lepissier A, Addy C, Hayes K, Noel S, Bui S, Burgel PR, et al. Inflammation biomarkers in sputum for clinical trials in cystic fibrosis: current understanding and gaps in knowledge. *J Cyst Fibros* 2022;21: 691-706.

6. Harris JK, Wagner BD, Zemanick ET, Robertson CE, Stevens MJ, Heltshe SL, et al. Changes in airway microbiome and inflammation with ivacaftor treatment in patients with cystic fibrosis and the G551D mutation. *Ann Am Thorac Soc* 2020;17:212-220.

7. Rowe SM, Heltshe SL, Gonska T, Donaldson SH, Borowitz D, Gelfond D, et al.; GOAL Investigators of the Cystic Fibrosis Foundation Therapeutics Development Network. Clinical mechanism of the cystic fibrosis transmembrane conductance regulator potentiator ivacaftor in G551D-mediated cystic fibrosis. *Am J Respir Crit Care Med* 2014;190:175-184.

8. Hisert KB, Heltshe SL, Pope C, Jorth P, Wu X, Edwards RM, et al. Restoring cystic fibrosis transmembrane conductance regulator function reduces airway bacteria and inflammation in people with cystic fibrosis and chronic lung infections. *Am J Respir Crit Care Med* 2017;195: 1617-1628.

9. Hayes E, Murphy MP, Pohl K, Browne N, McQuillan K, Saw LE, et al. Altered degranulation and pH of neutrophil phagosomes impacts antimicrobial efficiency in cystic fibrosis. *Front Immunol* 2020;11: 600033.

10. Sly PD, Gangell CL, Chen L, Ware RS, Ranganathan S, Mott LS, et al.; AREST CF Investigators. Risk factors for bronchiectasis in children with cystic fibrosis. *N Engl J Med* 2013;368:1963-1970.

Copyright © 2023 by the American Thoracic Society

<figure><img src="image_4.png" /></figure>

Check for updates

## Targeting Pulmonary Hypertension Caused by Pulmonary Fibrosis: A Promising Natural Killer T Cell-based Therapy

Zi-Hao Wang and Qiong Zhou

Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

ORCID IDs: 0000-0002-1069-4099 (Z.-H.W.); 0000-0002-3120-3017 (Q.Z.).

To the Editor:

We read the article by Jandl and colleagues, recently published in the *Journal*, with immense interest (1). In this study, Jandl and colleagues revealed the important role of natural killer T (NKT) cells in pulmonary hypertension (PH) caused by pulmonary fibrosis. Compared with patients who have interstitial lung disease (ILD) without PH and donor lung controls, patients with PH-ILD had significantly increased type I collagen deposition in lung tissues and decreased CD3⁺CD56⁺ NKT-like cells in pulmonary arteries (1). In a bleomycin-induced mouse model of pulmonary fibrosis, the authors showed potent antifibrotic efficacy of KRN7000

(α-Galactosylceramide; α-GalCer) (1), the classic agonist for invariant NKT (iNKT) cells (2).

The authors then detected decreased STAT1 expression in pulmonary artery smooth cells (PASMCs) and isolated pulmonary arteries from patients with PH-ILD. Coculture of α-GalCer-treated peripheral blood mononuclear cells and PASMCs significantly decreased (transforming growth factor (TGF)-β-induced type I collagen deposition in PASMCs. Jandl and colleagues found that CXCL9 expression was decreased in the plasma and lung tissue of patients with PH-ILD. Furthermore, the authors demonstrated that short-term treatment with α-GalCer significantly increased CXCL9 secretion in human NKT-like cells. *In vitro* CXCL9 treatment also inhibited TGF-β-induced type I collagen deposition in human PASMCs. The strengths of this study lie in the use of patient samples and mouse models for *in vivo* and *in vitro* experiments, as well as the potential for the translational treatment of PH-ILD with α-GalCer.

Human CD3⁺CD56⁺ NKT-like cells are known to be heterogenous, containing CD4⁺, CD8⁺, TCRγδ⁺, mucosal-associated invariant T-type cells, and a few iNKT cells (3). Also, murine CD3⁺NK1.1⁺ cells contain mucosal-associated invariant T-type cells and activated CD8⁺ T cells. iNKT cells in both mice and humans are defined as CD3⁺ T cells with semiinvariant T cell receptors (TCRs) that utilize invariant TCR-α chains based on mouse Vα14-Jα18 and human Vα24-Jα18 and recognize CD1d-presented lipids such as α-GalCer (4). In humans, they can be detected with anti-Vα24 antibodies or CD1d tetramers loaded with α-GalCer or an equivalent. In mice, there are no useful antibodies to Vα14 or invariant TCRs, and a convincing way to define them is by using CD1d tetramers loaded with α-GalCer or a homolog. Markers such as CD56 and CD161 in humans and NK1.1 in mice are also activation markers expressed on activated conventional T cells (5). Therefore, the expression of CD56 and NK1.1 molecules is present not only on NKT cells but also on other effector cells. A growing number of studies have shown that NKT cell-based therapy holds great promise in different diseases, especially cancer (6). The study by Jandl and colleagues is the first to demonstrate the essential role of NKT cells in the pathobiology of PH. However, the study still has some limitations. As the authors mentioned, NKT cell subpopulations should also be analyzed using more generally recognized markers, such as CD1d tetramers loaded with α-GalCer. Notably, this research enriches the field of NKT cells and provides a new NKT cell-based target for controlling the progression of PH. ■

**Author disclosures** are available with the text of this letter at www.atsjournals.org.

Correspondence and requests for reprints should be addressed to Qiong Zhou, M.D., Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Email: zhouqiongtj@126.com.

## References

1. Jandl K, Marsh LM, Mutgan AC, Crmkovic S, Valzano F, Zabini D, et al. Impairment of the NKT-STAT1-CXCL9 axis contributes to vessel fibrosis in pulmonary hypertension caused by lung fibrosis. *Am J Respir Crit Care Med* 2022;206:981-998.

2. Crosby CM, Kronenberg M. Tissue-specific functions of invariant natural killer T cells. *Nat Rev Immunol* 2018;18:559-574.

<footnote>ªThis article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern (dgern@thoracic.org).</footnote>

<footnote>Originally Published in Press as DOI: https://doi.org/10.1164/rccm. 202211-2134LE on January 13, 2023</footnote>