2.11.       Data and Statistical Analysis 
The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology 29. All data are expressed as mean ± SEM, where N is the number of independent measures. Data were analysed using GraphPad Prism 8 software (San Diego, California). Data distribution was determined using Shapiro-Wilk test. For comparisons of 2 independent groups, a Student’s t-test or Mann Whitney test was used. For comparison of multiple independent groups, one-way analysis of variance (ANOVA) or a Kruskal Wallis test was used, followed by a Tukey, Dunnett or Dunn post-hoctest. Differences were considered significant at p  0.05. 
3.    Results
3.1. The pulmonary phenotype during HF is characterised by vascular remodelling and myeloid cell infiltration
Twelve weeks after MI, mice presented with cardiac dysfunction evidenced by significantly reduced EF (HF: 37.5% ± 9.4% vs. sham: 63.8% ± 5.9%;Supplemental Table 4) and pulmonary structural alterations confined to the vasculature. HF mice exhibited markedly thicker blood vessel walls (Fig. 1A, B)and higher smooth muscle actin (SMA) mRNA (Supplemental Fig. 3)and protein levels (Fig. 1C) compared to sham-operated controls. HF lungs did not differ macroscopically nor showed signs of fibrosis demonstrated by the lack of collagen accumulation assessed by Masson trichrome staining (Fig. 1D, E) and hydroxyproline quantification (Fig. 1F). 
The apparent vascular remodelling was accompanied by higher monocyte/macrophage association with vascular structures in HF lungs as illustrated by monocyte/macrophage (MOMA) immunostaining in lung slices (Fig.2A, B). Flow cytometric immune cell profiling of the HF lung revealed significantly higher cell numbers of CD45hiLy6C+SiglecF-cells (Fig. 2C, E) and CD45hiLy6ChiSiglecF-cells in HF compared to sham mice (Fig. 2D, E),resembling infiltrating macrophages and pro-inflammatory monocytes. When analysing the activation profile of F4/80+macrophages, we observed significantly higher cell numbers of classically-activated CD80+macrophages in HF lungs (Fig. 2F), indicative of a shift to a pro-inflammatory phenotype within the macrophage population. This increase was mainly driven by non-alveolar (SiglecF-) macrophages (Fig. 2G, I) as no difference was observed in the alveolar (SiglecF+) macrophage population (Fig. 2H, I). 
 
3.2. Reduced pulmonary CFTR expression is a hallmark of the HF lung
The accumulation of non-alveolar classically-activated macrophages (CD80+SiglecF-) associated with markedly higher TNF-α protein levels during HF compared to sham lungs (Fig. 3A). Since TNF-α potently reduces CFTR surface expression in different cell types 5, 30, we determined cell surface-specific CFTR expression in the lung by performing a flow cytometry-based CFTR staining approach with an antibody targeting membrane-associated, mature CFTR protein 31(Supplemental Table 1). The overall number of surface-CFTR+cells was significantly reduced in lungs during HF (Fig. 3B, C), which coincided with significantly lower expression levels of membrane-bound CFTR assessed by western blotting (Fig. 3D). Interestingly, cell-specific CFTR assessment revealed lower CFTR positivity in SiglecF-non-alveolar macrophages (resembling an infiltrating pro-inflammatory subset) during HF (Fig. 3E, G), which coincided with higher CD80 positivity in this subset (see Fig. 2G). In contrast, no difference in the percentage of CFTR+SiglecF+alveolar macrophage population (resembling resident macrophages) was observed in HF (Fig. 3F, G). Different from their non-alveolar counterparts, alveolar macrophages did not upregulate CD80 after MI (see Fig. 2H).
 
3.3. Pharmacological CFTR correction mitigates structural changes in the HF lung
In attempt to improve altered CFTR expression in HF mice, we subjected a group of HF mice to CFTR corrector treatment using Lumacaftor (Lum). Lum acts as a chaperone improving CFTR protein folding and transport to the cell membrane and hence, increases cell surface CFTR protein expression 32, 33. Systemic (i.p.) Lum administration 10 weeks post-MI did not affect heart function (Supplemental Table 4), while significantly increasing the number of CFTR+cells in the HF lung (Fig. 4A, B). Similarly, western blot evaluation confirmed that the membrane-specific CFTR protein expression reached sham levels after two weeks of CFTR corrector treatment (Fig. 4C). The overall number of CFTR+cells was not further enhanced by lung-specific, orotracheal (o.t.) Lum instillation (Fig. 4D). However, o.t. treatment resulted in significantly higher CFTR expression on the cell surface of CFTR+lung cells as evidenced by increased median fluorescence intensity (MFI) in the o.t.-treated lungs compared to lungs from i.p.-treated HF mice (Fig. 4E, F). Although not significant, we noted higher CFTR protein expression by western blotting with o.t. application of Lum (Fig. 4G). 
CFTR correction attenuated alteration of the pulmonary vascular structure in the HF lung. Lum application mitigated the HF-associated thickening of pulmonary blood vessel walls (Fig 5A & Supplemental Fig. 4) and led to significantly lower SMA protein levels (Fig. 5B). This treatment effect was independent of application route supported by similar vessel wall thickness (Fig. 5C) and SMA protein expression (Fig. 5D) after both i.p. and o.t. Lum treatment. Immunofluorescent assessment of MOMA+cell distribution in lung slices verified an attenuation of the HF-associated elevation of monocytes/macrophages (see Fig. 2A) within and around the vessels after Lum treatment (Fig. 5E, F).  
 
3.4. Pharmacological CFTR correction promotes an anti-inflammatory phenotype of macrophages in the HF lung
Considering the high CFTR positivity of peripheral and pulmonary monocytes and macrophages (Supplemental Fig. 5A, 5B), we explored the effects of pharmacological CFTR correction on macrophages in the lung. Both systemic and lung-specific Lum administration significantly increased the overall number of pulmonary macrophages (Supplemental Fig. 6A, D) with larger effects after o.t. application. The treatment-associated increase of overall pulmonary macrophage counts was mainly mediated by increases of non-alveolar macrophages (Supplemental Fig. 6B, D), which were more pronounced after o.t. application. In contrast to systemic administration, o.t.-administered Lum markedly augmented the number of alveolar macrophages (Supplemental Fig. 6C, D). To understand whether this increase in macrophages was beneficial or rather detrimental, we explored macrophage activation profiles by determining the proportion of classically- (CD80+) and alternatively- (CD206+) activated cells within the pulmonary macrophage population. The HF-associated augmentation of classically-activated macrophages was alleviated by therapeutic Lum administration irrespective of application route (Fig. 6A). Likewise, therapeutic CFTR correction significantly attenuated the HF-associated increase of non-alveolar CD80+macrophages (Fig. 6B). Interestingly, o.t.-treated HF lungs presented with markedly higher proportions of CD80+alveolar macrophages (Fig. 6C), suggesting an application-induced pro-inflammatory response. In contrast to CD80+macrophages, Lum induced higher proportions of alternatively-activated (CD206+) macrophages overall (Fig. 6D) as well as alveolar (Fig. 6E) and non-alveolar (Fig. 6F) irrespective of treatment route (Fig. 6, Supplemental Fig. 7and Supplemental Table 5).This is corroborated by increased pulmonary IL-10 mRNA expression after systemic Lum administration (SupplementalFig. 8). In vitro, murine macrophages (RAW246.7 cells) presented with reduced CFTR positivity after PMA-induced activation, which was attenuated by CFTR correction with Lum (Supplemental Fig. 9), suggesting an interplay between CFTR surface expression and macrophage activation.
 
4.    Discussion
This study describes an apparent lung phenotype during experimental HF characterised by vascular remodelling and pronounced tissue inflammation. Specifically, our data suggest that the elevation of classically-activated non-alveolar macrophages coincides with a cell-specific reduction of pulmonary CFTR expression. In accordance, we show that pharmacological correction of CFTR, which increases the proportion of CFTR+cells in the lung, diminishes the HF-associated elevation of classically-activated non-alveolar macrophages, and normalises vessel wall thickness within the lungs of HF mice. Our data suggest pharmacological CFTR correction as promising approach to alleviate HF-induced inflammation in the lung.
The manifestation of HF in the lung is well-established. However, difficulties in the treatment of HF patients with chronic lung phenotypes remain, as standard therapies are often complicated by contraindications. Here, we verify a HF-mediated CFTR downregulation in the lung 5, 13, a concept that may provide new mechanism-based treatment options for HF patients with pulmonary complications. Given the increasing evidence for an acquired CFTR dysfunction not only during HF but also in classic chronic lung diseases such as COPD and asthma 34, the indication that CFTR modulators may be useful therapeutics in the treatment of acquired CFTR abnormalities is certainly of interest to the field. First trials verified efficacy of the CFTR potentiator ivacaftor in COPD patients with chronic bronchitis 35. Here, we describe beneficial effects of CFTR correction with Lum on lung inflammation and associated structural alterations during experimental HF. Specifically, Lum therapy attenuated the HF-associated increase in small vessel wall thickness, indicating beneficial effects on pulmonary arteriopathy, which often accompanies HF in patients with chronic left ventricular dysfunction 36, generally associating with increased risk of pulmonary complications and hence, overall poor disease outcome. Despite thickened vessel walls in the HF lung, we did not observe higher collagen accumulation within HF lungs or around the pulmonary vasculature. In our experiments, we aim at obtaining physiological values for animal ventilation during surgery to avoid ventilator-induced lung injury 37, which cannot be excluded from other studies that reported additional structural alterations and higher collagen content in HF lungs in mice with comparable EF 38, 39
         Inflammation is a key player in both chronic heart and lung diseases and critically contributes to vasculopathies. Here, we find increased numbers of pro-inflammatory monocytes/macrophages infiltrating the HF lung and an accumulation of monocytes/macrophages around the pulmonary vasculature, suggesting inflammation-associated vascular remodelling. Monocytes/macrophages have been shown to be among the primary effectors of inflammation in pulmonary lesions, and lung interstitial macrophages play a major role in lung inflammation and dysfunction in several diseases. Monocytes expressing certain chemokine receptors have been shown to differentiate into interstitial perivascular macrophages, which secrete pro-inflammatory cytokines and contribute to vascular remodelling 40. Whether changes in CFTR surface expression on circulating monocytes/macrophages mediates similar effects is an interesting question, especially considering their (1) relatively high CFTR positivity compared to other immune cells, (2) reported increased secretion of pro-inflammatory cytokines after pharmacological CFTR inhibition in macrophages 18,the herein observed (3) activation-induced CFTR surface reduction on macrophages and (4) reduction of CFTR+SiglecF-non-alveolar macrophages. Further cell type-specific investigations to characterise the CFTR expression in different cell types after MI could give insight into which cells are mainly affected and benefit from CFTR corrector treatment.
HF leads to systemic TNF-α elevation in mice and men 5, 25, 41, which negatively affects target organs, including the lung 41. We previously showed that TNF-α sequestration with Etanercept attenuated the HF-associated reduction of pulmonary CFTR protein expression 5. TNF-α was shown to mediate reduction of CFTR expression on the surface of different cell types 5, 30, suggesting that the herein detected HF-associated augmentation of pulmonary TNF-α might be directly linked to the observed overall CFTR downregulation in the HF lung. TNF-α, amongst other pro-inflammatory cytokines, induces M1-like macrophage phenotypes 42and is secreted by classically-polarised CD80+macrophages 43, which accumulate in the HF lung in our model. TNF-α sequestration using Etanercept was shown to reduce M1-type markers supported by decreases of CD40 and CD80 surface markers and increased expression of M2-type markers in human monocyte-derived macrophages 44. Here, we find a similar lowering of CD80+non-alveolar macrophages in the HF lung after Lum therapy, suggesting an intimate link between CFTR signalling and inflammation. Although direct Lum application to the lung resulted in higher CFTR expression on pulmonary CFTR+cells, supporting higher corrector efficacy, increased CD80+alveolar macrophage numbers that were observed with this treatment regimen may limit long-term benefits of lung-specific Lum application. CFTR corrector-induced increases of IL-10 in combination with the elevation of CD206+cells in our model are suggestive of an involvement of CFTR in macrophage phenotype switching that promote a more restorative environment 43. An alternative activation of human monocytes from CF patients after CFTR correction as evidenced by increased IL-10 secretion 45corroborate our findings. Since CFTR alterations in pulmonary macrophages and monocyte-derived macrophages present with an exaggerated cytokine response to bacterial lipopolysaccharide 20altered bactericidal activity 46, and adhesion 47, a direct role of CFTR in lung inflammation during HF is likely. 
 
5.    Summary and Conclusion
HF presents with an apparent lung phenotype characterised by inflammation and thickened walls of small vessels within the lung and an elevation of classically-activated non-alveolar macrophages that coincides with lower CFTR positivity in this immune cell subset. Pharmacological CFTR correction with Lum lowers HF-associated pro-inflammatory macrophage numbers, while promoting an alternatively-activated phenotype and attenuates vascular structural alterations within the HF lung. Collectively, these data suggest pharmacological CFTR correction as promising approach to mitigate HF-induced pulmonary inflammation and associated structural alterations. 
 
 
Funding: This work was supported by the following funding sources:Knut and Alice Wallenberg foundation [F 2015/2112 to A.M.];Swedish Research Council (VR) [2017-01243 to A.M.]; German Research Foundation (DFG) [ME 4667/2-1 to A.M.]; Åke Wibergs Stiftelse [M19-0380 to A.M.]; Albert Påhlssons Stiftelse [120482 to AM]; Inger Bendix Stiftelse [2019-10 to A.M.]; Stohnes Stiftelse [A.M.]; Crafoord Foundation [20190782to F.E.U.]; Royal Physiographic Society Lund [39716 and 40682to F.E.U.]; STINT [MG19-8469 to A.M.];and Lund University [to A.M.]. 
 
Author contributions: Conceptualization, AM; methodology, FEU and AM; validation, FEU and AM; formal analysis, FEU and LV; resources, AM; data curation, FEU and LV; writing—original draft preparation, FEU and AM; writing—review and editing, FEU, LV and AM.; visualization, FEU, LV and AM; supervision, AM; project administration, AM; funding acquisition, FEU and AM. All authors have read and agreed to the published version of the manuscript.
 
Ethics approval statement: This investigation conforms to the Guide for Care and Use of Laboratory Animals published by the European Union (Directive 2010/63/EU) and with the ARRIVE guidelines. All animal care and experimental protocols were approved by the institutional animal ethics committee at Lund University (Dnr.: 5.8.18-08003/2017, 5.8.18-04938/2021) and were conducted in accordance with European animal protection laws.
 
Acknowledgements
The authors thank the Knut and Alice Wallenberg foundation for generous support and the Lund University BioImaging Center (LBIC), Lund University is gratefully acknowledged for providing experimental resources. We further like to thank René In ’t Zandt and Michael Gottschalk from LBIC for help with the cardiac MRI analyses, Dr. Steffen-Sebastian Bolz and Dr. Darcy Lidington (both Department of Physiology, University of Toronto, Toronto, Canada) for providing lung tissue, Dr. Nicholas Don-Doncow (Department of Experimental Medical Science, Lund University, Lund, Sweden) for discussing FACS panel design, Dr. Gunilla Westergren-Thorsson (Department of Experimental Medical Science, Lund University, Lund, Sweden) for access to the microtome, Dr. Jonas Erjefält (Department of Experimental Medical Science, Lund University Sweden) for access to the paraffin embedding machine, Dr. Darcy Wagner (Department of Experimental Medical Science, Lund University, Lund Sweden) for access to the tissue processor, Dr. Ulrica Englund Johansson(Department of Clinical Sciences Section IV, Lund University, Lund Sweden) for access to the Zeiss Axio Imager, and Dr. Björn Olde (Department of Clinical Sciences Section II, Lund University Sweden) for the RAW246.7 cells.
 
Conflict of interest: The authors declare no conflict of interest.
 
Data availability: Data underlying this article are available in the article and in its online supplementary material or are available from the corresponding author upon reasonable request.
 
Permission to reproduce material from other sources: Only original material was used.
 
6.    References:
1.      Bueno H, Moura B, Lancellotti P, Bauersachs J. The year in cardiovascular medicine 2020: heart failure and cardiomyopathies. Eur Heart J2021;42:657-670.
2.      Savarese G, Lund LH. Global Public Health Burden of Heart Failure. Card Fail Rev 2017;3:7-11.
3.      Moradi M, Daneshi F, Behzadmehr R, Rafiemanesh H, Bouya S, Raeisi M. Quality of life of chronic heart failure patients: a systematic review and meta-analysis. Heart Fail Rev 2020;25:993-1006.
4.      Harjola VP, Mullens W, Banaszewski M, Bauersachs J, Brunner-La Rocca HP, Chioncel O, Collins SP, Doehner W, Filippatos GS, Flammer AJ, Fuhrmann V, Lainscak M, Lassus J, Legrand M, Masip J, Mueller C, Papp Z, Parissis J, Platz E, Rudiger A, Ruschitzka F, Schafer A, Seferovic PM, Skouri H, Yilmaz MB, Mebazaa A. Organ dysfunction, injury and failure in acute heart failure: from pathophysiology to diagnosis and management. A review on behalf of the Acute Heart Failure Committee of the Heart Failure Association (HFA) of the European Society of Cardiology (ESC). Eur J Heart Fail 2017;19:821-836.
5.      Meissner A, Yang J, Kroetsch JT, Sauve M, Dax H, Momen A, Noyan-Ashraf MH, Heximer S, Husain M, Lidington D, Bolz SS. Tumor necrosis factor-alpha-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure. Circulation 2012;125:2739-2750.
6.      Lidington D, Fares JC, Uhl FE, Dinh DD, Kroetsch JT, Sauve M, Malik FA, Matthes F, Vanherle L, Adel A, Momen A, Zhang H, Aschar-Sobbi R, Foltz WD, Wan H, Sumiyoshi M, Macdonald RL, Husain M, Backx PH, Heximer SP, Meissner A, Bolz SS. CFTR Therapeutics Normalize Cerebral Perfusion Deficits in Mouse Models of Heart Failure and Subarachnoid Hemorrhage. JACC Basic Transl Sci 2019;4:940-958.
7.      Meissner A, Visanji NP, Momen MA, Feng R, Francis BM, Bolz SS, Hazrati LN. Tumor Necrosis Factor-alpha Underlies Loss of Cortical Dendritic Spine Density in a Mouse Model of Congestive Heart Failure. J Am Heart Assoc 2015;4.
8.      Chung KF. Cytokines in chronic obstructive pulmonary disease. The European respiratory journal Supplement 2001;34:50s-59s.
9.      Mann DL. Inflammatory mediators and the failing heart: past, present, and the foreseeable future. Circ Res 2002;91:988-998.
10.    Mann DL. Innate immunity and the failing heart: the cytokine hypothesis revisited. Circ Res 2015;116:1254-1268.
11.    Mann DL, McMurray JJ, Packer M, Swedberg K, Borer JS, Colucci WS, Djian J, Drexler H, Feldman A, Kober L, Krum H, Liu P, Nieminen M, Tavazzi L, van Veldhuisen DJ, Waldenstrom A, Warren M, Westheim A, Zannad F, Fleming T. Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL). Circulation 2004;109:1594-1602.
12.    Yang J, Noyan-Ashraf MH, Meissner A, Voigtlaender-Bolz J, Kroetsch JT, Foltz W, Jaffray D, Kapoor A, Momen A, Heximer SP, Zhang H, van Eede M, Henkelman RM, Matthews SG, Lidington D, Husain M, Bolz SS. Proximal cerebral arteries develop myogenic responsiveness in heart failure via tumor necrosis factor-alpha-dependent activation of sphingosine-1-phosphate signaling. Circulation2012;126:196-206.
13.    Uhl FE, Vanherle L, Matthes F, Meissner A. Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure. Int J Mol Sci 2022;23.
14.    Dransfield MT, Wilhelm AM, Flanagan B, Courville C, Tidwell SL, Raju SV, Gaggar A, Steele C, Tang LP, Liu B, Rowe SM. Acquired cystic fibrosis transmembrane conductance regulator dysfunction in the lower airways in COPD. Chest 2013;144:498-506.
15.    Rab A, Rowe SM, Raju SV, Bebok Z, Matalon S, Collawn JF. Cigarette smoke and CFTR: implications in the pathogenesis of COPD.Am J Physiol Lung Cell Mol Physiol 2013;305:L530-541.
16.    Engelhardt JF, Zepeda M, Cohn JA, Yankaskas JR, Wilson JM. Expression of the cystic fibrosis gene in adult human lung. J Clin Invest 1994;93:737-749.
17.    Yoshimura K, Nakamura H, Trapnell BC, Chu CS, Dalemans W, Pavirani A, Lecocq JP, Crystal RG. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. Nucleic Acids Res 1991;19:5417-5423.
18.    Di A, Brown ME, Deriy LV, Li C, Szeto FL, Chen Y, Huang P, Tong J, Naren AP, Bindokas V, Palfrey HC, Nelson DJ. CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 2006;8:933-944.
19.    Zaman MM, Gelrud A, Junaidi O, Regan MM, Warny M, Shea JC, Kelly C, O'Sullivan BP, Freedman SD. Interleukin 8 secretion from monocytes of subjects heterozygous for the deltaF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered. Clin Diagn Lab Immunol 2004;11:819-824.
20.    Bruscia EM, Zhang PX, Ferreira E, Caputo C, Emerson JW, Tuck D, Krause DS, Egan ME. Macrophages directly contribute to the exaggerated inflammatory response in cystic fibrosis transmembrane conductance regulator-/- mice. Am J Respir Cell Mol Biol 2009;40:295-304.
21.    Raju SV, Jackson PL, Courville CA, McNicholas CM, Sloane PA, Sabbatini G, Tidwell S, Tang LP, Liu B, Fortenberry JA, Jones CW, Boydston JA, Clancy JP, Bowen LE, Accurso FJ, Blalock JE, Dransfield MT, Rowe SM. Cigarette smoke induces systemic defects in cystic fibrosis transmembrane conductance regulator function. Am J Respir Crit Care Med 2013;188:1321-1330.
22.    Marklew AJ, Patel W, Moore PJ, Tan CD, Smith AJ, Sassano MF, Gray MA, Tarran R. Cigarette Smoke Exposure Induces Retrograde Trafficking of CFTR to the Endoplasmic Reticulum. Sci Rep 2019;9:13655.
23.    Tadic M, Cuspidi C, Plein S, Belyavskiy E, Heinzel F, Galderisi M. Sex and Heart Failure with Preserved Ejection Fraction: From Pathophysiology to Clinical Studies. J Clin Med 2019;8.
24.    Savarese G, D'Amario D. Sex Differences in Heart Failure. Adv Exp Med Biol 2018;1065:529-544.
25.    Yagi K, Lidington D, Wan H, Fares JC, Meissner A, Sumiyoshi M, Ai J, Foltz WD, Nedospasov SA, Offermanns S, Nagahiro S, Macdonald RL, Bolz SS. Therapeutically Targeting Tumor Necrosis Factor-alpha/Sphingosine-1-Phosphate Signaling Corrects Myogenic Reactivity in Subarachnoid Hemorrhage. Stroke 2015;46:2260-2270.
26.    Hu L, Chen Z, Li L, Jiang Z, Zhu L. Resveratrol decreases CD45(+) CD206(-) subtype macrophages in LPS-induced murine acute lung injury by SOCS3 signalling pathway. J Cell Mol Med 2019;23:8101-8113.
27.    Huaux F, De Gussem V, Lebrun A, Yakoub Y, Palmai-Pallag M, Ibouraadaten S, Uwambayinema F, Lison D. New interplay between interstitial and alveolar macrophages explains pulmonary alveolar proteinosis (PAP) induced by indium tin oxide particles. Arch Toxicol 2018;92:1349-1361.
28.    Misharin AV, Morales-Nebreda L, Mutlu GM, Budinger GR, Perlman H. Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. Am J Respir Cell Mol Biol 2013;49:503-510.
29.    Curtis MJ, Alexander S, Cirino G, Docherty JR, George CH, Giembycz MA, Hoyer D, Insel PA, Izzo AA, Ji Y, MacEwan DJ, Sobey CG, Stanford SC, Teixeira MM, Wonnacott S, Ahluwalia A. Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. Br J Pharmacol 2018;175:987-993.
30.    Malik FA, Meissner A, Semenkov I, Molinski S, Pasyk S, Ahmadi S, Bui HH, Bear CE, Lidington D, Bolz SS. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity. PLoS One 2015;10:e0130313.
31.    Csanady L, Vergani P, Gadsby DC. Structure, Gating, and Regulation of the Cftr Anion Channel. Physiol Rev 2019;99:707-738.
32.    Gentzsch M, Mall MA. Ion Channel Modulators in Cystic Fibrosis. Chest 2018;154:383-393.
33.    Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, Decker CJ, Miller M, McCartney J, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu PA. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A 2011;108:18843-18848.
34.    Solomon GM, Fu L, Rowe SM, Collawn JF. The therapeutic potential of CFTR modulators for COPD and other airway diseases. Curr Opin Pharmacol 2017;34:132-139.
35.    Solomon GM, Hathorne H, Liu B, Raju SV, Reeves G, Acosta EP, Dransfield MT, Rowe SM. Pilot evaluation of ivacaftor for chronic bronchitis. Lancet Respir Med 2016;4:e32-33.
36.    Gerges M, Gerges C, Pistritto AM, Lang MB, Trip P, Jakowitsch J, Binder T, Lang IM. Pulmonary Hypertension in Heart Failure. Epidemiology, Right Ventricular Function, and Survival. Am J Respir Crit Care Med 2015;192:1234-1246.
37.    Hamlington KL, Smith BJ, Dunn CM, Charlebois CM, Roy GS, Bates JHT. Linking lung function to structural damage of alveolar epithelium in ventilator-induced lung injury. Respir Physiol Neurobiol 2018;255:22-29.
38.    Dayeh NR, Tardif JC, Shi Y, Tanguay M, Ledoux J, Dupuis J. Echocardiographic validation of pulmonary hypertension due to heart failure with reduced ejection fraction in mice. Sci Rep 2018;8:1363.
39.    Jasmin JF, Mercier I, Hnasko R, Cheung MW, Tanowitz HB, Dupuis J, Lisanti MP. Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression. Cardiovasc Res 2004;63:747-755.
40.    Florentin J, Coppin E, Vasamsetti SB, Zhao J, Tai YY, Tang Y, Zhang Y, Watson A, Sembrat J, Rojas M, Vargas SO, Chan SY, Dutta P. Inflammatory Macrophage Expansion in Pulmonary Hypertension Depends upon Mobilization of Blood-Borne Monocytes. J Immunol 2018;200:3612-3625.
41.    Schumacher SM, Naga Prasad SV. Tumor Necrosis Factor-alpha in Heart Failure: an Updated Review. Curr Cardiol Rep 2018;20:117.
42.    Arora S, Dev K, Agarwal B, Das P, Syed MA. Macrophages: Their role, activation and polarization in pulmonary diseases. Immunobiology 2018;223:383-396.
43.    Shapouri-Moghaddam A, Mohammadian S, Vazini H, Taghadosi M, Esmaeili SA, Mardani F, Seifi B, Mohammadi A, Afshari JT, Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 2018;233:6425-6440.
44.    Degboe Y, Rauwel B, Baron M, Boyer JF, Ruyssen-Witrand A, Constantin A, Davignon JL. Polarization of Rheumatoid Macrophages by TNF Targeting Through an IL-10/STAT3 Mechanism. Front Immunol 2019;10:3.
45.    Jarosz-Griffiths HH, Scambler T, Wong CH, Lara-Reyna S, Holbrook J, Martinon F, Savic S, Whitaker P, Etherington C, Spoletini G, Clifton I, Mehta A, McDermott MF, Peckham D. Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis. Elife 2020;9.
46.    Kopp BT, Abdulrahman BA, Khweek AA, Kumar SB, Akhter A, Montione R, Tazi MF, Caution K, McCoy K, Amer AO. Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients. Biochem Biophys Res Commun 2012;424:221-227.
47.    Sorio C, Montresor A, Bolomini-Vittori M, Caldrer S, Rossi B, Dusi S, Angiari S, Johansson JE, Vezzalini M, Leal T, Calcaterra E, Assael BM, Melotti P, Laudanna C. Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency. Am J Respir Crit Care Med 2016;193:1123-1133.
 
 
7.    Figure Legends
Fig. 1: Heart failure-associated structural changes in the lung are confined to blood vessels. A)Representative Haematoxylin and Eosin (H&E) staining of lungs from sham and heart failure (HF) mice. Arrows indicate vessel walls; scale bar 20 µm. B)Quantification of wall thickness of small vessels in H&E-stained lung slices. C)Representative western blot and quantification of the smooth muscle actin (SMA) protein expression in lung tissue from sham and HF mice. D)Masson Trichrome (MTC) staining of lungs from sham and HF mice. Arrows indicate collagen, stained in blue; scale bar 20 µm. E)Qualitative quantification of the amount of collagen staining in MTC-stained lung sections. F)Quantification of hydroxyproline content of lung tissue from sham and HF mice. Data expressed as mean ± SEM. * denotes p ≤ 0.05 for single, unpaired comparisons.
 
Fig. 2:Heart failure associates with lung infiltration of CD80+pro-inflammatory macrophages. A) Representative images of lung sections from sham and heart failure (HF) mice that were stained for monocyte/macrophages (MOMA) in red, smooth muscle actin (SMA) in green, DAPI stained nuclei in blue. Arrows indicate vessel wall-associated MOMA positivity; scale bar 20 µm. B)Quantification of the percentage of MOMA positive cells in lung vessel walls. C)Flow cytometry results representing the number of CD45hiLy6C+SiglecF-and D)CD45hiLy6ChiSiglecF-macrophages. E)Representative dot blots of Ly6Cand SiglecF expression of F4/80+macrophages in the lung of sham and HF mice. Flow cytometric assessment of F)F4/80+CD80+classically activated macrophages, G)F4/80+CD80+SiglecF-classically-activated non-alveolar macrophages, and H)F4/80+CD80+SiglecF+classically-activated alveolar macrophages in lung of sham and HF mice. I)Representative dot blots of SiglecF and CD80 expression of F4/80+macrophages in the lung of sham and HF mice. Data expressed as mean ± SEM. * denotes p ≤ 0.05 for single, unpaired comparisons.
 
Fig. 3: Pulmonary tumour necrosis factor alpha increase is accompanied by decreased cystic fibrosis transmembrane regulator expression in the heart failure lung. A)Representative western blot and quantification of tumour necrosis factor alpha (TNF-α) expression in the lungs of sham and heart failure (HF) mice. B)Percentage of cystic fibrosis transmembrane regulator (CFTR) positive cells in lungs of sham and HF mice and C)representative dot plots. D)Representative western blot and quantification of CFTR protein expression in lungs of sham and HF mice. Flow cytometric assessment of proportion of E)CFTR+F4/80+SiglecF-non-alveolar macrophages and F)CFTR+F4/80+SiglecF+alveolar macrophages inlung of sham and HF mice. G)Representative histograms of CFTR+SiglecF-and CFTR+SiglecF+cells from naïve and HF mice. Data expressed as mean ± SEM. * denotes p ≤ 0.05 for single, unpaired comparisons.
 
Fig. 4: Systemic application of cystic fibrosis transmembrane regulator (CFTR) correctors increases pulmonary CFTR expression. A)Percentage of CFTR+cells in lungs of sham, heart failure (HF), and Lumacaftor (Lum) treated (intraperitoneally (i.p.)) HF mice and B)representative dot plots. C)Representative western blot and quantification of the CFTR expression in the lungs of sham, HF, and Lumacaftor treated (i.p.) HF mice. D) Percentage of CFTR+cells in the lungs of HF mice treated with Lumacaftor either i.p. or orotracheally (o.t.). E)Median fluorescence intensity and F)representative histograms of CFTR+cells in the lungs of HF mice treated with Lumacaftor either i.p. or o.t. G)Representative western blot and quantification of the CFTR expression in the lungs of HF mice treated with Lumacaftor either i.p. or o.t.  Data expressed as mean ± SEM. In (A)* denotes p ≤ 0.05 relative to sham, $ denotes p ≤ 0.05 relative to HF after one-way ANOVA followed by Tukey´s post-hoctesting; in (C), * denotes p ≤ 0.05 relative to sham, after one-way ANOVA followed by Dunnett´s post-hoctesting; in (E), $ denotes p ≤ 0.05 for single, unpaired comparisons.
 
Fig. 5: Cystic fibrosis transmembrane regulator correction mitigates heart failure-associated alteration of pulmonary vascular structure. A) Quantification of the vessel wall thickness of smaller vessels in the lungs of sham, heart failure (HF), and Lumacaftor (Lum) treated HF mice.  B) Repr esentative western blot and quantification of the smooth muscle actin (SMA) expression in lung tissue from sham, HF, and Lum treated HF mice. C)Quantification of the vessel wall thickness of smaller vessels in the lungs of Lum treated (intraperitoneally (i.p.) or orotracheally (o.t.)) HF mice. The dotted line indicates the level of HF mice. D)Representative western blot and quantification of SMA expression in lung tissue from Lum treated (i.p. and o.t.) HF mice. The dotted line indicates the level of HF mice. E)Representative images of lung sections from Lum treated (i.p. or o.t.) HF mice that were stained for monocyte/macrophages (MOMA, red) and SMA (green). DAPI stained nuclei in blue. Arrows indicate vessel wall-associated MOMA positivity; scale bar 20 µm. F)Quantification of the percentage of MOMA positive cells in lung vessel walls. The dotted line indicates the level of HF mice. Data expressed as mean ± SEM. * denotes p ≤ 0.05 relative to sham, $ denotes p ≤ 0.05 relative to HF after one-way ANOVA followed by Tukey’s post-hoctesting.
 
Fig. 6: Cystic fibrosis transmembrane regulator correction normalizes levels of non-alveolar macrophages and increases CD206+alveolar macrophages. Proportion of pulmonary F4/80+-macrophages in sham, heart failure (HF), and Lumacaftor (Lum) treated ((intraperitoneally (i.p.) or orotracheally (o.t.)) HF micepositive for A) CD80 andD)CD206+. Percentageofpulmonary non-alveolar F4/80+and SiglecFmacrophages in sham, HF, and Lum treated (i.p. and o.t.) HF mice positive for B) CD80+and E)CD206+. Percentage of pulmonary alveolar F4/80+and SiglecFmacrophages in sham, HF, and Lum treated (i.p. and o.t.) HF mice positive for C) CD80+and F) CD206+. Data expressed as mean ± SEM. In (A, B, D, F)* denotes p ≤ 0.05 relative to sham, $ denotes P ≤ 0.05 relative to HF after one-way ANOVA followed by Dunnett’s post-hoctesting relative to HF; in (C-E),* denotes p ≤ 0.05 relative to sham, $ denotes P ≤ 0.05 relative to HF after Kruskal Wallis followed by Dunn’s post-hoctesting.