Primary graft dysfunction (PGD) and chronic lung allograft dysfunction (CLAD) are critical challenges in lung transplantation. Dysregulated gene expression and epigenomic states in lung mesenchymal cells (MCs) play a key role in these conditions, but further work is needed to elucidate the biomarkers and molecular drivers. Single-cell multi-omic technologies offer an unprecedented opportunity to address this gap by jointly measuring gene expression and chromatin accessibility in diseased and healthy cells. We performed single-cell multi-omic profiling and genetic demultiplexing on MCs isolated from human bronchoalveolar lavage samples of lung transplant recipients with CLAD and PGD, compared with time-matched controls. A classification model trained on these profiles classified the disease status of individual cells with > 98% accuracy using a small set of signature genes. We further identified 8 subtypes of MCs each characterized by distinct transcriptomic and epigenomic signatures, including a CLAD-enriched subtype and a PGD-enriched subtype. The CLAD-enriched subtype expresses markers of fibrosis, while the PGD-enriched subtype expresses pro-inflammatory genes. Finally, we used co-accessibility and co-expression to identify C/EBPD, SOX4, and FOXP2 as key regulators of the genes and chromatin accessibility peaks that are dysregulated in the CLAD-enriched cell population. Knocking down C/EBPD with siRNA partially reverted the CLAD gene expression signature, confirming the importance of this factor in the dysregulated molecular state of CLAD-associated MCs. Additional scRNA-seq analysis of MCs from human lung autopsy samples showed that cultured MCs retained key transcriptomic signatures from their in vivo counterparts. Sub-clustering analysis identified a CLAD-enriched MC sub-cluster with high C/EBPD expression, further linking these cells to the fibrotic state seen in vitro. Our results provide deeper insights into the transcriptomic and epigenomic changes in post-transplant MCs, nominating biomarkers and disease-associated factors with implications for future therapeutic efforts. ### Competing Interest Statement The authors have declared no competing interest.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with hallmarks of fibroblastic foci and exuberant matrix deposition, unknown etiology, and ineffective therapies. Several profibrotic/proinflammatory pathways are implicated in accelerating tissue remodeling toward a honeycombed end-stage disease. NFAT1 is a transcriptional factor activated in IPF tissues. Nfat1-deficient mice subjected to chronic injury are protected against fibrosis independent of immune influxes, with suppression of profibrotic mesenchymal phenotypes including proliferation, differentiation, resistance to apoptosis, and autotaxin-related migration.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the leading long-term cause of poor outcomes after transplant and manifests by fibrotic remodeling of small airways and/or pleuroparenchymal fibroelastosis. This study evaluated the effect of pirfenidone on quantitative radiographic and pulmonary function assessment in patients with CLAD. METHODS: We performed a single-center, 6-month, randomized, placebo-controlled trial of pirfenidone in patients with CLAD. Randomization was stratified by CLAD phenotype. The primary outcome for this study was change in radiographic assessment of small airways disease, quantified as percentage of lung volume using parametric response mapping analysis of computed tomography scans (PRMfSAD); fSAD ); secondary outcomes included change in forced expiratory volume in 1 second (FEV1), 1 ), change in forced vital capacity (FVC), and change in radiographic quantification of parenchymal disease (PRMPD). PD ). Linear mixed models were used to evaluate the treatment effect on outcome measures. RESULTS: The goal enrollment of 60 patients was not met due to the coronavirus disease of 2019 pandemic, with 23 patients included in the analysis. There was no significant difference over the study period between the pirfenidone vs placebo groups with regards to the observed change in PRM fSAD (+4.2% vs -0.4%; p = 0.22), FEV1 1 (-3.5% vs -3.6%; p = 0.97), FVC (-1.9% vs -4.6%; p = 0.41), or PRMPD PD (-0.6% vs -2.5%; p = 0.30). The study treatment tolerance and adverse events were generally similar between the pirfenidone and placebo groups. CONCLUSIONS: Pirfenidone had no apparent impact on radiographic evidence of allograft dysfunction or pulmonary function decline in a single-center randomized trial of CLAD patients that did not meet enrollment goals but had an acceptable tolerance and side-effect profile. J Heart Lung Transplant 2024;43:1468-1477 (c) 2024 International Society for Heart and Lung Transplantation. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Purpose: Quantitative computed tomography (CT) scan features in lung transplant recipients with allograft dysfunction are associated with subsequent progression and mortality, but the quantitative CT scan profile of patients without allograft dysfunction is unknown.

Rationale: Chronic lung allograft dysfunction (CLAD) is the leading cause of death after lung transplant, and azithromycin has variable efficacy in CLAD. The lung microbiome is a risk factor for developing CLAD, but the relationship between lung dysbiosis, pulmonary inflammation, and allograft dysfunction remains poorly understood. Whether lung microbiota predict outcomes or modify treatment response after CLAD is unknown. Objectives: To determine whether lung microbiota predict post-CLAD outcomes and clinical response to azithromycin. Methods: Retrospective cohort study using acellular BAL fluid prospectively collected from recipients of lung transplant within 90 days of CLAD onset. Lung microbiota were characterized using 16S rRNA gene sequencing and droplet digital PCR. In two additional cohorts, causal relationships of dysbiosis and inflammation were evaluated by comparing lung microbiota with CLAD-associated cytokines and measuring ex vivo P. aeruginosa growth in sterilized BAL fluid. Measurements and Main Results: Patients with higher bacterial burden had shorter post-CLAD survival, independent of CLAD phenotype, azithromycin treatment, and relevant covariates. Azithromycin treatment improved survival in patients with high bacterial burden but had negligible impact on patients with low or moderate burden. Lung bacterial burden was positively associated with CLAD-associated cytokines, and ex vivo growth of P. aeruginosa was augmented in BAL fluid from transplant recipients with CLAD. Conclusions: In recipients of lung transplants with chronic rejection, increased lung bacterial burden is an independent risk factor for mortality and predicts clinical response to azithromycin. Lung bacterial dysbiosis is associated with alveolar inflammation and may be promoted by underlying lung allograft dysfunction.

BACKGROUND:The ScanCLAD study reported a lower incidence of chronic lung allograft dysfunction (CLAD) with the use of once-daily tacrolimus vs twice-daily cyclosporine. Using the International Society for Heart and Lung Transplantation (ISHLT) Thoracic Organ Transplant (TTX) Registry data, we evaluated the hypothesis that tacrolimus is superior to cyclosporine in real-world clinical practice. METHODS:This study is a retrospective cohort study of adult lung transplant recipients in the ISHLT registry from January 1, 2000, to June 30, 2018, with known CLAD status. The primary exposure variable was patients' maintenance calcineurin inhibitor (CNI) regimen captured at posttransplant discharge. The primary outcome variables were time to CLAD development (with death/retransplantation analyzed as a competing risk) and allograft survival (i.e., time to death/retransplant). RESULTS:Of the 57,403 adult lung transplant recipients in the registry, 22,222 had both CNI and CLAD data available. Of these, 19,698 (88.6%) received tacrolimus immediate release (IR), 2,477 (11.2%) received cyclosporine, and 47 (0.2%) received tacrolimus extended release (XR) for maintenance CNI. Receiving cyclosporine for maintenance immunosuppression (vs tacrolimus IR) was associated with an increased risk of developing CLAD (hazard ratio [HR] 1.16, 95% confidence interval [CI] 1.08-1.23, p < 0.001) and with an increased overall risk for death/retransplant (HR 1.16, 95% CI 1.09-1.23, p < 0.001). Receiving tacrolimus XR vs tacrolimus IR was not associated with differences in long-term posttransplant outcomes, although these analyses were limited by a small sample size. CONCLUSIONS:Patients receiving cyclosporine vs tacrolimus IR for maintenance calcineurin inhibition had an increased risk of CLAD and decreased overall allograft survival in the ISHLT TTX registry.

BackgroundLung transplantation remains the sole curative option for patients with idiopathic pulmonary fibrosis (IPF), but donor organs remain scarce, and many eligible patients die prior to transplant. Tools to optimize the timing of transplant referral are urgently needed.MethodsLeast absolute shrinkage and selection operator (LASSO) was applied to clinical and proteomic data generated as part of a prospective cohort study of interstitial lung disease (ILD) to derive clinical, proteomic, and multi-dimensional logit models of near-term death or lung transplant within 18 months of blood draw. Model fitted values were dichotomized at the point of maximal sensitivity and specificity and decision curve analysis used to select the best performing classifier. We then applied this classifier to independent IPF and non-IPF ILD cohorts to determine test performance characteristics. Cohorts were restricted to patients aged ≤72 years with body mass index 18-32 to increase likelihood of transplant eligibility.ResultsIPF derivation, IPF validation and non-IPF ILD validation cohorts consisted of 314, 105 and 295 patients, respectively. A multi-dimensional model comprising two clinical variables and 20 proteins outperformed stand-alone clinical and proteomic models. Following dichotomization, the multi-dimensional classifier predicted near-term outcome with 70% sensitivity and 92% specificity in the IPF validation cohort and 70% sensitivity and 80% specificity in the non-IPF ILD validation cohort.ConclusionsA multi-dimensional classifier of near-term outcome accurately discriminated this endpoint with good test performance across independent IPF and non-IPF ILD cohorts. These findings support refinement and prospective validation of this classifier in transplant eligible individuals.

The incidence of chronic lung allograft dysfunction (CLAD; the official term for chronic rejection) has remained unchanged for decades, and its effects are devastating: patients with CLAD report significant dyspnoea and have a decreased quality of life, and CLAD is the leading cause of death for patients who survive at least 1 year post transplantation. 1 Verleden GM Glanville AR Lease ED et al. Chronic lung allograft dysfunction: definition, diagnostic criteria, and approaches to treatment—a consensus report from the Pulmonary Council of the ISHLT. J Heart Lung Transplant. 2019; 38: 493-503 Summary Full Text Full Text PDF PubMed Scopus (434) Google Scholar , 2 Singer JP Singer LG Quality of life in lung transplantation. Semin Respir Crit Care Med. 2013; 34: 421-430 Crossref PubMed Scopus (58) Google Scholar Despite recognition of CLAD as the primary problem facing the field of lung transplantation, effective strategies to prevent or treat CLAD have not been definitively established. Effect of once-per-day tacrolimus versus twice-per-day ciclosporin on 3-year incidence of chronic lung allograft dysfunction after lung transplantation in Scandinavia (ScanCLAD): a multicentre randomised controlled trialImmunosuppression based on use of tacrolimus once per day significantly reduced the incidence of CLAD compared with use of ciclosporin twice per day. These findings support the use of tacrolimus as the first choice of calcineurin inhibitor after lung transplantation. Full-Text PDF

BACKGROUND: Reducing racial disparities in lung transplant outcomes is a current priority of providers, policymakers, and lung transplant centers. It is unknown how the combined effect of race and ethnicity, gender, and diagnosis group is associated with differences in 1-year mortality and 5-year survival.METHODS: This is a longitudinal cohort study using Standard Transplant Analysis Research files from the United Network for organ sharing. A total of 25,444 patients undergoing first time lung transplanta-tion between 2006 and 2019 in the United States. The primary exposures were lung transplant recipient race and ethnicity, gender, and primary diagnosis group at listing. Multivariable regression models and cox-proportional hazards models were used to determine adjusted 1-year mortality and 5-year survival.RESULTS: Overall, 25,444 lung transplant patients were included in the cohort including 15,160 (59.6%) men, 21,345 (83.9%) White, 2,318 (9.1%), Black and Hispanic/Latino (7.0%). Overall, men had a significant higher 1-year mortality than women (11.87%; 95% CI 11.07-12.67 vs 12.82%; 95% CI 12.20%-13.44%). Black women had the highest mortality of all race and gender combinations (14.51%; 95% CI 12.15%-16.87%). Black patients with pulmonary vascular disease had the highest 1-year mortality (19.77%; 95% CI 12.46%-27.08%) while Hispanic/Latino patients with obstructive lung disease had the lowest (7.42%; 95% CI 2.8%-12.05%). 5-year adjusted survival was highest among Hispanic/Latino patients (62.32%) compared to Black (57.59%) and White patients (57.82%).CONCLUSIONS: There are significant differences in 1-year and 5-year mortality between and within racial and ethnic groups depending on gender and primary diagnosis. This demonstrates the impact of social and clinical factors on lung transplant outcomes. J Heart Lung Transplant 2023;42:985-992 & COPY; 2023 International Society for Heart and Lung Transplantation. All rights reserved.

"Pretransplant Antifibrotic Therapy Is Associated with Resolution of Primary Graft Dysfunction." Annals of the American Thoracic Society, 19(2), pp. 335–338

PurposeChronic lung allograft dysfunction (CLAD) is the leading long-term contributor to mortality after lung transplant. The lung microbiome predicts subsequent development of CLAD, but whether lung bacteria differ between CLAD phenotypes is unknown.MethodsUsing a biorepository of acellular bronchoalveolar lavage (BAL) fluid, we identified specimens which were collected within 90 days of CLAD onset. CLAD phenotype was assigned based on the presence of absence of obstruction, restriction, and computed tomography (CT) scan opacities, in accordance with ISHLT guidelines. Bacterial DNA burden was measured with BioRad QX200 Droplet Digital PCR and 16S rRNA gene sequencing was performed using the Illumina MiSeq platform. Bacterial burden was compared using Wilcoxon rank-sum. Community composition was compared using PERMANOVA and mvabund (a model-based approach to analysis of multivariable abundance data).ResultsEighty (80) patients had a BAL specimen from near CLAD onset available for analysis. Of these, 41 (51%) had BOS, 13 (16%) had RAS, 6 (8%) had Mixed CLAD, and 20 (25%) had an undefined/unclassifiable phenotype. There were no differences in lung bacterial burden (p=0.56, Panel A) or overall community composition between CLAD phenotypes (p=0.53, Panel C). Shannon diversity index did differ between CLAD phenotypes, with lower average diversity observed in the patients with an unclassifiable CLAD phenotype (1.0 ± 0.4) vs. BOS (2.2 ± 0.8), RAS (2.1 ± 0.8), Mixed (2.1 ± 0.7), or undefined CLAD (2.1 ± 0.4, overall p=0.02, Panel B). This difference persisted even after accounting for clinical evidence of infection (BAL neutrophilia and bacterial culture results did not differ between groups).ConclusionLung microbiome characteristics are largely similar across CLAD phenotypes, although lower within-specimen diversity is seen in patients with unclassified CLAD. Chronic lung allograft dysfunction (CLAD) is the leading long-term contributor to mortality after lung transplant. The lung microbiome predicts subsequent development of CLAD, but whether lung bacteria differ between CLAD phenotypes is unknown. Using a biorepository of acellular bronchoalveolar lavage (BAL) fluid, we identified specimens which were collected within 90 days of CLAD onset. CLAD phenotype was assigned based on the presence of absence of obstruction, restriction, and computed tomography (CT) scan opacities, in accordance with ISHLT guidelines. Bacterial DNA burden was measured with BioRad QX200 Droplet Digital PCR and 16S rRNA gene sequencing was performed using the Illumina MiSeq platform. Bacterial burden was compared using Wilcoxon rank-sum. Community composition was compared using PERMANOVA and mvabund (a model-based approach to analysis of multivariable abundance data). Eighty (80) patients had a BAL specimen from near CLAD onset available for analysis. Of these, 41 (51%) had BOS, 13 (16%) had RAS, 6 (8%) had Mixed CLAD, and 20 (25%) had an undefined/unclassifiable phenotype. There were no differences in lung bacterial burden (p=0.56, Panel A) or overall community composition between CLAD phenotypes (p=0.53, Panel C). Shannon diversity index did differ between CLAD phenotypes, with lower average diversity observed in the patients with an unclassifiable CLAD phenotype (1.0 ± 0.4) vs. BOS (2.2 ± 0.8), RAS (2.1 ± 0.8), Mixed (2.1 ± 0.7), or undefined CLAD (2.1 ± 0.4, overall p=0.02, Panel B). This difference persisted even after accounting for clinical evidence of infection (BAL neutrophilia and bacterial culture results did not differ between groups). Lung microbiome characteristics are largely similar across CLAD phenotypes, although lower within-specimen diversity is seen in patients with unclassified CLAD.

BACKGROUND:Small airway inflammation and fibrosis or bronchiolitis obliterans (BO) is the predominant presentation of chronic lung allograft dysfunction (CLAD) post-lung transplantation. Carbon monoxide (CO) is a critical endogenous signaling transducer with known anti-inflammatory and anti-fibrotic effects but its therapeutic potential in CLAD remains to be fully elucidated. METHODS:Here we investigate the effect of inhaled CO in modulating chronic lung allograft rejection pathology in a murine orthotopic lung transplant model of BO (B6D2F1/J→DBA/2J). Additionally, the effects of CO on the activated phenotype of mesenchymal cells isolated from human lung transplant recipients with CLAD were studied. RESULTS:Murine lung allografts treated with CO (250 ppm × 30 minutes twice daily from days 7 to 40 post-transplantation) demonstrated decreased immune cell infiltration, fibrosis, and airway obliteration by flow cytometry, trichrome staining, and morphometric analysis, respectively. Decreased total collagen, with levels comparable to isografts, was noted in CO-treated allografts by quantitative hydroxyproline assay. In vitro, CO (250 ppm × 16h) was effective in reversing the fibrotic phenotype of human CLAD mesenchymal cells with decreased collagen I and β-catenin expression as well as an inhibitory effect on ERK1/2 MAPK, and mTORC1/2 signaling. Sildenafil, a phosphodiesterase 5 inhibitor, partially mimicked the effects of CO on CLAD mesenchymal cells and was partially effective in decreasing collagen deposition in murine allografts, suggesting the contribution of cGMP-dependent and -independent mechanisms in mediating the effect of CO. CONCLUSION:These results suggest a potential role for CO in alleviating allograft fibrosis and mitigating chronic rejection pathology post-lung transplant.

Purpose Antifibrotic therapy, i.e. pirfenidone or nintedanib, is commonly used for idiopathic pulmonary fibrosis (IPF) patients awaiting lung transplantation. However, they are inferred to cause increased wound healing complications post-transplant. This study compares post-transplant outcomes in IPF patients on the basis of pre-transplant antifibrotic treatment. Methods IPF patients transplanted after January 2015 were included in a cohort study with three groups: no antifibrotic, pirfenidone, and nintedanib. Pre-specified outcomes of interest included: PGD grades immediately after transplant (T0) and on post-operative day 3 (T72), duration of mechanical ventilation, wound dehiscence, anastomosis complications, time to CLAD development, and overall survival. Results Of the 85 eligible patients, 26 received no pre-transplant antifibrotic medication, 31 received pirfenidone, and 28 received nintedanib. Fewer female patients received antifibrotic therapy, but the groups were otherwise similar with regards demographics and clinical variables (Table). There were no differences between groups in PGD status at T0 or T72, but patients who received antifibrotics pre-transplant were more likely resolve PGD between T0 and T72 than patients who did not (mean change in PGD grade: nintedanib -0.3±0.9 vs. pirfenidone -0.5±1.3 vs. no antifibrotic +0.3±1.5, p=0.026). There was a trend toward decreased time on mechanical ventilation in the patients receiving antifibrotic treatment (no-antifibrotic vs. any antifibrotic medication p=0.025, between all group p=0.07). Anastomosis stenosis was less common in the antifibrotic groups (p=0.020). No differences were observed for wound dehiscence, incidence of CLAD, or mortality. Conclusion Receiving any pre-transplant antifibrotic therapy is associated with increased resolution of PGD, shorter duration of mechanical ventilation, and decreased incidence of anastomosis stenosis.

Purpose Chronic lung allograft dysfunction (CLAD) is the leading long-term cause of mortality after lung transplant. The lung microbiome in healthy lung transplant recipients predicts subsequent development of CLAD, but whether lung bacteria differ between CLAD and healthy allografts is unknown. Methods We performed a case control study using a biorepository of acellular bronchoalveolar lavage (BAL) fluid. Controls were specimens collected from asymptomatic patients undergoing surveillance bronchoscopy one-year post-transplant; cases were specimens collected within 90 days of CLAD onset. Bacterial DNA burden was measured using BioRad QX200 Droplet Digital PCR and 16S rRNA gene sequencing was performed using Illumina MiSeq. Results We identified 131 specimens which met criteria for use as CLAD-free controls and 78 specimens which met critieria for use as CLAD cases. The lung microbiome in specimens collected at CLAD vs. CLAD-free controls was characterized by higher bacterial burden (median [IQR]16S gene copies per mL 4991 [2889-13075] vs. 3561 [2426-8131], p=0.028, Panel A). There were significant overall community composition differences using both PERMANOVA (p=0.032) and mvabund, a model-based approach to analysis of multivariable abundance data (p=0.014, Panel B). As shown in biplot analysis, lung bacterial communities from patients with CLAD had relatively more oral flora than CLAD-free controls; although no individual taxa were responsible for these overall community composition differences. Twenty-two patients had BAL available for microbiome analysis at both time points. In paired comparisons, there was no intra-patient differences in bacterial burden, community diversity or community composition between CLAD-free and CLAD onset samples. Conclusion The lung microbiome in patients with CLAD is characterized by higher bacterial burden and communities with increased predominance of taxa commonly found in the upper respiratory tract.

Histopathologic examination of lungs afflicted by chronic lung allograft dysfunction (CLAD) consistently shows both mononuclear cell (MNC) inflammation and mesenchymal cell (MC) fibroproliferation. We hypothesize that interleukin 6 (IL-6) trans-signaling may be a critical mediator of MNC-MC crosstalk and necessary for the pathogenesis of CLAD. Bronchoalveolar lavage (BAL) fluid obtained after the diagnosis of CLAD has approximately twofold higher IL-6 and soluble IL-6 receptor (sIL-6R) levels compared to matched pre-CLAD samples. Human BAL-derived MCs do not respond to treatment with IL-6 alone but have rapid and prolonged JAK2-mediated STAT3 Tyr705 phosphorylation when exposed to the combination of IL-6 and sIL-6R. STAT3 phosphorylation within MCs upregulates numerous genes causing increased invasion and fibrotic differentiation. MNC, a key source of both IL-6 and sIL-6R, produce minimal amounts of these proteins at baseline but significantly upregulate production when cocultured with MCs. Finally, the use of an IL-6 deficient recipient in a murine orthotopic transplant model of CLAD reduces allograft fibrosis by over 50%. Taken together these results support a mechanism where infiltrating MNCs are stimulated by resident MCs to release large quantities of IL-6 and sIL-6R which then feedback onto the MCs to increase invasion and fibrotic differentiation.

CASE PRESENTATION: A 30-year-old woman was referred with increasing shortness of breath and cough in the setting of GATA2 deficiency. She initially presented 9 years previously with recurrent episodes of pneumonia and sinusitis. Genetic testing revealed a heterozygous GATA2 mutation (c.988C>T). She has since had multiple infections that have included necrotizing fasciitis of the right thumb, recurrent pilonidal infections (which required 23 procedures), esophageal candidiasis, and human papillomavirus-positive high-grade squamous intraepithelial lesion of the cervix. Serial bone marrow biopsy specimens showed persistent hypocellularity (20% to 60%) with intermittent erythroid atypia and variable detection of trisomy 8, which were concerning for evolving myelodysplastic syndrome. One year before the current admission, she was diagnosed with disseminated Mycobacterium avium complex and was treated with rifabutin, ethambutol, and azithromycin. She was taking voriconazole, acyclovir, and trimethoprim-sulfamethoxazole prophylaxis.