Skeletal Muscle SIRT3 Deficiency Contributes to Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) is a major complication linked to adverse outcomes in heart failure with preserved ejection fraction (HFpEF), yet no specific therapies exist for PH associated with HFpEF (PH-HFpEF). We have recently reported on the role of skeletal muscle SIRT3 (sirtuin-3) in modulation of PH-HFpEF, suggesting a novel endocrine signaling pathway for skeletal muscle modulation of pulmonary vascular remodeling. In this study, we attempted to define the processes by which skeletal muscle SIRT3 defects affect pulmonary vascular health in PH-HFpEF. METHODS AND RESULTS: Skeletal muscle-specific Sirt3 knockout mice (Sirt3skm-/-) exhibited reduced pulmonary vascular density accompanied by pulmonary vascular proliferative remodeling and elevated pulmonary pressures. Using mass spectrometry-based comparative secretome analysis, we demonstrated elevated secretion of LOXL2 (lysyl oxidase homolog 2) in SIRT3-deficient skeletal muscle cells. Elevated circulation and protein expression levels of LOXL2 were also observed in plasma and skeletal muscle of Sirt3skm-/- mice, a rat model of PH-HFpEF, and humans with PH-HFpEF. In addition, expression levels of CNPY2 (canopy fibroblast growth factor signaling regulator 2), a known proliferative and angiogenic factor, were increased in pulmonary artery endothelial cells and pulmonary artery smooth muscle cells of Sirt3skm-/- mice and animal models of PH-HFpEF. CNPY2 levels were also higher in pulmonary artery smooth muscle cells of subjects with obesity compared with nonobese subjects. Moreover, treatment with recombinant LOXL2 protein promoted pulmonary artery endothelial cell migration/proliferation and pulmonary artery smooth muscle cell proliferation through regulation of CNPY2-p53 signaling. Last, skeletal muscle-specific Loxl2 deletion decreased pulmonary artery endothelial cell and pulmonary artery smooth muscle cell expression of CNPY2 and improved pulmonary pressures in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: This study demonstrates a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH-HFpEF. This study suggests a new endocrine signaling axis that links skeletal muscle health and SIRT3 deficiency to remote CNPY2 regulation in the pulmonary vasculature through myokine LOXL2. Our data also identify skeletal muscle SIRT3, myokine LOXL2, and CNPY2 as potential targets for the treatment of PH-HFpEF.

Comparison of characteristics of muscle magnetic resonance imaging findings in patients with antineutrophilic cytoplasmic antibody-associated vasculitis and polyarteritis nodosa.

AIM: This study aimed to analyze the muscle magnetic resonance imaging (MRI) findings of patients with antineutrophilic cytoplasmic antibody-associated vasculitis (AAV) and polyarteritis nodosa (PAN) presenting with clinical symptoms in the extremities. METHODS: Retrospective analysis was conducted on short tau inversion recovery MRI findings, with a focus on intramuscular vessels displaying abnormal perivascular signals, in 22 and eight patients with AAV and PAN, respectively. The number per unit area (4 cm2) and diameter of abnormal vessels on muscle MRI were compared between patients with AAV and those with PAN. Cut-off values, clinical sensitivity, and specificity for these indices were calculated from the receiver operating characteristic curves to distinguish between AAV and PAN, and the relationship between the indices and clinical findings in AAV was analyzed. RESULTS: The number of abnormal vessels per unit area was significantly higher in AAV compared to PAN (p < .05). Additionally, the diameter of the abnormal vessels was significantly higher in PAN than in AAV (p < .05). The presence of >6.44 abnormal vessels per unit area or ≤3.61 mm diameter of abnormal vessels was able to predict AAV (sensitivity, 0.955; specificity, 0.625). AAV patients with peripheral neuropathy exhibited a significantly higher number of abnormal vessels per unit area than those without peripheral neuropathy (p < .05). CONCLUSIONS: Muscle MRI can detect small- to medium-vessel vasculitis and be a valuable tool for distinguishing between patients with AAV and PAN experiencing clinical symptoms in the extremities.

Magnetic Resonance Imaging Patterns Revealing Muscle Pathology and Clinical Features in Idiopathic Inflammatory Myopathies.

OBJECTIVE: Idiopathic inflammatory myopathies (IIMs) are autoimmune disorders significantly impacting skeletal muscles; however, the precise correlation between muscle magnetic resonance imaging (MRI) findings, muscle pathology, disease subtypes, and clinical characteristics remains uncertain. Thus, we investigated the association of muscle MRI findings in IIMs with muscle pathology and clinical features. METHODS: New-onset IIM patients underwent proximal upper and/or lower limb muscle MRI. Patterns of muscle oedema on MRI were categorised into fascial, honeycomb, peripheral, foggy, dense, or coarse dot patterns and compared with inflammatory cell infiltration sites in corresponding muscle biopsies. The incidence of MRI patterns was examined in patient subgroups using myositis-specific antibodies (MSAs) and 2017 EULAR/ACR classification criteria. Univariate and multivariate analyses were conducted to determine the odds ratios (ORs) of MRI findings for clinical characteristics. RESULTS: Fifty-six of 85 patients underwent muscle biopsy. Foggy, honeycomb, and fascial patterns at biopsy sites correlated with inflammatory cell infiltration in the endomysium (OR 11.9, p= 0.005), perimysium (OR 6.0, p= 0.014), and fascia (OR 16.9, p< 0.001), respectively. Honeycomb and foggy patterns were characteristic of patients with anti-TIF1γ or anti-Mi2 antibodies and MSA-negative dermatomyositis, and those with anti-SRP or anti-HMGCR antibodies and MSA-negative polymyositis (PM), respectively. The honeycomb pattern positively correlated with malignancy (OR 6.87, p< 0.001) and Gottron sign (OR 8.05, p= 0.002); the foggy pattern correlated with muscle weakness (OR 11.24, p= 0.005). The dense dot pattern was associated with dysphagia (OR 6.27, p= 0.006) and malignancy (OR 8.49, p= 0.002). CONCLUSION: Muscle MRI holds promise in predicting muscle pathology, disease subtypes, and clinical manifestations of IIMs.

Olfactory Bulbs in Arthritis Model Mouse Persistently Express Interleukin-6 before the Onset of Arthritis: Relationship to Food Intake.

INTRODUCTION: Rheumatoid arthritis (RA) can be comorbid with psychiatric symptoms. Brain abnormalities in RA patients and in arthritis models have been reported. However, it remains unclear when these abnormalities occur and where they are distributed. In this study, we analyzed spatiotemporal changes in gene expression in the brains of mice with collagen-induced arthritis (CIA). METHODS: Mice were divided into three groups: (i) CIA (all mice developed arthritis on day 35): complete Freund's adjuvant (CFA) and type II collagen at initial immunization, and incomplete Freund's adjuvant (IFA) and type II collagen at booster immunization; (ii) C(+/-) (50% mice developed arthritis on day 35): only IFA at booster immunization; and (iii) C(-/-) (no arthritis): only CFA at initial immunization and only IFA at booster immunization. Whole brains were collected at ten stages of arthritis and divided into six sections. Real-time polymerase chain reaction was performed using RNA extracted from the brain, and the expression of proinflammatory cytokines and glial markers was semi-quantified. Arthritis score, body weight, and food and water intakes were recorded and analyzed for correlations with brain gene expression. We also investigated the effect of interleukin-6 (IL-6) injection in the olfactory bulbs (OBs) on the food intake. RESULTS: After booster immunization, a transient increase in Integrin subunit α-M and IL-1ß was observed in multiple areas in CIA. IL-6 is persistently expressed in the OB before the onset of arthritis, which is correlated with body weight loss and decreased food intake. This change in the OB was observed in the C(+/-) but not in the C(-/-) groups. In the C(+/-) group, non-arthritic mice showed the same changes in the OB as the arthritic mice. This elevation in IL-6 levels persisted throughout the chronic phase until day 84. In addition, IL-6 injection into the OB reduced food intake. CONCLUSION: Persistent elevation of IL-6 in the OB from the early stage of arthritis may be an important finding that might explain the neuropsychiatric pathophysiology of RA, including appetite loss, which is present in the early stages of the disease and manifests as a variety of symptoms over time.

Intramuscular lesions in musculoskeletal MRI as a favourable prognostic sign in patients with anti-MDA5 antibody-positive dermatomyositis.

OBJECTIVES: Anti-differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis, which has been described as clinically amyopathic dermatomyositis, complicates rapidly progressive interstitial lung disease (ILD). Owing to the absence of significant muscle symptoms, musculoskeletal MRI is often not performed. In this study, we aimed to devise a simple evaluation method using musculoskeletal MRI findings to elucidate the relationship between MRI findings and ILD prognosis and development. METHODS: The medical records and MRI scans of the proximal muscles at the time of diagnosis were retrospectively reviewed for 28 patients with anti-MDA5 antibody-positive dermatomyositis who were admitted to The Jikei University Hospital and The Jikei University Kashiwa Hospital between January 2008 and March 2022. Three observers evaluated nine proximal muscles for high signals on either short-tau inversion recovery images and/or fat-saturated gadolinium-enhanced T1-weighted images in the fascia and/or in the margins of the muscles in contact with the fascia (fascial pattern), and/or high signals in the muscles away from the fascia (intramuscular pattern), and a consensus was reached. RESULTS: Of the 28 patients, 15 presented with 'radiological myositis', where an intramuscular pattern was observed at any site. Patients with radiological myositis had significantly higher survival rates than those without radiological myositis, despite the lower rate of triple therapy with prednisolone, calcineurin inhibitors and cyclophosphamide. The spread of ILD on chest CT negatively and significantly correlated with the proportion of intramuscular lesions. CONCLUSION: The detection of intramuscular lesions on musculoskeletal MRI using our novel evaluation method could be clinically useful as a favourable prognostic marker.

Clinical features of newly diagnosed systemic lupus erythematosus after SARS-CoV-2 vaccination.

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes damage to multiple organs. Various factors, including vaccination, have been associated with SLE development. Vaccination for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in 2020, and there are a few reports on the exacerbation of SLE after SARS-CoV-2 vaccination. The influence of SARS-CoV-2 vaccination on SLE development remains unclear. We present the case of a 53-year-old man who developed peritonitis and was subsequently diagnosed with SLE on Day 9 after receiving a third dose of the messenger ribonucleic acid-1273 SARS-CoV-2 vaccine. This case and previous reports have shown that patients who developed SLE after SARS-CoV-2 vaccination are more likely to develop it within 2 weeks of vaccination, especially when they have a higher rate of immunological abnormalities or a family history of autoimmune diseases. Furthermore, these features suggest that type I interferon is involved in the pathogenesis of SLE after SARS-CoV-2 vaccination.

Newly synthesized glycoprotein profiling to identify molecular signatures of warm ischemic injury in donor lungs.

Despite recent technological advances such as ex vivo lung perfusion (EVLP), the outcome of lung transplantation remains unsatisfactory with ischemic injury being a common cause for primary graft dysfunction. New therapeutic developments are hampered by limited understanding of pathogenic mediators of ischemic injury to donor lung grafts. Here, to identify novel proteomic effectors underlying the development of lung graft dysfunction, using bioorthogonal protein engineering, we selectively captured and identified newly synthesized glycoproteins (NewS-glycoproteins) produced during EVLP with unprecedented temporal resolution of 4 h. Comparing the NewS-glycoproteomes in lungs with and without warm ischemic injury, we discovered highly specific proteomic signatures with altered synthesis in ischemic lungs, which exhibited close association to hypoxia response pathways. Inspired by the discovered protein signatures, pharmacological modulation of the calcineurin pathway during EVLP of ischemic lungs offered graft protection and improved posttransplantation outcome. In summary, the described EVLP-NewS-glycoproteomics strategy delivers an effective new means to reveal molecular mediators of donor lung pathophysiology and offers the potential to guide future therapeutic development.NEW & NOTEWORTHY This study developed and implemented a bioorthogonal strategy to chemoselectively label, enrich, and characterize newly synthesized (NewS-)glycoproteins during 4-h ex vivo lung perfusion (EVLP). Through this approach, the investigators uncovered specific proteomic signatures associated with warm ischemic injury in donor lung grafts. These signatures exhibit high biological relevance to ischemia-reperfusion injury, validating the robustness of the presented approach.

Autocitrullination confers monocyte chemotactic properties to peptidylarginine deiminase 4.

Peptidylarginine deiminase 4 (PAD4) contributes to the production of citrullinated proteins as autoantigens for anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA). PAD4 can also self-deiminate via autocitrullination. However, the role of this process in RA pathogenesis has not been elucidated. This study aimed to clarify PAD4 function before and after autocitrullination and identify citrullinated PAD4 in the synovial fluid of patients with RA. The autocitrullination of recombinant human PAD4 (rhPAD4) was catalyzed in vitro and determined using anti-modified citrulline immunoblotting. Monocyte chemotaxis was evaluated using Boyden chambers, and citrullinated rhPAD4's ability to induce arthritis was assessed in a C57BL/6J mouse model. Citrullinated PAD4 levels were measured in the synovial fluid of patients with RA and osteoarthritis using a novel enzyme-linked immunosorbent assay. Chemotactic findings showed that citrullinated rhPAD4 recruited monocytes in vitro, whereas unmodified rhPAD4 did not. Compared to unmodified rhPAD4, citrullinated rhPAD4 induced greater inflammation in mouse joints through monocyte migration. More citrullinated PAD4 was found in the synovial fluid of patients with RA than in those with osteoarthritis. Citrullinated PAD4 was even detected in ACPA-negative patients with RA. The autocitrullination of PAD4 amplified inflammatory arthritis through monocyte recruitment, suggesting an ACPA-independent role of PAD4 in RA pathogenesis.

Temperature Dependence of the Beating Frequency of hiPSC-CMs Using a MEMS Force Sensor.

It is expected that human iPS cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat serious heart diseases. However, the properties and functions of human adult cardiomyocytes and hiPSC-CMs, including cell maturation, differ. In this study, we focused on the temperature dependence of hiPSC-CMs by integrating the temperature regulation system into our sensor platform, which can directly and quantitatively measure their mechanical motion. We measured the beating frequency of hiPSC-CMs at different environmental temperatures and found that the beating frequency increased as the temperature increased. Although the rate at which the beating frequency increased with temperature varied, the temperature at which the beating stopped was relatively stable at approximately 20 °C. The stopping of beating at this temperature was stable, even in immature hiPSC-CMs, and was considered to be a primitive property of cardiomyocytes.

Endothelial protection in lung grafts through heparanase inhibition during ex vivo lung perfusion in rats.

BACKGROUND: We hypothesized that enhancing glycocalyx preservation would reduce endothelial damage in lung grafts during ex-vivo lung perfusion (EVLP) leading to better transplant outcomes. In this study, we characterized the effects of inhibiting heparanase (HPSE), an enzyme responsible for glycocalyx shedding, on lung quality during EVLP. METHODS: Human clinical EVLP perfusate from lung graft patients was utilized to identify a potential association between glycocalyx integrity in grafted lung tissue and clinical data. In addition, we performed pre-clinical studies in which rat lungs underwent normothermic EVLP for 4 hours with/without HPSE inhibitors, heparin (1,000-U/h) or heparastatin (SF4; 1-µM), added to the perfusate. After 4-hours EVLP, left lungs were transplanted into syngeneic rats then evaluated for graft quality 2-hours after reperfusion. RESULTS: Clinically, increased degradation of syndecan-1 was identified in dysfunctional grafts during EVLP. Levels of heparan sulfate in perfusate after EVLP were associated with incidence of graft dysfunction after transplantation. In the pre-clinical rat study, SF4 effectively inhibited HPSE activity, and significantly attenuated dissociated glycocalyx levels, endothelial dysfunction, edema, and inflammation in lungs during EVLP compared to both controls and heparin groups. High-doses of heparin demonstrated markedly increased perfusate syndecan-1 concentrations and deteriorated lung quality during EVLP compared with controls. Post-transplant graft function and inflammation were significantly improved in SF4-treated group compared to those in both control and heparin-treated groups. CONCLUSIONS: This study demonstrated that HPSE activity inhibition by SF4 can improve graft preservation during EVLP by protecting the glycocalyx and endothelial function, leading to better lung function following transplantation.

Effects of Intraoperative Support Strategies on Endothelial Injury and Clinical Lung Transplant Outcomes.

In lung transplantation, postoperative outcomes favor intraoperative use of extracorporeal membrane oxygenation (ECMO) over cardiopulmonary bypass (CBP). We investigated the effect of intraoperative support strategies on endothelial injury biomarkers and short-term posttransplant outcomes. Adults undergoing bilateral lung transplantation with No-Support, venoarterial (V-A) ECMO, or CPB were included. Plasma samples pre- and post-transplant were collected for Luminex assay to measure endothelial injury biomarkers including syndecan-1 (SYN-1), intercellular adhesion molecule-1 (ICAM-1), and matrix metalloprotease-9. Fifty five patients were included for analysis. The plasma level of SYN-1 at arrival in the intensive care unit was significantly higher with CPB compared to V-A ECMO and No-Support (P < 0.01). The rate of primary graft dysfunction grade 3 (PGD3) at 72 hours was 60.0% in CPB, 40.1% in V-A ECMO, and 15% in No-Support (P = 0.01). Postoperative plasma levels of SYN-1 and ICAM-1 were significantly higher in recipients who developed PGD3 at 72 hours. SYN-1 levels were also significantly higher in patients who developed acute kidney injury and hepatic dysfunction after transplant. Postoperative, SYN-1 upon intensive care arrival was found to be a significant predictive biomarker of PGD3, acute kidney injury, and hepatic dysfunction following lung transplantation. CPB is associated with higher plasma concentrations of SYN-1, a marker of endothelial glycocalyx degradation, upon arrival to the intensive care unit. Higher levels of SYN-1 are predictive of end-organ dysfunction following lung transplantation. Our data suggests that intraoperative strategies aimed at modulating endothelial injury will help improve lung transplantation outcomes.

Inhibitory effect of baricitinib on microglia and STAT3 in a region with a weak blood-brain barrier in a mouse model of rheumatoid arthritis.

OBJECTIVES: In patients with RA, baricitinib not only improves arthritis symptom severity, but also patients' neuropsychological symptoms, such as depression and fatigue. However, the cellular mechanisms through which baricitinib can affect neural activity is unexplored. While the blood-brain barrier (BBB) permeability of this drug remains unclear, Janus kinase inhibitors (JAKi) might reach the area postrema, which is a unique brain region with a weak BBB function. Our recent study demonstrated microglial activation during experimental arthritis in the area postrema. Therefore, we sought to assess the effect of baricitinib on microglia in the area postrema using the CIA mouse model. METHODS: Microglia number and morphology in the area postrema were assessed by immunostaining for ionized calcium-binding adaptor molecule-1 (Iba-1). Data were collected on post-immunization day 35 (early phase) and 84 (late phase), and compared between baricitinib- and vehicle-treated mice. The effect on signal transducers and activators of transcription (STAT3) in the area postrema was also immunohistochemically examined. Behavioural outcomes were assessed by examining feeding behaviours and sucrose preference tests. RESULTS: In the early phase, activated microglial levels in the area postrema were decreased by baricitinib, accompanied by the inhibition of phosphorylated-STAT3 and recovery of food intake and sucrose preference. On the other hand, baricitinib did not affect microglial morphology in the late phase. CONCLUSION: Our results demonstrate that baricitinib can affect brain cells, specifically microglia, in the brain region with a weak BBB and mitigate aberrant behaviours during autoimmune arthritis, pointing to the potential therapeutic effect of JAKi on brain pathologies underpinning RA.

Single-center experience of ex vivo lung perfusion and subsequent lung transplantation.

BACKGROUND: The safety of lung transplantation using ex vivo lung perfusion (EVLP) has been confirmed in multiple clinical studies; however, limited evidence is currently available regarding the potential effects of EVLP on posttransplant graft complications and survival with mid- to long-term follow-up. In this study, we reviewed our institutional data to better understand the impact of EVLP. METHODS: Lungs placed on EVLP from 2014 through 2020 and transplant outcomes were retrospectively analyzed. Data were compared between lungs transplanted and declined after EVLP, between patients with severe primary graft dysfunction (PGD3) and no PGD3 after EVLP, and between matched patients with lungs transplanted with and without EVLP. RESULTS: In total, 98 EVLP cases were performed. Changes in metabolic indicators during EVLP were correlated with graft quality and transplantability, but not changes in physiological parameters. Among 58 transplanted lungs after EVLP, PGD3 at 72 h occurred in 36.9% and was associated with preservation time, mechanical support prior to transplant, and intraoperative transfusion volume. Compared with patients without EVLP, patients who received lungs screened with EVLP had a higher incidence of PGD3 and longer ICU and hospital stays. Lung grafts placed on EVLP exhibited a significantly higher chance of developing airway anastomotic ischemic injury by 30 days posttransplant. Acute and chronic graft rejection, pulmonary function, and posttransplant survival were not different between patients with lungs screened on EVLP versus lungs with no EVLP. CONCLUSION: EVLP use is associated with an increase of early posttransplant adverse events, but graft functional outcomes and patient survival are preserved.

Expanding Donor Options for Lung Transplant: Extended Criteria, Donation After Circulatory Death, ABO Incompatibility, and Evolution of Ex Vivo Lung Perfusion.

Only using brain-dead donors with standard criteria, the existing donor shortage has never improved in lung transplantation. Currently, clinical efforts have sought the means to use cohorts of untapped donors, such as extended criteria donors, donation after circulatory death, and donors that are ABO blood group incompatible, and establish the evidence for their potential contribution to the lung transplant needs. Also, technical maturation for using those lungs may eliminate immediate concerns about the early posttransplant course, such as primary graft dysfunction or hyperacute rejection. In addition, recent clinical and preclinical advances in ex vivo lung perfusion techniques have allowed the safer use of lungs from high-risk donors and graft modification to match grafts to recipients and may improve posttransplant outcomes. This review summarizes recent trends and accomplishments and future applications for expanding the donor pool in lung transplantation.

Lung transplantation from donation after circulatory death, evolution, and current status in the United States.

BACKGROUND: The number of lung transplants from donors after circulatory death has increased over the last decade. This study aimed to describe the evolution and outcomes following lung transplantation donation after circulatory death (DCD) and report the practices and outcomes of ex vivo lung perfusion (EVLP) in this donor population. METHODS: This was a retrospective study using a prospectively collected national registry. The United Network for Organ Sharing (UNOS) database was queried to identify adult patients who underwent lung transplantation between May 1, 2005, and December 31, 2021. Kaplan-Meier analysis and Weibull regression were used to compare survival in four cohorts (donation after brain death [DBD] with or without EVLP, and DCD with or without EVLP). The primary outcome of interest was patient survival. RESULTS: Of the 21 356 recipients who underwent lung transplantation, 20 380 (95.4%) were from brain death donors and 976 (4.6%) from donors after circulatory death. Kaplan-Meier analysis showed no difference in the survival time between the two groups. In a multivariable analysis that controlled for baseline differences in donor and recipient characteristics, recipients who received lungs from cardiac death donors after EVLP had 28% shorter survival time relative to donor lungs after brain death without EVLP (hazard ratio [HR] 1.53, 95% confidence interval [CI] 1.10-2.15, p = .01). CONCLUSIONS: The early survival differences observed after lung transplants from donors after circulatory death in lungs evaluated with EVLP deserves further investigation.

Subcutaneous Panniculitis-like T-cell Lymphoma with a HAVCR2 Mutation Diagnosed after 10 Years of Treatment with Glucocorticoids and Cyclosporine as Lupus Panniculitis.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL) is a very rare cutaneous T cell lymphoma that has been reported to be associated with autoimmune disorders but is most commonly associated with systemic lupus erythematosus. We herein report a 26-year-old man thought to have lupus panniculitis (LP) treated for 10 years with corticosteroids and cyclosporine. After several relapses with panniculitis, he was finally diagnosed with SPTCL, which was confirmed to have a HAVCR2 mutation for p.Tyr82Cys. We emphasize that rheumatologists should be aware of the possibility of SPTCL, despite its rare appearance, when making a diagnosis of LP or when encountering clinical manifestations that are not consistent with LP.

Single center study investigating the clinical association of donor-derived cell-free DNA with acute outcomes in lung transplantation.

Background: Circulating donor-derived cell-free DNA (dd-cfDNA) levels have been proposed as a potential tool for the diagnosis of graft injury. In this study, we prospectively investigated dd-cfDNA plasma levels and their association with severe primary graft dysfunction (PGD) and graft rejection after lung transplant. Methods: A total of 40 subjects undergoing de-novo lung transplants at our institution were recruited in this study. Blood samples were collected at various time points before and after lung transplant for 1 year. Dd-cfDNA in samples was determined using AlloSure assay (CareDx Inc.). The correlation of the value of %dd-cfDNA was investigated with the incidence of PGD, acute cellular rejection (ACR), and donor-specific antibody. Results: We observed a rapid increase of %dd-cfDNA in the blood of recipients after lung transplantation compared to baseline. The levels of dd-cfDNA decreased during the first two weeks. The peak was observed within 72 h after transplantation. The peak values of %dd-cfDNA varied among subjects and did not correlate with severe PGD incidence. We observed an association between levels of %dd-cfDNA from blood collected at the time of transbronchial biopsy and the histological diagnosis of ACR at 3 weeks. Conclusion: Our data show that circulating dd-cfDNA levels are associated with ACR early after transplantation but not with severe PGD. Plasma levels of dd-cfDNA may be a less invasive tool to estimate graft rejection after lung transplantation however larger studies are still necessary to better identify thresholds.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.