Azithromycin, an antibiotic used for multiple infectious disorders, exhibits anti-inflammatory effects, but the molecular basis for this activity is not well characterized. Azithromycin inhibits ...IL-1β-mediated inflammation that is dependent, in part, on inflammasome activity. Here, we investigated the effects of azithromycin on the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) protein, which is the sensing component of the NALP3 inflammasome, in human monocytes.
THP-1 cells were treated with azithromycin alone, LPS alone, or both. NALP3 and IL-1β protein levels were determined by immunoblotting. NLRP3 gene (encoding NALP3) transcript levels were determined by quantitative qPCR. In order to measure NLRP3 transcript decay, actinomycin D was used to impair gene transcription. THP-1 Lucia cells which contain an NF-κB responsive luciferase element were used to assess NF-κB activity in response to azithromycin, LPS, and azithromycin/LPS by measuring luminescence. To confirm azithromycin's effects on NLRP3 mRNA and promoter activity conclusively, HEK cells were lipofected with luciferase reporter constructs harboring either the 5' untranslated region (UTR) of the NLRP3 gene which included the promoter, the 3' UTR of the gene, or an empty plasmid prior to treatment with azithromycin and/or LPS, and luminescence was measured.
Azithromycin decreased IL-1β levels and reduced NALP3 protein levels in LPS-stimulated THP-1 monocytes through a mechanism involving decreased mRNA stability of the NALP3 - coding NLRP3 gene transcript as well as by decreasing NF-κB activity. Azithromycin accelerated NLRP3 transcript decay confirmed by mRNA stability and 3'UTR luciferase reporter assays, and yet the antibiotic had no effect on NLRP3 promoter activity in cells containing a 5' UTR reporter.
These studies provide a unique mechanism whereby azithromycin exerts immunomodulatory actions in monocytes by destabilizing mRNA levels for a key inflammasome component, NALP3, leading to decreased IL-1β-mediated inflammation.
Cytomegalovirus (CMV)-related morbidities remain one of the most common complications after lung transplantation and have been linked to allograft dysfunction, but the factors that predict high risk ...for CMV complications and effective immunity are incompletely understood.
To determine if short telomeres in idiopathic pulmonary fibrosis (IPF) lung transplant recipients (LTRs) predict the risk for CMV-specific T-cell immunity and viral control.
We studied IPF-LTRs (n = 42) and age-matched non-IPF-LTRs (n = 42) and assessed CMV outcomes. We measured lymphocyte telomere length and DNA sequencing, and assessed CMV-specific T-cell immunity in LTRs at high risk for CMV events, using flow cytometry and fluorescence in situ hybridization.
We identified a high prevalence of relapsing CMV viremia in IPF-LTRs compared with non-IPF-LTRs (69% vs. 31%; odds ratio, 4.98; 95% confidence interval, 1.95-12.50; P < 0.001). Within this subset, IPF-LTRs who had short telomeres had the highest risk of CMV complications (P < 0.01) including relapsing-viremia episodes, end-organ disease, and CMV resistance to therapy, as well as shorter time to viremia versus age-matched non-IPF control subjects (P < 0.001). The short telomere defect in IPF-LTRs was associated with significantly impaired CMV-specific proliferative responses, T-cell effector functions, and induction of the major type-1 transcription factor T-bet (T-box 21;TBX21).
Because the short telomere defect has been linked to the pathogenesis of IPF in some cases, our data indicate that impaired CMV immunity may be a systemic manifestation of telomere-mediated disease in these patients. Identifying this high-risk subset of LTRs has implications for risk assessment, management, and potential strategies for averting post-transplant CMV morbidities.
Antibody-mediated rejection (AMR) has emerged as an important cause of lung graft failure. In the current issue of the JCI, a study by Li et al. identifies a critical role of Foxp3+ T cells residing ...within lung allografts in the regulation of AMR. This study not only provides new insights into the nature of lung allografts as a primary site where T and B cell priming and immune regulation can occur, but also introduces the mouse orthotopic lung transplant as a model for studying the immunobiology of AMR. Because AMR can be so difficult to effectively treat in lung transplant recipients, the development of an animal model is a major advance in understanding the immunopathogenesis of AMR.
Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome ...coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers.
We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable.
Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort.
In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4
lymphopenia predominated, with lower CD4
/CD8
ratios in severe COVID-19 compared with patients with mild disease (
< 0.0001). In severe disease, immunodominant CD4
T-cell responses to Spike-1 (S1) produced increased
TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4
TNF-α
T-cell responses inversely correlated with absolute CD4
counts from patients with severe COVID-19 (
= 76;
= -0.797;
< 0.0001).
TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4
T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (
< 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4
cells with infliximab treatment. We also evaluated BAL and lung explant CD4
T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells.
Together, our findings show CD4
dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.
Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long‐term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a ...paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush‐derived transcriptomic signatures in CLAD (n = 24) versus non‐CLAD (n = 21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type‐1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO‐1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type‐1 profile with detection of IFN‐γ, TNF‐α, and IL‐1β as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.
This cross‐sectional analysis of transbronchial brush samples from lung transplant recipients identifies a transcriptomic signature marked by type‐1 immunity in recipients with chronic lung allograft dysfunction compared to stable controls.
Background
Induction therapy is used in about 80% of lung transplant centers and is increasing globally. Currently, there are no standards or guidelines for the use of induction therapy. At our ...institution, we have two induction strategies, basiliximab, and alemtuzumab. The goal of this manuscript is to share our experience and practice since this is an area of controversy.
Methods
We retrospectively reviewed 807 lung transplants performed at our institution between 2011 and 2020. Indications for the use of the basiliximab protocol were as follows: patients over the age of 70 years, history of cancer, hepatitis C virus or human immunodeficiency virus infection history, and cytomegalovirus or Epstein-Barr virus (donor positive/ recipient negative). In the absence of these clinical factors, the alemtuzumab protocol was used.
Results
453 patients underwent alemtuzumab induction and 354 patients underwent basiliximab. There were significant differences in delayed chest closure (24.7% alemtuzumab vs 31.4% basiliximab, p = 0.037), grade 3 primary graft dysfunction observed within 72 hours (19.9% alemtuzumab vs 29.9% basiliximab, p = 0.002), postoperative hepatic dysfunction (8.8% alemtuzumab vs 14.7% basiliximab, p = 0.009), acute cellular rejection in first year (39.1% alemtuzumab vs 53.4% basiliximab, p < 0.001). The overall survival rate of the patients with alemtuzumab induction was significantly higher than those of the patients with basiliximab induction (5 years survival rate: 64.1% alemtuzumab vs 52.3%, basiliximab, p < 0.001). Multivariate Cox regression analysis confirmed lower 5-year survival for basiliximab induction (HR = 1.41, p = 0.02), recipient cytomegalovirus positive (HR = 1.49, p = 0.01), postoperative hepatic dysfunction (HR = 2.20, p < 0.001), and acute kidney injury requiring renal replacement therapy (HR = 2.27, p < 0.001).
Conclusions
In this single center retrospective review, there was a significant difference in survival rates between induction strategies. This outcome may be attributable to differences in recipient characteristics between the groups. However, the Alemtuzumab group experienced less episodes of acute cellular rejection within the first year.
Epstein‐Barr virus (EBV)–associated posttransplant lymphoproliferative disorder (EBV‐PTLD) is a serious complication in lung transplant recipients (LTRs) associated with significant mortality. We ...performed a single‐center retrospective study to evaluate the risks for PTLD in LTRs over a 7‐year period. Of 611 evaluable LTRs, we identified 28 cases of PTLD, with an incidence of 4.6%. Kaplan‐Meier analysis showed a decreased freedom from PTLD in idiopathic pulmonary fibrosis (IPF)‐LTRs (P < .02). Using a multivariable Cox proportional hazards model, we found IPF (hazard ratio HR 3.51, 95% confidence interval CI 1.33‐8.21, P = .01) and alemtuzumab induction therapy (HR 2.73, 95% CI 1.10‐6.74, P = .03) as risk factors for PTLD, compared to EBV mismatch (HR: 34.43, 95% CI 15.57‐76.09, P < .0001). Early PTLD (first year) was associated with alemtuzumab use (P = .04), whereas IPF was a predictor for late PTLD (after first year) (P = .002), after controlling for age and sex. Kaplan‐Meier analysis revealed a shorter time to death from PTLD in IPF LTRs compared to other patients (P = .04). The use of alemtuzumab in EBV mismatch was found to particularly increase PTLD risk. Together, our findings identify IPF LTRs as a susceptible population for PTLD. Further studies are required to understand the mechanisms driving PTLD in IPF LTRs and develop strategies to mitigate risk.
The authors report a single‐center, retrospective cohort study that finds an increased hazard of Epstein Barr virus–associated posttransplant lymphoproliferative disorder in lung transplant recipients transplanted for idiopathic pulmonary fibrosis and that EBV mismatch serostatus and lymphodepleting induction immunosuppression are also associated with increased risk.
Antibody-mediated rejection (AMR) has emerged as an important cause of lung graft failure. In the current issue of theJCI, a study by Li et al. identifies a critical role of Foxp3.sup.+ T cells ...residing within lung allografts in the regulation of AMR. This study not only provides new insights into the nature of lung allografts as a primary site where T and B cell priming and immune regulation can occur, but also introduces the mouse orthotopic lung transplant as a model for studying the immunobiology of AMR. Because AMR can be so difficult to effectively treat in lung transplant recipients, the development of an animal model is a major advance in understanding the immunopathogenesis of AMR.
Occupational lung diseases (OLD) including silicosis, asbestosis, and pneumoconiosis progress to end stage lung disease requiring lung transplantation (LT). Prognosis and treatment of OLDs are poorly ...understood and a paucity of data exists regarding LT outcomes. Additionally, transplant operative complexity for patients with OLD is high. A single center retrospective review of all single and bilateral LT recipients between May 2005 and Oct 2016 was performed. Patients were grouped by OLD, and nearest neighbor matching was performed at a ratio of 1:3 cases to controls. Thirty cases were matched to 88 controls. Seventeen patients (57%) with OLD required intraoperative support with either extra‐corporeal membrane oxygenation (ECMO) or cardiopulmonary bypass (P = 0.02), and 5 (17%) required delayed chest closure (P = 0.05) which was more frequent than matched controls. In addition, operative time was significantly longer in patients with OLD (P = 0.03). Despite these factors, there were no significant differences in immediate post‐operative outcomes including mechanical ventilator support, post‐operative ECMO, and tracheostomy. Chronic lung allograft dysfunction and long‐term survival were also similar between cases and controls. OLDs should not preclude LT. The operation should be performed at experienced centers.
Current immunosuppressive regimens with calcineurin inhibitors have improved the management of patients after transplantation. However, their adverse effects are linked to increased morbidity and ...limit the long‐term survival of heart and lung transplant recipients. Belatacept, a costimulation inhibitor interfering with the interaction between CD28 on T cells and the B7 ligands on antigen presenting cells, has shown success and is currently approved for use in renal transplant recipients. Furthermore, it lacks many of the cardiovascular, metabolic, neurologic, and renal adverse of effects of calcineurin inhibitors that have the largest impact on long‐term survival in cardiothoracic transplant. Additionally, it requires no therapeutic drug monitoring and is only administered once a month. Limitations to belatacept use have been observed that must be considered when comparing immunosuppression options. Despite this, maintenance immunosuppression with belatacept has the potential to improve outcomes in cardiothoracic transplant recipients, as it has with kidney transplant recipients. However, no large clinical trials investigating belatacept for maintenance immunosuppression in heart and lung transplant recipients exist. There is a large need for focused research of belatacept in cardiothoracic transplantation. Belatacept is a viable treatment option for maintenance immunosuppression, and it is reasonable to pursue more evidence in cardiothoracic transplant recipients.
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