Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

Airway pepsinogen A4 identifies lung transplant recipients with microaspiration and predicts chronic lung allograft dysfunction.

BACKGROUND: Aspiration is a known risk factor for adverse outcomes post-lung transplantation. Airway bile acids are the gold-standard biomarker of aspiration; however, they are released into the duodenum and likely reflect concurrent gastrointestinal dysmotility. Previous studies investigating total airway pepsin have found conflicting results on its relationship with adverse outcomes post-lung transplantation. These studies measured total pepsin and pepsinogen in the airways. Certain pepsinogens are constitutively expressed in the lungs, while others, such as pepsinogen A4 (PGA4), are not. We sought to evaluate the utility of measuring airway PGA4 as a biomarker of aspiration and predictor of adverse outcomes in lung transplant recipients (LTRs) early post-transplant. METHODS: Expression of PGA4 was compared to other pepsinogens in lung tissue. Total pepsin and PGA4 were measured in large airway bronchial washings and compared to preexisting markers of aspiration. Two independent cohorts of LTRs were used to assess the relationship between airway PGA4 and chronic lung allograft dysfunction (CLAD). Changes to airway PGA4 after antireflux surgery were assessed in a third cohort of LTRs. RESULTS: PGA4 was expressed in healthy human stomach but not lung. Airway PGA4, but not total pepsin, was associated with aspiration. Airway PGA4 was associated with an increased risk of CLAD in two independent cohorts of LTRs. Antireflux surgery was associated with reduced airway PGA4. CONCLUSIONS: Airway PGA4 is a marker of aspiration that predicts CLAD in LTRs. Measuring PGA4 at surveillance bronchoscopies can help triage high-risk LTRs for anti-reflux surgery.

Interferon-stimulated and metallothionein-expressing macrophages are associated with acute and chronic allograft dysfunction after lung transplantation.

BACKGROUND: Lung transplant recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). ALAD episodes are a risk factor for chronic lung allograft dysfunction (CLAD), the major cause of death after lung transplantation. Our objective was to determine key cellular elements in dysfunctional lung allografts, with a focus on macrophages. METHODS: We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage cells from stable and ALAD patients and to cells from explanted CLAD lung tissue. RESULTS: We identified 2 alveolar macrophage (AM) subsets uniquely represented in ALAD. Using pathway analysis and differentially expressed genes, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) AMs. Functional analysis of an independent set of AMs in vitro revealed that ALAD AMs exhibited a higher expression of CXCL10, a marker of ISG AMs, and increased secretion of pro-inflammatory cytokines compared to AMs from stable patients. Using publicly available bronchoalveolar lavage scRNAseq datasets, we found that ISG and MT AMs are associated with more severe inflammation in COVID-19 patients. Analysis of cells from 4 explanted CLAD lungs revealed similar macrophage populations. Donor and recipient cells were identified using expressed single nucleotide variations. We demonstrated contributions of donor and recipient cells to all AM subsets early post-transplant, with loss of donor-derived cells over time. CONCLUSIONS: Our data reveal extensive heterogeneity among lung macrophages after lung transplantation and indicates that specific sub-populations may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.

Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.

Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.

Association between the renin-angiotensin system and chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) is the major cause of death after lung transplantation. Angiotensin II (AngII), the main effector of the renin-angiotensin system, elicits fibrosis in both kidney and lung. We identified six AngII-regulated proteins (Ras homolog family member B (RHOB), bone marrow stromal cell antigen 1 (BST1), lysophospholipase 1 (LYPA1), glutamine synthetase (GLNA), thrombospondin 1 (TSP1) and laminin subunit ß2 (LAMB2)) that were increased in urine of patients with kidney allograft fibrosis. We hypothesised that the renin-angiotensin system is active in CLAD and that AngII-regulated proteins are increased in bronchoalveolar lavage fluid (BAL) of CLAD patients.We performed immunostaining of AngII receptors (AGTR1 and AGTR2), TSP1 and GLNA in 10 CLAD lungs and five controls. Using mass spectrometry, we quantified peptides corresponding to AngII-regulated proteins in BAL of 40 lung transplant recipients (stable, acute lung allograft dysfunction (ALAD) and CLAD). Machine learning algorithms were developed to predict CLAD based on BAL peptide concentrations.Immunostaining demonstrated significantly more AGTR1+ cells in CLAD versus control lungs (p=0.02). TSP1 and GLNA immunostaining positively correlated with the degree of lung fibrosis (R2=0.42 and 0.57, respectively). In BAL, we noted a trend towards higher concentrations of AngII-regulated peptides in patients with CLAD at the time of bronchoscopy, and significantly higher concentrations of BST1, GLNA and RHOB peptides in patients that developed CLAD at follow-up (p<0.05). The support vector machine classifier discriminated CLAD from stable and ALAD patients at the time of bronchoscopy (area under the curve (AUC) 0.86) and accurately predicted subsequent CLAD development (AUC 0.97).Proteins involved in the renin-angiotensin system are increased in CLAD lungs and BAL. AngII-regulated peptides measured in BAL may accurately identify patients with CLAD and predict subsequent CLAD development.

Use of the Posterior Lumbar Approach for Psoas Major Injection in Hip Flexor Spasticity.

BACKGROUND: Hip flexor spasticity in patients with upper motor neuron syndrome of multiple etiologies has been managed with botulinum neurotoxin injections mainly targeting the "iliopsoas" muscle. A lumbar approach to target psoas major (PM) has been described; however, it has not been incorporated into clinical practice due to perceived risk of injury to surrounding structures. This study will investigate the feasibility and accuracy of ultrasound-guided (UG)-PM injection using a lumbar approach by assessing the intra/extramuscular injectate spread in cadaveric specimens. METHODS: In eight lightly embalmed specimens, toluidine blue dye/saline was injected into PM using a UG-posterior lumbar approach. The posterior abdominal wall was exposed, and dye spread and surrounding structures digitized and modeled in 3D. The area and vertebral level of dye spread, distance of dye to the inferior vena cava (IVC), and abdominal aorta (AA) and dye spread to adjacent organs were quantified. RESULTS: The models enabled visualization of the dye spread in 3D. Mean area of dye spread was 24.4 ± 2.8 cm2; most commonly between L2 and L4 vertebral levels. Mean distance of the dye to AA was 3.2 ± 1.2 cm and to IVC was 1.8 ± 0.4 cm. Dye spread remained intramuscular in all but one specimen. No dye spread occurred to any adjacent organs. CONCLUSIONS: The injection of PM using the UG-posterior lumbar approach was consistent and without spread to surrounding structures. This technique alone or in addition to the anterior approach is expected to have better clinical outcomes in the treatment of hip flexor spasticity. Further clinical studies are required.