Intravital Two-Photon Microscopy of the Transplanted Mouse Lung.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in naïve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Nanoparticle targeting of neutrophil glycolysis prevents lung ischemia-reperfusion injury.

Neutrophils exacerbate pulmonary ischemia-reperfusion injury (IRI) resulting in poor short and long-term outcomes for lung transplant recipients. Glycolysis powers neutrophil activation, but it remains unclear if neutrophil-specific targeting of this pathway will inhibit IRI. Lipid nanoparticles containing the glycolysis flux inhibitor 2-deoxyglucose (2-DG) were conjugated to neutrophil-specific Ly6G antibodies (NP-Ly6G[2-DG]). Intravenously administered NP-Ly6G(2-DG) to mice exhibited high specificity for circulating neutrophils. NP-Ly6G(2-DG)-treated neutrophils were unable to adapt to hypoglycemic conditions of the lung airspace environment as evident by the loss of demand-induced glycolysis, reductions in glycogen and ATP content, and an increased vulnerability to apoptosis. NP-Ly6G(2-DG) treatment inhibited pulmonary IRI following hilar occlusion and orthotopic lung transplantation. IRI protection was associated with less airspace neutrophil extracellular trap generation, reduced intragraft neutrophilia, and enhanced alveolar macrophage efferocytotic clearance of neutrophils. Collectively, our data show that pharmacologically targeting glycolysis in neutrophils inhibits their activation and survival leading to reduced pulmonary IRI.

Eosinophils restrain humoral alloimmunity after lung transplantation.

While the function of many leukocytes in transplant biology has been well defined, the role of eosinophils is controversial and remains poorly explored. Conflicting data exist regarding eosinophils' role in alloimmunity. Due to their prevalence in the lung, and their defined role in other pulmonary pathologies such as asthma, we set out to explore the role of eosinophils in the long-term maintenance of the lung allograft. We noted that depletion of eosinophils results in the generation of donor-specific antibodies. Eosinophil depletion increased memory B cell, plasma cell, and antibody-secreting cell differentiation and resulted in de novo generation of follicular germinal centers. Germinal center formation depended on the expansion of CD4+Foxp3-Bcl6+CXCR5+PD-1+ T follicular helper (Tfh) cells, which increase in number after eosinophil depletion. Mechanistically, we demonstrate that eosinophils prevent Tfh cell generation by acting as the dominant source of IFN-γ in an established lung allograft, thus facilitating Th1 rather than Tfh polarization of naive CD4+ T cells. Our data thus describe what we believe is a unique and previously unknown role for eosinophils in maintaining allograft tolerance and suggest that indiscriminate administration of eosinophil-lytic corticosteroids for treatment of acute cellular rejection may inadvertently promote humoral alloimmunity.

Smoking exposure-induced bronchus-associated lymphoid tissue in donor lungs does not prevent tolerance induction after transplantation.

The presence of bronchus-associated lymphoid tissue (BALT) in donor lungs has been suggested to accelerate graft rejection after lung transplantation. Although chronic smoke exposure can induce BALT formation, the impact of donor cigarette use on alloimmune responses after lung transplantation is not well understood. Here, we show that smoking-induced BALT in mouse donor lungs contains Foxp3+ T cells and undergoes dynamic restructuring after transplantation, including recruitment of recipient-derived leukocytes to areas of pre-existing lymphoid follicles and replacement of graft-resident donor cells. Our findings from mouse and human lung transplant data support the notion that a donor's smoking history does not predispose to acute cellular rejection or prevent the establishment of allograft acceptance with comparable outcomes to nonsmoking donors. Thus, our work indicates that BALT in donor lungs is plastic in nature and may have important implications for modulating proinflammatory or tolerogenic immune responses following transplantation.

Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are critical mediators of type 2 respiratory inflammation, releasing IL-5 and IL-13 and promoting the pulmonary eosinophilia associated with allergen provocation. Although ILC2s have been shown to promote eosinophil activities, the role of eosinophils in group 2 innate lymphoid cell (ILC2) responses is less well defined. OBJECTIVE: We sought to investigate the role of eosinophils in activation of ILC2s in models of allergic asthma and in vitro. METHODS: Inducible eosinophil-deficient mice were exposed to allergic respiratory inflammation models of asthma, such as ovalbumin or house dust mite challenge, or to innate models of type 2 airway inflammation, such as inhalation of IL-33. Eosinophil-specific IL-4/13-deficient mice were used to address the specific roles for eosinophil-derived cytokines. Direct cell interactions between ILC2s and eosinophils were assessed by in vitro culture experiments. RESULTS: Targeted depletion of eosinophils resulted in significant reductions of total and IL-5+ and IL-13+ lung ILC2s in all models of respiratory inflammation. This correlated with reductions in IL-13 levels and mucus in the airway. Eosinophil-derived IL-4/13 was necessary for both eosinophil and ILC2 accumulation in lung in allergen models. In vitro, eosinophils released soluble mediators that induced ILC2 proliferation and G protein-coupled receptor-dependent chemotaxis of ILC2s. Coculture of ILC2s and IL-33-activated eosinophils resulted in transcriptome changes in both ILC2s and eosinophils, suggesting potential novel reciprocal interactions. CONCLUSION: These studies demonstrate that eosinophils play a reciprocal role in ILC2 effector functions as part of both adaptive and innate type 2 pulmonary inflammatory events.

Regadenoson Reduces Soluble Receptor for Advanced Glycation End-Products in Lung Recipients.

BACKGROUND: The selective adenosine A2A receptor (A2AR) agonist regadenoson reduces inflammation due to lung ischemia-reperfusion injury (IRI). The objective of this study was to investigate molecular and cellular mechanisms by which regadenoson reduces IRI in lung transplant recipients. METHODS: Fourteen human lung transplant recipients were infused for 12 hours with regadenoson and 7 more served as untreated controls. Plasma levels of high mobility group box 1 and its soluble receptor for advanced glycation end-products (sRAGE) were measured by Luminex. Matrix metalloproteinase (MMP) 2 and 9 were measured by gelatin zymography. Tissue inhibitor of metalloproteinase 1 was measured by mass spectroscopy. A2AR expression on leukocytes was analyzed by flow cytometry. MMP-9-mediated cleavage of RAGE was evaluated using cultured macrophages in vitro. RESULTS: Regadenoson treatment during lung transplantation significantly reduced levels of MMP-9 (P < .05), but not MMP-2, and elevated levels of tissue inhibitor of metalloproteinase 1 (P < .05), an endogenous selective inhibitor of MMP-9. Regadenoson infusion significantly reduced plasma levels of sRAGE (P < .05) during lung reperfusion compared with control subjects. A2AR expression was highest on invariant natural killer T cells and higher on monocytes than other circulating immune cells (P < .05). The shedding of RAGE from cultured monocytes/macrophages was increased by MMP-9 stimulation and reduced by an MMP inhibitor or by A2AR agonists, regadenoson or ATL146e. CONCLUSIONS: In vivo and in vitro studies suggest that A2AR activation reduces sRAGE in part by inhibiting MMP-9 production by monocytes/macrophages. These results suggest a novel molecular mechanism by which A2AR agonists reduce primary graft dysfunction.

Full-length IL-33 augments pulmonary fibrosis in an ST2- and Th2-independent, non-transcriptomic fashion.

Mature IL-33 (MIL33) acting through its receptor, ST2, is known to regulate fibrosis. The precursor, full-length IL-33 (FLIL33), may function differently from MIL33 and independently of ST2. Here we report that genetic deletion of either IL-33 or ST2 attenuates pulmonary fibrosis in the bleomycin model, as does Cre-induced IL-33 deficiency in response to either acute or chronic bleomycin challenge. However, adenovirus-mediated gene delivery of FLIL33, but not MIL33, to the lungs of either wild-type or ST2-deficient mice potentiates the profibrotic effect of bleomycin without inducing a Th2 phenotype. In cultured mouse lung cells, FLIL33 overexpression induces moderate and distinct transcriptomic changes compared with a robust response induced by MIL33, whereas ST2 deletion abrogates the effects of both IL-33 forms. Thus, FLIL33 may contribute to fibrosis in an ST2-independent, Th2-independent, non-transcriptomic fashion, suggesting that pharmacological targeting of both FLIL33 and MIL33 may prove efficacious in patients with pulmonary fibrosis.

Successful lung transplantation using an allograft from a COVID-19-recovered donor: a potential role for subgenomic RNA to guide organ utilization.

Although the risk of SARS-CoV-2 transmission through lung transplantation from acutely infected donors is high, the risks of virus transmission and long-term lung allograft outcomes are not as well described when using pulmonary organs from COVID-19-recovered donors. We describe successful lung transplantation for a COVID-19-related lung injury using lungs from a COVID-19-recovered donor who was retrospectively found to have detectable genomic SARS-CoV-2 RNA in the lung tissue by multiple highly sensitive assays. However, SARS-CoV-2 subgenomic RNA (sgRNA), a marker of viral replication, was not detectable in the donor respiratory tissues. One year after lung transplantation, the recipient has a good functional status, walking 1 mile several times per week without the need for supplemental oxygen and without any evidence of donor-derived SARS-CoV-2 transmission. Our findings highlight the limitations of current clinical laboratory diagnostic assays in detecting the persistence of SARS-CoV-2 RNA in the lung tissue. The persistence of SARS-CoV-2 RNA in the donor tissue did not appear to represent active viral replication via sgRNA testing and, most importantly, did not negatively impact the allograft outcome in the first year after lung transplantation. sgRNA is easily performed and may be a useful assay for assessing viral infectivity in organs from donors with a recent infection.

The impact of the American Association for Thoracic Surgery on National Institutes of Health grant funding for cardiothoracic surgeons.

OBJECTIVES: The American Association for Thoracic Surgery, through its annual meeting, pilot grant funding, Scientific Affairs and Government Relations Committee activity, and academic development programs (Grant Writing Workshop, Clinical Trials Course, Innovation Summit), has aimed to develop the research careers of cardiothoracic surgeons. We hypothesized that American Association for Thoracic Surgery activities have helped increase National Institutes of Health grants awarded to cardiothoracic surgeons. METHODS: A database of 1869 academic cardiothoracic surgeons in the United States was created in December 2020. National Institutes of Health grant records from 1985 to 2020 were obtained for each surgeon using National Institutes of Health Research Portfolio Online Reporting Tools Expenditures and Results. Analyses were normalized to the number of active surgeons per year, based on the year of each surgeon's earliest research publication on Scopus. RESULTS: A total of 346 cardiothoracic surgeons have received 696 National Institutes of Health grants totaling more than $1.5 billion in funding, with 48 surgeons actively serving as principal investigator of 66 R01 grants in 2020. The prevalence of research grants (7.4 vs 5.6 grants per 100 active surgeons, P < .0001), percentage of surgeons with a research grant (5.3% vs 4.7%, P = .0342), and number of research grants per funded surgeon (1.4 vs 1.2 grants, P < .0001) were significantly greater during the Scientific Affairs and Government Relations era (2003-2020) than the pre-Scientific Affairs and Government Relations era (1985-2002). The incidence of new research grants after surgeon participation in an American Association for Thoracic Surgery academic development program was significantly greater than that in the absence of participation (3.5 vs 1.1 new grants per 100 surgeons per year, P < .0001). CONCLUSIONS: Through dedicated efforts and programs, the American Association for Thoracic Surgery has provided effective support to help increase National Institutes of Health grant funding awarded to cardiothoracic surgeons.

Reprogramming alveolar macrophage responses to TGF-ß reveals CCR2+ monocyte activity that promotes bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients, but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-ß bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-ß and reduce TGF-ß bioavailability through secretion of the TGF-ß antagonist decorin. In untreated recipients, high airway TGF-ß activity stimulated AMs to express CCL2, leading to CCR2+ monocyte-driven BOS development. Moreover, we found TGF-ß receptor 2-dependent differentiation of CCR2+ monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8+ T cells that inflicted airway injury through Blimp-1-mediated granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-ß-dependent network that couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS.

The role of neutrophil extracellular traps in acute lung injury.

Acute lung injury (ALI) is a heterogeneous inflammatory condition associated with high morbidity and mortality. Neutrophils play a key role in the development of different forms of ALI, and the release of neutrophil extracellular traps (NETs) is emerging as a common pathogenic mechanism. NETs are essential in controlling pathogens, and their defective release or increased degradation leads to a higher risk of infection. However, NETs also contain several pro-inflammatory and cytotoxic molecules than can exacerbate thromboinflammation and lung tissue injury. To reduce NET-mediated lung damage and inflammation, DNase is frequently used in preclinical models of ALI due to its capability of digesting NET DNA scaffold. Moreover, recent advances in neutrophil biology led to the development of selective NET inhibitors, which also appear to reduce ALI in experimental models. Here we provide an overview of the role of NETs in different forms of ALI discussing existing gaps in our knowledge and novel therapeutic approaches to modulate their impact on lung injury.

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Updated Views on Neutrophil Responses in Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury is an inevitable event during organ transplantation and represents a primary risk factor for the development of early graft dysfunction in lung, heart, liver, and kidney transplant recipients. Recent studies have implicated recipient neutrophils as key mediators of this process and also have found that early innate immune responses after transplantation can ultimately augment adaptive alloimmunity and affect late graft outcomes. Here, we discuss signaling pathways involved in neutrophil recruitment and activation after ischemia-mediated graft injury in solid organ transplantation with an emphasis on lung allografts, which have been the focus of recent studies. These findings suggest novel therapeutic interventions that target ischemia-reperfusion injury-mediated graft dysfunction in transplant recipients.

Role of tertiary lymphoid organs in the regulation of immune responses in the periphery.

Tertiary lymphoid organs (TLOs) are collections of immune cells resembling secondary lymphoid organs (SLOs) that form in peripheral, non-lymphoid tissues in response to local chronic inflammation. While their formation mimics embryologic lymphoid organogenesis, TLOs form after birth at ectopic sites in response to local inflammation resulting in their ability to mount diverse immune responses. The structure of TLOs can vary from clusters of B and T lymphocytes to highly organized structures with B and T lymphocyte compartments, germinal centers, and lymphatic vessels (LVs) and high endothelial venules (HEVs), allowing them to generate robust immune responses at sites of tissue injury. Although our understanding of the formation and function of these structures has improved greatly over the last 30 years, their role as mediators of protective or pathologic immune responses in certain chronic inflammatory diseases remains enigmatic and may differ based on the local tissue microenvironment in which they form. In this review, we highlight the role of TLOs in the regulation of immune responses in chronic infection, chronic inflammatory and autoimmune diseases, cancer, and solid organ transplantation.

A reengineered common chain cytokine augments CD8+ T cell-dependent immunotherapy.

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3-mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell-based immunotherapy.

Necroptosis triggers spatially restricted neutrophil-mediated vascular damage during lung ischemia reperfusion injury.

Ischemia reperfusion injury represents a common pathological condition that is triggered by the release of endogenous ligands. While neutrophils are known to play a critical role in its pathogenesis, the tissue-specific spatiotemporal regulation of ischemia-reperfusion injury is not understood. Here, using oxidative lipidomics and intravital imaging of transplanted mouse lungs that are subjected to severe ischemia reperfusion injury, we discovered that necroptosis, a nonapoptotic form of cell death, triggers the recruitment of neutrophils. During the initial stages of inflammation, neutrophils traffic predominantly to subpleural vessels, where their aggregation is directed by chemoattractants produced by nonclassical monocytes that are spatially restricted in this vascular compartment. Subsequent neutrophilic disruption of capillaries resulting in vascular leakage is associated with impaired graft function. We found that TLR4 signaling in vascular endothelial cells and downstream NADPH oxidase 4 expression mediate the arrest of neutrophils, a step upstream of their extravasation. Neutrophil extracellular traps formed in injured lungs and their disruption with DNase prevented vascular leakage and ameliorated primary graft dysfunction. Thus, we have uncovered mechanisms that regulate the initial recruitment of neutrophils to injured lungs, which result in selective damage to subpleural pulmonary vessels and primary graft dysfunction. Our findings could lead to the development of new therapeutics that protect lungs from ischemia reperfusion injury.

Influence of broader geographic allograft sharing on outcomes and cost in smaller lung transplant centers.

OBJECTIVE: On November 24, 2017, Organ Procurement and Transplantation Network implemented a change to lung allocation replacing donor service area with a 250 nautical mile radius around donor hospitals. We sought to evaluate the experience of a small to medium size center following implementation. METHODS: Patients (47 pre and 54 post) undergoing lung transplantation were identified from institutional database from January 2016 to October 2019. Detailed chart review and analysis of institutional cost data was performed. Univariate analysis was performed to compare eras. RESULTS: Similar short-term mortality and primary graft dysfunction were observed between groups. Decreased local donation (68% vs 6%; P < .001), increased travel distance (145 vs 235 miles; P = .004), travel cost ($8626 vs $14,482; P < .001), and total procurement cost ($60,852 vs $69,052; P = .001) were observed postimplementation. We also document an increase in waitlist mortality postimplementation (6.9 vs 31.6 per 100 patient-years; P < .001). CONCLUSIONS: Following implementation of the new allocation policy in a small to medium size center, several changes were in accordance with policy intention. However, concerning shifts emerged, including increased waitlist mortality and resource utilization. Continued close monitoring of transplant centers stratified by size and location are paramount to maintaining global availability of lung transplantation to all Americans regardless of geographic residence or socioeconomic status.

Innate immunity in lung transplantation.

Innate immune pathways early after pulmonary transplantation have been shown to cause primary graft dysfunction (PGD) and also predispose to late graft failure. Recent studies in animal models have elucidated critical mechanisms governing such innate immune responses. Here, we discuss pathways of inflammatory cell death, triggers for sterile and infectious inflammation, and signaling cascades that mediate lung injury early after transplantation. These studies highlight potential avenues for lung-specific therapies early following lung transplantation to dampen innate immune responses and improve outcomes.

Ex Vivo Lung Perfusion: Current Achievements and Future Directions.

There is a severe shortage in the availability of donor organs for lung transplantation. Novel strategies are needed to optimize usage of available organs to address the growing global needs. Ex vivo lung perfusion has emerged as a powerful tool for the assessment, rehabilitation, and optimization of donor lungs before transplantation. In this review, we discuss the history of ex vivo lung perfusion, current evidence on its use for standard and extended criteria donors, and consider the exciting future opportunities that this technology provides for lung transplantation.