Gold Standard RNAscope ISH Technology

The purpose of this review is to analyze the most recent and relevant literature involving lung transplantation for coronavirus disease 2019 (COVID-19) associated acute respiratory distress syndrome (ARDS), the pathological mechanisms of lung injury, selection criteria and outcomes.Pathological analysis of lungs after COVID-19 ARDS has shown architectural distortion similar to that observed in explanted lungs from patients undergoing lung transplantation for end-stage lung diseases such as emphysema. Short-term outcomes after lung transplantation for COVID-19 associated respiratory failure are comparable to those performed for other indications.Lung transplantation after COVID-19 ARDS is a potentially life-saving procedure for appropriately selected patients with no evidence of lung function recovery despite maximal treatment. Lung transplantation should be ideally performed in high-volume centers with expertise.

Primary graft dysfunction (PGD) is the leading cause of postoperative mortality in lung transplant recipients and the most important risk factor for development of chronic lung allograft dysfunction. The mechanistic basis for the variability in the incidence and severity of PGD between lung transplant recipients is not known. Using a murine orthotopic vascularized lung transplant model, we found that redundant activation of Toll-like receptors 2 and 4 (TLR2 and -4) on nonclassical monocytes activates MyD88, inducing the release of the neutrophil attractant chemokine CXCL2. Deletion of Itgam (encodes CD11b) in nonclassical monocytes enhanced their production of CXCL2 and worsened PGD, while a CD11b agonist, leukadherin-1, administered only to the donor lung prior to lung transplantation, abrogated CXCL2 production and PGD. The damage-associated molecular pattern molecule HMGB1 was increased in peripheral blood samples from patients undergoing lung transplantation after reperfusion and induced CXCL2 production in nonclassical monocytes via TLR4/MyD88. An inhibitor of HMGB1 administered to the donor and recipient prior to lung transplantation attenuated PGD. Our findings suggest that CD11b acts as a molecular brake to prevent neutrophil recruitment by nonclassical monocytes following lung transplantation, revealing an attractive therapeutic target in the donor lung to prevent PGD in lung transplant recipients.

There have been over 177 million cases of COVID-19 worldwide, many of whom could be organ donors. Concomitantly, there is an anticipated increase in the need for donor lungs due to expanding indications. Given that the respiratory tract is most commonly affected by COVID-19, there is an urgent need to develop donor assessment criteria while demonstrating safety and "efficacy" of lung donation following COVID-19 infection. Accordingly, we report an intentional transplant using lungs from a donor with recent, microbiologically confirmed, COVID-19 infection into a recipient suffering from COVID-19 induced ARDS and pulmonary fibrosis. In addition to the standard clinical assays, both donor and recipient lungs were analyzed using RNAscope, which confirmed that tissues were negative for SARS-CoV-2. Immunohistochemistry demonstrated colocalized KRT17+ basaloid-like epithelium and COL1A1+ fibroblasts, a marker suggestive of lung fibrosis in COVID-19 associated lung disease, in the explanted recipient lungs but absent in the donor lungs. We demonstrate that following a thorough assessment, lung donation following resolved COVID-19 infection is safe and feasible.

Objective The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS- or bleomycin-induced acute lung injury, sheddases (membrane-bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV-induced shedding of the glycocalyx. Hypothesis We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases. Methods We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP-7, -2, -9 and their inhibitors TIMP-1 and -2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT-qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE-12 mouse lung epithelial cells were infected with viable or heat-inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT-qPCR. Results Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP-7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP-7 is only expressed in cells that are directly infected by IAV. Expression of the MMP-7 inhibitor TIMP-1 is similar to uninfected lungs on day 1, but increases 50-fold on day 3. In contrast, MMP-2 and MMP-9, as well as their inhibitor TIMP-2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat-inactivated IAV fails to upregulate MMP-7. Conclusions Together, these data suggest that localized IAV infection increases MMP-7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP-7 may modulate IAV-induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV-ARDS pathogenesis.

In the progression phase of idiopathic pulmonary fibrosis (IPF) the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a co-culture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing (sc-RNA-seq) revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB cell populations adopted distinct pro-fibrotic cell differentiation states upon co-cultivation, resembling specific cell populations that were previously identified in lungs of IPF patients. Transcriptomic analysis revealed active nuclear factor NF-kappa-B (NF-κB) signaling early in the co-cultured EC and FB cells and the identified NF-κB expression signatures were also found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6 (IL6), interleukin-8 (IL-8) and C-X-C motif chemokine ligand 6 (CXCL6) cytokine secretion, as well as collagen alpha-1(I) chain (COL1A1) and alpha-smooth muscle actin (α-SMA) accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

Ventilatory support, such as supplemental oxygen, used to save premature infants impairs the growth of the pulmonary microvasculature and distal alveoli, leading to bronchopulmonary dysplasia (BPD). Although lung cellular composition changes with exposure to hyperoxia in neonatal mice, most human BPD survivors are weaned off oxygen within the first weeks to months of life, yet they may have persistent lung injury and pulmonary dysfunction as adults. We hypothesized that early-life hyperoxia alters the cellular landscape in later life and predicts long-term lung injury. Using single-cell RNA sequencing, we mapped lung cell subpopulations at postnatal day (pnd)7 and pnd60 in mice exposed to hyperoxia (95% O2) for 3 days as neonates. We interrogated over 10,000 cells and identified a total of 45 clusters within 32 cell states. Neonatal hyperoxia caused persistent compositional changes in later life (pnd60) in all five type II cell states with unique signatures and function. Premature infants requiring mechanical ventilation with different durations also showed similar alterations in these unique signatures of type II cell states. Pathologically, neonatal hyperoxic exposure caused alveolar simplification in adult mice. We conclude that neonatal hyperoxia alters the lung cellular landscape in later life, uncovering neonatal programing of adult lung dysfunction.

This study compared the immune response in mild versus fatal SARS-CoV2 infection. Forty nasopharyngeal swabs with either productive mild infection (n = 20) or negative for SARS-CoV2 (n = 20) were tested along with ten lung sections from people who died of COVID-19 which contained abundant SARS-CoV2 and ten controls. There was a 25-fold increase in the CD3+T cell numbers in the viral positive nasopharyngeal swabs compared to the controls (p < 0.001) and no change in the CD3+T cell count in the fatal COVID-19 lungs versus the controls. CD11b + and CD206+ macrophage counts were significantly higher in the mild versus fatal disease (p = 0.002). In situ analysis for SARS-CoV2 RNA found ten COVID-19 lung sections that had no/rare detectable virus and also lacked the microangiopathy typical of the viral positive sections. These viral negative lung tissues when compared to the viral positive lung samples showed a highly significant increase in CD3+ and CD8 T cells (p < 0.001), equivalent numbers of CD163+ cells, and significantly less PDL1, CD11b and CD206+ cells (p = 0.002). It is concluded that mild SARS-CoV2 infection is marked by a much stronger CD3/CD8 T cell, CD11b, and CD206 macrophage response than the fatal lung disease where viral RNA is abundant.

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF) have worse outcomes following COVID-19. SARSCoV-2 (2019-nCoV) spike protein (S1) harbors an RGD motif in its receptor-binding domain (RBD). Although SARS-CoV-2 is to exploit human Angiotensin Converting Enzyme-2 (ACE2) receptors for cell entry. Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 with very low expression (1.5%) in Alveolar type 2 epithelial cells. It is possible that SARS-CoV-2 needs a cellular co-receptor, which could include integrins, to promote alveolar cell internalization and pneumonitis.METHODS: Solid-phase binding assays were used to investigate S1 binding to ACE2 or αv containing integrins. Pseudovirus entry assays were used to measure the internalization of SARS-CoV-2 into Human embryonic kidney 293T cells expressing different combinations of potential receptors. RNAscope was used to visualize the co-localization of SARS-CoV-2, ACE2, and integrin mRNAs. Immunohistochemistry was used to evaluate the expression of αvβ6 integrins and ACE2 in lung tissue.RESULTS: Binding assays demonstrated that the RGD containing αvβ3 and αvβ6 integrins bound robustly to the SARS-CoV-2 S1 subunit of Spike protein and overexpression of the αvβ6 integrin modestly augments ACE2 mediated SARS-CoV-2 pseudoviral entry into epithelial cells. In COVID-19 damaged lung ACE2 levels are low but the αvβ6 integrin levels are increased in alveolar epithelium whereas both ACE2 and αvβ6 integrin are increased in lung sections from idiopathic pulmonary fibrosis compared with normal lung samples. CONCLUSION: The SARS-CoV-2 S1 subunit can bind αvβ6 integrins augmenting ACE2-dependent internalization of pseudovirus. In IPF patients, ACE2 levels and αvβ6 integrin levels are increased. Increased binding of the SARS-CoV-2 to ACE2 and the αvβ6 integrin within fibrotic lung may explain the increased risk of severe COVID in patients with IPF.

Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.