Inflammasome activation in neutrophils of patients with severe COVID-19.

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

Divergent Function of Programmed Death-Ligand 1 in Donor Tissue versus Recipient Immune System in a Murine Model of Bronchiolitis Obliterans.

Costimulatory molecules, such as the programmed death ligand (PD-L1), might exert differential effects on T-cell function, depending on the clinical setting and/or immunological environment. Given the impact of T cells on bronchiolitis obliterans (BO) in lung transplantation, we used an established tracheal transplant model inducing BO-like lesions to investigate the impact of PD-L1 on alloimmune responses and histopathological outcome in BO. In contrast to other transplant models in which PD-L1 generally shows protective functions, we demonstrated that PD-L1 has divergent effects depending on its location in donor versus recipient tissue. Although PD-L1 deficiency in donor tissue worsened histopathological outcome, and increased systemic inflammatory response, recipient PD-L1 deficiency induced opposite effects. Mechanistic studies revealed PD-L1-deficient recipients were hyporesponsive toward alloantigen, despite increased numbers of CD8+ effector T cells. The function of PD-L1 on T cells after unspecific stimulation was dependent on both cell type and strength of stimulation. This novel function of recipient PD-L1 may result from the high degree of T-cell activation within the highly immunogenic milieu of the transplanted tissue. In this model, both decreased T-cell alloimmune responses and the reduction of BO in PD-L1-deficient recipients suggest a potential therapeutic role of selectively blocking PD-L1 in the recipient. Further investigation is warranted to determine the impact of this finding embedded in the complex pathophysiological context of BO.

Bone marrow-derived multipotent stromal cells attenuate inflammation in obliterative airway disease in mouse tracheal allografts.

Obliterative bronchiolitis (OB) remains the most significant cause of death in long-term survival of lung transplantation. Using an established murine heterotopic tracheal allograft model, the effects of different routes of administration of bone marrow-derived multipotent stromal cells (MSCs) on the development of OB were evaluated. Tracheas from BALB/c mice were implanted into the subcutaneous tissue of major histocompatibility complex- (MHC-) disparate C57BL/6 mice. At the time of transplant, bone marrow-derived MSCs were administered either systemically or locally or via a combination of the two routes. The allografts were explanted at various time points after transplantation and were evaluated for epithelial integrity, inflammatory cell infiltration, fibrosis, and luminal obliteration. We found that the most effective route of bone marrow-derived MSC administration is the combination of systemic and local delivery. Treatment of recipient mice with MSCs suppressed neutrophil, macrophage, and T-cell infiltration and reduced fibrosis. These beneficial effects were observed despite lack of significant MSC epithelial engraftment or new epithelial cell generation. Our study suggests that optimal combination of systemic and local delivery of MSCs may ameliorate the development of obliterative airway disease through modulation of immune response.

An interesting case of dysautonomia presenting with dyspnea.

Dysautonomia such as POTS syndrome presenting with respiratory symptoms can often be misdiagnosed for other common pulmonary conditions. It can be diagnosed with a comprehensive history and orthostatic vital measurement. Simple diagnostic test such as diffusing capacity in supine and standing position can emerge as a noninvasive tool to guide the long-term monitoring and treatment response.

Green tea epigallo-catechin-galleate ameliorates the development of obliterative airway disease.

Lung transplantation has the worst outcome compared to all solid organ transplants due to chronic rejection known as obliterative bronchiolitis (OB). Pathogenesis of OB is a complex interplay of alloimmune-dependent and -independent factors, which leads to the development of inflammation, fibrosis, and airway obliteration that have been resistant to therapy. The alloimmune-independent inflammatory pathway has been the recent focus in the pathogenesis of rejection, suggesting that targeting this may offer therapeutic benefits. As a potent anti-inflammatory agent, epigallo-catechin-galleate (EGCG), a green tea catechin, has been very effective in ameliorating inflammation in a variety of diseases, providing the rationale for its use in this study in a murine heterotopic tracheal allograft model of OB. Mice treated with EGCG had reduced inflammation, with significantly less neutrophil and macrophage infiltration and significantly reduced fibrosis. On further investigation into the mechanisms, inflammatory cytokines keratinocyte (KC), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α), involved in neutrophil recruitment, were reduced in the EGCG-treated mice. In addition, monocyte chemokine monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by EGCG treatment. Antifibrotic cytokine interferon-γ-inducible protein-10 (IP-10) was increased and profibrotic cytokine transforming growth factor-ß (TGF-ß) was reduced, further characterizing the antifibrotic effects of EGCG. These findings suggest that EGCG has great potential in ameliorating the development of obliterative airway disease.

MMP-8 promotes polymorphonuclear cell migration through collagen barriers in obliterative bronchiolitis.

Increased levels of MMP-8 (neutrophil collagenase) have been reported in OB, but the biological role of MMP-8 in OB is not known. MMP-8 is an interstitial collagenase highly expressed by polymorphonuclear leukocytes, which are prominent in early OB. Here, we show that MMP-8 promotes migration of PMNs through the collagen-rich matrix in a mouse heterotopic airway transplant model of OB. Overall, MMP-8(-/-) mice had significantly fewer PMNs in the airway lumen 2 and 14 days post-transplantation, and the percentage of PMNs traversing the matrix to the lumen was decreased markedly in the MMP-8(-/-) compared with WT mice at 14 days. There were significantly more PMNs outside of the lumen in the ECM in the MMP-8(-/-) mice compared with WT mice. In vitro, significantly fewer MMP-8(-/-) PMNs migrated through 3D cross-linked collagen gels than WT PMNs. MMP inhibitor GM6001 was also able to impede migration of WT PMNs through collagen gels. The decreased migration was likely a result of pericollagenase activity of MMP-8, as WT PMNs expressing MMP-8 were not able to migrate effectively through collagen that was resistant to the collagenase. Protection from OB was seen in the MMP-8(-/-) mice, as the airway lumen had significantly less obliteration and collagen deposition, suggesting that MMP-8 plays an important role in the pathogenesis of OB.

Centrifugal migration of mesenchymal cells in embryonic lung.

Murine lung development begins at embryonic day (E) 9.5. Normal lung structure and function depend on the patterns of localization of differentiated cells. Pulmonary mesenchymal cell lineages have been relatively unexplored. Importantly, there has been no prior evidence of clonality of any lung cells. Herein we use a definitive genetic approach to demonstrate a common origin for proximal and distal pulmonary mesenchymal cells. A retroviral library with 3,400 unique inserts was microinjected into the airway lumen of E11.5 lung buds. After 7-11 days of culture, buds were stained for placental alkaline phosphatase (PLAP). Most PLAP+ cells are peribronchial smooth muscle cells, initially localized laterally near the hilum, then migrating down airways to the subpleural region. Laser-capture microdissection and polymerase chain reaction confirm the clonal identities of PLAP+ cells proximally and distally. Our observation of this fundamental process during lung development opens new avenues for investigation of maladaptive mesenchymal responses in lung diseases.

Bombesin-like peptides modulate alveolarization and angiogenesis in bronchopulmonary dysplasia.

RATIONALE: The incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, is paradoxically rising despite medical advances. We demonstrated elevated bombesin-like peptide levels in infants that later developed BPD. In the 140-day hyperoxic baboon model of BPD, anti-bombesin antibody 2A11 abrogated lung injury. OBJECTIVES: To test the hypothesis that bombesin-like peptides mediate BPD in extremely premature baboons (born at Gestational Day 125 and given oxygen pro re nata [PRN], called the 125-day PRN model), similar to "modern-day BPD." METHODS: The 125-day animals were treated with 2A11 on Postnatal Day 1 (P1), P3, and P6. On P14 and P21, lungs were inflation-fixed for histopathologic analyses of alveolarization. Regulation of angiogenesis by bombesin was evaluated using cultured pulmonary microvascular endothelial cells. MEASUREMENTS AND MAIN RESULTS: In 125-day PRN animals, urine bombesin-like peptide levels at P2-3 are directly correlated with impaired lung function at P14. Gastrin-releasing peptide (the major pulmonary bombesin-like peptide) mRNA was elevated eightfold at P1 and remained high thereafter. At P14, 2A11 reduced alveolar wall thickness and increased the percentage of secondary septa containing endothelial cells. At P21, 2A11-treated 125-day PRN animals had improved alveolarization according to mean linear intercepts and number of branch points per millimeter squared. Bombesin promoted tubulogenesis of cultured pulmonary microvascular endothelial cells, but cocultured fetal lung mesenchymal cells abrogated this effect. CONCLUSIONS: Early bombesin-like peptide overproduction in 125-day PRN animals predicted alveolarization defects weeks later. Bombesin-like peptide blockade improved septation, with the greatest effects at P21. This could have implications for preventing BPD in premature infants.

Immunomodulatory functions of the diffuse neuroendocrine system: implications for bronchopulmonary dysplasia.

Pulmonary neuroendocrine (NE) cells are believed to be the precursor of NE lung carcinomas, including well-differentiated (carcinoids) and moderately/poorly differentiated (atypical carcinoids and small-cell carcinomas, SCLCs) subtypes. In early studies, we determined mechanisms by which NE cell-derived peptides such as bombesin-like peptide (BLP) promote normal fetal lung development. Postnatally, BLP may normally regulate perinatal adaptation of the pulmonary circulation. However, elevated BLP levels in premature infants shortly after birth predict which infants are at high risk for developing bronchopulmonary dysplasia (BPD, chronic lung disease of newborns). An anti-BLP blocking antibody abrogates clinical and pathological evidence of lung injury in two baboon models of BPD. These observations indicate that BLP mediates lung injury in BPD, supporting a role for BLP as pro-inflammatory cytokines. We have directly tested the effects of BLP on eliciting inflammatory cell infiltrates in vivo. Surprisingly, mast cells are the major responding cell population. These data suggest that the diffuse NE system may be a newly recognized component of innate immunity in multiple organ systems. We speculate that overproduction of NE cell-derived peptides such as BLP may be responsible for a variety of chronic inflammatory disorders.

Functional diversity of notch family genes in fetal lung development.

In Drosophila, developmental signaling via the transmembrane Notch receptor modulates branching morphogenesis and neuronal differentiation. To determine whether the notch gene family can regulate mammalian organogenesis, including neuroendocrine cell differentiation, we evaluated developing murine lung. After demonstrating gene expression for notch-1, notch-2, notch-3, and the Notch ligands jagged-1 and jagged-2 in embryonic mouse lung, we tested whether altering expression of these genes can modulate branching morphogenesis. Branching of embryonic day (E) 11.5 lung buds increased when they were treated with notch-1 antisense oligodeoxynucleotides in culture compared with the corresponding sense controls, whereas notch-2, notch-3, jagged-1, or jagged-2 antisense oligos had no significant effect. To assess cell differentiation, we immunostained lung bud cultures for the neural/neuroendocrine marker PGP9.5. Antisense to notch-1 or jagged-1 markedly increased numbers of PGP9.5-positive neuroendocrine cells alone without affecting neural tissue, whereas only neural tissue was promoted by notch-3 antisense in culture. There was no significant effect on cell proliferation or apoptosis in these antisense experiments. Cumulatively, these observations suggest that interactions between distinct Notch family members can have diverse tissue-specific regulatory functions during development, arguing against simple functional redundancy.

Bombesin-like peptides and mast cell responses: relevance to bronchopulmonary dysplasia?

Bombesin-like peptides (BLPs) are elevated in newborns who later develop bronchopulmonary dysplasia (BPD). In baboon models, anti-BLP blocking antibodies abrogate BPD. We now demonstrate hyperplasia of both neuroendocrine cells and mast cells in lungs of baboons with BPD, compared with non-BPD controls or BLP antibody-treated BPD baboons. To determine whether BLPs are proinflammatory, bombesin was administered intratracheally to mice. Forty-eight hours later, we observed increased numbers of lung mast cells. We analyzed murine mast cells for BLP receptor gene expression, and identified mRNAs encoding bombesin receptor subtype 3 and neuromedin-B receptor (NMB-R), but not gastrin-releasing peptide receptor. Only NMB-R-null mice accumulated fewer lung mast cells after bombesin treatment. Bombesin, gastrin-releasing peptide, NMB, and a bombesin receptor subtype 3-specific ligand induced mast cell proliferation and chemotaxis in vitro. These observations support a role for multiple BLPs in promoting mast cell responses, suggesting a mechanistic link between BLPs and chronic inflammatory lung diseases.

A catalytic antioxidant attenuates alveolar structural remodeling in bronchopulmonary dysplasia.

Superoxide anion and other oxygen-free radicals have been implicated in the pathogenesis of bronchopulmonary dysplasia. We tested the hypothesis that a catalytic antioxidant metalloporphyrin AEOL 10113 can protect against hyperoxia-induced lung injury using a fetal baboon model of bronchopulmonary dysplasia. Fetal baboons were delivered by hysterotomy at 140 days of gestation (term = 185 days) and given 100% oxygen for 10 days. Morphometric analysis of alveolar structure showed that fetal baboons on 100% oxygen alone had increased parenchymal mast cells and eosinophils, increased alveolar tissue volume and septal thickness, and decreased alveolar surface area compared with animals given oxygen as needed. Treatment with AEOL 10113 (continuous intravenous infusion) during 100% oxygen exposure partially reversed these oxygen-induced changes. Hyperoxia increased the number of neuroendocrine cells in the peripheral lung, which was preceded by increased levels of urine bombesin-like peptide at 48 hours of age. AEOL 10113 inhibited the hyperoxia-induced increases in urine bombesin-like peptide and numbers of neuroendocrine cells. An increasing trend in oxygenation index over time was observed in the 100% oxygen group but not the mimetic-treated group. These results suggest that AEOL 10113 might reduce the risk of pulmonary oxygen toxicity in prematurely born infants.