Christina Sul

Expression of the redox enzyme EC-SOD limits PH severity. Using vascular-selective EC-SOD depletion and SOD mimetic treatment in chronic hypoxic PH, we demonstrated that EC-SOD limits the hypoxia-induced accumulation of IMs. IMs from mice with low vascular EC-SOD were proinflammatory at baseline and enhanced ECM remodeling pathway activation in response to hypoxia. We identified Lyve1⁺ IMs as a perivascular, ECM-interacting subset that accumulate in hypoxia and could contribute to vascular remodeling in PH.

Acute respiratory distress syndrome is a serious illness accounting for 10% of ICU admissions and has a high mortality of 31–45%, with a paucity of pharmacologic treatment options. Dysregulated inflammation and oxidative stress are hallmark features of acute respiratory distress syndrome. We previously showed that transgenic mice expressing a naturally occurring polymorphism of the antioxidant enzyme EC-SOD (extracellular superoxide dismutase) are protected against Staphylococcus aureus pneumonia, acute lung injury, and pulmonary neutrophilia. In this mouse strain, an R213G amino acid substitution leads to lower tissue-binding affinity and elevated alveolar and plasma EC-SOD amounts, although the redox-regulated mechanisms responsible for protection against S. aureus are not yet elucidated. Neutrophils are recruited to the areas of injury and inflammation, in part by activated platelets, which contain multiple redox-sensitive targets. Thus, we hypothesize that increased circulating EC-SOD due to the EC-SOD R213G variant protects against S. aureus pneumonia by reducing platelet activation and subsequent neutrophil recruitment to the lung. We demonstrate that, compared with wild-type mice with S. aureus pneumonia, platelet activation, formation of platelet–neutrophil aggregates, and influx of neutrophils and platelet–neutrophil aggregates into the lung are decreased in the infected R213G mice. Furthermore, pretreatment with a MnTE-2-PyP SOD mimetic protects against S. aureus–induced platelet activation, pulmonary neutrophilia, and acute lung injury. Our data highlight the redox regulation of platelet activation as a driver of S. aureus–induced acute lung injury.

Inflammation drives the initiation and progression of pulmonary hypertension (PH). Platelets, increasingly recognized as immune cells, are activated and increased in the lungs of patients with PH. Platelet activation leads to the release of α-granule chemokines, many of which are implicated in PH. We hypothesized that hypoxia-induced secretion of platelet α-granule–stored proteins and PH would be prevented in Neurobeachin-like 2 knockout (Nbeal2−/−) α-granule–deficient mice. Wild-type (WT) and Nbeal2−/− mice were maintained in normoxia or exposed to 10% hypobaric hypoxia for 3, 14, 21, or 35 days. We observed macrothrombocytopenia, increased circulating neutrophils and monocytes, and increased lung interstitial macrophages (IMs) in Nbeal2−/− mice at baseline. Hypoxia-induced platelet activation was attenuated, and hypoxia-induced increase in lung platelet factor 4 (PF4) and platelets was delayed in Nbeal2−/− mice compared with in WT mice. Finally, although pulmonary vascular remodeling (PVR) and PH were attenuated at day 21, Nbeal2−/− mice were not protected against hypoxia-induced PVR and PH at day 35. Although this mutation also affected circulating monocytes, neutrophils, and lung IMs, all of which are critical in the development of experimental PH, we gained further support for the role of platelets and α-granule proteins, such as PF4, in PH progression and pathogenesis and made several observations that expand our understanding of α-granule–deficient mice in chronic hypoxia.

Redistributed expression of SOD3 out of tissue ECM due to the human R213G SNP exacerbates chronic hypoxic PH. Highlighting the importance of macrophage phenotype, our findings reveal that the R213G SNP does not exacerbate pulmonary macrophage accumulation in response to hypoxia but influences their metabolic and phenotypic reprogramming. We demonstrate a deficiency in the metabolic response to hypoxic stress in R213G macrophages, associated with weakened inflammatory resolution and activation of profibrotic pathways implicated in PH.

Pulmonary hypertension (PH) is a progressive disease marked by pulmonary vascular remodeling and right ventricular failure. Inflammation and oxidative stress are critical in PH pathogenesis, with early pulmonary vascular inflammation preceding vascular remodeling. Extracellular superoxide dismutase (EC-SOD), a key vascular antioxidant enzyme, mitigates oxidative stress and protects against inflammation and fibrosis in diverse lung and vascular disease models. This study utilizes a murine hypobaric hypoxia model to investigate the role of lung EC-SOD on hypoxia-induced platelet activation and platelet lung accumulation, a critical factor in PH-related inflammation. We found that lung EC-SOD overexpression blocked hypoxia-induced platelet activation and platelet accumulation in the lung. Though lung EC-SOD overexpression increased lung EC-SOD content, it did not impact plasma extracellular SOD activity. However, ex vivo, exogenous extracellular SOD treatment specifically blunted convulxin-induced platelet activation but did not blunt platelet activation with thrombin or ADP. Our data identify platelets as a novel target of EC-SOD in response to hypoxia, providing a foundation to advance the understanding of dysregulated redox signaling and platelet activation in PH and other chronic hypoxic lung diseases.

Alveolar inflammation is a hallmark of acute lung injury (ALI), and its clinical correlate is acute respiratory distress syndrome—and it is as a result of interactions between alveolar type II cells (ATII) and alveolar macrophages (AM). In the setting of acute injury, the microenvironment of the intra‐alveolar space is determined in part by metabolites and cytokines and is known to shape the AM phenotype. In response to ALI, increased glycolysis is observed in AT II cells, mediated by the transcription factor hypoxia‐inducible factor (HIF) 1α, which has been shown to decrease inflammation. We hypothesized that in acute lung injury, lactate, the end product of glycolysis, produced by ATII cells shifts AMs toward an anti‐inflammatory phenotype, thus mitigating ALI. We found that local intratracheal delivery of lactate improved ALI in two different mouse models. Lactate shifted cytokine expression of murine AMs toward increased IL‐10, while decreasing IL‐1 and IL‐6 expression. Mice with ATII‐specific deletion of Hif1a and mice treated with an inhibitor of lactate dehydrogenase displayed exacerbated ALI and increased inflammation with decreased levels of lactate in the bronchoalveolar lavage fluid; however, all those parameters improved with intratracheal lactate. When exposed to LPS (to recapitulate an inflammatory stimulus as it occurs in ALI), human primary AMs co‐cultured with alveolar epithelial cells had reduced inflammatory responses. Taken together, these studies reveal an innate protective pathway, in which lactate produced by ATII cells shifts AMs toward an anti‐inflammatory phenotype and dampens excessive inflammation in ALI.

Acute respiratory distress syndrome (ARDS) has approximately 40% in-hospital mortality, and treatment is limited to supportive care. Pneumonia is the underlying etiology in many cases with unrestrained inflammation central to the pathophysiology. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA(miR)-146a, reduces bleomycin- and endotoxin-induced acute lung injury (ALI) by decreasing inflammation. We therefore hypothesized that CNP-miR146a would decrease inflammation in murine infectious ALI. Mice were injured with intratracheal (IT) MRSA or saline followed by treatment with IT CNP-miR146a or saline control. Twenty-four hours post-infection, bronchoalveolar lavage fluid (BALF) and whole lungs were analyzed for various markers of inflammation. Compared to controls, MRSA infection significantly increased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1β; p < 0.05), BALF proinflammatory cytokines (IL-6, IL-8, TNFα, IL-1β; p < 0.01), and inflammatory cell infiltrate (p = 0.03). CNP-miR146a treatment significantly decreased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1β; p < 0.05), bronchoalveolar proinflammatory protein leak (IL-6, IL-8, TNFα; p < 0.05), and inflammatory infiltrate (p = 0.01). CNP-miR146a decreases inflammation and improves alveolar–capillary barrier integrity in the MRSA-infected lung and has significant promise as a potential therapeutic for ARDS.

Acute respiratory distress syndrome (ARDS) remains a significant cause of morbidity and mortality in critically ill patients. Oxidative stress and inflammation play a crucial role in the pathogenesis of ARDS. Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is an important enzymatic defense against superoxide. Human single-nucleotide polymorphism in matrix binding region of EC-SOD leads to the substitution of arginine to glycine at position 213 (R213G) and results in release of EC-SOD into alveolar fluid, without affecting enzyme activity. We hypothesized that R213G EC-SOD variant protects against lung injury and inflammation via the blockade of neutrophil recruitment in infectious model of methicillin-resistant S. aureus (MRSA) pneumonia. After inoculation with MRSA, wild-type (WT) mice had impaired integrity of alveolar-capillary barrier and increased levels of IL-1β, IL-6, and TNF-α in the broncho-alveolar lavage fluid (BALF), while infected mice expressing R213G EC-SOD variant maintained the integrity of alveolar-capillary interface and had attenuated levels of proinflammatory cytokines. MRSA-infected mice expressing R213G EC-SOD variant also had attenuated neutrophil numbers in BALF and decreased expression of neutrophil chemoattractant CXCL1 by the alveolar epithelial ATII cells, compared with the infected WT group. The decreased neutrophil numbers in R213G mice were not due to increased rate of apoptosis. Mice expressing R213G variant had a differential effect on neutrophil functionality—the generation of neutrophil extracellular traps (NETs) but not myeloperoxidase (MPO) levels were attenuated in comparison with WT controls. Despite having the same bacterial load in the lung as WT controls, mice expressing R213G EC-SOD variant were protected from extrapulmonary dissemination of bacteria.