Pulmonary vascular inflammation with fatal coronavirus disease 2019 (COVID-19): possible role for the NLRP3 inflammasome.

BACKGROUND: Pulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs. METHODS: Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1. RESULTS: In lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs. CONCLUSION: Vascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

Inhibition of Peroxiredoxin 6 PLA2 Activity Decreases Oxidative Stress and the Severity of Acute Lung Injury in the Mouse Cecal Ligation and Puncture Model.

The use of agents to inhibit the production of reactive oxygen species (ROS) has been proposed for the treatment of Acute Lung Injury (ALI). However, this approach also inhibits the bactericidal activity of polymorphonuclear leucocytes (PMN) and other cells, raising the possibility of aggravating lung injury in ALI associated with bacterial infection. We used the cecal ligation and puncture (CLP) model of ALI associated with sepsis to investigate the effect of inhibiting NADPH oxidase 2 (NOX2)-derived ROS production, the main source of ROS in lungs. A phospholipase A2 inhibitor called peroxiredoxin 6 inhibitory peptide-2 (PIP-2) was used to inhibit NOX2 activation; the peptide prevents liberation of Rac, a necessary NOX2 co-factor. At 18 h after intravenous treatment with 2 µg PIP-2 /gram body weight (wt), the number of colony-forming bacteria in lungs and peritoneal fluid of mice with CLP was approximately doubled as compared to untreated mice. Treatment with 10 µg PIP-2/g body wt resulted in 100% mortality within 18 h. Antibiotic treatment abolished both the increase in lung bacteria with low dose PIP-2 and the increased mortality with high dose PIP-2. Treatment with PIP-2 plus antibiotics resulted in significantly improved lung histology, decreased PMN infiltration, decreased lung fluid accumulation, and decreased oxidative lung injury compared to antibiotics alone. We conclude that the administration of PIP-2 provides partial protection against lung injury in a model of ALI due to bacterial infection, while concurrent antibiotic treatment abolishes the deleterious effects of PIP-2 on lung bacterial clearance. These results suggest that addition of PIP-2 to the antibiotic regimen is beneficial for treatment of ALI associated with bacterial infection.

Vascular Inflammation in Lungs of Patients with Fatal Coronavirus Disease 2019 (COVID-19): Possible Role for the NLRP3 Inflammasome.

Background: Hyperinflammation is a key event that occurs with SARS-CoV-2 infection. In the lung, hyperinflammation leads to structural damage to tissue. To date, numerous lung histological studies have shown extensive alveolar damage, but there is scarce documentation of vascular inflammation in postmortem lung tissue. Methods: Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects [of which 8 were acute respiratory disease syndrome (ARDS) affected and 3 were from subjects with non-respiratory diseases] were stained for H & E to ascertain histopathological features including presence of thrombi/microthrombi. Inflammation along the vessel wall was also monitored by quantification of the expression of moieties of the NLRP3 inflammasome pathway (NLRP3 and caspase-1). Results: In lungs from "fatal COVID-19", vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from "non-COVID-19 non respiratory disease" affected subjects. The NLRP3 pathway components were significantly higher in lungs from COVID-19 subjects as compared to non-COVID-19 fatal cases without respiratory disease. No significant differences were observed between COVID-19 lungs and non-COVID-19 ARDS lungs. Conclusion: We posit that inflammasome formation along the vessel wall is a characteristic of lung inflammation that accompanies COVID-19. Thus, the NLRP3 inflammasome pathway seems to be probable candidate that drives amplification of inflammation post SARS-CoV-2 infection.

Vascular Inflammation in Lungs of Patients with Fatal Coronavirus Disease 2019 (COVID-19) Infection: Possible role for the NLRP3 inflammasome.

Hyperinflammation is a key event that occurs with SARS-CoV-2 infection. In the lung, hyperinflammation leads to structural damage to tissue. To date, numerous lung histological studies have shown extensive alveolar damage, but there is scarce documentation of vascular inflammation in postmortem lung tissue. Here we document histopathological features and monitor the NLRP3 inflammasome in fatal cases of disease caused by SARS Cov2 (COVID-19). We posit that inflammasome formation along the vessel wall is a characteristic of lung inflammation that accompanies COVID-19 and that it is a probable candidate that drives amplification of inflammation post infection.

Acute e-cig inhalation impacts vascular health: a study in smoking naïve subjects.

This study was designed to investigate the acute effects of nonnicotinized e-cigarette (e-cig) aerosol inhalation in nonsmokers both in terms of blood-based markers of inflammation and oxidative stress and evaluate their association with hemodynamic-metabolic MRI parameters quantifying peripheral vascular reactivity, cerebrovascular reactivity, and aortic stiffness. Thirty-one healthy nonsmokers were subjected to two blood draws and two identical MRI protocols, each one before and after a standardized e-cig vaping session. After vaping, the serum levels of C-reactive protein, soluble intercellular adhesion molecule, and the danger signal machinery high-mobility group box 1 (HMGB1) and its downstream effector and the NLR family pyrin domain containing 3 (NLRP3) inflammasome (as monitored by its adaptor protein ASC) increased significantly relative to the respective baseline (prevaping) values. Moreover, nitric oxide metabolites and reactive oxygen species production decreased and increased, respectively. These observations were paralleled by impaired peripheral vascular reactivity (with reduced flow-mediated dilation and attenuated hyperemic response after a cuff-occlusion test) and metabolic alterations expressed by decreased venous oxygen saturation, postvaping. The current results suggest propagation of inflammation signaling via activation of the danger signaling axis (HMGB1-NLRP3). The findings indicate that a single episode of vaping has adverse impacts on vascular inflammation and function.NEW & NOTWORTHY Endothelial cell signaling and blood biomarkers were found to correlate with functional vascular changes in a single episode e-cigarettes inhalation in healthy adults. This is indicative of the potential of e-cigarettes (even when inhaled acutely) to lead of vascular dysfunction.

Acute exposure to e-cigarettes causes inflammation and pulmonary endothelial oxidative stress in nonsmoking, healthy young subjects.

The effects of e-cigarette (e-cig) aerosol inhalation by nonsmokers have not been examined to date. The present study was designed to evaluate the acute response to aerosol inhalation of non-nicotinized e-cigarettes in terms of oxidative stress and indices of endothelial activation in human pulmonary microvascular endothelial cells (HPMVEC). Ten smoking-naïve healthy subjects (mean age ± SD = 28.7 ± 5.5 yr) were subjected to an e-cig challenge, following which their serum was monitored for markers of inflammation [C-reactive protein (CRP) and soluble intercellular adhesion molecule (sICAM)] and nitric oxide metabolites (NOx). The oxidative stress and inflammation burden of the circulating serum on the vascular network was also assessed by measuring reactive oxygen species (ROS) production and induction of ICAM-1 expression on HPMVEC. Our results show that serum indices of oxidative stress and inflammation increased significantly (P < 0.05 as compared with baseline), reaching a peak at approximately 1-2 h post-e-cig aerosol inhalation and returning to baseline levels at 6 h. The circulatory burden of the serum (ICAM-1 and ROS) increased significantly at 2 h and returned to baseline values 6 h post-e-cig challenge. ROS production by HPMVEC was found to occur via activation of the NADPH oxidase 2 (NOX2) pathways. These findings suggest that even in the absence of nicotine, acute e-cig aerosol inhalation leads to a transient increase in oxidative stress and inflammation. This can adversely affect the vascular endothelial network by promoting oxidative stress and immune cell adhesion. Thus e-cig inhalation has the potential to drive the onset of vascular pathologies.

Detection of lung transplant rejection in a rat model using hyperpolarized [1-13 C] pyruvate-based metabolic imaging.

The current standard for noninvasive imaging of acute rejection consists of X-ray/CT, which derive their contrast from changes in ventilation, inflammation and edema, as well as remodeling during rejection. We propose the use of hyperpolarized [1-13 C] pyruvate MRI-which provides real-time metabolic assessment of tissue-as an early biomarker for tissue rejection. In this preliminary study, we used µCT-derived parameters and HP 13 C MR-derived biomarkers to predict rejection in an orthotopic left lung transplant model in both allogeneic and syngeneic rats. On day 3, the normalized lung density-a parameter that accounts for both lung volume (mL) and density (HU)-was -0.335 (CI: -0.598, -0.073) and - 0.473 (CI: -0.726, -0.220) for the allograft and isograft, respectively (not significant, 0.40). The lactate-to-pyruvate ratios-derived from the HP 13 C MRI-for the allograft and isograft were 0.200 (CI: 0.161, 0.240) and 0.114 (CI: 0.074, 0.153), respectively (significant, 0.020). Both techniques showed tissue rejection on day 7. A separate sub-study revealed CD8+ cells as the primary source of the lactate-to-pyruvate signal. Our study suggests that hyperpolarized (HP) [1-13 C] pyruvate MRI is a promising early biomarker for tissue rejection that provides metabolic assessment in real time based on changes in cellularity and metabolism of lung tissue and the infiltrating inflammatory cells, and may be able to predict tissue rejection earlier than X-ray/CT.

Non-Mammalian Prdx6 Enzymes (Proteins with 1-Cys Prdx Mechanism) Display PLA2 Activity Similar to the Human Orthologue.

Mammalian peroxiredoxin class 6 (Prdx6) are bifunctional enzymes. Non-mammalian Prdx6 enzymes display Cys-based peroxidase activity, but to date their putative phospholipase A2 (PLA2 activities) has not been experimentally investigated. Initially, we observed that five non-mammalian Prdx6 enzymes (enzymes from Arabidopsis thaliana (AtPER1), Triticum aestivum (TaPER1), Pseudomonas aeruginosa (PaLsfA) and Aspergillus fumigatus (AfPrx1 and AfPrxC)) present features compatible with PLA2 activities in mammalian Prdx6 by amino acid sequences alignment and tertiary structure modeling. Employing unilamellar liposomes with tracer amounts of [³H]-1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and thin layer chromatography, all the tested non-mammalian Prdx6 enzymes displayed PLA2 activities, with values ranging from 3.4 to 6.1 nmol/min/mg protein. It was previously shown that Thr177 phosphorylation of human Prdx6 increases its PLA2 activity, especially at neutral pH. Therefore, we investigated if human Erk2 kinase could also phosphorylate homologous Thr residues in non-mammalian Prdx6 proteins. We observed phosphorylation of the conserved Thr in three out of the five non-mammalian Prdx enzymes by mass spectrometry. In the case of the mitochondrial Prdx6 from A. fumigatus (AfPrxC), we also observed phosphorylation by western blot, and as a consequence, the PLA2 activity was increased in acidic and neutral conditions by the human Erk2 kinase treatment. The possible physiological meanings of these PLA2 activities described open new fields for future research.

LGM2605 Reduces Space Radiation-Induced NLRP3 Inflammasome Activation and Damage in In Vitro Lung Vascular Networks.

Updated measurements of charged particle fluxes during the transit from Earth to Mars as well as on site measurements by Curiosity of Martian surface radiation fluxes identified potential health hazards associated with radiation exposure for human space missions. Designing mitigation strategies of radiation risks to astronauts is critical. We investigated radiation-induced endothelial cell damage and its mitigation by LGM2605, a radioprotector with antioxidant and free radical scavenging properties. We used an in vitro model of lung vascular networks (flow-adapted endothelial cells; FAECs), exposed to gamma rays, low/higher linear energy transfer (LET) protons (3⁻4 or 8⁻10 keV/µm, respectively), and mixed field radiation sources (gamma and protons), given at mission-relevant doses (0.25 gray (Gy)⁻1 Gy). We evaluated endothelial inflammatory phenotype, NLRP3 inflammasome activation, and oxidative cell injury. LGM2605 (100 µM) was added 30 min post radiation exposure and gene expression changes evaluated 24 h later. Radiation induced a robust increase in mRNA levels of antioxidant enzymes post 0.25 Gy and 0.5 Gy gamma radiation, which was significantly decreased by LGM2605. Intercellular cell adhesion molecule-1 (ICAM-1) and NOD-like receptor protein 3 (NLRP3) induction by individual or mixed-field exposures were also significantly blunted by LGM2605. We conclude that LGM2605 is a likely candidate to reduce tissue damage from space-relevant radiation exposure.

Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude.

Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases.

Peroxiredoxin 6 phospholipid hydroperoxidase activity in the repair of peroxidized cell membranes.

Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A2 and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H2O2 and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O2). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H2O2 or short chain hydroperoxides does not play a role in the recovery process.

Mutation of Serine 32 to Threonine in Peroxiredoxin 6 Preserves Its Structure and Enzymatic Function but Abolishes Its Trafficking to Lamellar Bodies.

Peroxiredoxin 6 (Prdx6), a bifunctional protein with phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid turnover. Prdx6 has been localized to both cytosol and lamellar bodies (LB) in lung epithelium, and its organellar targeting sequence has been identified. We propose that Prdx6 LB targeting facilitates its role in the metabolism of lung surfactant phosphatidylcholine (PC). Ser-32 has been identified as the active site in Prdx6 for aiPLA2 activity, and this activity was abolished by the mutation of serine 32 to alanine (S32A). However, aiPLA2 activity was unaffected by mutation of serine 32 in Prdx6 to threonine (S32T). Prdx6 protein expression and aiPLA2 activity were normal in the whole lung of a "knock-in" mouse model carrying an S32T mutation in the Prdx6 gene but were absent from isolated LB. Analyses by proximity ligation assay in lung sections demonstrated the inability of S32T Prdx6 to bind to the chaperone protein, 14-3-3ϵ, that is required for LB targeting. The content of total phospholipid, PC, and disaturated PC in lung tissue homogenate, bronchoalveolar lavage fluid, and lung LB was increased significantly in Prdx6-S32T mutant lungs, whereas degradation of internalized [(3)H]dipalmitoyl-PC was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for LB Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine.

Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress.

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O(2)). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA(2) activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA(2) activity each contribute to the recovery process.

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium.

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1(-/-) lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1(-/-) PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1(-/-) mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1(-/-) lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.

Pulmonary abnormalities in animal models due to Niemann-Pick type C1 (NPC1) or C2 (NPC2) disease.

Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies (LB, mean area 2-fold larger), while NPC2-mutant cells had predominantly smaller lamellar bodies (mean area 50% of normal) than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities.

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung.

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.

Rab38 targets to lamellar bodies and normalizes their sizes in lung alveolar type II epithelial cells.

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.

Early alveolar epithelial dysfunction promotes lung inflammation in a mouse model of Hermansky-Pudlak syndrome.

RATIONALE: The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. OBJECTIVES: To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. METHODS: Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. MEASUREMENTS AND MAIN RESULTS: EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1. CONCLUSIONS: Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.

Role of the PI3-kinase signaling pathway in trafficking of the surfactant protein A receptor P63 (CKAP4) on type II pneumocytes.

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

Pathway to lamellar bodies for surfactant protein A.

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled (35)S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [(35)S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [(35)S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50-60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [(35)S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [(35)S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.