Tau protein, a microtubule-associated protein expressed in lung capillary endothelium, is detectable early in lung development and has been implicated in systemic dysfunction during adult respiratory infections. Its role in neonatal lung injury, however, remains undefined. In this study, we analyzed tracheal aspirates from 67 mechanically ventilated infants (51 preterm and 16 term) and compared them with 20 cord blood samples and five healthy adult plasma samples. Preterm infants were further stratified by histological chorioamnionitis status and severity of bronchopulmonary dysplasia (BPD) at 36-wk postmenstrual age (Jensen's 2019 criteria). Total tau concentrations were measured using a Meso Scale Discovery assay, and neuronal tau seeding assays assessed the presence of cytopathic tau variants. Tau was detected in all samples, with significantly higher concentrations in neonatal tracheal aspirates and cord blood compared with adult plasma. In both cord blood and tracheal aspirates, preterm infants exhibited elevated total tau levels relative to term infants. The tau protein present in preterm tracheal aspirates induced neuronal tau aggregation across gestational ages. Although total tau concentrations did not differ, the cytopathic activity of tracheal aspirates was increased in infants with chorioamnionitis. Neither total tau nor seeding activity correlated with BPD severity. Our study findings demonstrate that tau is abundant in the airways of mechanically ventilated preterm infants and exhibits cytopathic properties, suggesting a potential role in both neonatal lung injury and development. Further studies are needed to clarify the mechanistic contribution of tau to lung pathology in this highly vulnerable population.NEW & NOTEWORTHY In mechanically ventilated preterm infants, endogenous airway molecules may influence lung injury and development. Tau, a microtubule-associated protein expressed in lung endothelium, was abundant in neonatal tracheal aspirates and elevated in preterm infants. Tau in preterm samples also showed cytopathic seeding activity, which was further increased in infants with histological chorioamnionitis. These findings identify tau as a previously unrecognized bioactive airway component that may contribute to early lung pathology.

Influenza A virus (IAV) remains a significant public health challenge, with seasonal influenza accounting for approximately 43 million cases of illness in the U.S. during the 2024-2025 season. While IAV primarily manifests as an upper respiratory tract infection, its most severe complications and associated mortality arise from lower respiratory tract involvement. During lower respiratory tract infection, IAV replicates in alveolar epithelium. Virions are symmetrically released from apical and basolateral aspects of epithelial cells and consequently presented to capillary endothelial cells where additional viral replication cycles can occur. The objective of this study was to determine the extent to which pulmonary microvascular endothelial cells (PMVECs) support IAV infection and to test whether the microtubule-associated protein tau is required for efficient viral replication in these cells. We hypothesized that tau stabilizes peripheral microtubules to support early endosomal trafficking of the IAV genome to the nucleus for replication, and further, that loss of tau limits viral replication and endothelial injury. To test this idea, wild-type PMVECs were infected with the A/WSN/1933 (H1N1) strain of IAV and viral particle accumulation was measured by reverse transcription quantitative polymerase chain reaction across a 48-hour time course. Tau phosphorylation was also assessed over this time course by Western blot. IAV particle numbers began to increase in as little as 3-hours post infection of PMVECs; whereas 12-hours was required for particle numbers to increase in A549 cells, a Gold-standard cell line for IAV infection. By 48-hours, widespread gap formation and cell death were evident in both A549 cells and PMVECs. To determine whether tau contributes to viral replication in PMVECs, IAV infection was tested in both tau knockout cells and tau knockout cells in which tau expression was rescued (i.e., tau-rescue). Tau-knockout PMVECs produced approximately 2.5 log fewer viral particles than the wild-type controls, and remarkably, endothelial barrier integrity was preserved over the entire 48-hour time course. In stark contrast, tau-rescue cells supported IAV infection at a rate that was equivalent to wild type cells, and widespread gap formation and death were also evident. In tau-expressing cells, IAV infection promoted tau phosphorylation at serine-214 within 30 minutes of exposure to IAV, indicating rapid modification of tau during the period of viral entry and trafficking. Together, these results demonstrate that PMVECs support IAV infection and provide evidence that endothelial tau plays a role in IAV replication kinetics and endothelial cell injury. Studies are ongoing to determine the extent to which tau represents a critical host factor that regulates endothelial infection with IAV; however, our preliminary results suggest that tau phosphorylation during IAV infection may represent a previously unrecognized endothelial mechanism contributing to the progression of IAV-induced pneumonia. Supported by HL140182, HL167997, and HL148069. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Pseudomonas aeruginosa utilizes a type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote interendothelial gap formation, impair motility, and arrest cell growth. The disruptive activities of cAMP and cGMP during the P. aeruginosa infection are established; however, little is known about the function of cUMP. Here, we tested the hypothesis that cUMP contributes to endothelial cell barrier disruption during P. aeruginosa infection. Using a membrane permeable cUMP analog, cUMP-AM, we revealed that during infection with catalytically inactive ExoY, cUMP promotes interendothelial gap formation in cultured pulmonary microvascular endothelial cells (PMVECs) and contributes to increased filtration coefficient in the isolated perfused lung. These findings indicate that cUMP contributes to endothelial permeability during P. aeruginosa lung infection.NEW & NOTEWORTHY During pneumonia, bacteria utilize a virulence arsenal to communicate with host cells. The Pseudomonas aeruginosa T3SS directly introduces virulence molecules into the host cell cytoplasm. These molecules are enzymes that trigger interkingdom communication. One of the exoenzymes is a nucleotidyl cyclase that produces noncanonical cyclic nucleotides like cUMP. Little is known about how cUMP acts in the cell. Here we found that cUMP instigates pulmonary edema during Pseudomonas aeruginosa infection of the lung.

Patients who recover from hospital-acquired pneumonia exhibit a high incidence of end-organ dysfunction following hospital discharge, including cognitive deficits. We have previously demonstrated that pneumonia induces the production and release of cytotoxic oligomeric tau from pulmonary endothelial cells, and these tau oligomers can enter the circulation and may be a cause of long-term morbidities. Endothelial-derived oligomeric tau is hyperphosphorylated during infection. The purpose of these studies was to determine whether Ser-214 phosphorylation of tau is a necessary stimulus for generation of cytotoxic tau variants. The results of these studies demonstrate that Ser-214 phosphorylation is critical for the cytotoxic properties of infection-induced oligomeric tau. In the lung, Ser-214 phosphorylated tau contributes to disruption of the alveolar-capillary barrier, resulting in increased permeability. However, in the brain, both the Ser-214 phosphorylated tau and the mutant Ser-214-Ala tau, which cannot be phosphorylated, disrupted hippocampal long-term potentiation suggesting that inhibition of long-term potentiation was relatively insensitive to the phosphorylation status of Ser-214. Nonetheless, phosphorylation of tau is essential to its cytotoxicity since global dephosphorylation of the infection-induced cytotoxic tau variants rescued long-term potentiation. Collectively, these data demonstrate that multiple forms of oligomeric tau are generated during infectious pneumonia, with different forms of oligomeric tau being responsible for dysfunction of distinct end-organs during pneumonia.

Patients who recover from nosocomial pneumonia oftentimes exhibit long-lasting cognitive impairment comparable to what is observed in Alzheimer's Disease patients. We previously hypothesized that the lung endothelium contributes to infection-related neurocognitive dysfunction, since bacteria-exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long-term potentiation in the hippocampus. However, lung endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection-induced endothelial cytotoxic tau can trigger neuronal tau aggregation which is the major hallmark of several neuropathologies. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule-binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, cells were infected with P. aeruginosa. The infection-induced tau released from endothelium into the medium induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule-binding repeat domains or the full-length tau. Infection-induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial-derived tau to cause neuronal tau aggregation, was abolished using tau knockout cells. After bacterial lung infection, brain homogenates from wild-type mice, but not from tau knockout mice, initiated tau aggregation. Notably, plasma samples obtained from pneumonia-positive patients showed significantly higher tau aggregation activity when compared to pneumonia-negative plasma samples. Thus, bacterial pneumonia initiates the release of lung endothelial-derived cytotoxic tau into the circulation, which is capable of propagating a neuronal tauopathy.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel—a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Patients who recover from nosocomial pneumonia oftentimes exhibit long-lasting cognitive impairment comparable with what is observed in Alzheimer's disease patients. We previously hypothesized that the lung endothelium contributes to infection-related neurocognitive dysfunction, because bacteria-exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long-term potentiation in the hippocampus. However, the full-length lung and endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection-induced endothelial cytotoxic tau triggers neuronal tau aggregation. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule-binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, the cells were infected with Pseudomonas aeruginosa. The infection-induced tau released from endothelium into the medium-induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule-binding repeat domains or the full-length tau. Infection-induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial-derived tau to cause neuronal tau aggregation, was abolished in tau knockout cells. After bacterial lung infection, brain homogenates from WT mice, but not from tau knockout mice, initiated tau aggregation. Thus, we conclude that bacterial pneumonia initiates the release of lung endothelial-derived cytotoxic tau, which is capable of propagating a neuronal tauopathy.

Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. CA (carbonic anhydrase) IX is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics, and angiogenesis. We hypothesized that CA IX is important for PMVEC survival and that CA IX expression and release from PMVECs are increased during infection. Although the plasma concentration of CA IX was unchanged in human and rat pneumonia, there was a trend toward increasing CA IX in the bronchoalveolar fluid of mechanically ventilated critically ill patients with pneumonia and a significant increase in CA IX in the lung tissue lysates of pneumonia rats. To investigate the functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. By using these cells, we found that infection promotes intracellular (IC) expression, release, and MMP (metalloproteinase)-mediated extracellular cleavage of CA IX in PMVECs. IC domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX IC domain promoted cell death after infection, suggesting that the IC domain has an important role in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report 1) that CA IX increases in the lungs of pneumonia rats and 2) that the CA IX IC domain and hypoxia promote PMVEC survival during infection.

Amyloids have autofluorescence properties that may be exploited for their detection in patients recovering from hospital acquired pneumonia (HAP). Recovering HAP patients have cytotoxic amyloids in their bronchoalveolar lavage fluid, blood, and cerebrospinal fluid, and these amyloids may be the cause of the cognitive decline and secondary organ failure patients encounter after HAP. Infection of pulmonary microvascular endothelial cells (PMVECs) produces bacterial strain‐dependent cytotoxic or antimicrobial amyloids. Currently, there are no detection methods for pulmonary amyloids at the bedside. Here, we hypothesize that cytotoxic and antimicrobial amyloids can be detected and distinguished by their autofluorescence properties. PMVECS were infected with mutants of gram‐negative bacterium Pseudomonas aeruginosa, including virulent ExoY+ and avirulent ∆PcrV, to generate amyloids with distinctive phenotypes. Infection with ExoY+ for 7 hours produced cytotoxic amyloids, while infection with ∆PcrV for 4 hours yielded antimicrobial amyloids. To resolve whether the autofluorescence properties are concentration‐dependent, protein concentrations of the supernatants were standardized to 50 μg/mL. To assess whether fibrillar or oligomeric amyloids are responsible for the autofluorescent signature of ExoY+ and ∆PcrV supernatants, the supernatants were immunodepleted with the amyloid fibril‐specific OC or amyloid oligomer‐specific A11 antibody. Fluorescence spectroscopy was performed to obtain the fluorescence spectra of the unstandardized, standardized, and immunodepleted ExoY+ and ΔPcrV supernatants. The unstandardized fluorescence spectra of ExoY+ and ΔPcrV were indistinguishable; however, when standardized to 50 μg/mL, the fluorescence intensity of ΔPcrV supernatant containing antimicrobial amyloids was significantly greater than ExoY+ supernatant containing cytotoxic amyloids. Immunodepleting the supernatant with the OC antibody eliminated the fluorescence intensity of ΔPcrV supernatant but did not diminish the fluorescence intensity of the ExoY+ supernatant. However, fluorescence of the ExoY+ supernatant was eradicated after immunodepletion with the A11 antibody. Our work suggests that the intrinsic fluorescence of amyloids can be developed as a point‐of‐care diagnostic tool in HAP. Antimicrobial amyloids may be detected by the immunodepletion of amyloid fibrils whereas cytotoxic amyloids can be distinguished by immunodepleting oligomeric amyloids. In the future we will apply this method to patient samples to assess its use as a diagnostic tool.