Lung endothelium, tau, and amyloids in health and disease.

Lung endothelia in the arteries, capillaries, and veins are heterogeneous in structure and function. Lung capillaries in particular represent a unique vascular niche, with a thin yet highly restrictive alveolar-capillary barrier that optimizes gas exchange. Capillary endothelium surveys the blood while simultaneously interpreting cues initiated within the alveolus and communicated via immediately adjacent type I and type II epithelial cells, fibroblasts, and pericytes. This cell-cell communication is necessary to coordinate the immune response to lower respiratory tract infection. Recent discoveries identify an important role for the microtubule-associated protein tau that is expressed in lung capillary endothelia in the host-pathogen interaction. This endothelial tau stabilizes microtubules necessary for barrier integrity, yet infection drives production of cytotoxic tau variants that are released into the airways and circulation, where they contribute to end-organ dysfunction. Similarly, beta-amyloid is produced during infection. Beta-amyloid has antimicrobial activity, but during infection it can acquire cytotoxic activity that is deleterious to the host. The production and function of these cytotoxic tau and amyloid variants are the subject of this review. Lung-derived cytotoxic tau and amyloid variants are a recently discovered mechanism of end-organ dysfunction, including neurocognitive dysfunction, during and in the aftermath of infection.

Coronavirus Lung Infection Impairs Host Immunity against Secondary Bacterial Infection by Promoting Lysosomal Dysfunction.

Postviral bacterial infections are a major health care challenge in coronavirus infections, including COVID-19; however, the coronavirus-specific mechanisms of increased host susceptibility to secondary infections remain unknown. In humans, coronaviruses, including SARS-CoV-2, infect lung immune cells, including alveolar macrophages, a phenotype poorly replicated in mouse models of SARS-CoV-2. To overcome this, we used a mouse model of native murine ß-coronavirus that infects both immune and structural cells to investigate coronavirus-enhanced susceptibility to bacterial infections. Our data show that coronavirus infection impairs the host ability to clear invading bacterial pathogens and potentiates lung tissue damage in mice. Mechanistically, coronavirus limits the bacterial killing ability of macrophages by impairing lysosomal acidification and fusion with engulfed bacteria. In addition, coronavirus-induced lysosomal dysfunction promotes pyroptotic cell death and the release of IL-1ß. Inhibition of cathepsin B decreased cell death and IL-1ß release and promoted bacterial clearance in mice with postcoronavirus bacterial infection.

Cytotoxic tau released from lung microvascular endothelial cells upon infection with Pseudomonas aeruginosa promotes neuronal tauopathy.

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.

Gamma secretase activating protein promotes end-organ dysfunction after bacterial pneumonia.

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.

Pneumonia initiates a tauopathy.

Pneumonia causes short- and long-term cognitive dysfunction in a high proportion of patients, although the mechanism(s) responsible for this effect are unknown. Here, we tested the hypothesis that pneumonia-elicited cytotoxic amyloid and tau variants: (1) are present in the circulation during infection; (2) lead to impairment of long-term potentiation; and, (3) inhibit long-term potentiation dependent upon tau. Cytotoxic amyloid and tau species were recovered from the blood and the hippocampus following pneumonia, and they were present in the extracorporeal membrane oxygenation oxygenators of patients with pneumonia, especially in those who died. Introduction of immunopurified blood-borne amyloid and tau into either the airways or the blood of uninfected animals acutely and chronically impaired hippocampal information processing. In contrast, the infection did not impair long-term potentiation in tau knockout mice and the amyloid- and tau-dependent disruption in hippocampal signaling was less severe in tau knockout mice. Moreover, the infection did not elicit cytotoxic amyloid and tau variants in tau knockout mice. Therefore, pneumonia initiates a tauopathy that contributes to cognitive dysfunction.

Carbonic Anhydrase IX and Hypoxia Promote Rat Pulmonary Endothelial Cell Survival during Infection.

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.

Virulent Pseudomonas aeruginosa infection converts antimicrobial amyloids into cytotoxic prions.

Pseudomonas aeruginosa infection elicits the production of cytotoxic amyloids from lung endothelium, yet molecular mechanisms of host-pathogen interaction that underlie the amyloid production are not well understood. We examined the importance of type III secretion system (T3SS) effectors in the production of cytotoxic amyloids. P aeruginosa possessing a functional T3SS and effectors induced the production and release of cytotoxic amyloids from lung endothelium, including beta amyloid, and tau. T3SS effector intoxication was sufficient to generate cytotoxic amyloid release, yet intoxication with exoenzyme Y (ExoY) alone or together with exoenzymes S and T (ExoS/T/Y) generated the most virulent amyloids. Infection with lab and clinical strains engendered cytotoxic amyloids that were capable of being propagated in endothelial cell culture and passed to naïve cells, indicative of a prion strain. Conversely, T3SS-incompetent P aeruginosa infection produced non-cytotoxic amyloids with antimicrobial properties. These findings provide evidence that (1) endothelial intoxication with ExoY is sufficient to elicit self-propagating amyloid cytotoxins during infection, (2) pulmonary endothelium contributes to innate immunity by generating antimicrobial amyloids in response to bacterial infection, and (3) ExoY contributes to the virulence arsenal of P aeruginosa through the subversion of endothelial amyloid host-defense to promote a lung endothelial-derived cytotoxic proteinopathy.

Infection-induced endothelial amyloids impair memory.

Patients with nosocomial pneumonia exhibit elevated levels of neurotoxic amyloid and tau proteins in the cerebrospinal fluid (CSF). In vitro studies indicate that pulmonary endothelium infected with clinical isolates of either Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus produces and releases cytotoxic amyloid and tau proteins. However, the effects of the pulmonary endothelium-derived amyloid and tau proteins on brain function have not been elucidated. Here, we show that P. aeruginosa infection elicits accumulation of detergent insoluble tau protein in the mouse brain and inhibits synaptic plasticity. Mice receiving endothelium-derived amyloid and tau proteins via intracerebroventricular injection exhibit a learning and memory deficit in object recognition, fear conditioning, and Morris water maze studies. We compared endothelial supernatants obtained after the endothelia were infected with P. aeruginosa possessing an intact [P. aeruginosa isolated from patient 103 (PA103) supernatant] or defective [mutant strain of P. aeruginosa lacking a functional type 3 secretion system needle tip complex (ΔPcrV) supernatant] type 3 secretion system. Whereas the PA103 supernatant impaired working memory, the ΔPcrV supernatant had no effect. Immunodepleting amyloid or tau proteins from the PA103 supernatant with the A11 or T22 antibodies, respectively, overtly rescued working memory. Recordings from hippocampal slices treated with endothelial supernatants or CSF from patients with or without nosocomial pneumonia indicated that endothelium-derived neurotoxins disrupted the postsynaptic synaptic response. Taken together, these results establish a plausible mechanism for the neurologic sequelae consequent to nosocomial bacterial pneumonia.-Balczon, R., Pittet, J.-F., Wagener, B. M., Moser, S. A., Voth, S., Vorhees, C. V., Williams, M. T., Bridges, J. P., Alvarez, D. F., Koloteva, A., Xu, Y., Zha, X.-M., Audia, J. P., Stevens, T., Lin, M. T. Infection-induced endothelial amyloids impair memory.

Tau and Aß42 in lavage fluid of pneumonia patients are associated with end-organ dysfunction: A prospective exploratory study.

BACKGROUND: Bacterial pneumonia and sepsis are both common causes of end-organ dysfunction, especially in immunocompromised and critically ill patients. Pre-clinical data demonstrate that bacterial pneumonia and sepsis elicit the production of cytotoxic tau and amyloids from pulmonary endothelial cells, which cause lung and brain injury in naïve animal subjects, independent of the primary infection. The contribution of infection-elicited cytotoxic tau and amyloids to end-organ dysfunction has not been examined in the clinical setting. We hypothesized that cytotoxic tau and amyloids are present in the bronchoalveolar lavage fluid of critically ill patients with bacterial pneumonia and that these tau/amyloids are associated with end-organ dysfunction. METHODS: Bacterial culture-positive and culture-negative mechanically ventilated patients were recruited into a prospective, exploratory observational study. Levels of tau and Aß42 in, and cytotoxicity of, the bronchoalveolar lavage fluid were measured. Cytotoxic tau and amyloid concentrations were examined in comparison with patient clinical characteristics, including measures of end-organ dysfunction. RESULTS: Tau and Aß42 were increased in culture-positive patients (n = 49) compared to culture-negative patients (n = 50), independent of the causative bacterial organism. The mean age of patients was 52.1 ± 16.72 years old in the culture-positive group and 52.78 ± 18.18 years old in the culture-negative group. Males comprised 65.3% of the culture-positive group and 56% of the culture-negative group. Caucasian culture-positive patients had increased tau, boiled tau, and Aß42 compared to both Caucasian and minority culture-negative patients. The increase in cytotoxins was most evident in males of all ages, and their presence was associated with end-organ dysfunction. CONCLUSIONS: Bacterial infection promotes the generation of cytotoxic tau and Aß42 within the lung, and these cytotoxins contribute to end-organ dysfunction among critically ill patients. This work illuminates an unappreciated mechanism of injury in critical illness.

Pneumonia-induced endothelial amyloids reduce dendritic spine density in brain neurons.

Pseudomonas aeruginosa pneumonia elicits endothelial cell release of cytotoxic amyloids that can be recovered from the bronchoalveolar lavage and cerebrospinal fluids of critically ill patients. Introduction of these cytotoxic amyloids into the lateral ventricle impairs learning and memory in mice. However, it is unclear whether the amyloids of lung origin (1) are neurotropic, and (2) cause structural remodeling of hippocampal dendrites. Thus, we used electrophysiological studies in brain slices and structural analysis of post-mortem tissues obtained from animals exposed to endothelium-derived amyloids to assess these issues. The amyloids were administered via three different routes, by intracerebroventricular, intratracheal, and intraperitoneal injections. Synaptic long-term potentiation was abolished following intracerebroventricular amyloid injection. Fluorescence dialysis or Golgi-impregnation labeling showed reduced dendritic spine density and destabilized spines of hippocampal pyramidal neurons 4 weeks after intracerebroventricular amyloid injection. In comparison, endothelial amyloids introduced to the airway caused the most prominent dendritic spine density reduction, yet intraperitoneal injection of these amyloids did not affect spine density. Our findings indicate that infection-elicited lung endothelial amyloids are neurotropic and reduce neuronal dendritic spine density in vivo. Amyloids applied into the trachea may either be disseminated through the circulation and cross the blood-brain barrier to access the brain, initiate feed-forward amyloid transmissibility among cells of the blood-brain barrier or access the brain in other ways. Nevertheless, lung-derived amyloids suppress hippocampal signaling and cause injury to neuronal structure.

Cystatin C regulates the cytotoxicity of infection-induced endothelial-derived ß-amyloid.

Infection of rat pulmonary microvascular endothelial cells with the bacterium Pseudomonas aeruginosa induces the production and release of cytotoxic oligomeric tau and beta amyloid (Aß). Here, we characterized these cytotoxic amyloids. Cytotoxic behavior and oligomeric tau were partially resistant to digestion with proteinase K, but cytotoxicity was abolished by various denaturants including phenol, diethylpyrocarbonate (DEPC), and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP). Ultracentrifugation for 8 h at 150 000 g was required to remove cytotoxic activity from the supernatant. Ultracentrifugation, DEPC treatment, and immunodepletion using antibodies against Aß also demonstrated that cytoprotective protein(s) are released from endothelial cells during P. aeruginosa infection. Mass spectrometry of endothelial cell culture media following P. aeruginosa infection allowed identification of multiple potential secreted modulators of Aß, including cystatin C, gelsolin, and ApoJ/clusterin. Immunodepletion, co-immunoprecipitation, and ultracentrifugation determined that the cytoprotective factor released during infection of endothelial cells by P. aeruginosa is cystatin C, which appears to be in a complex with Aß. Cytoprotective cystatin C may provide a novel therapeutic avenue for protection against the long-term consequences of infection with P. aeruginosa.