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Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NOX2 (nicotinamide adenine dinucleotide phosphate hydrogen [NADPH] oxidase 2), involving the pneumococcal virulence factor PLY (pneumolysin). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of ENaC (epithelial sodium channel) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function—measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans blue dye incorporation in mouse lungs—after infection with pneumococci. PLY-induced hyperpermeability in human lung microvascular endothelial cell monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47phox subunit (Western blotting) in vitro and by colocalization of CD31 and gp91phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α knockout (enENaC-α knockout) mice develop increased capillary leak upon intratracheal instillation with PLY or pneumococci, compared with wild-type animals. TIP peptide diminishes capillary leak in Streptococcus pneumoniae–infected wild-type mice, without significantly increasing lung bacterial load. Lung slices from S. pneumoniae–infected enENaC-α knockout mice have significantly increased endothelial NOX2 expression, compared with infected cyclization recombination mice. In conclusion, enENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in acute respiratory distress syndrome, without impairing host defense.

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection. The human pathogen Neisseria meningitidis colonizes blood vessels and causes vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, Manriquez et al. use a humanized mouse model to show that pathogen colonization of capillaries and arterioles creates an intravascular niche that precludes an effective action of neutrophils.

Introduction The microcirculation supplies oxygen (O 2 ) and nutrients to all cells with the red blood cell (RBC) acting as both a deliverer and sensor of O 2 . In sepsis, a proinflammatory disease with microvascular complications, small blood vessel alterations are associated with multi-organ dysfunction and poor septic patient outcome. We hypothesized that microvascular autoregulation—existing at three levels: over the entire capillary network, within a capillary and within the erythrocyte—was impaired during onset of sepsis. This study had three objectives: 1) measure capillary response time within hypoxic capillaries, 2) test the null hypothesis that RBC O 2 -dependent adenosine triphosphate (ATP) efflux was not altered by sepsis and 3) develop a framework of a pathophysiological model. Methods This was an animal study, comparing sepsis with control, set in a university laboratory. Acute hypotensive sepsis was studied using cecal ligation and perforation (CLP) with a 6-hour end-point. Rat hindlimb skeletal muscle microcirculation was imaged, and capillary RBC supply rate (SR = RBC/s), RBC hemoglobin O 2 saturation (SO 2 ) and O 2 supply rate (qO 2  = pLO 2 /s) were quantified. Arterial NOx (nitrite + nitrate) and RBC O 2 -dependent ATP efflux were measured using a nitric oxide (NO) analyzer and gas exchanger, respectively. Results Sepsis increased capillary stopped-flow ( p  = 0.001) and increased plasma lactate ( p  < 0.001). Increased plasma NOx ( p  < 0.001) was related to increased capillary RBC supply rate ( p  = 0.027). Analysis of 30-second SR–SO 2 –qO 2 profiles revealed a shift towards decreased ( p  < 0.05) O 2 supply rates in some capillaries. Moreover, we detected a three- to fourfold increase ( p  < 0.05) in capillary response time within hypoxic capillaries (capillary flow states where RBC SO 2  < 20 %). Additionally, sepsis decreased the erythrocyte’s ability to respond to hypoxic environments, as normalized RBC O 2 -dependent ATP efflux decreased by 62.5 % ( p  < 0.001). Conclusions Sepsis impaired microvascular autoregulation at both the individual capillary and erythrocyte level, seemingly uncoupling the RBC acting as an “O 2 sensor” from microvascular autoregulation. Impaired microvascular autoregulation was manifested by increased capillary stopped-flow, increased capillary response time within hypoxic capillaries, decreased capillary O 2 supply rate and decreased RBC O 2 -dependent ATP efflux. This loss of local microvascular control was partially off-set by increased capillary RBC supply rate, which correlated with increased plasma NOx.

Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne bacterial infection that is transmitted through the bite of an infected tick. If not treated with antibiotics during the early stages of infection, disseminated infection can spread to the central nervous system (CNS). In non-human primates (NHPs) it has been demonstrated that the leptomeninges are among the tissues colonized by B. burgdorferi spirochetes. Although the NHP model parallels aspects of human borreliosis, a small rodent model would be ideal to study the trafficking of spirochetes and immune cells into the CNS. Here we show that during early and late disseminated infection, B. burgdorferi infects the meninges of intradermally infected mice, and is associated with concurrent increases in meningeal T cells. We found that the dura mater was consistently culture positive for spirochetes in transcardially perfused mice, independent of the strain of B. burgdorferi used. Within the dura mater, spirochetes were preferentially located in vascular regions, but were also present in perivascular, and extravascular regions, as late as 75 days post-infection. At the same end-point, we observed significant increases in the number of CD3+ T cells within the pia and dura mater, as compared to controls. Flow cytometric analysis of leukocytes isolated from the dura mater revealed that CD3+ cell populations were comprised of both CD4 and CD8 T cells. Overall, our data demonstrate that similarly to infection in peripheral tissues, spirochetes adhere to the dura mater during disseminated infection, and are associated with increases in the number of meningeal T cells. Collectively, our results demonstrate that there are aspects of B. burgdorferi meningeal infection that can be modelled in laboratory mice, suggesting that mice may be useful for elucidating mechanisms of meningeal pathogenesis by B. burgdorferi.

The lung is the terminal target of Bacillus anthracis before death, whatever the route of infection (cutaneous, inhalational, or digestive). During a cutaneous infection in absence of toxins, we observed encapsulated bacteria colonizing the alveolar capillary network, bacteria and hemorrhages in alveolar and bronchiolar spaces, and hypoxic foci in the lung (endothelial cells) and brain (neurons and neuropil). Circulating encapsulated bacteria were as chains of approximately 13 μm in length. Bacteria of such size were immediately trapped within the lung capillary network, but bacteria of shorter length were not. Controlling lung-targeted pathology would be beneficial for anthrax treatment.

BackgroundCentral nervous system (CNS) tuberculosis is a serious, often fatal disease that disproportionately affects young children. It is thought to develop when Mycobacterium tuberculosis breaches the blood-brain barrier (BBB), which is composed of tightly apposed brain microvascular endothelial cells. However, the mechanism(s) involved in this process are poorly understood Methods To better understand these processes, we developed an in vitro model of M. tuberculosis BBB infection using primary human brain microvascular endothelial cells ResultsM. tuberculosis was found to both invade and traverse the model BBB significantly more than did M. smegmatis (a nonpathogenic mycobacterium). Invasion by M. tuberculosis across the BBB required host-cell actin cytoskeletal rearrangements. By microarray expression profiling, we found 33 M. tuberculosis genes to be highly up-regulated during the early stages of invasion of the BBB by M. tuberculosis; 18 of them belong to a previously described in vivo–expressed genomic island (Rv0960–Rv1001). Defined M. tuberculosis isogenic transposon mutants for the up-regulated genes Rv0980c, Rv0987, Rv0989c and Rv1801 were found to be deficient in their ability to invade the BBB model ConclusionsWe developed an in vitro model of M. tuberculosis BBB infection and identified M. tuberculosis genes that may be involved in CNS invasion

Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB.

Introduction Query fever (Q fever), a zoonotic disease, caused by Coxiella burnetii , is an infectious disease that has long been considered a rare and regionally restricted disease. It can be responsible for endocarditis and endovascular infections. Systemic capillary leak syndrome (SCLS), a rare disease of unknown etiology that most commonly develops in adults 50–70 years of age, is diagnosed clinically based on a characteristic symptomatic triad of hypotension, hemoconcentration (elevated hemoglobin or hematocrit), and serum hypoalbuminemia resulting from fluid extravasation. Although Q fever has increasingly been recognized and reported in recent years, the treatment of Q fever complicated by SCLS, with an etiological diagnosis aided by metagenomic next-generation sequencing (mNGS), remains uncommon. Case presentation This report describes a case of acute Q fever with concurrent SCLS in a 54-year-old male who worked in a slaughterhouse. The patient presented with fever, chest tightness, and shortness of breath, accompanied by severe headache. His condition rapidly deteriorated, leading to acute fever, generalized weakness, and hypotension. Due to respiratory failure and shock, he was admitted to the intensive care unit (ICU) for treatment. Despite empirical antibiotic therapy along with fluid resuscitation, his blood pressure continued to decline, and metabolic acidosis and respiratory distress worsened. As his condition failed to improve, tracheal intubation was performed. mNGS detected both Coxiella burnetii in his BALF and blood samples. Based on the mNGS results, he was started on doxycycline, alongside penicillin antibiotics, vasopressors, and continuous renal replacement therapy (CRRT). The patient’s condition gradually improved, and he was discharged home after 12 days of treatment. At his 90-day follow-up, he had nearly fully recovered to his pre-illness status. Conclusions mNGS plays a crucial role in assisting the diagnosis of Q fever, which enables the timely treatment of the underlying disease triggering SCLS. This, combined with restrictive fluid resuscitation strategies, is essential for improving patient outcomes.

Bacterial sepsis is one of the most frequent and dreaded causes of death in intensive care units. According to the current understanding of sepsis, bacterial components activate innate immune responses via pattern-recognition receptors that stimulate signaling pathways, thereby leading to activation of NF-κB and the release of cytokines, alarming the organism and coordinating appropriate defense mechanisms. The resulting “cytokine storm” not only restricts bacterial invasion; it also harms the host by triggering a hemodynamic collapse with a drop in blood pressure, which could lead to death. One of the cytokines released during sepsis is interleukin-6 (IL-6). Originally described as a B-cell–stimulating factor, this cytokine has since been shown to have multiple additional functions. Interestingly, there is emerging evidence of IL-6 trans-signaling in the pathogenesis of sepsis. We review recent findings and discuss whether therapeutic interference with IL-6 trans-signaling may be beneficial in this important clinical scenario.

Background. The ability of Staphylococcus aureus to adhere to endothelial cells is a major prerequisite for the tissue-invasive stage of bacterial infection. Methods. To develop a model for the study of endothelial attachment and detachment kinetics of S. aureus within the host's microvasculature in vivo, we labeled inactivated staphylococci with fluorescein isothiocyanate and investigated their interaction with the vascular endothelium of arterioles, capillaries, and venules in the dorsal skin-fold chamber of untreated and tumor necrosis factor (TNF)-α-treated hamsters by use of intravital fluorescence microscopy. Results. During the first 20 min after injection, >99% of the bacteria were removed from the microvascular bloodstream. In parallel, single bacteria and bacterial clusters adhered to the endothelial lining of postcapillary venules and to nutritive capillaries. Bacterial adherence to the endothelium of arterioles was only rarely observed. TNF-α treatment significantly accelerated bacterial clearance and resulted in a significant increase of venular, but not arteriolar and capillary, bacterial adherence, indicating the venular endothelium to be the target structure for bacterial recruitment. Conclusion. The insights into host-pathogen interaction gained with this new in vivo model offer highly promising novel aspects of the understanding of infections caused by S. aureus.