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Francisella tularensis, the causative agent of tularemia, is a highly virulent organism, especially when exposure occurs by inhalation. Recent data suggest that Francisella interacts directly with alveolar epithelial cells. Although F. tularensis causes septicemia and can live extracellulariy in a murine infection model, there is little information about the role of the vascular endothelium in the host response. We hypothesized that F. tularensis would interact with pulmonary endothelial cells as a prerequisite to the clinically observed recruitment of neutrophils to the lung. Using an in vitro Transwell model system, we studied interactions between F. tularensis live vaccine strain (Ft LVS) and a pulmonary microvascular endothelial cell (PMVEC) monolayer. Organisms invaded the endothelium and were visualized within individual endothelial cells by confocal microscopy. Although these bacteria-endothelial cell interactions did not elicit production of the proinflammatory chemokines, polymorphonuclear leukocytes (PMN) were stimulated to transmigrate across the endothelium in response to Ft LVS. Moreover, transendothelial migration altered the phenotype of recruited PMN; i.e., the capacity of these PMN to activate NADPH oxidase and release elastase in response to subsequent stimulation was reduced compared with PMN that traversed PMVEC in response to Streptococcus pneumoniae. The blunting of PMN responsiveness required PMN transendothelial migration but did not require PMN uptake of Ft LVS, was not dependent on the presence of serum-derived factors, and was not reproduced by Ft LVS-conditioned medium. We speculate that the capacity of Ft LVS-stimulated PMVEC to support transendothelial migration of PMN without triggering release of IL-8 and monocyte chemotactic protein-1 and to suppress the responsiveness of transmigrated PMN to subsequent stimulation could contribute to the dramatic virulence during inhalational challenge with Francisella. tularemia; NADPH oxidase; pulmonary; polymorphonuclear leukocyte

Type 1 diabetes (T1D) is a chronic inflammatory condition sometimes complicated by acute diabetic ketoacidosis (DKA). A subset of patients with T1D develop DKA independent of known risk factors. This study tested the hypothesis that circulating polymorphonuclear leukocytes (PMN) from children with T1D and DKA would exhibit a primed phenotype and that the signature would be unique in patients predisposed to have DKA. Using a prospective cohort study design, neutrophil phenotype was assessed in 30 patients with T1D seen in endocrinology clinic for routine care, 30 patients with acute DKA, and 36 healthy donors. Circulating PMN from patients with DKA display a primed phenotype with increased basal cell‐surface CD11b, L‐selectin shedding, and enhanced fMLF‐elicited reactive oxygen species (ROS) production. Moreover, PMN from T1D patients both with and without DKA lack the capacity to be further primed by incubation with TNF‐α, a classic priming stimulus. Primed PMN phenotypic signatures demonstrated are independent of hemoglobin A1c, the premier biological marker for DKA risk, and are consistent with a hyperinflammatory state. A single nucleotide polymorphism in TLR‐1 (1805G>T), known to be associated with a hyperinflammatory PMN phenotype, correlated with DKA. This study elucidated a novel phenotypic signature in circulating PMN from children with T1D with and without DKA, and suggests the possibility of a previously unrecognized PMN phenotype with potential clinical implications. Immunophenotype and genotype may be applicable as biomarkers for DKA risk stratification in patients with T1D. [ABSTRACT FROM AUTHOR]

Neutrophils, polymorphonuclear (PMN) leukocytes, play an important role in the early innate immune response to Mycobacterium tuberculosis infection in the lung. Interactions between PMN and mycobacterial lipids impact the activation state of these migrated cells with consequences for the surrounding tissue in terms of resolution versus ongoing inflammation. We hypothesized that lipoarabinomannan from M. tuberculosis (Mtb LAM) would prime human PMN in a TLR2-dependent manner and investigated this with specific comparison with the purified synthetic TLR2 agonists, Pam3CSK4 and FSL-1. In contrast to Pam3CSK4 and FSL-1, we found Mtb LAM did not induce any of the classical PMN priming phenotypes, including enhancement of NADPH oxidase activity, shedding of L-selectin, or mobilization of CD11b. However, exposure of PMN to Mtb LAM did elicit pro- and antiinflammatory cytokine production and release in a TLR2/1-dependent manner, using the TLR1 single-nucleotide polymorphism rs5743618 (1805G/T) as a marker for TLR2/1 specificity. Moreover, Mtb LAM did not elicit p38 MAPK phosphorylation or endocytosis, although these processes occurred with Pam3CSK4 stimulation, and were necessary for the early priming events to occur. Interestingly, Mtb LAM did not abrogate priming responses elicited by Pam3CSK4. Notably, subfractionation of light membranes from Pam3CSK4 versus Mtb LAM-stimulated cells demonstrated differential patterns of exocytosis. In summary, Mtb LAM activates PMN via TLR2/1, resulting in the production of cytokines but does not elicit early PMN priming responses, as seen with Pam3CSK4. We speculate that the inability of Mtb LAM to prime PMN may be due to differential localization of TLR2/1 signaling. [ABSTRACT FROM AUTHOR]

Systemic inflammatory response syndrome (SIRS) is a common clinical condition in patients in intensive care units that can lead to complications, including multiple organ dysfunction syndrome (MODS). MODS carries a high mortality rate, and it is unclear why some patients resolve SIRS, whereas others develop MODS. Although oxidant stress has been implicated in the development of MODS, several recent studies have demonstrated a requirement for NADPH oxidase 2 (NOX2)-derived oxidants in limiting inflammation. We recently demonstrated that NOX2 protects against lung injury and mortality in a murine model of SIRS. In the present study, we investigated the role of NOX2-derived oxidants in the progression from SIRS to MODS. Using a murine model of sterile systemic inflammation, we observed significantly greater illness and subacute mortality in gp91(phox-/y) (NOX2-deficient) mice compared with wild-type mice. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histological examination showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation., (Copyright © 2014 the American Physiological Society.)