Immunomodulatory cytotoxins are prominent virulence factors produced by Staphylococcus aureus, a leading cause of bacterial sepsis, skin infection, and pneumonia. S. aureus α-toxin is a pore-forming toxin that utilizes a widely expressed receptor, ADAM10, to injure the host epithelium, endothelium, and immune cells. As each host tissue is characterized by a unique composition of resident cells and recruited immune cells, the outcome of α-toxin-mediated injury may depend on the infected tissue environment. Utilizing myeloid lineage-specific Adam10 knockout mice, we show that α-toxin exerts tissue-specific effects on innate immunity to staphylococcal infection. Loss of ADAM10 expression exacerbates skin infection, yet affords protection against lethal pneumonia. These diverse outcomes are not related to altered immune cell recruitment, but rather correlate with a defect in toxin-induced IL-1β production. Extension of these studies through analysis of ADAM10 double-knockout mice affecting both the myeloid lineage and either the skin or lung epithelium highlight the prominence of toxin-induced injury to the epithelium in governing the outcome of infection. Together, these studies provide evidence of tissue specificity of pore-forming cytotoxin action in the modulation of host immunity, and illustrate that the outcome of infection is a collective manifestation of all effects of the toxin within the tissue microenvironment.

Staphyococcus aureus frequently causes recurrent skin and soft-tissue infection (SSTI). In the pediatric population, elevated serum antibody targeting S. aureus α-toxin is correlated with a reduced incidence of recurrent SSTI. Using a novel model of recurrent SSTI, we demonstrated that expression of α-toxin during primary infection increases the severity of recurrent disease. Antagonism of α-toxin by either a dominant-negative toxin mutant or a small molecule inhibitor of the toxin receptor ADAM10 during primary infection reduces reinfection abscess severity. Early neutralization of α-toxin activity during S. aureus SSTI therefore offers a new therapeutic strategy to mitigate primary and recurrent disease.

Staphylococcus aureus is a leading cause of human bacterial infection, causing a wide spectrum of disease ranging from skin and soft tissue infections to life-threatening pneumonia and sepsis. S. aureus toxins play an essential role in disease pathogenesis, contributing to both immunomodulation and host tissue injury. Prominent among these toxins are the membrane-active poreforming cytolysin alpha-toxin (Hla) and the amphipathic ␣-helical phenol-soluble modulin (PSM) peptides. As deletion of either the hla or psm locus leads to a phenotypically similar virulence defect in skin and soft tissue infection, we sought to determine the relative contribution of each locus to disease pathogenesis. Here we show that production of Hla can be modulated by PSM expression. An S. aureus mutant lacking PSM expression exhibits a transcriptional delay in hla mRNA production and therefore fails to secrete normal levels of Hla at early phases of growth. This leads to attenuation of virulence in vitro and in murine skin and lung models of infection, correlating with reduced recovery of Hla from host tissues. Production of Hla and restoration of staphylococcal virulence can be achieved in the psm mutant by plasmid-driven overexpression of hla. Our study suggests the coordinated action of Hla and PSMs in host tissue during early pathogenesis, confirming a major role for Hla in epithelial injury during S. aureus infection. These findings highlight the possibility that therapeutics targeting PSM production may simultaneously prevent Hla-mediated tissue injury.

The effect of nicotine exposure on the subsequent self-administration of amphetamine, extinction of this behavior, and amphetamine-induced reinstatement of drug seeking was assessed with particular attention to the contribution of contextual stimuli paired or unpaired with nicotine during exposure. Rats were exposed to five injections, one injection every third day, of either saline or nicotine (0.4 mg/kg, IP, base) in three experiments. In one, exposure injections were administered in the home cage. In another, they were administered in the self-administration chambers with the levers retracted. In a third, nicotine was administered either explicitly paired or unpaired with the self-administration chambers using a discrimination learning procedure. Starting 13-15 days later, rats were trained to self-administer amphetamine (100 μg/kg/infusion, IV), tested under a progressive ratio (PR) schedule for 6 days, subjected to up to 20 days of extinction training, and were then tested for reinstatement by non-contingent injections of amphetamine (0, 0.2, 0.4, and 0.75 mg/kg, IP). Nicotine enhanced the self-administration of amphetamine under the PR schedule and amphetamine-induced reinstatement but only when rats were tested in the chamber in which they were previously exposed to nicotine. These effects were not observed in rats exposed to nicotine in the home cage or in rats exposed to nicotine explicitly unpaired with the self-administration chambers. Exposure to nicotine also rendered rats resistant to extinction when amphetamine was withheld but this effect was observed regardless of nicotine exposure context, suggesting a separate consequence of drug exposure. Together, these results show that previous exposure to nicotine can enhance the incentive motivational effects of other psychostimulants like amphetamine and indicate a critical role for nicotine-associated contextual stimuli in the mediation of this effect. These findings have important implications for the treatment of addictions in humans.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

Introduction S. aureus is a major cause of morbidity globally, and in the United States it contributes significantly to both hospital admissions and in-hospital morbidity [1, 2]. The increasing incidence of antibiotic-resistant strains increases the urgency of understanding the mechanisms by which this infection exerts its toxic acute effects, as well as potential longterm impact on infected patients, especially those with comorbid conditions. The major virulent toxin secreted by S. aureus is α-hemolysin (Hla). A Disintegrin And Metalloproteinase domain-containing protein-10 (ADAM10), which is involved in ectodomain shedding, is the eukaryotic receptor for Hla [3-5], and mediates vascular injury caused by Hla [6]. Almost all isolates of S. aureus express Hla, including methicillinresistant strains [7]. Recently, Hla has been shown to mediate VE-cadherin degradation in endothelial cells (EC) via ADAM10, affecting permeability [6]. Importantly, the Notch1 and 2 receptors are known ADAM10 targets [8]. Notch proteins are highly evolutionarily conserved. In mammals, the Notch pathway is comprised of the Jagged and Delta-like ligands, and the receptors Notch1 through Notch4. Both ligands and receptors are membrane-bound: in order for activation to take place, the ligand and receptor must be expressed in adjacent cells. Notch ligands are cleaved at Site 1 (S1) and can be post-translationally modified by glycosyltransferases, such as Fringe. Upon

Contextual stimuli present during nicotine exposure can come to act as conditioned stimuli and have been shown to play an important role in ongoing nicotine self-administration. In the present study, we characterized the effects of contextual stimuli previously paired with non-contingent nicotine exposure injections on subsequent nicotine self-administration. Rats were exposed to five injections of either saline or nicotine (0.4 mg/kg, IP) in either their home cage or a self-administration chamber with the levers retracted. Two weeks later, they were allowed to self-administer nicotine (30 μg/kg/infusion, IV) under fixed ratio (FR) schedules of reinforcement across 12 consecutive sessions. Lastly, responding under a progressive ratio (PR) schedule was assessed. Rats exposed to nicotine in the self-administration chamber subsequently increased their intake of nicotine across the FR test days, obtaining more infusions on average by days 7–12 compared to their saline exposed controls. This increase was not due to nicotine exposure alone as rats exposed to nicotine in the home cage did not show this effect. It was also not due to differences in the final ratio achieved between nicotine and saline exposed rats. Although rats exposed to nicotine in the self-administration chambers displayed reduced discrimination between the active and inactive levers during FR testing, they showed increased motivation to self-administer nicotine under the PR schedule. These results indicate that exposure to nicotine can enhance its subsequent self-administration and highlight the contribution of nicotine-associated contextual stimuli to the work output rats ultimately emit to obtain the drug.

Staphylococcus aureus is the leading cause of infection in the setting of critical illness and injury. This pathogen causes life-threatening infection in otherwise healthy individuals and also complicates the clinical course of patients requiring intensive care as a result of their primary medical or surgical disease processes. S. aureus infection in the intensive care unit (ICU) most commonly manifests as sepsis, ventilator-associated pneumonia, and infection of surgical sites and indwelling medical devices. With the epidemic spread of methicillin-resistant S. aureus, many cases of staphylococcal infection in the ICU are now classified as drug resistant, prompting hospital-based screening for methicillin-resistant S. aureus and implementation of both isolation practices and decolonization strategies in ICU patients. The genetic adaptability of S. aureus, heterogeneity of disease presentation, clinical course, and outcome between individual S. aureus-infected ICU patients remains enigmatic, suggesting a need to define disease classification subtypes that inform disease progression and therapy. We propose that S. aureus infection in the ICU now presents a unique opportunity for individualized risk stratification coupled with the investigation of novel approaches to mitigate disease. Given our increasing knowledge of the molecular pathogenesis of S. aureus disease, we suggest that the application of molecular pathological epidemiology to S. aureus infection can usher in a new era of highly focused personalized therapy that may be particularly beneficial in the setting of critical illness and injury.