Assessment of the Glycan-Binding Profile of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen that can establish acute and chronic infections in individuals who lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance of P. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible to P. aeruginosa infection, thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate the binding and subsequent phagocytosis of P. aeruginosa cells. However, the suite of glycans that P. aeruginosa cells bind to on host phagocytic cells remains poorly characterized. Here, we demonstrate, with the use of exogenous N-linked glycans and a glycan array, that P. aeruginosa PAO1 cells preferentially attach to a subset of glycans, including a bias toward monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and disaccharide glycans. We discuss our findings in the context of previous reports of P. aeruginosa glycan binding. IMPORTANCE P. aeruginosa cells bind to a variety of glycans as part of their interaction with host cells, and a number of P. aeruginosa-encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here, we extend this work by studying the glycans used by P. aeruginosa PAO1 cells to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that can facilitate host cell binding by this microbe. This study provides an increased understanding of the glycans bound by P. aeruginosa and furthermore provides a useful data set for future studies of P. aeruginosa-glycan interactions.

Assessment of the Glycan-Binding Profile of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen that can establish acute and chronic infections in individuals that lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance of P. aeruginosa . Individuals with neutropenia or cystic fibrosis are highly susceptible to P. aeruginosa infection thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate binding and subsequent phagocytosis of P. aeruginosa . However, the suite of glycans that P. aeruginosa binds to on host phagocytic cells remains poorly characterized. Here we demonstrate, with the use of exogenous N-linked glycans and a glycan array, that P. aeruginosa PAO1 preferentially attaches to a subset of glycans, including a bias towards monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and di-saccharide glycans. We discuss of findings in the context of previous reports of P. aeruginosa glycan binding. IMPORTANCE: P. aeruginosa binds to a variety of glycans as part of its interaction with host cells, and a number of P. aeruginosa- encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here we extend this work by studying the glycans used by P. aeruginosa PAO1 to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that could facilitate host cell-binding by this microbe. This study provides an increased understanding of the glycans bound by P. aeruginosa , and furthermore, provides a useful dataset for future studies of P. aeruginosa- glycan interactions.

Cisplatin increases immune activity of monocytes and cytotoxic T-cells in a murine model of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is an immunologically active malignancy, but thus far immune therapy has had limited success in clinical trials. One barrier to implementation of efficacious immune therapies is a lack of knowledge of the effect of chemotherapy on the monocyte-derived component of the immune infiltrate within the tumor. We utilized the ID8 murine EOC model to investigate alterations within tumor ascites that occur following administration of platinum chemotherapy. Cisplatin treatment resulted in a significant increase in monocytes within the ascites of tumor bearing mice. We identified that CD11b+ cells from the ascites of mice that have been treated with cisplatin elicits an increase in IFN-É£ expression from CD8+ T-cells compared to CD11b+ cells from a mouse treated with vehicle control (604.0 pg/mL v. 4328.0 pg/mL; p < .0001). Splenocytes derived from tumor bearing mice released increase levels of IFN-É£ after treatment with cisplatin when incubated with dendritic cells (DCs) and tumor antigen (62.0 v. 92.1 pg/mL; p = .03). Cisplatin induced an increase in T-cell and monocyte/macrophage activation markers (CD62L and CD301). Levels of IL-10, IL-6, and VEGF in the cell free ascites of mice treated with cisplatin decreased (p > .05). These results indicate that treatment with cisplatin leads to an increase of anti-tumor activity within the ascites related to alterations in the ascites monocytes. Further investigation of these findings in humans is necessary to identify how these cells behave in different patient subgroups and if there is a role for monocyte directed therapy in conjunction with T-cell directed therapy and/or chemotherapy.

Identification of cell-surface glycans that mediate motility-dependent binding and internalization of Pseudomonas aeruginosa by phagocytes.

Phagocytic cells are critical to host defense against Pseudomonas aeruginosa, a Gram-negative bacterium that is an opportunistic pathogen. Accordingly, susceptible individuals frequently have impaired innate immune responses, including those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation, or loss, of bacterial flagellar motility enables bacteria to evade interactions with phagocytic cells that result in phagocytic uptake of the bacteria. However, the mechanistic bases for motility-dependent interactions between P. aeruginosa and host cell surfaces that lead to phagocytic uptake of the bacteria are poorly understood. A recent insight is that exogenous addition of a negatively charged phospholipid, phosphatidylinositol-(3,4,5)-triphosphate (PIP3), promotes the engagement of non-motile strains of P. aeruginosa with phagocytes leading to uptake of the bacteria. Thus, we hypothesized that the engagement of P. aeruginosa by phagocytic cells is mediated by motility-dependent interactions with cell-surface polyanions. Here we report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific interactions resulted in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1ß response of host innate immune cells. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Identifying in vivo inflammation using magnetic nanoparticle spectra.

We are developing magnetic nanoparticle (NP) methods to characterize inflammation and infection in vivo. Peritoneal infection in C57BL/6 mice was used as a biological model. An intraperitoneal NP injection was followed by measurement of magnetic nanoparticle spectroscopy of Brownian rotation (MSB) spectra taken over time. MSB measures the magnetization of NPs in a low frequency alternating magnetic field. Two groups of three mice were studied; each group had two infected mice and one control with no infection. The raw MSB signal was compared with two derived metrics: the NP relaxation time and number of NPs present in the sensitive volume of the receive coil. A four compartment dynamic model was used to relate those physical properties to the relevant biological processes including phagocytic activity and migration. The relaxation time increased over time for all of the mice as the NPs were absorbed. The NP number decreased over time as the NPs were cleared from the sensitive volume of the receive coil. The composite p-values for all three rate constants were significant: raw signal, 0.0002, relaxation, <10-16 and local NP clearance, <10-16. However, not all the individual mice had significant changes: Only half the infected mice had significantly different rate constants for raw signal reduction. All infected mice had significantly smaller relaxation time constants. All but one of the infected mice had significantly lower rate constants for local clearance. Relaxation is affected by both phagocytic activity, edema and temperature changes and it should be possible to better isolate those effects to more completely characterize inflammation using more advanced MSB methods. The MSB NP signal can be used to identify inflammation in vivo because it has the unique ability to monitor phagocytic absorption through relaxation measurements.

Distinct Contributions of CD18 Integrins for Binding and Phagocytic Internalization of Pseudomonas aeruginosa.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Regulation of Pseudomonas aeruginosa-Mediated Neutrophil Extracellular Traps.

Pseudomonas aeruginosa is the most prevalent opportunistic pathogen in the airways of cystic fibrosis (CF) patients. The pulmonary disorder is characterized by recurrent microbial infections and an exaggerated host inflammatory immune response led primarily by influx of neutrophils. Under these conditions, chronic colonization with P. aeruginosa is associated with diminished pulmonary function and increased morbidity and mortality. P. aeruginosa has a wide array of genetic mechanisms that facilitate its persistent colonization of the airway despite extensive innate host immune responses. Loss of function mutations in the quorum sensing regulatory gene lasR have been shown to confer survival advantage and a more pathogenic character to P. aeruginosa in CF patients. However, the strategies used by LasR-deficient P. aeruginosa to modulate neutrophil-mediated bactericidal functions are unknown. We sought to understand the role of LasR in P. aeruginosa-mediated neutrophil extracellular trap (NET) formation, an important anti-microbial mechanism deployed by neutrophils, the first-line responder in the infected airway. We observe mechanistic and phenotypic differences between NETs triggered by LasR-sufficient and LasR-deficient P. aeruginosa strains. We uncover that LasR-deficient P. aeruginosa strains fail to induce robust NET formation in both human and murine neutrophils, independently of bacterial motility or LPS expression. LasR does not mediate NET release via downstream quorum sensing signaling pathways but rather via transcriptional regulation of virulence factors, including, but not restricted to, LasB elastase and LasA protease. Finally, our studies uncover the differential requirements for NADPH oxidase in NET formation triggered by different P. aeruginosa strains.

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates.

Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections.IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

A Syngeneic Mouse Model of Epithelial Ovarian Cancer Port Site Metastases.

Epithelial ovarian cancer (EOC) is a deadly gynecologic malignancy, but animal models for the study of EOC pathophysiology and drug efficacy are limited. Based on the finding that women with EOC are at risk for metastasis at a trocar site after laparoscopy, we developed a syngeneic murine model of port-site metastasis of EOC. We leveraged the ID8 murine EOC cell line to induce intra-peritoneal tumors in mice. Once durable intraperitoneal tumor was confirmed with bioluminescence imaging, intra-abdominal wall tumors were induced by abdominal wall puncture with a hollow bore needle. This resulted in a robust system in which C57BL/6 mice developed metastatic deposits at a rate of 66.7% ±â€¯10.77; no intra-abdominal wall metastases were seen in control samples (P = .0003, CI 41.16-90.84). Immunodeficient NOD SCID gamma mice developed puncture site metastases in 70% ±â€¯10.0 of mice and also had no metastases documented in control sites (P = .002, CI 42.24-97.76). In addition we were able to demonstrate the presence of immune infiltrates within the metastatic deposits of C57BL/6 mice via IHC. Therefore, in this study we demonstrate the predictable development of invasive abdominal wall metastases in a syngeneic mouse model of EOC. This model enables studies of the metastatic process and provides a novel system in which to test the effect of therapies on a clinically-relevant model in an immune competent mouse.

Inflammasomes: An Emerging Mechanism Translating Environmental Toxicant Exposure Into Neuroinflammation in Parkinson's Disease.

Evidence indicates that complex gene-environment interactions underlie the incidence and progression of Parkinson's disease (PD). Neuroinflammation is a well-characterized feature of PD widely believed to exacerbate the neurodegenerative process. Environmental toxicants associated with PD, such as pesticides and heavy metals, can cause cellular damage and stress potentially triggering an inflammatory response. Toxicant exposure can cause stress and damage to cells by impairing mitochondrial function, deregulating lysosomal function, and enhancing the spread of misfolded proteins. These stress-associated mechanisms produce sterile triggers such as reactive oxygen species (ROS) along with a variety of proteinaceous insults that are well documented in PD. These associations provide a compelling rationale for analysis of sterile inflammatory mechanisms that may link environmental exposure to neuroinflammation and PD progression. Intracellular inflammasomes are cytosolic assemblies of proteins that contain pattern recognition receptors, and a growing body of evidence implicates the association between inflammasome activation and neurodegenerative disease. Characterization of how inflammasomes may function in PD is a high priority because the majority of PD cases are sporadic, supporting the widely held belief that environmental exposure is a major factor in disease initiation and progression. Inflammasomes may represent a common mechanism that helps to explain the strong association between exposure and PD by mechanistically linking environmental toxicant-driven cellular stress with neuroinflammation and ultimately cell death.

Guanylate binding proteins facilitate caspase-11-dependent pyroptosis in response to type 3 secretion system-negative Pseudomonas aeruginosa.

Detection of bacterial ligands is a pre-requisite for inflammasome activation. During Pseudomonas aeruginosa infection, flagellin which is secreted through the T3SS is detected by the NLRC4 inflammasome. Activation of the NLRC4 inflammasome is believed to contribute to high IL-1ß production and pathogenicity in cystic fibrosis patients with chronic P. aeruginosa infection. Interestingly, the majority of P. aeruginosa isolated from cystic fibrosis patients with chronic airway infection are non-motile and T3SS-negative, suggesting that yet un-characterized inflammasome pathways regulate IL-1ß production in cystic fibrosis patients. Here we demonstrate the role of guanylate-binding proteins (GBPs) in regulating bacterial proliferation and inflammasome activation in response to T3SS-negative P. aeruginosa. Bacterial ligands liberated by the action of GBP2 and IRGB10 activate caspase-11 and regulate non-canonical NLRP3 inflammasome activation and IL-1ß release. Overall, our results reveal the role of caspase-11 in inhibiting bacterial proliferation and promoting IL-1ß secretion during T3SS-negative P. aeruginosa infection. This study suggests that non canonical inflammasomes might have co-evolved to detect Gram-negative bacterial pathogens that have evolved to bypass detection by canonical NLRs.

Phosphatidylinositol-(3,4,5)-Trisphosphate Induces Phagocytosis of Nonmotile Pseudomonas aeruginosa.

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.

Evaluating blood clot progression using magnetic particle spectroscopy.

PURPOSE: To evaluate the thrombus maturity noninvasively providing the promise of much earlier and more accurate diagnosis of diseases ranging from stroke to myocardial infarction to deep vein thrombosis. METHODS: Magnetic spectroscopy of nanoparticle Brownian rotation (MSB), a form of magnetic particle spectroscopy sensitive to Brownian rotation of magnetic nanoparticles, was used for the detection and characterization of blood clots. The nanoparticles' relaxation time was quantified by scaling the MSB spectra in frequency to match the spectra from nanoparticles in a reference state. The nanoparticles' relaxation time, in the bound state, was used to characterize the nanoparticle binding to thrombin on the blood clot. The number of nanoparticles bound to the clot was also estimated. Both the relaxation time and the weight of bound nanoparticles were obtained for clots of several ages, reflecting different stages of development and organization. The impact of clot development was explored using functionalized nanoparticles present during clot formation. RESULTS: The relaxation time of the bound nanoparticles decreases for more mature, organized clots. The number of nanoparticles able to bind the clot diminishes quantitatively with clot age. On mature clots, the nanoparticles bind the thrombin on the surface while for developing clots the nanoparticles bind several thrombin molecules or become trapped in the clot matrix during formation. CONCLUSIONS: By estimating the magnetic nanoparticles' relaxation time the clot age and organization can be predicted. The purposed methods are quick and minimally invasive for in vivo applications.

Acidosis exacerbates in vivo IL-1-dependent inflammatory responses and neutrophil recruitment during pulmonary Pseudomonas aeruginosa infection.

Acidic microenvironments commonly occur at sites of inflammation and bacterial infections. In the context of a Pseudomonas aeruginosa infection, we previously demonstrated that acidosis enhances the cellular proinflammatory interleukin (IL)-1ß response in vitro. However, how pH alterations affect in vivo IL-1ß responses and subsequent IL-1-driven inflammation during infection with P. aeruginosa is unclear. Here, we report that acidosis enhances in vivo IL-1ß production and downstream IL-1 receptor-dependent responses during infection with P. aeruginosa in models of acute pneumonia and peritonitis. Importantly, we demonstrate that infection with P. aeruginosa within an acidic environment leads to enhanced production of a subset of proinflammatory cytokines, including chemokine (C-X-C) motif ligand 1, IL-6, and chemokine (C-C motif) ligand 2, and increased neutrophil recruitment. Furthermore, with the use of IL-1 receptor type 1-deficient mice, we identify the contribution of the IL-1 signaling pathway to the acidosis-enhanced inflammatory response and pathology. These data provide insights into the potential benefit of pH regulation during bacterial infections to control disease progression and immunopathology.

Lumacaftor (VX-809) restores the ability of CF macrophages to phagocytose and kill Pseudomonas aeruginosa.

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by chronic bacterial lung infection and excessive inflammation, which lead to progressive loss of lung function and premature death. Although ivacaftor (VX-770) alone and ivacaftor in combination with lumacaftor (VX-809) improve lung function in CF patients with the Gly551Asp and del508Phe mutations, respectively, the effects of these drugs on the function of human CF macrophages are unknown. Thus studies were conducted to examine the effects of lumacaftor alone and lumacaftor in combination with ivacaftor (i.e., ORKAMBI) on the ability of human CF ( del508Phe/ del508Phe) monocyte-derived macrophages (MDMs) to phagocytose and kill Pseudomonas aeruginosa. Lumacaftor alone restored the ability of CF MDMs to phagocytose and kill P. aeruginosa to levels observed in MDMs obtained from non-CF (WT-CFTR) donors. This effect contrasts with the partial (~15%) correction of del508Phe Cl- secretion of airway epithelial cells by lumacaftor. Ivacaftor reduced the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa. Lumacaftor had no effect on P. aeruginosa-stimulated cytokine secretion by CF MDMs. Ivacaftor (5 µM) alone and ivacaftor in combination with lumacaftor reduced secretion of several proinflammatory cytokines. The clinical efficacy of ORKAMBI may be related in part to the ability of lumacaftor to stimulate phagocytosis and killing of P. aeruginosa by macrophages.

The effect of loss of O-antigen ligase on phagocytic susceptibility of motile and non-motile Pseudomonas aeruginosa.

The bacterial pathogen Pseudomonas aeruginosa undergoes adaptation and selection over the course of chronic respiratory tract infections which results in repeatedly-observed phenotypic changes that are proposed to enable its persistence. Two of the clinically significant P. aeruginosa phenotypic changes are loss of flagellar motility and modifications to LPS structure, including loss of O-antigen expression. The effect of loss of O-antigen, frequently described as conversion from smooth to rough LPS, and the combined effect of loss of motility and O-antigen on phagocytic susceptibility by immune cells remain unknown. To address this, we generated genetic deletion mutants of waaL, which encodes the O-antigen ligase responsible for linking O-antigen to lipid A-core oligosaccharide, in both motile and non-motile P. aeruginosa strains. With the use of these bacterial strains we provide the first demonstration that, despite a progressive selection for P. aeruginosa with rough LPS during chronic pulmonary infections, loss of the LPS O-antigen does not confer phagocytic resistance in vitro. However, use of the waaLmotABmotCD mutant revealed that loss of motility confers resistance to phagocytosis regardless of the smooth or rough LPS phenotype. These findings reveal how the O-antigen of P. aeruginosa can influence bacterial clearance during infection and expand our current knowledge about the impact of bacterial phenotypic changes during chronic infection.

Editor's Highlight: Nlrp3 Is Required for Inflammatory Changes and Nigral Cell Loss Resulting From Chronic Intragastric Rotenone Exposure in Mice.

Complex interactions between genetic and environmental factors are widely believed to underlie the incidence and progression of Parkinson's disease (PD). Rotenone is a naturally occurring metabolic toxin employed as an insecticide and piscicide identified as a risk factor for the development of PD in agricultural workers. The Nlrp3 inflammasome is an intracellular mediator that can initiate an inflammatory cascade in response to cellular stress. Reports by others indicating that NLRP3 expression was detectable in tissues obtained from Alzheimer's disease patients and that the PD-associated protein α-synuclein could activate inflammasomes in cultured glial cells, prompted us to test the prediction that Nlrp3 was required for the development of Parkinson's-like changes resulting from rotenone exposure in mice. We exposed wild type and Nlrp3-/- mice to chronic low doses of intragastric rotenone and conducted longitudinal behavioral and serum cytokine analysis followed by evaluation of neuroinflammatory and neurodegenerative endpoints in brain tissues. We observed progressive rotenone-dependent changes in serum cytokine levels and circulating leukocytes in wild type mice not observed in Nlrp3-/- mice. Analysis of brain tissues revealed Nlrp3-dependent neuroinflammation and nigral cell loss in mice exposed to rotenone as compared with mice exposed to vehicle alone. Together, our findings provide compelling evidence of a role for Nlrp3 in nigral degeneration and neuroinflammation resulting from systemic rotenone exposure and suggest that the suppression of NLRP3 activity may be a rational neuroprotective strategy for toxin-associated PD.

Acidosis increases the susceptibility of respiratory epithelial cells to Pseudomonas aeruginosa-induced cytotoxicity.

Bacterial infection can lead to acidosis of the local microenvironment, which is believed to exacerbate disease pathogenesis; however, the mechanisms by which changes in pH alter disease progression are poorly understood. We test the hypothesis that acidosis enhances respiratory epithelial cell death in response to infection with Pseudomonas aeruginosa Our findings support the idea that acidosis in the context of P. aeruginosa infection results in increased epithelial cell cytotoxicity due to ExoU intoxication. Importantly, enforced maintenance of neutral pH during P. aeruginosa infection demonstrates that cytotoxicity is dependent on the acidosis. Investigation of the underlying mechanisms revealed that host cell cytotoxicity correlated with increased bacterial survival during an acidic infection that was due to reduced bactericidal activity of host-derived antimicrobial peptides. These findings extend previous reports that the activities of antimicrobial peptides are pH-dependent and provide novel insights into the consequences of acidosis on infection-derived pathology. Therefore, this report provides the first evidence that physiological levels of acidosis increase the susceptibility of epithelial cells to acute Pseudomonas infection and demonstrates the benefit of maintaining pH homeostasis during a bacterial infection.

Blood clot detection using magnetic nanoparticles.

Deep vein thrombosis, the development of blood clots in the peripheral veins, is a very serious, life threatening condition that is prevalent in the elderly. To deliver proper treatment that enhances the survival rate, it is very important to detect thrombi early and at the point of care. We explored the ability of magnetic particle spectroscopy (MSB) to detect thrombus via specific binding of aptamer functionalized magnetic nanoparticles with the blood clot. MSB uses the harmonics produced by nanoparticles in an alternating magnetic field to measure the rotational freedom and, therefore, the bound state of the nanoparticles. The nanoparticles' relaxation time for Brownian rotation increases when bound [A.M. Rauwerdink and J. B. Weaver, Appl. Phys. Lett. 96, 1 (2010)]. The relaxation time can therefore be used to characterize the nanoparticle binding to thrombin in the blood clot. For longer relaxation times, the approach to saturation is more gradual reducing the higher harmonics and the harmonic ratio. The harmonic ratios of nanoparticles conjugated with anti-thrombin aptamers (ATP) decrease significantly over time with blood clot present in the sample medium, compared with nanoparticles without ATP. Moreover, the blood clot removed from the sample medium produced a significant MSB signal, indicating the nanoparticles are immobilized on the clot. Our results show that MSB could be a very useful non-invasive, quick tool to detect blood clots at the point of care so proper treatment can be used to reduce the risks inherent in deep vein thrombosis.

Monomethylarsonous Acid (MMAIII) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas aeruginosa.

UNLABELLED: Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. POPULATION: Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa.