Lipopolysaccharides directly decrease Ca2+ oscillations and the hyperpolarization-activated nonselective cation current If in immortalized HL-1 cardiomyocytes.

Lipopolysaccharide (LPS) has been implicated in sepsis-mediated heart failure and chronic cardiac myopathies. We determined that LPS directly and reversibly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration ([Ca2+]i) in immortalized cardiomyocytes, HL-1 cells. [Ca2+]i oscillated (<0.4 Hz), displaying slow and transient components. LPS (1 microg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca2+]i by 30-40 nM. HL-1 cells expressed Toll-like receptors, i.e., TLR-2 and TLR-4. Thus, we differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of utlrapure LPS, a TLR-4 ligand. Ultrapure LPS had no effect on basal [Ca2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca2+ parameter, and the effect of ultrapure LPS and Pam3CSK4 combined was similar to that of utlrapure LPS alone. Thus, unpurified LPS directly inhibits HL-1 calcium metabolism via TLR-4 and non-TLR-4-dependent mechanisms. Since others have shown that endotoxin impairs the hyperpolarization-activated, nonselective cationic pacemaker current (I(f)), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 microg/ml) reduced I(f) in HL-1 cells. This inhibition was marginal at physiologic membrane potentials and significant at very negative potentials (P < 0.05 at -140, -150, and -160 mV). So, we also evaluated effects of LPS on tail currents of fully activated I(f). LPS reduced the slope conductance of the tail currents from 498 +/- 140 pS/pF to 223 +/- 65 pS/pF (P < 0.05) without affecting reversal potential of -11 mV. Ultrapure LPS had similar effect on I(f), whereas Pam3CSK4 had no effect on I(f). We conclude that LPS inhibits activation of I(f), enhances its deactivation, and impairs regulation of [Ca2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.

Leukocyte Dectin-1 expression is differentially regulated in fungal versus polymicrobial sepsis.

OBJECTIVE: To examine peripheral leukocyte Dectin-1 regulation in clinically relevant models of fungal and polymicrobial sepsis. DESIGN: Prospective animal study. SETTING: University medical school research laboratory. SUBJECTS: Age, weight, and sex matched ICR/HSD mice. INTERVENTIONS: Mice were infected with Candida albicans (1 x 10, intravenously) or were subjected to cecal ligation and puncture to induce polymicrobial sepsis. MEASUREMENTS: Blood, spleen, and peritoneal exudate were harvested and leukocytes were isolated. Leukocytes were evaluated for membrane-associated Dectin-1 expression and cell phenotype by flow cytometry. MAIN RESULTS: In C. albicans infection, Dectin-1-positive blood and splenic leukocytes were increased from 23.5% to 58.9% over the course of infection. The increased percentage of Dectin-1-expressing cells was primarily attributable to neutrophilia. However, the amount of Dectin-1 expressed by blood and splenic neutrophils in C. albicans-infected mice was decreased by a range of 49.0% to 53.3%. C. albicans infection also resulted in an infiltration of Dectin-1-positive macrophages and neutrophils into the kidney. In contrast, polymicrobial sepsis decreased blood leukocyte Dectin-1-expressing cells by up to 51.4%. This reduction was due to a decrease in Dectin-1-positive neutrophils in the periphery. However, the percentage of Dectin-1-expressing cells in the peritoneal cavity increased by 774% with cecal ligation and puncture. Treatment of isolated neutrophils with three soluble glucans, mannan, lipopolysaccharide, or a variety of cytokines revealed that glucans, alone or in combination, were the only treatment that resulted in a decrease in Dectin-1-positive neutrophils. CONCLUSIONS: We conclude that peripheral leukocyte Dectin-1 expression is differentially regulated in fungal vs. polymicrobial sepsis. These data demonstrate that leukocyte Dectin-1 levels are modulated in response to infections of fungal and nonfungal origin.

Vav1 and PI3K are required for phagocytosis of beta-glucan and subsequent superoxide generation by microglia.

Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. beta-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by beta-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of beta-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with beta-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to beta-glucans.

Prolonged reduction of leukocyte membrane-associated Dectin-1 levels following beta-glucan administration.

Dectin-1 is the primary pattern recognition receptor for fungal glucans. Dectin-1 mediates the internalization and biological response to glucans. We examined the effect of i.v. or i.p. glucan phosphate (GP) administration on Dectin-1 membrane expression in murine peripheral blood leukocytes, splenocytes, bone marrow, and peritoneal cells from 3 h to 10 days after injection. Circulating leukocytes were also examined for uptake and internalization of glucans from the blood. Fluorescent-labeled GP was taken up from the systemic circulation by circulating peripheral leukocytes, splenocytes, and peritoneal cells. Following internalization, glucan colocalized with Dectin-1 in an intracellular vesicle. A single parenteral injection of GP resulted in a significant reduction (approximately 33-85%) in peripheral leukocyte membrane-associated Dectin-1 positivity that lasted for up to 7 days. The loss of leukocyte membrane-associated Dectin-1 after GP administration was primarily due to decreased levels of Dectin-1 on neutrophil and monocyte membranes with no significant changes in the percentage of neutrophils or monocytes circulating in the blood. Administration of control carbohydrate polymers, i.e., mannan or pullulan, which are not ligands for Dectin-1, did not decrease Dectin-1 leukocyte positivity, indicating that the effect on Dectin-1 is specific to glucans. In fact, mannan administration increased leukocyte Dectin-1 positivity, thus demonstrating a differential effect on leukocyte Dectin-1, compared with GP. We conclude that systemic administration of GP has a specific and prolonged effect on loss of leukocyte membrane Dectin-1 positivity. These data may have important implications for developing dosing regimens for immunomodulatory carbohydrates.