Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Gαi proteins

AbstractHeterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Gαi2 (−/−) or Gαi1/3 (−/−) mice were investigated. LPS- or SA-induced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GM-CSF, MIP-1α, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p<0.05) in splenocytes harvested from Gαi2(−/−) mice compared with WT mice. The effect of Gαi protein depletion was remarkably isoform specific. In splenocytes from Gαi1/3 (−/−) mice relative to WT mice, SA-induced IL-6, IFNγ, GM-CSF, and IP-10 levels were decreased (59% to 86%, p<0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Gαi2 (−/−) and Gαi1/3 (−/−) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Gαi2 (−/−) and Gαi1/3 (−/−) mice relative to WT mice. The disparate response of splenocytes from the Gαi2 (−/−) relative to Gαi1/3 (−/−) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that Gi2 and Gi1/3 proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli

Scavenger Receptor Class A Plays a Central Role in Mediating Mortality and the Development of the Pro-Inflammatory Phenotype in Polymicrobial Sepsis

Sepsis is a frequent complication in critical illness. The mechanisms that are involved in initiation and propagation of the disease are not well understood. Scavenger receptor A (SRA) is a membrane receptor that binds multiple polyanions such as oxidized LDL and endotoxin. Recent studies suggest that SRA acts as a pattern recognition receptor in the innate immune response. The goal of the present study was to determine the role of SRA in polymicrobial sepsis. SRA deficient (SRA-/-) and C57BL/6JB/6J (WT) male mice were subjected to cecal ligation and puncture (CLP) to induce polymicrobial sepsis. NFκB activity, myeloperoxidase activity, and co-association of SRA with toll like receptor (TLR) 4 and TLR2 was analyzed in the lungs. Spleens were analyzed for apoptosis. Serum cytokines and chemokines were assayed. Blood and peritoneal fluid were cultured for aerobic and anaerobic bacterial burdens. Long term survival was significantly increased in SRA-/- septic mice (53.6% vs. 3.6%, p\u3c0.05) when compared to WT mice. NFκB activity was 45.5% lower in the lungs of SRA-/- septic mice versus WT septic mice (p\u3c0.05). Serum levels of interleukin (IL)-5, IL-6, IL-10 and monocyte chemoattractant protein -1 were significantly lower in septic SRA-/- mice when compared to septic WT mice (p\u3c0.05). We found that SRA immuno-precipitated with TLR4, but not TLR2, in the lungs of WT septic mice. We also found that septic SRA-/- mice had lower bacterial burdens than WT septic mice. SRA deficiency had no effect on pulmonary neutrophil infiltration or splenocyte apoptosis during sepsis. We conclude that SRA plays a pivotal, and previously unknown, role in mediating the pathophysiology of sepsis/septic shock in a murine model of polymicrobial sepsis. Mechanistically, SRA interacts with TLR4 to enhance the development of the pro-inflammatory phenotype and mediate the morbidity and mortality of sepsis/septic shock

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p30 days (

Effect of Platelet Activating Factor on Embryonic Development and Implantation in the Mouse

Platelet activating factor (PAF) was administered to female mice in order to investigate its effect on ovulation rate and on oocyte quality including their in-vitro embryonic development, implantation and uterine receptivity. In experiment 1, 4-week-old female mice were assigned to receive PAF or phosphate buffered saline for 4 consecutive days. On the second day of this treatment, pregnant mares\u27 serum gonadotrophin was administered and human chorionic gonadotrophin (HCG) 48 h later, after which copulation occurred. Oocytes were collected on the following day and evaluated. The mean number of oocytes and zygotes (two pronuclear stage embryos) recovered from the PAF-treated group was not diffferent from the control group (31 versus 27), but the proportion of zygotes was higher in PAF-treated group than in controls (83 versus 68%, P \u3c 0.05, PAF versus controls). Although the rate of in-vitro first cleavage was not different in the two groups (82 versus 69% respectively), hatching was higher in the PAF-treated group than control mice (99 versus 83%, P \u3c 0.01). In experiment 2, the in-vitro developed blastocysts from experiment 1 were transferred into the uterus of day 3 pseudopregnant PAF-treated or control recipients. Three different combinations of intrauterine transfer were performed; PAF embryo to control recipient (PAF → C: n = 19), control embryo to PAF recipient (C → PAF: n = 19), and control embryo to control recipient (C → C: n = 22). Implantation and abortion were assessed on day 19 post-transfer. The implantation rate of C → PAF (23.7%) was lower than C → C (31.1%, P \u3c 0.05), but was not different from PAF → C (31.2%). Further, C → PAF showed a higher abortion rate per embryo (29.6%) than PAF → C (12.7%, P \u3c 0.05), but was not different from C → C (24.4%). In the present study, PAF administration enables females to produce oocytes with a higher potential for fertilization, in vitro development and implantation, but has a detrimental effect on uterine receptivity to embryos

Aspirin Dose Dependently Inhibits the Interleukin-1β-Stimulated Increase in Inducible Nitric Oxide Synthase, Nitric Oxide, and Prostaglandin E\u3csub\u3e2\u3c/sub\u3e Production in Rat Ovarian Dispersates Cultured in Vitro

Objective: Determine if aspirin inhibits the IL-1β-stimulated expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and prostaglandin E2 (PGE2) in rat ovarian dispersates cultured in vitro. Design: Prospective, controlled in vitro study. Setting: Academic research laboratory. Animals: Ovaries collected from immature rats. Intervention(s): Ovaries were collected from immature rats and enzymatically dispersed. Ovarian dispersates were placed into plates containing media alone or media supplemented with IL-1β (100 U/mL) and varying concentrations of aspirin (0, 1, 3, 5 and 10 mM). Ovarian dispersates were cultured in a humidified environment of 5% CO2 in air at 37°C for 24 or 48 hours. Main Outcome Measure(s): Twenty-four- and 48-hour iNOS, nitrite (a stable metabolite of NO), and PGE2 levels were determined from ovarian dispersates cultured in vitro. Result(s): Administration of IL-1β increased nitrite and PGE2 levels over that observed in the control group after culture of ovarian dispersates for 24 and 48 hours. Aspirin dose dependently reduced the IL-1β-stimulated increase in nitrite production from ovarian dispersates after culture for 24 and 48 hours. Aspirin completely (24 hours) or dose dependently (48 hours) prevented the IL-β-stimulated increase in PGE2. Coadministration of IL-1β and aspirin (10 mM) attenuates IL-1β-stimulated iNOS expression after culture for 24 and 48 hours. Conclusion(s): Aspirin significantly inhibits the IL-1β-stimulated expression of iNOS, NO, and PGE2 in ovarian dispersates cultured in vitro

In Vivo 4-androstene-3,17-dione and 4-androstene-3β, 17β-diol Supplementation in Young Men

To determine if known androgenic hormone precursors for testosterone in the androgen pathway would be readily transformed to testosterone, eight male subjects [mean age 23.8 (SEM 3) years, bodymass 83.1 (SEM 8.7) kg, height 175.6 (SEM 8.5) cm] underwent a randomized, double-blind, cross-over, placebo-controlled oral treatment with 200 mg of 4-androstene-3,17-dione (Δ4), 4-androstene-3β,17β-diol (Δ4Diol), and placebo (PL). The periods of study were separated by 7 days of washout. Blood was drawn at baseline and subsequently every 30 min for 90 min after treatment. Analysis revealed mean area-under-the-curve (AUC) serum Δ4 concentrations to be higher during Δ4 treatment [2177 (SEM 100) nmol.l-1] than Δ4Diol [900 (SEM 96) nmol.l-1] or PL [484 (SEM 82) nmol.l-1; P \u3c 0.0001]. The Δ4 treatment also revealed a significant effect on total testosterone with a mean AUC [1632.5 (SEM 121) nmol.l-1] that was greater than PL [1418.5 (SEM 131) nmol.l-1; P \u3c 0.05] but not significantly different from those observed after Δ4Diol treatment [1602.9 (SEM 119) nmol.l-1; P = 0.77]. Free testosterone concentrations followed a similar pattern where mean AUC for the Δ4 treatment [6114.0 (SEM 600) pmol.l-1] was greater than after PL [4974.6 (SEM 565) pmol.l-1; P \u3c 0.06] but not significantly different from those observed after Δ4Diol [5632.0 (SEM 389) pmol.l-1; P = 0.48]. The appearance and apparent conversion to total and free testosterone over 90 min was stronger for the Δ4 treatment (r = 0.91, P \u3c 0.045) than for Δ4Diol treatment (r = 0.69, NS) and negatively correlated for PL (r = -0.90, P \u3c 0.02). These results would suggest that Δ4, and perhaps Δ4Diol, taken by month are capable of producing in vivo increases in testosterone concentrations in apparently healthy young men as has already been observed in women after treatment with Δ4

Secretion of Anti-Müllerian Hormone in the Florida manatee Trichechus manatus Latirostris, With Implications for Assessing Conservation Status

Environmental and anthropogenic stressors can affect wildlife populations in a number of ways. For marine mammals (e.g. the Florida manatee Trichechus manatus latirostris), certain stressors or conservation risk factors have been identified, but sublethal effects have been very difficult to assess using traditional methods. The development of \u27biomarkers\u27 allows us to correlate effects, such as impaired reproduction, with possible causes. A recently developed biomarker (anti-Müllerian hormone, AMH) provides an enzyme-linked immunosorbent assay of gonadal function. The study objective was to determine AMH levels in wild manatees. In total, 28 male and 17 female manatee serum samples were assayed. Animal demographics included collection date, body weight (kg) and total length (cm). In certain cases, age of individuals was also known. AMH levels ranged from 160 to 2451.85 ng ml -1 (mean = 844.65 ng ml -1) in males and 0.00 to 0.38 ng ml -1 (mean = 0.10 ng ml -1) in females. Linear regression analyses revealed a significant relationship between male AMH levels and body weight (R 2 = 0.452; p \u3c 0.001) and length (R 2 = 0.338; p \u3c 0.001). Due to the small sample size, regression analyses for female AMH and body weight and length were not significant. This represents the first report of AMH detection in a marine mammal. AMH levels in male manatees are the highest of any species observed to date, whereas levels in females are within reported ranges. Further studies will promote improved conservation decision by assessing AMH levels in the manatee as a function of various stressors including, but not limited to, nutritional status, serious injuries (e.g. watercraft collisions), exposure to biotoxins or contaminants, or disease

G\u3csub\u3eI\u3c/sub\u3e Proteins Regulate Lipopolysaccharide and Staphylococcus aureus Induced Cytokine Production but Not (1→3)-Beta-D-Glucan Induced Cytokine Suppression

Previous studies have demonstrated that bacterial lipopolysaccharide (LPS) and heat killed Staphylococcus aureus (SA) activation of inflammatory cells depended in part upon activation of heterotrimeric Gi proteins. It has also been shown that (1→3) beta-D-glucan can suppress inflammatory cell activation by microbial products although the cellular mechanism of the glucan effect remains to be clearly defined. We hypothesized that Gi proteins function as a common convergent signaling pathway for both LPS and SA leading to monocyte mediator production. Additionally, we hypothesized that soluble glucan suppresses LPS and SA induced cytokine production via Gi protein coupled signaling. Human THP-1 promonocytic cells were pretreated with pertussis toxin (PTx, 100ng/ml or 1 microgram/ml) 6 hours prior to stimulation with LPS (10 microgram/ml) and SA (10 microgram/ml) and/or soluble glucan (10 microgram/ml). Both LPS and SA significantly (p\u3c0.05) induced cytokine production IL-6 \u3eTNF alpha \u3eIL-1 beta \u3eGM-CSF \u3eIL-10 \u3eIFN gamma. The induction of these cytokines was significantly (p\u3c0.05) suppressed by PTx. Glucan treatment alone had no effect on cytokine production but suppressed (P\u3c0.05) LPS and SA induced cytokines. PTx further augmented (p\u3c0.05) the inhibitory effect of glucan on the LPS and SA induced cytokine expression. The data support the hypothesis that Gi proteins function as a common signaling protein for both LPS and SA induction of pro-and anti-inflammatory cytokines and that soluble glucan effectively suppresses cytokine production to the microbial stimuli. In contrast, the effect of soluble glucan on inhibiting cellular activation by LPS and SA is Gi protein independent

Differential Regulation of Cytokine and Chemokine Production in Lipopolysaccharide-Induced Tolerance and Priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFα production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFα production. The pro-inflammatory cytokine, IFNγ, also abolishes suppression of TNFα in LPS tolerance. The effect of LPS tolerance on HKSa and IFNγ-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNγ differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24h with LPS (100ng/ml) or LPS (100ng/ml)+IFNγ (1μg/ml). Cells were subsequently stimulated with LPS or HKSa (10μg/ml) for 24h. The production of the cytokines TNFα, IL-6, IL-1β, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFα (3070±711pg/ml and 217±9pg/ml, respectively) and IL-6 (237±8.9pg/ml and 56.2±2.9pg/ml, p\u3c0.05, n=3, respectively) in control cells compared to basal levels (\u3c25pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p\u3c0.05, n=3) reduction in TNFα. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFα (2.7 fold, from 217 to 580pg/ml, p\u3c0.05, n=3). In contrast to suppressed TNFα, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076pg/ml, p\u3c0.01, n=3) and also primed to HKSa stimulation (62 fold, from 56 to 3470pg/ml, p\u3c0.01, n=3). LPS induced IL-8 production and to a lesser extent IL-1β and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1β, although HKSa stimulation augmented both mediators. In addition, IFNγ pretreatment reversed LPS tolerance as evidenced by increased TNFα levels while IL-6, IL-1β, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNγ. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis

At Low Serum Glucan Concentrations There Is an Inverse Correlation Between Serum Glucan and Serum Cytokine Levels in ICU Patients With Infections

Glucans are fungal cell wall glucose polymers that are released into the blood of infected patients. The role of glucans in infection is unknown. We examined serum glucan and cytokine levels in intensive care unit (ICU) patients with infections. There was an inverse correlation (p\u3c0.001) between serum glucan levels and interleukin (IL)-2), IL-4, tumor necrosis factorα (TNFα) and granulocyte macrophage-colony stimulating factor (GM-CSF) levels in infected ICU patients. The correlation between serum cytokines and serum glucan was only observed at glucan concentrations \u3c40 pg/ml. No change was observed at serum glucan levels of \u3e40 pg/ml. There was no correlation between serum glucan levels and systemic levels of IL-1β, IL-5, IL-6, IL-8, IL-10 or IFNγ. Interestingly, blood borne glucans did not suppress systemic cytokine levels in infected ICU patients, instead they were maintained at control levels. We conclude that circulating glucans may prevent cytokine upregulation in response to infection. This may represent an adaptive response to septic injury