Aranya Bagchi

MyD88-Dependent and MyD88-Independent Pathways in Synergy, Priming, and Tolerance between TLR Agonists

TLRs sense components of microorganisms and are critical host mediators of inflammation during infection. Different TLR agonists can profoundly alter inflammatory effects of one another, and studies suggest that the sequence of exposure to TLR agonists may importantly impact on responses during infection. We tested the hypothesis that synergy, priming, and tolerance between TLR agonists follow a pattern that can be predicted based on differential engagement of the MyD88-dependent (D) and the MyD88-independent (I) intracellular signaling pathways. Inflammatory effects of combinations of D and I pathway agonists were quantified in vivo and in vitro. Experiments used several D-specific agonists, an I-specific agonist (poly(I:C)), and LPS, which acts through both the D and I pathways. D-specific agonists included: peptidoglycan-associated lipoprotein, Pam3Cys, flagellin, and CpG DNA, which act through TLR2 (peptidoglycan-associated lipoprotein and Pam3Cys), TLR5, and TLR9, respectively. D and I agonists were markedly synergistic in inducing cytokine production in vivo in mice. All of the D-specific agonists were synergistic with poly(I:C) in vitro in inducing TNF and IL-6 production by mouse bone marrow-derived macrophages. Pretreatment of bone marrow-derived macrophages with poly(I:C) led to a primed response to subsequent D-specific agonists and vice versa, as indicated by increased cytokine production, and increased NF-κB translocation. Pretreatment with a D-specific agonist augmented LPS-induced IFN-β production. All D-specific agonists induced tolerance to one another. Thus, under the conditions studied here, simultaneous and sequential activation of both the D and I pathways causes synergy and priming, respectively, and tolerance is induced by agonists that act through the same pathway.

MyD88-Dependent Immune Activation Mediated by Human Immunodeficiency Virus Type 1-Encoded Toll-Like Receptor Ligands

ABSTRACT Immune activation is a major characteristic of human immunodeficiency virus type 1 (HIV-1) infection and a strong prognostic factor for HIV-1 disease progression. The underlying mechanisms leading to immune activation in viremic HIV-1 infection, however, are not fully understood. Here we show that, following the initiation of highly active antiretroviral therapy, the immediate decline of immune activation is closely associated with the reduction of HIV-1 viremia, which suggests a direct contribution of HIV-1 itself to immune activation. To propose a mechanism, we demonstrate that the single-stranded RNA of HIV-1 encodes multiple uridine-rich Toll-like receptor 7/8 (TLR7/8) ligands that induce strong MyD88-dependent plasmacytoid dendritic cell and monocyte activation, as well as accessory cell-dependent T-cell activation. HIV-1-encoded TLR ligands may, therefore, directly contribute to the immune activation observed during viremic HIV-1 infection. These data provide an initial rationale for inhibiting the TLR pathway to directly reduce the chronic immune activation induced by HIV-1 and the associated immune pathogenesis.

Bacterial Peptidoglycan-Associated Lipoprotein: A Naturally Occurring Toll-Like Receptor 2 Agonist That Is Shed into Serum and Has Synergy with Lipopolysaccharide

Sepsis is initiated by interactions between microbial products and host inflammatory cells. Toll-like receptors (TLRs) are central innate immune mediators of sepsis that recognize different components of microorganisms. Peptidoglycan-associated lipoprotein (PAL) is a ubiquitous gram-negative bacterial outer-membrane protein that is shed by bacteria into the circulation of septic animals. We explored the inflammatory effects of purified PAL and of a naturally occurring form of PAL that is shed into serum. PAL is released into human serum by Escherichia coli bacteria in a form that induces cytokine production by macrophages and is tightly associated with lipopolysaccharide (LPS). PAL activates inflammation through TLR2. PAL and LPS synergistically activate macrophages. These data suggest that PAL may play an important role in the pathogenesis of sepsis and imply that physiologically relevant PAL and LPS are shed into serum and act in concert to initiate inflammation in sepsis.

Upregulation of Pd-l1 on monocytes and dendritic cells by Hiv-1 derived Tlr ligands

Increased PD-L1 expression has been reported in HIV-1-infected individuals, but the mechanisms leading to PD-L1 upregulation remain to be elucidated. Here we demonstrate that HIV-1-derived Toll-like receptor (TLR)7/8 ligands can induce MyD88-dependent upregulation of PD-L1 on plasmacytoid dendritic cells, myeloidic dendritic cells and monocytes. These data suggest a mechanism through which HIV-1-derived TLR ligands might contribute to the functional impairment of virus-specific PD-1-positive T cells by inducing the upregulation of PD-L1 on antigen-presenting cells.

Bacterial Lipoprotein TLR2 Agonists Broadly Modulate Endothelial Function and Coagulation Pathways In Vitro and In Vivo

TLR2 activation induces cellular and organ inflammation and affects lung function. Because deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells to produce IL-6 and IL-8 and to express E-selectin on their surface, including HUVEC, human lung microvascular endothelial cells, and human coronary artery endothelial cells. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen activator inhibitor-1 (PAI-1) and tissue factor, as well as decreased production of tissue plasminogen activator and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. Peptidoglycan-associated lipoprotein induced IL-6 production by endothelial cells from wild-type mice but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged i.v. with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and they had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.

Toll-like receptor 2 activation by bacterial peptidoglycan–associated lipoprotein activates cardiomyocyte inflammation and contractile dysfunction

Objective:Although cardiac dysfunction plays an important role in the pathogenesis of sepsis, the mechanisms that underlie cardiac dysfunction in sepsis remain poorly understood. Bacterial peptidoglycan-associated lipoprotein (PAL), an outer-membrane protein of Gram-negative bacteria, was recently found to be released into the bloodstream in sepsis and to cause inflammation and death in mice. The present studies assessed the effects of PAL on cardiomyocyte function and its signal transduction in cardiomyocytes. Design:Randomized prospective animal study. Setting:Research laboratory. Subjects:Male C57BL/6 mice, B6;129S-Tnfrsf1atm1ImxTnfrsf1btm1Imx/ J knockout mice, Toll-like receptor 2 (TLR2) knockout mice, and myeloid differentiation factor 88 (MyD88) knockout mice. Interventions:None. Measurements and Results:Immunohistochemical staining and immunoblot analysis indicated that intravenously injected PAL bound to myocardium. Injection of PAL decreased cardiac function in vivo. Challenge with PAL altered cell shortening and Ca2+ transients in isolated mouse cardiomyocytes but not in cardiomyocytes isolated from TLR2−/− and MyD88−/− mice. Cytokine profiling arrays demonstrated that tumor necrosis factor-&agr; (TNF&agr;), granulocyte colony-stimulating factor, and interferon-&ggr; production were elevated in PAL-treated cardiomyocytes. Increased TNF&agr; production was abolished in MyD88−/− cardiomyocytes but restored by adenovirally mediated expression of MyD88. PAL did not affect cell shortening and Ca2+ cycling in cardiomyocytes obtained from mice deficient for TNF&agr; receptor (TNFR) 1 and TNFR2 (TNFR1/2−/−). Conclusion:Our data reveal that PAL uses the TLR2/MyD88 signaling cascade to induce cardiomyocyte dysfunction and inflammatory responses and that TNF&agr; is a major mediator of PAL-induced dysfunction in cardiomyocytes. These studies suggest that circulating PAL and other TLR2 agonists may contribute to cardiac dysfunction in sepsis.

Pulmonary Hypertension in Lambs Transfused with Stored Blood is Prevented by Breathing Nitric Oxide

Background: During extended storage, erythrocytes undergo functional changes. These changes reduce the viability of erythrocytes leading to release of oxyhemoglobin, a potent scavenger of nitric oxide. We hypothesized that transfusion of ovine packed erythrocytes (PRBC) stored for prolonged periods would induce pulmonary vasoconstriction in lambs, and that reduced vascular nitric oxide concentrations would increase this vasoconstrictor effect. Methods: We developed a model of autologous stored blood transfusion in lambs (n = 36). Leukoreduced blood was stored for either 2 days (fresh PRBC) or 40 days (stored PRBC). Fresh or stored PRBC were transfused into donors instrumented for awake hemodynamic measurements. Hemodynamic effects of PRBC transfusion were also studied after infusion of NG-nitro-L-arginine methyl-ester (25 mg/kg) or during inhalation of nitric oxide (80 ppm). Results: Cell-free hemoglobin levels were higher in the supernatant of stored PRBC than in supernatant of fresh PRBC (Mean ± SD, 148 ± 20 vs. 41 ± 13 mg/dl, respectively, P < 0.001). Pulmonary artery pressure during transfusion of stored PRBC transiently increased from 13 ± 1 to 18 ± 1 mmHg (P < 0.001) and was associated with increased plasma hemoglobin concentrations. NG-nitro-L-arginine methyl-ester potentiated the increase in pulmonary arterial pressure induced by transfusing stored PRBC, whereas inhalation of nitric oxide prevented the vasoconstrictor response. Conclusions: Our results suggest that patients with reduced vascular nitric oxide levels because of endothelial dysfunction may be more susceptible to adverse effects of transfusing blood stored for prolonged periods. These patients might benefit from transfusion of fresh PRBC, when available, or inhaled nitric oxide supplementation to prevent the pulmonary hypertension associated with transfusion of stored PRBC.

The association of postoperative pulmonary complications in 109,360 patients with pressure-controlled or volume-controlled ventilation.

We thought that the rate of postoperative pulmonary complications might be higher after pressure‐controlled ventilation than after volume‐controlled ventilation. We analysed peri‐operative data recorded for 109,360 adults, whose lungs were mechanically ventilated during surgery at three hospitals in Massachusetts, USA. We used multivariable regression and propensity score matching. Postoperative pulmonary complications were more common after pressure‐controlled ventilation, odds ratio (95%CI) 1.29 (1.21–1.37), p < 0.001. Tidal volumes and driving pressures were more varied with pressure‐controlled ventilation compared with volume‐controlled ventilation: mean (SD) variance from the median 1.61 (1.36) ml.kg−1 vs. 1.23 (1.11) ml.kg−1, p < 0.001; and 3.91 (3.47) cmH2O vs. 3.40 (2.69) cmH2O, p < 0.001. The odds ratio (95%CI) of pulmonary complications after pressure‐controlled ventilation compared with volume‐controlled ventilation at positive end‐expiratory pressures < 5 cmH2O was 1.40 (1.26–1.55) and 1.20 (1.11–1.31) when ≥ 5 cmH2O, both p < 0.001, a relative risk ratio of 1.17 (1.03–1.33), p = 0.023. The odds ratio (95%CI) of pulmonary complications after pressure‐controlled ventilation compared with volume‐controlled ventilation at driving pressures of < 19 cmH2O was 1.37 (1.27–1.48), p < 0.001, and 1.16 (1.04–1.30) when ≥ 19 cmH2O, p = 0.011, a relative risk ratio of 1.18 (1.07–1.30), p = 0.016. Our data support volume‐controlled ventilation during surgery, particularly for patients more likely to suffer postoperative pulmonary complications.

Thiamine as a neuroprotective agent after cardiac arrest

AIMS Reduction of pyruvate dehydrogenase (PDH) activity in the brain is associated with neurological deficits in animals resuscitated from cardiac arrest. Thiamine is an essential co-factor of PDH. The objective of this study was to examine whether administration of thiamine improves outcomes after cardiac arrest in mice. Secondarily, we aimed to characterize the impact of cardiac arrest on PDH activity in mice and humans. METHODS Animal study: Adult mice were subjected to cardiac arrest whereupon cardiopulmonary resuscitation was performed. Thiamine or vehicle was administered 2min before resuscitation and daily thereafter. Mortality, neurological outcome, and metabolic markers were evaluated. Human study: In a convenience sample of post-cardiac arrest patients, we measured serial PDH activity from peripheral blood mononuclear cells and compared them to healthy controls. RESULTS Animal study: Mice treated with thiamine had increased 10-day survival (48% versus 17%, P<0.01) and improved neurological function when compared to vehicle-treated mice. In addition, thiamine markedly improved histological brain injury compared to vehicle. The beneficial effects of thiamine were accompanied by improved oxygen consumption in mitochondria, restored thiamine pyrophosphate levels, and increased PDH activity in the brain at 10 days. Human study: Post-cardiac arrest patients had lower PDH activity in mononuclear cells than did healthy volunteers (estimated difference: -5.8O.D./min/mg protein, P<0.001). CONCLUSIONS The provision of thiamine after cardiac arrest improved neurological outcome and 10-day survival in mice. PDH activity was markedly depressed in post-cardiac arrest patients suggesting that this pathway may represent a therapeutic target.

Mashed potatoes and maize: are the starches safe?

244 February 2013 I N 1831 during the European cholera epidemic, Latta1 was the first to report on a patient who was successfully resuscitated with intravenous fluids. He injected 60 ounces of warm saline intravenously to a pulseless cholera patient. According to the author’s report, on receiving the fluid resuscitation, every symptom of cholera was removed. About 80 yr later, fluid resuscitation with colloids was introduced to clinical medicine for treatment of severe hemorrhage. In his case series report published in JAMA in 1915, Hogan2 noted that although salt solutions give a temporary rise in blood pressure (and improvement in the general symptoms resulting from hemorrhage), he could obtain a more permanent rise with gelatin, a hydrophilic colloidal solution. In the same report, Dr. Hogan included the caveat that resuscitation with colloids is insufficient to treat toxemic shock despite the initial effects of colloid resuscitation on blood pressure. This differential effect on outcome of patients presenting with hemorrhagic and septic shock should be kept in mind when interpreting the results from current trials. Hydroxyethyl starches (HES) are the most commonly used colloids in many parts of the world;3however, recent studies suggest that HES may be associated with worse outcomes, when given for fluid resuscitation to patients with sepsis.4,5 Outcome data on the topic of colloid resuscitation are sparse, which is probably why it still raises strong opinions from key opinion leaders in the field. In this issue of ANEStHESIOLOgy, two groups of researchers provide important new data on the safety and potential benefits of modern 6% HES. Silva et al. 6 show in a preclinical model of hemorrhage and lung injury that potato-derived 6% HES resuscitation compares favorably with crystalloid and gelatin–based fluid resuscitation in terms of variables reflecting pulmonary and renal injury. The meta-analysis of Martin et al.7 reports on the absence of renal toxicity of maize-derived HES given to 1,230 patients undergoing a variety of surgical procedures.