Katerina Vaporidi

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2−/− mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1−/− mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2−/− macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2−/− macrophages, and its target C/EBPβ appear to play a key role in this process. C/EBPβ, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Proportional assist ventilation with load-adjustable gain factors in critically ill patients: comparison with pressure support.

ObjectivesIt is not known if proportional assist ventilation with load-adjustable gain factors (PAV+) may be used as a mode of support in critically ill patients. The aim of this study was to examine the effectiveness of sustained use of PAV+ in critically ill patients and compare it with pressure support ventilation (PS).Design and settingRandomized study in the intensive care unit of a university hospital.MethodsA total of 208 critically ill patients mechanically ventilated on controlled modes for at least 36 h and meeting certain criteria were randomized to receive either PS (n = 100) or PAV+ (n = 108). Specific written algorithms were used to adjust the ventilator settings in each mode. PAV+ or PS was continued for 48 h unless the patients met pre-defined criteria either for switching to controlled modes (failure criteria) or for breathing without ventilator assistance.ResultsFailure rate was significantly lower in PAV+ than that in PS (11.1 vs. 22.0%, P = 0.040, OR 0.443, 95% CI 0.206–0.952). The proportion of patients exhibiting major patient–ventilator dyssynchronies at least during one occasion and after adjusting the initial ventilator settings, was significantly lower in PAV+ than in PS (5.6 vs. 29.0%, P < 0.001, OR 0.1, 95% CI 0.06–0.4). The proportion of patients meeting criteria for unassisted breathing did not differ between modes.ConclusionsPAV+ may be used as a useful mode of support in critically ill patients. Compared to PS, PAV+ increases the probability of remaining on spontaneous breathing, while it considerably reduces the incidence of patient–ventilator asynchronies.

Akt2 Deficiency Protects from Acute Lung Injury via Alternative Macrophage Activation and miR-146a Induction in Mice

Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2−/− mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2−/− mice compared with WT mice. Alveolar macrophages from acid-injured Akt2−/− mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2−/− mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2−/− mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2−/− macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

Pulmonary microRNA profiling in a mouse model of ventilator-induced lung injury.

The aim of this study was to investigate the changes induced by high tidal volume ventilation (HVTV) in pulmonary expression of micro-RNAs (miRNAs) and identify potential target genes and corresponding miRNA-gene networks. Using a real-time RT-PCR-based array in RNA samples from lungs of mice subjected to HVTV for 1 or 4 h and control mice, we identified 65 miRNAs whose expression changed more than twofold upon HVTV. An inflammatory and a TGF-β-signaling miRNA-gene network were identified by in silico pathway analysis being at highest statistical significance (P = 10(-43) and P = 10(-28), respectively). In the inflammatory network, IL-6 and SOCS-1, regulated by miRNAs let-7 and miR-155, respectively, appeared as central nodes. In TGF-β-signaling network, SMAD-4, regulated by miR-146, appeared as a central node. The contribution of miRNAs to the development of lung injury was evaluated in mice subjected to HVTV treated with a precursor or antagonist of miR-21, a miRNA highly upregulated by HVTV. Lung compliance was preserved only in mice treated with anti-miR-21 but not in mice treated with pre-miR-21 or negative-control miRNA. Both alveolar-arterial oxygen difference and protein levels in bronchoalveolar lavage were lower in mice treated with anti-miR-21 than in mice treated with pre-miR-21 or negative-control miRNA (D(A-a): 66 ± 27 vs. 131 ± 22, 144 ± 10 mmHg, respectively, P < 0.001; protein concentration: 1.1 ± 0.2 vs. 2.3 ± 1, 2.1 ± 0.4 mg/ml, respectively, P < 0.01). Our results show that HVTV induces changes in miRNA expression in mouse lungs. Modulation of miRNA expression can affect the development of HVTV-induced lung injury.

Protective and Detrimental Effects of Sodium Sulfide and Hydrogen Sulfide in Murine Ventilator-induced Lung Injury.

Background: The antiinflammatory effects of hydrogen sulfide (H2S) and sodium sulfide (Na2S) treatment may prevent acute lung injury induced by high tidal volume (HVT) ventilation. However, lung protection may be limited by direct pulmonary toxicity associated with H2S inhalation. Therefore, the authors tested whether the inhalation of H2S or intravascular Na2S treatment can protect against ventilator-induced lung injury in mice. Methods: Anesthetized mice continuously inhaled 0, 1, 5, or 60 ppm H2S or received a single bolus infusion of Na2S (0.55 mg/kg) or vehicle and were then subjected to HVT (40 ml/kg) ventilation lasting 4 h (n = 4–8 per group). Results: HVT ventilation increased the concentrations of protein and interleukin-6 in bronchoalveolar lavage fluid, contributing to reduced respiratory compliance and impaired arterial oxygenation, and caused death from lung injury and pulmonary edema. Inhalation of 1 or 5 ppm H2S during HVT ventilation did not alter lung injury, but inhalation of 60 ppm H2S accelerated the development of ventilator-induced lung injury and enhanced the pulmonary expression of the chemoattractant CXCL-2 and the leukocyte adhesion molecules CD11b and L-selectin. In contrast, pretreatment with Na2S attenuated the expression of CXCL-2 and CD11b during HVT ventilation and reduced pulmonary edema. Moreover, Na2S enhanced the pulmonary expression of Nrf2-dependent antioxidant genes (NQO1, GPX2, and GST-A4) and prevented oxidative stress-induced depletion of glutathione in lung tissue. Conclusions: The data suggest that systemic intravascular treatment with Na2S represents a novel therapeutic strategy to prevent both ventilator-induced lung injury and pulmonary glutathione depletion by activating Nrf2-dependent antioxidant gene transcription.

Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization.

Macrophages become activated initiating innate immune responses. Depending on the signals, macrophages obtain an array of activation phenotypes, described by the broad terms of M1 or M2 phenotype. The PI3K/Akt/mTOR pathway mediates signals from multiple receptors including insulin receptors, pathogen-associated molecular pattern receptors, cytokine receptors, adipokine receptors, and hormones. As a result, the Akt pathway converges inflammatory and metabolic signals to regulate macrophage responses modulating their activation phenotype. Akt is a family of three serine-threonine kinases, Akt1, Akt2, and Akt3. Generation of mice lacking individual Akt, PI3K, or mTOR isoforms and utilization of RNA interference technology have revealed that Akt signaling pathway components have distinct and isoform-specific roles in macrophage biology and inflammatory disease regulation, by controlling inflammatory cytokines, miRNAs, and functions including phagocytosis, autophagy, and cell metabolism. Herein, we review the current knowledge on the role of the Akt signaling pathway in macrophages, focusing on M1/M2 polarization and highlighting Akt isoform–specific functions.

Nitric oxide synthase 3 contributes to ventilator-induced lung injury

Nitric oxide synthase (NOS) depletion or inhibition reduces ventilator-induced lung injury (VILI), but the responsible mechanisms remain incompletely defined. The aim of this study was to elucidate the role of endothelial NOS, NOS3, in the pathogenesis of VILI in an in vivo mouse model. Wild-type and NOS3-deficient mice were ventilated with high-tidal volume (HV(T); 40 ml/kg) for 4 h, with and without adding NO to the inhaled gas. Additional wild-type mice were pretreated with tetrahydrobiopterin and ascorbic acid, agents that can prevent NOS-generated superoxide production. Arterial blood gas tensions, histology, and lung mechanics were evaluated after 4 h of HV(T) ventilation. The concentration of protein, IgM, cytokines, malondialdehyde, and 8-isoprostane were measured in bronchoalveolar lavage fluid (BALF). Myeloperoxidase activity, total and oxidized glutathione levels, and NOS-derived superoxide production were measured in lung tissue homogenates. HV(T) ventilation induced VILI in wild-type mice, as reflected by decreased lung compliance, increased concentrations of protein and cytokines in BALF, and oxidative stress. All indices of VILI were ameliorated in NOS3-deficient mice. Augmenting pulmonary NO levels by breathing NO during mechanical ventilation did not increase lung injury in NOS3-deficient mice. HV(T) ventilation increased NOS-inhibitable superoxide production in lung extracts from wild-type mice but not in those from NOS3-deficient mice. Administration of tetrahydrobiopterin and ascorbic acid ameliorated VILI in wild-type mice. Our results indicate that NOS3 contributes to ventilator-induced lung injury via increased production of superoxide.

Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells

The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2−/− genetic background. Surprisingly, the TCR2Ctg/tg/Tpl2−/− mice developed T cell lymphomas with a latency of 4–6 months. The tumor cells were consistently TCR2C+CD8+CD4−, suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8+ ISP cells was not expanded in the thymus of TCR2Ctg/tg/Tpl2−/− mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2−/− mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

Effects of respiratory rate on ventilator-induced lung injury at a constant Paco2 in a mouse model of normal lung

Objective:The aim of this study was to evaluate the effects of respiratory rate (RR) at a constant Paco2 and conventional tidal volume (VT) on the development of ventilator-induced lung injury in normal lungs. Design:Prospective, randomized, experimental study. Setting:University research laboratory. Subjects:Adult male C57BL/6 mice. Interventions:Four groups of anesthetized mice were exposed to mechanical ventilation with different RRs and VTs. Three groups were assigned to one of three RRs (80, 120, and 160 breaths/min), and VT was set to 12, 10, and 8 mL/kg, respectively (RR80VT12, RR120VT10, and RR160VT8), to achieve normal Paco2. A fourth group was ventilated at 160 breaths/min and VT of 10 mL/kg (RR160VT10) with adjustment of dead space. All animals were ventilated for 120 mins with a positive end-expiratory pressure of 1.5 cm H2O and Fio2 of 1. Nonventilated animals were also studied. Measurements and Main Results:Arterial blood gases and static pressure–volume curves were not different among groups at the end of the experiment. Independent of ventilator settings, mechanical ventilation was associated with increased bronchoalveolar lavage protein and increased bronchoalveolar lavage and serum interleukin-6. Total bronchoalveolar lavage protein and interleukin-6 were significantly lower in RR80VT12 and RR160VT8 compared with RR120VT10 and RR160VT10. In all experimental conditions, mechanical ventilation was associated with activation of AKT and ERK1/2 kinases, known to be activated on stretch. Phosphorylation both of AKT and ERK1/2 was lower in RR80VT12 compared with other groups of ventilated animals. Histologic injury did not differ among nonventilated, RR80VT12, and RR160VT8 animals; however, it increased significantly and progressively in RR120VT10 and RR160VT10 animals. Conclusions:Mechanical ventilation with conventional VT induces lung injury in normal lungs, even without alteration in lung mechanics. Reduction of RR and VT ameliorates lung inflammation and injury.

Short-term cardiorespiratory effects of proportional assist and pressure-support ventilation in patients with acute lung injury/acute respiratory distress syndrome

Background: Recent data indicate that assisted modes of mechanical ventilation improve pulmonary gas exchange in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Proportional assist ventilation (PAV) is a new mode of support that amplifies the ventilatory output of the patient effort and improves patient–ventilator synchrony. It is not known whether this mode may be used in patients with ALI/ARDS. The aim of this study was to compare the effects of PAV and pressure-support ventilation on breathing pattern, hemodynamics, and gas exchange in a homogenous group of patients with ALI/ARDS due to sepsis. Methods: Twelve mechanically ventilated patients with ALI/ARDS (mean ratio of partial pressure of arterial oxygen to fractional concentration of oxygen 190 ± 49 mmHg) were prospectively studied. Patients received pressure-support ventilation and PAV in random order for 30 min while maintaining mean airway pressure constant. With both modes, the level of applied positive end-expiratory pressure (7.1 ± 2.1 cm H2O) was kept unchanged throughout. At the end of each study period, cardiorespiratory data were obtained, and dead space to tidal volume ratio was measured. Results: With both modes, none of the patients exhibited clinical signs of distress. With PAV, breathing frequency and cardiac index were slightly but significantly higher than the corresponding values with pressure-support ventilation (24.5 ± 6.9 vs. 21.4 ± 6.9 breaths/min and 4.4 ± 1.6 vs. 4.1 ± 1.3 l · min−1 · m−2, respectively). None of the other parameters differ significantly between modes. Conclusions: In patients with ALI/ARDS due to sepsis, PAV and pressure-support ventilation both have clinically comparable short-term effects on gas exchange and hemodynamics.