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

Circulating extracellular vesicles are associated with the clinical outcomes of sepsis

IntroductionSepsis is associated with endothelial cell (EC) dysfunction, increased vascular permeability and organ injury, which may lead to mortality, acute respiratory distress syndrome (ARDS) and acute renal failure (ARF). There are no reliable biomarkers to predict these sepsis complications at present. Recent evidence suggests that circulating extracellular vesicles (EVs) and their content caspase-1 and miR-126 may play a critical role in modulating vascular injury in sepsis; however, the association between circulating EVs and sepsis outcomes remains largely unknown.MethodsWe obtained plasma samples from septic patients (n=96) within 24 hours of hospital admission and from healthy controls (n=45). Total, monocyte- or EC-derived EVs were isolated from the plasma samples. Transendothelial electrical resistance (TEER) was used as an indicator of EC dysfunction. Caspase-1 activity in EVs was detected and their association with sepsis outcomes including mortality, ARDS and ARF was analyzed. In another set of experiments, total EVs were isolated from plasma samples of 12 septic patients and 12 non-septic critical illness controls on days 1, and 3 after hospital admission. RNAs were isolated from these EVs and Next-generation sequencing was performed. The association between miR-126 levels and sepsis outcomes such as mortality, ARDS and ARF was analyzed.ResultsSeptic patients with circulating EVs that induced EC injury (lower transendothelial electrical resistance) were more likely to experience ARDS (p&lt;0.05). Higher caspase-1 activity in total EVs, monocyte- or EC-derived EVs was significantly associated with the development of ARDS (p&lt;0.05). MiR-126-3p levels in EC EVs were significantly decreased in ARDS patients compared with healthy controls (p&lt;0.05). Moreover, a decline in miR-126-5p levels from day 1 to day 3 was associated with increased mortality, ARDS and ARF; while decline in miR-126-3p levels from day 1 to day 3 was associated with ARDS development.ConclusionsEnhanced caspase-1 activity and declining miR-126 levels in circulating EVs are associated with sepsis-related organ failure and mortality. Extracellular vesicular contents may serve as novel prognostic biomarkers and/or targets for future therapeutic approaches in sepsis

Effects of repeated sleep deprivation on brain pericytes in mice

Abstract The damaging effects of sleep deprivation (SD) on brain parenchyma have been extensively studied. However, the specific influence of SD on brain pericytes, a primary component of the blood–brain barrier (BBB) and the neurovascular unit (NVU), is still unclear. The present study examined how acute or repeated SD impairs brain pericytes by measuring the cerebrospinal fluid (CSF) levels of soluble platelet-derived growth factor receptor beta (sPDGFRβ) and quantifying pericyte density in the cortex, hippocampus, and subcortical area of the PDGFRβ-P2A-CreERT2/tdTomato mice, which predominantly express the reporter tdTomato in vascular pericytes. Our results showed that a one-time 4 h SD did not significantly change the CSF sPDGFRβ level. In contrast, repeated SD (4 h/day for 10 consecutive days) significantly elevated the CSF sPDGFRβ level, implying explicit pericyte damages due to repeated SD. Furthermore, repeated SD significantly decreased the pericyte densities in the cortex and hippocampus, though the pericyte apoptosis status remained unchanged as measured with Annexin V-affinity assay and active Caspase-3 staining. These results suggest that repeated SD causes brain pericyte damage and loss via non-apoptosis pathways. These changes to pericytes may contribute to SD-induced BBB and NVU dysfunctions. The reversibility of this process implies that sleep improvement may have a protective effect on brain pericytes

Proteomic Analysis of Exosomes Secreted from Human Alpha-1 Antitrypsin Overexpressing Mesenchymal Stromal Cells

Extracellular vesicles (EVs) mediate many therapeutic effects of stem cells during cellular therapies. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) were manufactured to overexpress the human antiprotease alpha-1 antitrypsin (hAAT) and studied to compare the EV production compared to lentivirus treated control MSCs. The goal of this study was to compare protein profiles in the EVs/exosomes of control and hAAT-MSCs using unbiased, high resolution liquid chromatography and mass spectrometry to explore differences. Nanoparticle tracking analysis (NTA) showed that the particle size of the EVs from control MSCs or hAAT-MSCs ranged from 30 to 200 nm. Both MSCs and hAAT-MSCs expressed exosome-associated proteins, including CD63, CD81, and CD9. hAAT-MSCs also expressed high levels of hAAT. We next performed proteomic analysis of EVs from three healthy donor cell lines. Exosomes collected from cell supernatant were classified by GO analysis which showed proteins important to cell adhesion and extracellular matrix organization. However, there were differences between exosomes from control MSCs and hAAT-MSCs in cytokine signaling of the immune system, stem cell differentiation, and carbohydrate metabolism (p &lt; 0.05). These results show that hAAT-MSC exosomes contain a different profile of paracrine effectors with altered immune function, impacts on MSC stemness, differentiation, and prevention of cell apoptosis and survival that could contribute to improved therapeutic functions

Peroxisome proliferator activated receptor γ is not necessary for the development of LPS-induced tolerance in macrophages

Peroxisome proliferator activated receptor-γ (PPARγ) has been reported to exert anti-inflammatory properties in endotoxic shock and sepsis. One phenomenon that alters the inflammatory response to endotoxin [lipopolysaccharide (LPS)] is endotoxin tolerance, which is caused by previous exposure to endotoxin. Here, we investigate whether changes in endogenous PPARγ function regulate this phenomenon using three different models of LPS-induced tolerance in macrophages. In a first in vitro model, previous LPS exposure of murine J774.2 macrophages suppressed tumour necrosis factor-α (TNF-α) release in response to subsequent LPS challenge. Treatment of J774.2 cells with the PPARγ inhibitor GW9662 did not alter tolerance induction because these cells were still hyporesponsive to the secondary LPS challenge. In a second ex vivo model, primary rat peritoneal macrophages from LPS-primed rats exhibited suppression of thromboxane B2 and TNF-α production, while maintaining nitrite production in response to in vitro LPS challenge. Pretreatment of rats with the PPARγ inhibitor GW9662 in vivo failed to alter the tolerant phenotype of these primary macrophages. In a third ex vivo model, primary peritoneal macrophages with conditional deletion of PPARγ were harvested from LPS-primed Cre-lox mice (Cre+/+ PPARγ−/−) and exhibited significant suppression of TNF-α production in response to in vitro LPS challenge. Furthermore, both LPS-primed PPARγ-deficient Cre+/+ PPARγ−/− mice and wild-type Cre−/− PPARγ+/+ mice exhibited reduced plasma TNF-α levels in response to a high dose of LPS in vivo. These data demonstrate that PPARγ does not play a role in the LPS-induced tolerant phenotype in macrophages

Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury

Abstract Background The acute respiratory distress syndrome (ARDS) is characterized by disruption of the alveolar-capillary barrier resulting in accumulation of proteinaceous edema and increased inflammatory cells in the alveolar space. We previously found that endothelial progenitor cell (EPC) exosomes prevent endothelial dysfunction and lung injury in sepsis in part due to their encapsulation of miRNA-126. However, the effects of EPC exosomes in acute lung injury (ALI) remain unknown. Methods To determine if EPC exosomes would have beneficial effects in ALI, intratracheal administration of lipopolysaccharide (LPS) was used to induce ALI in mice. Lung permeability, inflammation, and the role of miRNA-126 in the alveolar-epithelial barrier function were examined. Results The intratracheal administration of EPC exosomes reduced lung injury following LPS-induced ALI at 24 and 48 h. Compared to placebo, intratracheal administration of EPC exosomes significantly reduced the cell number, protein concentration, and cytokines/chemokines in the bronchoalveolar lavage fluid (BALF), indicating a reduction in permeability and inflammation. Further, EPC exosomes reduced myeloperoxidase (MPO) activity, lung injury score, and pulmonary edema, demonstrating protection against lung injury. Murine fibroblast (NIH3T3) exosomes, which do not contain abundant miRNA-126, did not provide these beneficial effects. In human small airway epithelial cells (SAECs), we found that overexpression of miRNA-126-3p can target phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2), while overexpression of miRNA-126-5p inhibits the inflammatory alarmin HMGB1 and permeability factor VEGFα. Interestingly, both miR-126-3p and 5p increase the expression of tight junction proteins suggesting a potential mechanism by which miRNA-126 may mitigate LPS-induced lung injury. Conclusions Our data demonstrated that human EPC exosomes are beneficial in LPS-induced ALI mice, in part through the delivery of miRNA-126 into the injured alveolus

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