Adiponectin-deficiency exaggerates sepsis-induced microvascular dysfunction in the mouse brain

Obesity increases circulating cell-endothelial cell interactions; an early marker of inflammation in laboratory model of sepsis, but little is known about the effect of different adipokines. Adiponectin is an anti-inflammatory adipokine secreted by adipocytes. Adiponectin deficiency is implicated in exaggerated proinflammatory phenotype in both obesity and sepsis via increased proinflammatory cytokine expression. However the effect of adiponectin deficiency on circulating cell-endothelial cell interactions in polymicrobial sepsis is unknown. Furthermore although brain dysfunction in septic patients is a known predictor of death, the pathophysiology involved is unknown. In the current study, we examined the effects of adiponectin deficiency on leukocyte (LA) and platelet adhesion (PA) in cerebral microcirculation of septic mice. Adiponectin deficient (Adipoq(-/-): Adko) and background strain C57Bl/6 (wild type (WT)) mice were used. Sepsis was induced using cecal ligation and puncture (CLP). We studied LA and PA in the cerebral microcirculation using intravital fluorescent video microscopy (IVM), blood brain barrier (BBB) dysfunction using Evans Blue (EB) leakage method and E-selectin expression using dual radiolabeling technique in different WT and Adko mice with CLP. Adiponectin deficiency significantly exaggerated LA (WT-CLP:201 ± 17; Adko-CLP: ± 53 cells/mm(2); P < 0.05) and PA (WT-CLP:125 ± 17; Adko-CLP:188 ± 20 cells/mm(2); P < 0.05) in cerebral microcirculation, EB leakage (WT-CLP:10 ± 3.7; Adko-CLP:24 ± 4.3 ng/g × μl plasma; P < 0.05) and E-selectin expression (WT-CLP:0.06 ± 0.11; Adko-CLP:0.44 ± 0.053 ng/g; P < 0.05) in the brain tissue of the mice with CLP. Furthermore, E-selectin monoclonal antibody (mAb) treatment attenuated cell adhesion and BBB dysfunction of Adko-CLP mice. Adiponectin deficiency is associated with exaggerated leukocyte and PA in cerebral microcirculation of mice with CLP via modulation of E-selectin expression.     

A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.     

CRTH2 is a critical regulator of neutrophil migration and resistance to polymicrobial sepsis

Although arachidonic acid cascade has been shown to be involved in sepsis, little is known about the role of PGD(2) and its newly found receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), on the septic response. Severe sepsis is associated with the failure of neutrophil migration. To investigate whether CRTH2 influences neutrophil recruitment and the lethality during sepsis, sepsis was induced by cecal ligation and puncture (CLP) surgery in mice. CRTH2 knockout (CRTH2(-/-)) mice were highly resistant to CLP-induced sepsis, which was associated with lower bacterial load and lower production of TNF-α, IL-6, and CCL3. IL-10, an anti-inflammatory cytokine, was higher in CRTH2(-/-) mice, blunting CLP-induced lethality in CRTH2(-/-) mice. Neutrophil accumulation in the peritoneum was more pronounced after CLP in CRTH2(-/-) mice, which was associated with higher CXCR2 levels in circulating neutrophils. Furthermore, sepsis caused a decrease in the level of acetylation of histone H3, an activation mark, at the CXCR2 promoter in wild-type neutrophils, suggesting that CXCR2 expression levels are epigenetically regulated. Finally, both pharmacological depletion of neutrophils and inhibition of CXCR2 abrogated the survival benefit in CRTH2(-/-) mice. These results demonstrate that genetic ablation of CRTH2 improved impaired neutrophil migration and survival during severe sepsis, which was mechanistically associated with epigenetic-mediated CXCR2 expression. Thus, CRTH2 is a potential therapeutic target for polymicrobial sepsis.     

Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.     

Cecal ligation and puncture-induced impairment of innate immune function does not occur in the absence of caspase-1

Mice that have been subjected to cecal ligation and puncture (CLP) have an impaired ability to clear a subsequent Pseudomonas aeruginosa challenge compared with that of sham CLP controls. We hypothesized that this outcome is dependent upon a caspase-1 mechanism and tested this hypothesis by measuring caspase-1 after CLP and by measuring clearance of a bacterial challenge in caspase-1-deficient mice after CLP. Wild-type mice subjected to CLP had increased caspase-1 activity as well as increased IL-1β and increased IL-18 production in splenocytes stimulated with heat-killed Pseudomonas and had increased plasma concentrations of IL-1β and IL-18 and impaired clearance of a P. aeruginosa challenge compared with sham controls. Healthy, uninjured caspase-1(-\-) mice did not differ from wild-type mice in their ability to clear a Pseudomonas challenge. However, unlike wild-type mice, caspase-1(-/-) mice subjected to CLP had no impairment of bacterial clearance of the Pseudomonas challenge, suggesting that caspase-1 induction after CLP played a role in impairment of bacterial clearance. This was further substantiated by the use of a specific caspase-1 inhibitor, Ac-YVAD-CMK. Wild-type mice treated with Ac-YVAD-CMK (10 mg/kg s.c. twice daily, initiated at time of CLP) did not have impaired clearance of a Pseudomonas challenge compared with that of sham mice and had significantly improved bacterial clearance compared with that of untreated CLP mice. Increased caspase-1 expression and activity after CLP injury appears to contribute to diminished innate immune function.     

Adiponectin blocks interleukin-18-mediated endothelial cell death via APPL1-dependent AMP-activated protein kinase (AMPK) activation and IKK/NF-kappaB/PTEN suppression

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IkappaB kinase (IKK)-NF-kappaB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-kappaB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation.     

Hydrogen sulfide up-regulates substance P in polymicrobial sepsis-associated lung injury

Hydrogen sulfide (H2S) has been shown to induce the activation of neurogenic inflammation especially in normal airways and urinary bladder. However, whether endogenous H2S would regulate sepsis-associated lung inflammation via substance P (SP) and its receptors remains unknown. Therefore, the aim of the study was to investigate the effect of H2S on the pulmonary level of SP in cecal ligation and puncture (CLP)-induced sepsis and its relevance to lung injury. Male Swiss mice or male preprotachykinin-A gene knockout (PPT-A-/-) mice and their wild-type (PPT-A+/+) mice were subjected to CLP-induced sepsis. DL-propargylglycine (50 mg/kg i.p.), an inhibitor of H2S formation was administered either 1 h before or 1 h after the induction of sepsis, while NaHS, an H2S donor, was given at the same time as CLP. L703606, an inhibitor of the neurokinin-1 receptor was given 30 min before CLP. DL-propargylglycine pretreatment or posttreatment significantly decreased the PPT-A gene expression and the production of SP in lung whereas administration of NaHS resulted in a further rise in the pulmonary level of SP in sepsis. PPT-A gene deletion and pretreatment with L703606 prevented H2S from aggravating lung inflammation. In addition, septic mice genetically deficient in PPT-A gene or pretreated with L703606 did not exhibit further increase in lung permeability after injection of NaHS. The present findings show for the first time that in sepsis, H2S up-regulates the generation of SP, which contributes to lung inflammation and lung injury mainly via activation of the neurokinin-1 receptor.     

Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated

Recent studies from our laboratory demonstrated that mucosal lymphoid tissue such as Peyer's patch cells and lamina propria (LP) B lymphocytes from mice shows evidence of increased apoptosis after sepsis that is associated with localized inflammation/activation. The mechanism for this is poorly understood. Endotoxin as well as Fas/Fas ligand (FasL) have been shown to augment lymphocyte apoptosis; however, their contribution to the increase of apoptosis in LP B-cells during sepsis is not known. To study this, sepsis was induced by cecal ligation and puncture (CLP) in endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasL(gld) (FasL(-)) mice and LP lymphocytes were isolated 24 h later. Phenotypic, apoptotic, and functional indexes were assessed. The number of LP B cells decreased markedly in C3H/HeJ mice but not in FasL-deficient animals at 24 h after CLP. This was associated with comparable alteration in apoptosis and Fas antigen expression in the B cells of these mice. Septic LP lymphocytes also showed increased IgA production, which was absent in the FasL-deficient CLP mice. Furthermore, Fas ligand deficiency appeared to improve survival of septic challenge. These data suggest that the increase in B cell apoptosis in septic animals is partially due to a Fas/FasL-mediated process but not endotoxin.     

A3 adenosine receptor activation decreases mortality and renal and hepatic injury in murine septic peritonitis

The role of A3 adenosine receptors (ARs) in sepsis and inflammation is controversial. In this study, we determined the effects of A3AR modulation on mortality and hepatic and renal dysfunction in a murine model of sepsis. To induce sepsis, congenic A3AR knockout mice (A3AR KO) and wild-type control (A3AR WT) mice were subjected to cecal ligation and double puncture (CLP). A3AR KO mice had significantly worse 7-day survival compared with A3AR WT mice. A3AR KO mice also demonstrated significantly higher elevations in plasma creatinine, alanine aminotransferase, aspartate aminotransferase, keratinocyte-derived chemokine, and TNF-alpha 24 h after induction of sepsis compared with A3AR WT mice. Renal cortices from septic A3AR KO mice exhibited increased mRNA encoding proinflammatory cytokines and enhanced nuclear translocation of NF-kB compared with samples from A3AR WT mice. A3AR WT mice treated with N6-(3-iodobenzyl)ADO-5'N-methyluronamide (IB-MECA; a selective A3AR agonist) or 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; a selective A3AR antagonist) had improved or worsened 7-day survival after induction of sepsis, respectively. Moreover, A3AR WT mice treated with IB-MECA or MRS-1191 showed acutely improved or worsened, respectively, renal and hepatic function following CLP. IB-MECA significantly reduced mortality in mice lacking the A1AR or A2aAR but not the A3AR, demonstrating specificity of IB-MECA in activating A3ARs and mediating protection against sepsis-induced mortality. We conclude that endogenous or exogenous A3AR activation confers significant protection from murine septic peritonitis primarily by attenuating the hyperacute inflammatory response in sepsis.     

Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI^-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI^-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI^-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.     

IL-12, but not IL-18, is critical to neutrophil activation and resistance to polymicrobial sepsis induced by cecal ligation and puncture

Sepsis is a systemic inflammatory response resulting from local infection due, at least in part, to impaired neutrophil migration. IL-12 and IL-18 play an important role in neutrophil migration. We have investigated the mechanism and relative role of IL-12 and IL-18 in polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. Wild-type (WT) and IL-18(-/-) mice were resistant to sublethal CLP (SL-CLP) sepsis. In contrast, IL-12(-/-) mice were susceptible to SL-CLP sepsis with high bacteria load in peritoneal cavity and systemic inflammation (serum TNF-alpha and lung neutrophil infiltration). The magnitude of these events was similar to those observed in WT mice with lethal CLP sepsis. The inability of IL-12(-/-) mice to restrict the infection was not due to impairment of neutrophil migration, but correlated with decrease of phagocytosis, NO production, and microbicidal activities of their neutrophils, and with reduction of systemic IFN-gamma synthesis. Consistent with this observation, IFN-gamma(-/-) mice were as susceptible to SL-CLP as IL-12(-/-) mice. Moreover, addition of IFN-gamma to cultures of neutrophils from IL-12(-/-) mice restored their phagocytic, microbicidal activities and NO production. Mortality of IL-12(-/-) mice to SL-CLP was prevented by treatment with IFN-gamma. Thus we show that IL-12, but not IL-18, is critical to an efficient host defense in polymicrobial sepsis. IL-12 acts through induction of IFN-gamma and stimulation of phagocytic and microbicidal activities of neutrophils, rather than neutrophil migration per se. Our data therefore provide further insight into the defense mechanism against this critical area of infectious disease.     

Short-term high fat feeding increases organ injury and mortality after polymicrobial sepsis

The purpose of this study was to examine the effect of short-term high fat feeding on the inflammatory response in polymicrobial sepsis. Male C57BL/6 mice at 6 weeks of age were randomized to a high-fat diet (HFD) (60% kcal fat) or control diet (CD) (16% kcal fat) for 3 weeks. After 3 weeks of feeding, sepsis was induced by cecal ligation and puncture (CLP) and animals were monitored for survival. In a separate experiment, after 3 weeks of feeding mice underwent CLP and were sacrificed at various time points thereafter. Tissue was collected for biochemical studies. Mice fed a HFD gained more weight and had a greater fat mass compared to CD-fed mice. Mice on a HFD had a lower probability of survival and more severe lung injury compared with CD-fed mice following sepsis. Myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, was increased in the lung and liver after CLP in HFD-fed mice compared with CD (P < 0.05). The plasma cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were increased in both groups after CLP, however, TNF-α and IL-6 levels were lower in HFD mice at 3 h after CLP compared with CD and consistent with lung, but not liver, messenger RNA (mRNA) expression. Leptin levels were higher in HFD-fed mice at 18 h after sepsis compared to baseline levels (P < 0.05). Polymicrobial sepsis increased hepatic nuclear factor-κB (NF-κB) activation in HFD-fed mice after CLP vs. CD-fed mice. Short duration high fat feeding increases mortality and organ injury following polymicrobial sepsis. These effects correspond to changes in NF-κB.     

Negative impact of tissue inhibitor of metalloproteinase-3 null mutation on lung structure and function in response to sepsis

Matrix metalloproteinases (MMPs) are degradative enzymes, which act to remodel tissue. Their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). An imbalance in the degradation/inhibition activities has been associated with many diseases, including sepsis. We have previously shown that TIMP-3 knockout animals develop spontaneous, progressive air space enlargement. The objectives of this study were to determine the effects of a septic lung stress induced by cecal ligation and perforation (CLP) on lung function, structure, pulmonary surfactant, and inflammation in TIMP-3 null mice. Knockout and wild-type animals were randomized to either sham or CLP surgery, allowed to recover for 6 h, and then euthanized. TIMP-3 null animals exposed to sham surgery had a significant increase in lung compliance when compared with sham wild-type mice. Additionally, the TIMP-3 knockout mice showed a significant increase in compliance following CLP. Rapid compliance changes were accompanied by significantly decreased collagen and fibronectin levels and increased gelatinase (MMP-2 and -9) abundance and activation. Additionally, in situ zymography showed increased airway-associated gelatinase activity in the knockout animals enhanced following CLP. In conclusion, exposing TIMP-3 null animals to sepsis rapidly enhances the phenotypic abnormalities of these mice, due to increased MMP activity induced by CLP.     

Effects of HSP70.1/3 gene knockout on acute respiratory distress syndrome and the inflammatory response following sepsis

Heat shock response has been implicated in attenuating NF-kappaB activation and inflammation following sepsis. Studies utilizing sublethal heat stress or chemical enhancers to induce in vivo HSP70 expression have demonstrated survival benefit after experimental sepsis. However, it is likely these methods of manipulating HSP70 expression have effects on other stress proteins. The aim of this study was to evaluate the role of specific deletion of HSP70.1/3 gene expression on ARDS, NF-kappaB activation, inflammatory cytokine expression, and survival following sepsis. To address this question, we induced sepsis in HSP70.1/3 KO and HSP70.1/3 WT mice via cecal ligation and puncture (CLP). We evaluated lung tissue NF-kappaB activation and TNF-alpha protein expression at 1 and 2 h, IL-6 protein expression at 1, 2, and 6, and lung histopathology 24 h after sepsis initiation. Survival was assessed for 5 days post-CLP. NF-kappaB activation in lung tissue was increased in HSP70.1/3((-/-)) mice at all time points after sepsis initiation. Deletion of HSP70.1/3 prolonged NF-kappaB binding/activation in lung tissue. Peak expression of lung TNF-alpha at 1 and 2 h was also significantly increased in HSP70.1/3((-/-)) mice. Expression of IL-6 was significantly increased at 2 and 6 h, and histopathology revealed a significant increase in lung injury in HSP70.1/3((-/-)) mice. Last, deletion of the HSP70 gene led to increased mortality 5 days after sepsis initiation. These data reveal that absence of HSP70 alone can significantly increase ARDS, activation of NF-kappaB, and inflammatory cytokine response. The specific absence of HSP70 gene expression also leads to increased mortality after septic insult.     

Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.     

Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression

IL-6 is known to be an important pro- and anti-inflammatory cytokine, which is up-regulated during sepsis. Our previous work has suggested a role for IL-6 in the up-regulation of C5aR in sepsis. We reported earlier that interception of C5a or C5aR results in improved outcomes in experimental sepsis. Using the cecal ligation/puncture (CLP) model in mice, we now demonstrate that treatment with anti-IL-6 Ab (anti-IL-6) results in significantly improved survival, dependent on the amount of Ab infused. CLP animals showed significantly increased binding of 125I-labeled anti-C5aR to organs when compared to either control mice at 0 h or CLP animals infused with normal rabbit 125I-labeled IgG. Binding of 125I-labeled anti-C5aR to lung, liver, kidney, and heart was significantly decreased in anti-IL-6-treated animals 6 h after CLP. RT-PCR experiments with mRNA isolated from various organs obtained 3, 6, and 12 h after CLP demonstrated increased C5aR mRNA expression during the onset of sepsis, which was greatly suppressed in CLP mice treated with anti-IL-6. These data suggest that IL-6 plays an important role in the increased expression of C5aR in lung, liver, kidney, and heart during the development of sepsis in mice and that interception of IL-6 leads to reduced expression of C5aR and improved survival.     

N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation

Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.