Leukotriene Pathway Activation Associates with Poor Glycemic Control and with Cardiovascular Autonomic Neuropathy in Type 1 Diabetes.

BACKGROUND AND AIMS: Since hyperglycemia promotes inflammation by different pathways and inflammation participates in the development of chronic diabetes complications, we investigated the association between the leukotriene (LT) pathway and microvascular diabetes complications. METHODS AND RESULTS: Quantitative polymerase chain reaction was employed to quantify the expression of ALOX5 (encodes 5-lipoxygenase), LTB4R (encodes one of the LTB4 receptors), and MYD88 in peripheral blood mononuclear cells from 164 type 1 diabetes (T1D) individuals presenting or not diabetes kidney disease, retinopathy, peripheral neuropathy, and cardiovascular autonomic neuropathy (CAN); 26 nondiabetic subjects were included as controls. LTB4 plasmatic concentrations were also evaluated. The expression of LTB4R was significantly higher in T1D individuals than in controls. T1D individuals with microvascular complications presented lower MYD88 mRNA expression when compared to those without microvascular complications. Higher LTB4 concentrations were found in individuals with CAN versus without CAN. The observation of two distinct subgroups of T1D individuals in the correlation analyses motivated us to evaluate the characteristics of each one of these groups separately. The group presenting higher expression of ALOX5 and of LTB4R also presented higher values of HbA1C, of fructosamine, and of plasmatic LTB4. CONCLUSION: In the diabetes setting, the LT pathway is not only activated by hyperglycemia but is also modulated by the status of the autonomic nervous system.

Site-Specific Reprogramming of Macrophage Responsiveness to Bacterial Lipopolysaccharide in Obesity.

The mechanisms by which obesity may alter immune responses to pathogens are poorly understood. The present study assessed whether the intrinsic responsiveness of resident macrophages to bacterial lipopolysaccharide (LPS) is reprogrammed in high-fat diet (HFD)-induced obesity. Macrophages from adipose tissue, lung alveoli, and the peritoneal cavity were extracted from obese rats on a HFD or from their lean counterparts, and subsequently studied in culture under identical conditions. CD45+/CD68+ cells (macrophages) were abundant in all cultures, and became the main producers of TNF-α upon LPS stimulation. But although all macrophage subpopulations responded to LPS with an M1-like profile of cytokine secretion, the TNF-α/IL-10 ratio was the lowest in adipose tissue macrophages, the highest in alveolar macrophages, and intermediary in peritoneal macrophages. What is more, diet exerted qualitatively distinct effects on the cytokine responses to LPS, with obesity switching adipose tissue macrophages to a more pro-inflammatory program and peritoneal macrophages to a less pro-inflammatory program, while not affecting alveolar macrophages. Such reprogramming was not associated with changes in the inflammasome-dependent secretion of IL-1ß. The study further shows that the effects of diet on TNF-α/IL-10 ratios were linked to distinct patterns of NF-κB accumulation in the nucleus: while RelA was the NF-κB subunit most impacted by obesity in adipose tissue macrophages, cRel was the subunit affected in peritoneal macrophages. It is concluded that obesity causes dissimilar, site-specific changes in the responsiveness of resident macrophages to bacterial LPS. Such plasticity opens new avenues of investigation into the mechanisms linking obesity to pathogen-induced immune responses.

Leukotriene-B4 modulates macrophage metabolism and fat loss in type 1 diabetic mice.

Serum levels of leukotriene-B4 (LTB4) are increased in type 1 diabetes (T1D) and it mediates systemic inflammation and macrophage reprogramming associated with this condition. Herein, we investigated the involvement of LTB4 in adiposity loss, hyperlipidemia, and changes in macrophage metabolism in a mouse model of streptozotocin-induced T1D. LTB4 receptor (BLT1) antagonist u75302 was employed to block LTB4 effects. As expected, hypoinsulinemia in T1D was associated with hyperglycemia, low levels of glucagon, hyperlipidemia, significant body fat loss, and increased white adipose tissue expression of Fgf21, a marker for lipolysis. With the exception of hyperglycemia and hypoglucagonemia, blockade of LTB4 signaling reverted these parameters in T1D mice. Along with hyperlipidemia, macrophages from T1D mice exhibited higher lipid uptake and accumulation. These cells also had enhanced glycolysis and oxidative metabolism and these parameters were dependent on the mitochondrial uncoupling respiration, as evidenced by elevated expression of oxidation markers carnitine palmitoyltransferase and uncoupling protein 1. Interestingly, all these parameters were at least partially reverted in T1D mice treated with u75302. Altogether, these findings suggest that in T1D mice LTB4/BLT1 is involved in the fat loss, hyperlipidemia, and increased macrophage lipid uptake and metabolism with an important involvement of mitochondrial uncoupling activity. These previously unrecognized LTB4/BLT1 functions may be explored in future to therapeutically alleviate severity of hyperlipidemia and systemic inflammation in T1D.

Aedes aegypti saliva impairs M1-associated proinflammatory phenotype without promoting or affecting M2 polarization of murine macrophages.

BACKGROUND: During the feeding process, the mouthparts of hematophagous mosquitoes break the skin barrier and probe the host tissue to find the blood. The saliva inoculated in this microenvironment modulates host hemostasis, inflammation and adaptive immune responses. However, the mechanisms involved in these biological activities remain poorly understood and few studies explored the potential roles of mosquito saliva on the individual cellular components of the immune system. Here, we report the immunomodulatory activities of Aedes aegypti salivary cocktail on murine peritoneal macrophages. RESULTS: The salivary gland extract (SGE) of Ae. aegypti inhibited the production of nitric oxide and inflammatory cytokines such as interleukin-6 (IL-6) and IL-12, as well as the expression of inducible nitric oxide synthase and NF-κB by murine macrophages stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). The spare respiratory capacity, the phagocytic and microbicidal activities of these macrophages were also reduced by Ae. aegypti SGE. These phenotypic changes are consistent with SGE suppressing the proinflammatory program of M1 macrophages. On the other hand, Ae. aegypti SGE did not influence M2-associated markers (urea production, arginase-1 and mannose receptor-1 expression), either in macrophages alternatively activated by IL-4 or in those classically activated by LPS plus IFN-γ. In addition, Ae. aegypti SGE did not display any cytokine-binding activity, nor did it affect macrophage viability, thus excluding supposed experimental artifacts. CONCLUSIONS: Given the importance of macrophages in a number of biological processes, our findings help to enlighten how vector saliva modulates vertebrate host immunity.

Leukotriene Involvement in the Insulin Receptor Pathway and Macrophage Profiles in Muscles from Type 1 Diabetic Mice.

Type 1 diabetes (T1D) is a metabolic disease associated with systemic low-grade inflammation and macrophage reprogramming. There is evidence that this inflammation depends on the increased systemic levels of leukotriene (LT) B4 found in T1D mice, which shifts macrophages towards the proinflammatory (M1) phenotype. Although T1D can be corrected by insulin administration, over time T1D patients can develop insulin resistance that hinders glycemic control. Here, we sought to investigate the role of leukotrienes (LTs) in a metabolically active tissue such as muscle, focusing on the insulin signaling pathway and muscle-associated macrophage profiles. Type 1 diabetes was induced in the 129/SvE mouse strain by streptozotocin (STZ) in mice deficient in the enzyme responsible for LT synthesis (5LO-/-) and the LT-sufficient wild type (WT). The response to insulin was evaluated by the insulin tolerance test (ITT), insulin concentration by ELISA, and Akt phosphorylation by western blotting. The gene expression levels of the insulin receptor and macrophage markers Stat1, MCP-1, Ym1, Arg1, and IL-6 were evaluated by qPCR, and that of IL-10 by ELISA. We observed that after administration of a single dose of insulin to diabetic mice, the reduction in glycemia was more pronounced in 5LO-/- than in WT mice. When muscle homogenates were analyzed, diabetic 5LO-/- mice showed a higher expression of the insulin receptor gene and higher Akt phosphorylation. Moreover, in muscle homogenates from diabetic 5LO-/- mice, the expression of anti-inflammatory macrophage markers Ym1, Arg1, and IL-10 was increased, and the relative expression of the proinflammatory cytokine IL-6 was reduced compared with WT diabetic mice. These results suggest that LTs have an impact on the insulin receptor signaling pathway and modulate the inflammatory profile of muscle-resident macrophages from T1D mice.

Leukotriene B4 modulation of murine dendritic cells affects adaptive immunity.

Dendritic cells (DCs) link innate and adaptive immunity. The microenvironment generated during the innate immunity affects DCs and the type of adaptive immunity generated. Lipid mediators are released early in inflammation and could modify the functional state of DCs. Leukotriene B4 (LTB4) has a wide range of effects on macrophages and in the present study we investigated if it also affects DCs. Murine bone marrow-derived DCs were employed and it was found that stimulation of DCs with LTB4 (10 nM) increased the gene expression of the high affinity receptor BLT-1 but not of BLT-2. It also increased the co-stimulatory molecule CD86 expression but did not affect CD80 and CD40. LTB4-stimulated DCs acquired the capacity to present antigen to T lymphocytes, evidenced by antigen-specific proliferation of CD4+ lymphocytes in co-cultures of ovalbumin-loaded DCs with DO11.10 splenocytes. LTB4-stimulated DCs induced Treg proliferation and increased Th2 cytokine IL-13 in the co-cultures. Expression of transcription factor genes, Gata3 and Foxp3 (Th2 and Treg, respectively) were also found increased. However, the expression of Th1 transcription factor (Tbet) and Th17 (RorγT) were not affected. These results indicate that LTB4 affects DCs and modulates the type of adaptive immune response.

Impaired wound healing in type 1 diabetes is dependent on 5-lipoxygenase products.

Type 1 diabetes is associated with systemic low grade inflammation (LGI). We have previously shown that LGI in diabetic mice depends on systemic circulation of leukotriene (LTB4) which potentiates the toll-like/IL1ß receptors response in macrophages. Impaired wound healing is an important co-morbidity in diabetes, and macrophages play a key role in this process. Here, we investigated the role of leukotrienes on monocytes and macrophages phenotype and in the impaired wound healing in diabetic mice. Type 1 diabetes was induced with streptozotocin in 129SvE wild-type (WT) and leukotrienes-deficient 5LO-/- (5-lipoxygenase knockout) mice. In diabetics, the systemic levels of LTB4, TNF-α, IL-6, IL-10, IL-12 and IFNγ were increased as well as the frequency of pro-inflammatory monocytes (CD11b+Ly6ChighLy6G-) compared to healthy mice. In diabetic 5LO-/- mice, these parameters were similar to those in healthy mice. Resident peritoneal macrophages from diabetic WT mice showed a classically activated M1-like phenotype (high Nos2, Stat and Il12 expression, and nitrite levels). Macrophages from diabetic 5LO-/- mice presented alternatively activated M2-macrophages markers (high Arg1 and Chi3l3 expression and arginase activity) and when stimulated with IL4, enhanced phosphorylated-STAT6. Cutaneous wound healing in diabetic WT mice was impaired, which correlated with the decreased frequency of M2-macrophages (CD45+F4/80+CD206+) in the lesions. In diabetic 5LO-/- mice, the frequency of M2-macrophages in the wound was similar to that in healthy mice, suggesting that the impaired healing of diabetic mice depends on 5LO products. The inhibition of leukotrienes or antagonism of its receptors could be a therapeutic alternative for diabetic patients with impaired healing.

Excessive localized leukotriene B4 levels dictate poor skin host defense in diabetic mice.

Poorly controlled diabetes leads to comorbidities and enhanced susceptibility to infections. While the immune components involved in wound healing in diabetes have been studied, the components involved in susceptibility to skin infections remain unclear. Here, we examined the effects of the inflammatory lipid mediator leukotriene B4 (LTB4) signaling through its receptor B leukotriene receptor 1 (BLT1) in the progression of methicillin-resistant Staphylococcus aureus (MRSA) skin infection in 2 models of diabetes. Diabetic mice produced higher levels of LTB4 in the skin, which correlated with larger nonhealing lesion areas and increased bacterial loads compared with nondiabetic mice. High LTB4 levels were also associated with dysregulated cytokine and chemokine production, excessive neutrophil migration but impaired abscess formation, and uncontrolled collagen deposition. Both genetic deletion and topical pharmacological BLT1 antagonism restored inflammatory response and abscess formation, followed by a reduction in the bacterial load and lesion area in the diabetic mice. Macrophage depletion in diabetic mice limited LTB4 production and improved abscess architecture and skin host defense. These data demonstrate that exaggerated LTB4/BLT1 responses mediate a derailed inflammatory milieu that underlies poor host defense in diabetes. Prevention of LTB4 production/actions could provide a new therapeutic strategy to restore host defense in diabetes.

Nuclear PTEN enhances the maturation of a microRNA regulon to limit MyD88-dependent susceptibility to sepsis.

Sepsis-induced organ damage is caused by systemic inflammatory response syndrome (SIRS), which results in substantial comorbidities. Therefore, it is of medical importance to identify molecular brakes that can be exploited to dampen inflammation and prevent the development of SIRS. We investigated the role of phosphatase and tensin homolog (PTEN) in suppressing SIRS, increasing microbial clearance, and preventing lung damage. Septic patients and mice with sepsis exhibited increased PTEN expression in leukocytes. Myeloid-specific Pten deletion in an animal model of sepsis increased bacterial loads and cytokine production, which depended on enhanced myeloid differentiation primary response gene 88 (MyD88) abundance and resulted in mortality. PTEN-mediated induction of the microRNAs (miRNAs) miR125b and miR203b reduced the abundance of MyD88. Loss- and gain-of-function assays demonstrated that PTEN induced miRNA production by associating with and facilitating the nuclear localization of Drosha-Dgcr8, part of the miRNA-processing complex. Reconstitution of PTEN-deficient mouse embryonic fibroblasts with a mutant form of PTEN that does not localize to the nucleus resulted in retention of Drosha-Dgcr8 in the cytoplasm and impaired production of mature miRNAs. Thus, we identified a regulatory pathway involving nuclear PTEN-mediated miRNA generation that limits the production of MyD88 and thereby limits sepsis-associated mortality.

Adipose tissue inflammation in insulin resistance: review of mechanisms mediating anti-inflammatory effects of omega-3 polyunsaturated fatty acids.

Obesity is an increasingly costly and widespread epidemic, effecting 1 in 10 adults worldwide. It has been causally linked with both the metabolic syndrome and insulin resistance, both of which are associated with increased chronic inflammation. The exact mechanisms through which inflammation may contribute to both MetS and IR are numerous and their details are still largely unknown. Recently, micro-RNAs (miRNAs) have emerged as potential interventional targets due to their potential preventive roles in the pathogenesis of several diseases, including MetS and obesity. The purpose of this review paper is to discuss some of the known roles of miRNAs as mediators of inflammation-associated obesity and IR and how omega-3 polyunsaturated fatty acids may be used as a nutritional intervention for these disorders.

Imbalance between HDAC and HAT activities drives aberrant STAT1/MyD88 expression in macrophages from type 1 diabetic mice.

AIMS: To investigate the hypothesis that alteration in histone acetylation/deacetylation triggers aberrant STAT1/MyD88 expression in macrophages from diabetics. Increased STAT1/MyD88 expression is correlated with sterile inflammation in type 1 diabetic (T1D) mice. METHODS: To induce diabetes, we injected low-doses of streptozotocin in C57BL/6 mice. Peritoneal or bone marrow-differentiated macrophages were cultured in 5mM (low) or 25mM (high glucose). ChIP analysis of macrophages from nondiabetic or diabetic mice was performed to determine acetylation of lysine 9 in histone H3 in MyD88 and STAT1 promoter regions. Macrophages from diabetic mice were treated with the histone acetyltransferase inhibitor anacardic acid (AA), followed by determination of mRNA expression by qPCR. RESULTS: Increased STAT1 and MyD88 expression in macrophages from diabetic but not naive mice cultured in low glucose persisted for up to 6days. Macrophages from diabetic mice exhibited increased activity of histone acetyltransferases (HAT) and decreased histone deacetylases (HDAC) activity. We detected increased H3K9Ac binding to Stat1/Myd88 promoters in macrophages from T1D mice and AA in vitro treatment reduced STAT1 and MyD88 mRNA expression. CONCLUSIONS/INTERPRETATION: These results indicate that histone acetylation drives elevated Stat1/Myd88 expression in macrophages from diabetic mice, and this mechanism may be involved in sterile inflammation and diabetes comorbidities.

PAFR activation of NF-κB p65 or p105 precursor dictates pro- and anti-inflammatory responses during TLR activation in murine macrophages.

Platelet-activating factor receptor (PAFR) is a G protein-coupled receptor (GPCR) implicated in many diseases. Toll-like receptors (TLRs) play a critical role in shaping innate and adaptive immune responses. In this study, we investigated whether PAFR signaling changes the macrophages responsiveness to agonists of TLR2 (Pam3Cys), TLR4 (LPS), and TLR3 agonist Poly(I:C). Exogenous PAF inhibited the production of pro-inflammatory cytokines (IL-12p40, IL-6, and TNF-α) and increased anti-inflammatory IL-10 in macrophages challenged with Pam3Cys and LPS, but not with Poly (I:C). PAF did not affect mRNA expression of MyD88, suggesting that PAF acts downstream the adaptor. PAF inhibited LPS-induced phosphorylation of NF-κB p65 and increased NF-κB p105 phosphorylation, which is processed in the proteasome to generate p50 subunit. The PAF potentiation of IL-10 production was dependent on proteasome processing but independent of NF-κB transactivation domain. Inhibition of p50 abolished the PAF-induced IL-10 production. These findings indicate that the impaired transcriptional activity of the p65 subunit and the enhanced p105 phosphorylation induced by PAF are responsible for down regulation of pro-inflammatory cytokines and up regulation of IL-10, respectively, in LPS-challenged macrophages. Together, our data unveil a heretofore unrecognized role for PAFR in modulating activation of NF-κB in macrophages.

Gamma-Terpinene Modulation of LPS-Stimulated Macrophages is Dependent on the PGE2/IL-10 Axis.

Gamma-terpinene is a monoterpene present in the essential oils of several plants, including those from the Eucalyptus genus. This molecule was recently described as anti-inflammatory and microbiocidal, but little is known about the mechanisms behind its effects. The aim of the present study was to investigate the effect of gamma-terpinene on the lipopolysaccharide-induced production of cytokines by murine peritoneal macrophages. Gamma-terpinene treatment was found to reduce the production of proinflammatory cytokines, such as interleukin-1ß and interleukin-6, and enhance that of the anti-inflammatory cytokine interleukin-10. This was accompanied by increased levels of the enzyme cycloxygenase-2 and its product, the lipid mediator prostaglandin E2. Inhibition of cycloxygenase-2 with nimesulide abolished the potentiating effect of gamma-terpinene on interleukin-10 production. Moreover, nimesulide treatment also abrogated the inhibitory effect of gamma-terpinene on interleukin-1ß and interleukin-6. Furthermore, in macrophages from mice deficient in the interleukin-10 gene, gamma-terpinene failed to inhibit interleukin-1ß and interleukin-6 production. These results suggest that this monoterpene promotes the prostaglandin E2/interleukin-10 axis, which inhibits the production of these proinflammatory cytokines.

PAFR in adipose tissue macrophages is associated with anti-inflammatory phenotype and metabolic homoeostasis.

Metabolic dysfunction is associated with adipose tissue inflammation and macrophage infiltration. PAFR (platelet-activating factor receptor) is expressed in several cell types and binds to PAF (platelet-activating factor) and oxidized phospholipids. Engagement of PAFR in macrophages drives them towards the anti-inflammatory phenotype. In the present study, we investigated whether genetic deficiency of PAFR affects the phenotype of ATMs (adipose tissue macrophages) and its effect on glucose and insulin metabolism. PARFKO (PAFR-knockout) and WT (wild-type) mice were fed on an SD (standard diet) or an HFD (high-fat diet). Glucose and insulin tolerance tests were performed by blood monitoring. ATMs were evaluated by FACS for phenotypic markers. Gene and protein expression was investigated by real-time reverse transcription-quantitative PCR and Western blotting respectively. Results showed that the epididymal adipose tissue of PAFRKO mice had increased gene expression of Ccr7, Nos2, Il6 and Il12, associated with pro-inflammatory mediators, and reduced expression of the anti-inflammatory Il10. Moreover, the adipose tissue of PAFRKO mice presented more pro-inflammatory macrophages, characterized by an increased frequency of F4/80(+)CD11c(+) cells. Blood monocytes of PAFRKO mice also exhibited a pro-inflammatory phenotype (increased frequency of Ly6C(+) cells) and PAFR ligands were detected in the serum of both PAFRKO and WT mice. Regarding metabolic parameters, compared with WT, PAFRKO mice had: (i) higher weight gain and serum glucose concentration levels; (ii) decreased insulin-stimulated glucose disappearance; (iii) insulin resistance in the liver; (iv) increased expression of Ldlr in the liver. In mice fed on an HFD, some of these changes were potentiated, particularly in the liver. Thus it seems that endogenous ligands of PAFR are responsible for maintaining the anti-inflammatory profile of blood monocytes and ATMs under physiological conditions. In the absence of PAFR signalling, monocytes and macrophages acquire a pro-inflammatory phenotype, resulting in adipose tissue inflammation and metabolic dysfunction.

Leukotriene B4-mediated sterile inflammation promotes susceptibility to sepsis in a mouse model of type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is associated with chronic systemic inflammation and enhanced susceptibility to systemic bacterial infection (sepsis). We hypothesized that low insulin concentrations in T1DM trigger the enzyme 5-lipoxygenase (5-LO) to produce the lipid mediator leukotriene B4 (LTB4), which triggers systemic inflammation that may increase susceptibility to polymicrobial sepsis. Consistent with chronic inflammation, peritoneal macrophages from two mouse models of T1DM had greater abundance of the adaptor MyD88 (myeloid differentiation factor 88) and its direct transcriptional effector STAT-1 (signal transducer and activator of transcription 1) than macrophages from nondiabetic mice. Expression of Alox5, which encodes 5-LO, and the concentration of the proinflammatory cytokine interleukin-1ß (IL-1ß) were also increased in peritoneal macrophages and serum from T1DM mice. Insulin treatment reduced LTB4 concentrations in the circulation and Myd88 and Stat1 expression in the macrophages from T1DM mice. T1DM mice treated with a 5-LO inhibitor had reduced Myd88 mRNA in macrophages and increased abundance of IL-1 receptor antagonist and reduced production of IL-ß in the circulation. T1DM mice lacking 5-LO or the receptor for LTB4 also produced less proinflammatory cytokines. Compared to wild-type or untreated diabetic mice, T1DM mice lacking the receptor for LTB4 or treated with a 5-LO inhibitor survived polymicrobial sepsis, had reduced production of proinflammatory cytokines, and had decreased bacterial counts. These results uncover a role for LTB4 in promoting sterile inflammation in diabetes and the enhanced susceptibility to sepsis in T1DM.

Sepsis-induced lung inflammation is modulated by insulin.

BACKGROUND: We have previously shown that diabetic rats are more susceptible to sepsis, but that the Acute lung injury (ALI) secondary to sepsis is less intense than in non-diabetics. In the present study, we further investigated the ALI-secondary to sepsis in diabetic rats and the effect of insulin treatment. METHODS: Diabetes was induced in male Wistar rats by alloxan and sepsis by cecal ligation and puncture surgery (CLP). Some diabetic rats were given neutral protamine Hagedorn (NPH) insulin (4 IU, s.c.) 2 h before CLP. Six h later, the lungs were examined for edema, cell infiltration and prostaglandin-E2 (PGE2) levels in the bronchoalveolar lavage (BAL). RESULTS: The results confirmed that leukocyte infiltration and edema were milder in diabetic rats with sepsis. After insulin treatment, the lung inflammation in diabetics increased to levels comparable to the non-diabetics. The BAL concentration of PGE2 was also lower in diabetics with sepsis, and increased after insulin treatment. Sepsis was followed by early fibroblast activation in the lung parenchyma, evaluated by increased transforming growth factor (TGF)-ß and smooth muscle actin (α-SMA) expression, as well as an elevated number of cells with myofibroblasts morphology. These events were significantly lower in diabetic rats and increased after insulin treatment. CONCLUSION: The results show that insulin modulates the early phase of inflammation and myofibroblast differentiation in diabetic rats.

PPAR-γ/IL-10 axis inhibits MyD88 expression and ameliorates murine polymicrobial sepsis.

Polymicrobial sepsis induces organ failure and is accompanied by overwhelming inflammatory response and impairment of microbial killing. Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear receptor with pleiotropic effects on lipid metabolism, inflammation, and cell proliferation. The insulin-sensitizing drugs thiazolidinediones (TZDs) are specific PPAR-γ agonists. TZDs exert anti-inflammatory actions in different disease models, including polymicrobial sepsis. The TZD pioglitazone, which has been approved by the U.S. Food and Drug Administration, improves sepsis outcome; however, the molecular programs that mediate its effect have not been determined. In a murine model of sepsis, we now show that pioglitazone treatment improves microbial clearance and enhances neutrophil recruitment to the site of infection. We also observed reduced proinflammatory cytokine production and high IL-10 levels in pioglitazone-treated mice. These effects were associated with a decrease in STAT-1-dependent expression of MyD88 in vivo and in vitro. IL-10R blockage abolished PPAR-γ-mediated inhibition of MyD88 expression. These data demonstrate that the primary mechanism by which pioglitazone protects against polymicrobial sepsis is through the impairment of MyD88 responses. This appears to represent a novel regulatory program. In this regard, pioglitazone provides advantages as a therapeutic tool, because it improves different aspects of host defense during sepsis, ultimately enhancing survival.

Leukotriene B4 enhances the generation of proinflammatory microRNAs to promote MyD88-dependent macrophage activation.

MicroRNAs are known to control TLR activation in phagocytes. We have shown that leukotriene (LT) B4 (LTB4) positively regulates macrophage MyD88 expression by decreasing suppressor of cytokine signaling-1 (SOCS-1) mRNA stability. In this study, we investigated the possibility that LTB4 control of MyD88 expression involves the generation of microRNAs. Our data show that LTB4, via its receptor B leukotriene receptor 1 (BLT1) and Gαi signaling, increased macrophage expression of inflammatory microRNAs, including miR-155, miR-146b, and miR-125b. LTB4-mediated miR-155 generation was attributable to activating protein-1 activation. Furthermore, macrophage transfection with antagomirs against miR-155 and miR-146b prevented both the LTB4-mediated decrease in SOCS-1 and increase in MyD88. Transfection with miR-155 and miR-146b mimics decreased SOCS-1 levels, increased MyD88 expression, and restored TLR4 responsiveness in both wild type and LT-deficient macrophages. To our knowledge, our data unveil a heretofore unrecognized role for the GPCR BLT1 in controlling expression of microRNAs that regulate MyD88-dependent activation of macrophages.

Sepsis-induced acute lung injury (ALI) is milder in diabetic rats and correlates with impaired NFkB activation.

Acute lung injury (ALI) develops in response to a direct insult to the lung or secondarily to a systemic inflammatory response, such as sepsis. There is clinical evidence that the incidence and severity of ALI induced by direct insult are lower in diabetics. In the present study we investigated whether the same occurs in ALI secondarily to sepsis and the molecular mechanisms involved. Diabetes was induced in male Wistar rats by alloxan and sepsis by caecal ligation and puncture surgery (CLP). Six hours later, the lungs were examined for oedema and cell infiltration in bronchoalveolar lavage. Alveolar macrophages (AMs) were cultured in vitro for analysis of IκB and p65 subunit of NFκB phosphorylation and MyD88 and SOCS-1 mRNA. Diabetic rats were more susceptible to sepsis than non-diabetics. In non-diabetic rats, the lung presented oedema, leukocyte infiltration and increased COX2 expression. In diabetic rats these inflammatory events were significantly less intense. To understand why diabetic rats despite being more susceptible to sepsis develop milder ALI, we examined the NFκB activation in AMs of animals with sepsis. Whereas in non-diabetic rats the phosphorylation of IκB and p65 subunit occurred after 6 h of sepsis induction, this did not occur in diabetics. Moreover, in AMs from diabetic rats the expression of MyD88 mRNA was lower and that of SOCS-1 mRNA was increased compared with AMs from non-diabetic rats. These results show that ALI secondary to sepsis is milder in diabetic rats and this correlates with impaired activation of NFκB, increased SOCS-1 and decreased MyD88 mRNA.