Publications

Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells

No abstract is provided for this article.

TRIF is essential for the clearance of Pseudomonas aeruginosa from the mouse lung

Toll-IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF) and MyD88 are adaptor molecules that mediate two distinct Toll-like receptor (TLR) signaling pathways. The MyD88 pathway is critical for the defense against a number of bacteria. Using a MyD88-deficient animal model, we showed an important role of the MyD88 pathway in the host defense against Pseudomonas aeruginosa lung infection. Roles of TRIF in the host defense have largely been associated with virus infections. Here we investigated a role of TRIF in P. aeruginosa lung infection. TRIF deficient mice showed severe impairment in the clearance of P. aeruginosa from the lung when compared with wild type mice. A TRIF deficiency impairs production of a selective profile of cytokines and chemokines following P. aeruginosa lung infection. Interestingly, TRIF deficient and TRIF-MyD88 double deficient mice showed similar levels of impairment in the clearance of P. aeruginosa from the lung and cytokine production, suggesting a major role of TRIF in P. aeruginosa lung infection. This study demonstrates for the first time that TRIF pathway is involved in the host defense against P. aeruginosa lung infection. Thus, the full development of host responses to P. aeruginosa lung infection requires both TRIF- and MyD88-dependent mechanisms. Research funded by grants from CIHR, CCFF amd the IWK Health Centre

[Control of inflammatory responses by a novel RNase, Zc3h12a].

Innate immune recognition of nucleic acids: Beyond toll-like receptors

During infection or tissue damage, the innate immune system detects and responds to nucleic acids released from pathogens or damaged host cells. Accumulating evidence has showed that specific sequences, modifications or structures of nucleic acids influence their immunomodulatory activities. Resulting innate immune modulations are regulated by Toll-like receptor (TLR)-dependent or -independent signaling pathways. The first step in host defense against foreign or unwelcome self nucleic acids may play important roles in immune responses against infectious organisms, as well as in clearance of unnecessary tissues, which may be linked to autoimmune diseases and possibly to other immunological disorders. Elucidating mechanisms of innate immune activation by nucleic acids will help future development of more efficient or safer nucleic acid-based immunotherapies and gene therapies. © 2005 Wiley-Liss, Inc.

Mycobacterial Infection in TLR2 and TLR6 Knockout Mice

To investigate the role of TLR in the development of murine tuberculosis in vivo , TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild‐type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF‐α, TGF‐β, IL‐1β, and IL‐2 were significantly lower, but levels of the mRNAs for IL‐4 and IL‐6 were higher, than in wild‐type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF‐κB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.

Cutting Edge: TLR2-Deficient and MyD88-Deficient Mice Are Highly Susceptible to<i>Staphylococcus aureus</i>Infection

Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns. We previously showed that TLR2 recognizes Gram-positive bacterial components whereas TLR4 recognizes LPS, a component of Gram-negative bacteria. MyD88 is shown to be an adaptor molecule essential for TLR family signaling. To investigate the role of TLR family in host defense against Gram-positive bacteria, we infected TLR2- and MyD88-deficient mice with Staphylococcus aureus. Both TLR2- and MyD88-deficient mice were highly susceptible to S. aureus infection, with more enhanced susceptibility in MyD88-deficient mice. Peritoneal macrophages from MyD88-deficient mice did not produce any detectable levels of cytokines in response to S. aureus. In contrast, TLR2-deficient macrophages produced reduced, but significant, levels of the cytokines, and TLR4-deficient macrophages produced the same amounts as wild-type cells, indicating that S. aureus is recognized not only by TLR2, but also by other TLR family members except for TLR4.

U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7

Abstract Plasmacytoid dendritic cells (PDCs), which produce IFN-α in response to autoimmune complexes containing nuclear antigens, are thought to be critically involved in the pathogenesis of systemic lupus erythematosus (SLE). One of the immunostimulatory components of SLE immune complexes (SLE-ICs) is self DNA, which is recognized through Tlr9 in PDCs and B cells. Small nuclear ribonucleoproteins (snRNPs) are another major component of SLE-ICs in 30% to 40% of patients. In this study, we show that murine PDCs are activated by purified U1snRNP/anti-Sm ICs to produce IFN-α and proinflammatory cytokines and to up-regulate costimulatory molecules. The induction of IFN-α and IL-6 by U1snRNPs in murine bone marrow–derived PDCs required the presence of intact U1RNA and was largely dependent on Tlr7 but independent of Tlr3. Intracellularly delivered isolated U1snRNA and oligoribonucleotides derived from the stem loop regions and the Sm-binding site of U1snRNA efficiently induced IFN-α and IL-6 in Flt3L-cultured DCs in a Tlr7-dependent manner. The U1snRNA component of U1snRNP immune complexes, found in patients with SLE, acts as an endogenous “self” ligand for Tlr7 and triggers IFN-α and IL-6 production in PDCs.

Innate immunity in allergy

Innate immune system quickly responds to invasion of microbes and foreign substances through the extracellular and intracellular sensing receptors, which recognize distinctive molecular and structural patterns. The recognition of innate immune receptors leads to the induction of inflammatory and adaptive immune responses by activating downstream signaling pathways. Allergy is an immune‐related disease and results from a hypersensitive immune response to harmless substances in the environment. However, less is known about the activation of innate immunity during exposure to allergens. New insights into the innate immune system by sensors and their signaling cascades provide us with more important clues and a framework for understanding allergy disorders. In this review, we will focus on recent advances in the innate immune sensing system.

Prevention of Intestinal Allergy in Mice by rflaA:Ova Is Associated with Enforced Antigen Processing and TLR5-Dependent IL-10 Secretion by mDC

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.

Erratum: TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway (The Journal of Biological Chemistry (2008) vol. 283 (36211-36220) 10.1074/jbc.A806576200)

lung injury induced by hypoxia and LPS Adrenomedullin expression in a rat model of acute

No abstract is provided for this article.

ZIP Kinase Triggers Apoptosis from Nuclear PML Oncogenic Domains

PML oncogenic domains (PODs), also referred to as nuclear dot 10 bodies, Kreb's bodies, or nuclear bodies, represent nuclear structures implicated in the regulation of a variety of cellular processes, including transcription, tumor suppression, and apoptosis.ZIP kinase (ZIPK) is a proapoptotic protein kinase with homology to DAP kinase, a protein kinase implicated in apoptosis.We show here that ZIPK is present in PODs, where it colocalizes with and binds to proapoptotic protein Daxx.Arsenic trioxide (As 2 O 3 ) and gamma interferon (IFN-␥), which accentuate POD formation, increased the association of ZIPK with PODs.In contrast, the kinase-inactive ZIPK resides in nuclei with a diffuse pattern and significantly prevents the association of Daxx with PODs, implying that ZIPK recruits Daxx to PODs via its catalytic activity.ZIPK also binds and phosphorylates proapoptotic protein Par-4.Association of ZIPK with Daxx was enhanced by coexpression of Par-4.Activation of caspases and induction of apoptosis were also observed in cells overexpressing these proteins.Conversely, small-interfering RNA-mediated reduction of ZIPK, Daxx, or Par-4 expression decreased activation of caspase and apoptosis induced by As 2 O 3 and IFN-␥.These results suggest that ZIPK, in collaboration with Daxx and Par-4, mediates a novel nuclear pathway for apoptosis.

TAK1 Prevents Endothelial Apoptosis and Maintains Vascular Integrity

TNF-α is a pleiotropic cytokine that has the potential to induce apoptosis under inflammation. How endothelial cells (ECs) are spared from this fate in inflammatory environments where TNF-α is present is not known. Here, we show that TGF-β-activated kinase 1 (TAK1) ensures EC survival and maintains vascular integrity upon TNF-α stimulation. Endothelial-specific TAK1 knockout mice exhibit intestinal and liver hemorrhage due to EC apoptosis, leading to vascular destruction and rapid death. This EC apoptosis was induced by TNF-α from myeloid cells responding to intestinal microbiota. TNF-α secretion associated with inflammation also induced vascular defects in inflamed organs. Additionally, we determined that TAK1 deletion in tumor ECs resulted in blood vessel and hence tumor regression. Our results illuminate mechanisms ensuring survival of intestinal and liver ECs under physiological conditions and ECs of other organs under inflammatory conditions that could be exploited for anti-angiogenic therapy to treat cancer.

Recognition of lipopeptides by Toll-like receptors

Toll-like receptors (TLRs) recognize specific molecular patterns present only in micro-organisms and thereby activate innate immune cells. TLR2 is essential for the recognition of peptidoglycan and lipoprotein/lipopeptides. Lipoprotein/lipopeptides are observed in cell walls of a variety of micro-organisms. Host immune cells recognize the specific patterns of lipoprotein/lipopeptides through the association of TLR2 with other TLRs. TLR1 and TLR6 are highly homologous to TLR2 in structure. TLR6-deficient mice showed an impaired response to mycoplasmal lipopeptides that are diacylated, whereas TLR1-deficient mice were defective in their response to bacterial lipopeptides that are triacylated. TLR2-deficient mice did not show any inflammatory response to either type of lipopeptide. The functional association of TLR2 with TLR1 or TLR6 has been demonstrated. Thus, TLR1 and TLR6 are involved in the discrimination of a subtle difference between triacyl and diacyl lipopeptides through interaction with TLR2.

Signal Transducer and Activator of Transcription-3 Is Critical for Uterine Implantation and Decidualization.

Infertility rates in humans have increased to approximately 10-15% in recent years. A major cause of infertility is implantation failure. Decidualization, mediated by the ovarian steroids estrogen and progesterone, is required to sustain the post-implantation phase. Mitogen inducible gene 6 (Mig-6) is an essential mediator of progesterone receptor (PR) function in the uterus. Signal transducer and activator of transcription-3 (Stat3) has been identified as a MIG-6-associated molecule, and interaction of STAT3 with PR has been detected in decidual lysates. To identify the interaction of STAT3 with PR and MIG-6 and its effect on uterine function, we performed immunoprecipitation using transient co-transfection and mouse uteri treated with estrogen and progesterone. STAT3 interacted with PR-A and MIG-6 but not with PR-B. The interaction of STAT3 with MIG-6 was also identified by co-immunoprecipitation in uterine lysate of control mice but not in PRcre/+Mig-6f/f mice. These results indicate that STAT3, MIG-6 and PR-A form a complex and may regulate each other's function. Stat3 activation during the implantation period has been shown to be important for uterine receptivity and decidualization. However, research on the role of Stat3 has been restricted to transient Stat3 knockdown because Stat3-deficient mice are embryo lethal. Therefore, a Stat3 conditional knockout mouse model is critical for understanding the role of Stat3 in vivo. In order to investigate the role of Stat3 in uterine function, we have conditionally ablated Stat3 in the PR-positive cells (PRcre/+Stat3f/f; Stat3d/d). Female Stat3d/d mice developed normal uterine and ovarian morphology and structure. Ovulation and fertilization rate of Stat3d/d mice did not differ from control Stat3f/f mice. Stat3d/d mice have fertility defects. Analysis of the uterus on day 5.5 of pregnancy showed disrupted blastocyst implantation in Stat3d/d mice. Stat3d/d mice also showed a defect of the uterus to respond to the artificial induction of the decidual response. In conclusion, our study suggests that Stat3 regulates decidualization and implantation which is a requisite for fertility, through interaction of PR and Mig-6 in the mouse uterus. This work was supported by National Institutes of Health Grant R01HD057873.

The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Inhibition of microsomal prostaglandin E synthase-1 facilitates liver repair after hepatic injury in mice

Background & Aims Liver repair following hepatic ischemia/reperfusion (I/R) injury is crucial to survival. This study aims to examine the role of endogenous prostaglandin E2 (PGE2) produced by inducible microsomal PGE synthase-1 (mPGES-1), a terminal enzyme of PGE2 generation, in liver injury and repair following hepatic I/R. Methods mPGES-1 deficient (Ptges −/−) mice or their wild-type (WT) counterparts were subjected to partial hepatic ischemia followed by reperfusion. The role of E prostanoid receptor 4 (EP4) was then studied using a genetic knockout model and a selective antagonist. Results Compared with WT mice, Ptges −/− mice exhibited reductions in alanine aminotransferase (ALT), necrotic area, neutrophil infiltration, chemokines, and proinflammatory cytokine levels. Ptges −/− mice also showed promoted liver repair and increased Ly6Clow macrophages (Ly6Clow/CD11bhigh/F4/80high-cells) with expression of anti-inflammatory and reparative genes, while WT mice exhibited delayed liver repair and increased Ly6Chigh macrophages (Ly6Chigh/CD11bhigh/F4/80low-cells) with expression of proinflammatory genes. Bone marrow (BM)-derived mPGES-1-deficient macrophages facilitated liver repair with increases in Ly6Clow macrophages. In vitro, mPGES-1 was expressed in macrophages polarized toward the proinflammatory profile. Mice treated with the mPGES-1 inhibitor Compound III displayed increased liver protection and repair. Hepatic I/R enhanced the hepatic expression of PGE receptor subtype, EP4, in WT mice, which was reduced in Ptges −/− mice. A selective EP4 antagonist and genetic deletion of Ptger4, which codes for EP4, accelerated liver repair. The proinflammatory gene expression was upregulated by stimulation of EP4 agonist in WT macrophages but not in EP4-deficient macrophages. Conclusions These results indicate that mPGES-1 regulates macrophage polarization as well as liver protection and repair through EP4 signaling during hepatic I/R. Inhibition of mPGES-1 could have therapeutic potential by promoting liver repair after acute liver injury. Lay summary Hepatic ischemia/reperfusion injury is a serious complication that occurs in liver surgery. Herein, we demonstrated that inducible prostaglandin E2 synthase (mPGES-1), an enzyme involved in synthesizing prostaglandin E2, worsens the injury and delays liver repair through accumulation of proinflammatory macrophages. Inhibition of mPGES-1 offers a potential therapy for both liver protection and repair in hepatic ischemia/reperfusion injury.

Myeloid Differentiation Factor 88 (MyD88)-Deficiency Increases Risk of Diabetes in Mice

Background Multiple lines of evidence suggest innate immune response pathways to be involved in the development of obesity-associated diabetes although the molecular mechanism underling the disease is unknown. Recent observations suggest that saturated fatty acids can act as a ligand for toll-like receptor (TLR) 4, which is thought to mediate obesity-associated insulin resistance. Myeloid differentiation factor 88 (MyD88) is an adapter protein for TLR/IL-1 receptor signaling, which is involved in the activation of inflammatory pathways. To evaluate molecular mechanisms linking obesity-associated diabetes down-stream of TLR4, we investigated physiological role of MyD88 in high-fat diet (HFD)-induced obesity. Methodology/Principal Findings In the present study, we found MyD88-deficient mice fed a HFD had increased circulating levels of insulin, leptin and cholesterol, as well as liver dysfunction (increased induction of ALT levels, increased activation of JNK and cleavage of PARP), which were linked to the onset of severe diabetes. On the other hand, TNF-α would not be involved in HFD-induced diabetes in MyD88-deficient mice, because TNF-α level was attenuated in MyD88-deficient mice fed with HFD. Conclusions/Significance The present finding of an unexpected role for MyD88 in preventing diabetes may provide a potential novel target/strategy for treating metabolic syndrome.