Publications

STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch

No abstract is provided for this article.

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

The TRAF-associated protein TANK facilitates cross-talk within the IκB kinase family during Toll-like receptor signaling

Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFNβ (TRIF) activate the IκB kinase (IKK)-related kinases, TRAF associated NFκB activator (TANK)-binding kinase-1 (TBK1) and IKKε, which then phosphorylate IRF3 and induce the production of IFNβ. Here we show that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKKε is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKKε then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKKε is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK −/− macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFNβ production was normal in TANK −/− macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.

Faculty Opinions recommendation of A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3.

[TIR domain--containing adaptors regulate TLR-mediated signaling pathways].

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (TIR) domain-containing adaptors such as MyD88, TIRAP, and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.

Intestinal mast cell progenitors function as innate lymphoid cells (P497) (71.3)

Small intestinal innate lymphoid cells (mainly NKp46+ILCs) are known to have a protective role from infection of pathogenic bacteria through the production of IL-22. The proportions of ILCs localized intestinal lamina propria are higher than those in the others lymphoid tissues, such as mesenteric lymph node, spleen, liver, and bone marrow. In this study, we did a microarray experiment to study whether small intestinal ILCs (Lin-c-Kit+Sca-1- cells) highly express which genes as compared with non-ILCs (Lin-c-Kit-Sca-1- cells). We selected 251 up-regulated genes and 219 down-regulated genes in Lin-c-Kit+Sca-1- cells. Especially, Lin-c-Kit+Sca-1- cells highly expressed genes coding to Il22, Csf2rb2, mast cell proteases (Mcpts), and Rorc. To address whether which ILC populations are main source of IL-22, we more detailed divided Lin-c-Kit+Sca-1- cells into the two groups [Lin-c-Kit+NKp46+ (NKp46+ILCs) and Lin-c-Kit+NKp46-CD4-]. Only Lin-c-Kit+NKp46-CD4- cells expressed Csf2rb2 and Mcpts transcripts. These cells expressed higher level of Il22 mRNA, IL-22 protein, and IL-17F protein in physiological condition and in IL-23 stimulated condition compared with NKp46+ILCs. Moreover, these cells could differentiate into mast cells in the presence of mIL-3 and mSCF. Taken together, Lin-c-Kit+NKp46-CD4- cells as mast cell progenitors may regulate small intestinal homeostasis through the production of Mcpts, IL-17F, and IL-22 in physiological condition and in pathophysiological condition.

Intestine and Innate Immunity

While the intestinal immune system coexists[DPM1] with commensal bacterial flora through immunological tolerance, invading microorganisms are recognized and properly eliminated. However, it remains unknown what kinds of cells in the intestine initiate immune responses and how they activate host immunity. Recently, we identified a subset of CD11chiCD11bhi lamina propria (LP) dendritic cells (DCs) as TLR5-expressing cells, which have the ability to activate adaptive immune responses. The LPDCs induced antigen-specific Th17 cells as well as Th1 cells in a TLR5-dependent manner. In addition, they acted on naïve B cells to induce their development to immunoglobulin A (IgA)+ plasma cells in response to flagellin, and such IgA+ plasma cell generation took place in a gut-associated lymphoid tissue (GALT)-independent fashion. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine. We also generated and examined mutant mice of ATG16L1. ATG16L1 is a component of autophagy machinery and has been reported to be a candidate gene responsible for susceptibility to Crohn's disease. We discuss a novel role for autophagy in the regulation of the inflammatory immune responses in the intestine.

Lipid A Receptor TLR4-Mediated Signaling Pathways

No abstract is provided for this article.

Hepatic lipocalin 2 promotes liver fibrosis and portal hypertension

Advanced fibrosis and portal hypertension influence short-term mortality. Lipocalin 2 (LCN2) regulates infection response and increases in liver injury. We explored the role of intrahepatic LCN2 in human alcoholic hepatitis (AH) with advanced fibrosis and portal hypertension and in experimental mouse fibrosis. We found hepatic LCN2 expression and serum LCN2 level markedly increased and correlated with disease severity and portal hypertension in patients with AH. In control human livers, LCN2 expressed exclusively in mononuclear cells, while its expression was markedly induced in AH livers, not only in mononuclear cells but also notably in hepatocytes. Lcn2 −/− mice were protected from liver fibrosis caused by either ethanol or CCl 4 exposure. Microarray analysis revealed downregulation of matrisome, cell cycle and immune related gene sets in Lcn2 −/− mice exposed to CCl 4 , along with decrease in Timp1 and Edn1 expression. Hepatic expression of COL1A1 , TIMP1 and key EDN1 system components were elevated in AH patients and correlated with hepatic LCN2 expressio n. In vitro , recombinant LCN2 induced COL1A1 expression. Overexpression of LCN2 increased HIF1A that in turn mediated EDN1 upregulation. LCN2 contributes to liver fibrosis and portal hypertension in AH and could represent a new therapeutic target.

Dual Role of MyD88 in Rapid Clearance of Relapsing Fever<i>Borrelia</i>spp

Relapsing fever Borrelia spp. undergo antigenic variation, achieve high levels in blood, and require rapid production of immunoglobulin M (IgM) for clearance. MyD88-deficient mice display defective clearance of many pathogens; however, the IgM response to persistent infection is essentially normal. Therefore, MyD88 −/− mice provided a unique opportunity to study the effect of nonantibody, innate host defenses to relapsing fever Borrelia . Infected MyD88 −/− mice harbored extremely high levels of B. hermsii in the blood compared to wild-type littermates. In the comparison of MyD88 −/− mice and B- and T-cell-deficient scid mice, two features stood out: (i) bacterial numbers in blood were at least 10-fold greater in MyD88 −/− mice than scid mice, even though the production of IgM still occurred in MyD88 −/− mice; and (ii) many of the MyD88 −/− mice were able to exert partial clearance, although with delayed kinetics relative to wild-type mice, a feature not seen in scid mice. Further analysis revealed a delay in the IgM response to lipoproteins expressed by the original inoculum; however, by 6 days of infection antibodies were produced in MyD88 −/− mice that could clear spirochetemia in scid mice. While these results indicated that the production of IgM was delayed in MyD88 −/− mice, they also point to a second, antibody-independent role for MyD88 signaling in host defense to relapsing fever Borrelia . This second defect was apparent only when antibody levels were limiting.

Macrophages and Myeloid Dendritic Cells, but Not Plasmacytoid Dendritic Cells, Produce IL-10 in Response to MyD88- and TRIF-Dependent TLR Signals, and TLR-Independent Signals

We have previously reported that mouse plasmacytoid dendritic cells (DC) produce high levels of IL-12p70, whereas bone marrow-derived myeloid DC and splenic DC produce substantially lower levels of this cytokine when activated with the TLR-9 ligand CpG. We now show that in response to CpG stimulation, high levels of IL-10 are secreted by macrophages, intermediate levels by myeloid DC, but no detectable IL-10 is secreted by plasmacytoid DC. MyD88-dependent TLR signals (TLR4, 7, 9 ligation), Toll/IL-1 receptor domain-containing adaptor-dependent TLR signals (TLR3, 4 ligation) as well as non-TLR signals (CD40 ligation) induced macrophages and myeloid DC to produce IL-10 in addition to proinflammatory cytokines. IL-12p70 expression in response to CpG was suppressed by endogenous IL-10 in macrophages, in myeloid DC, and to an even greater extent in splenic CD8α− and CD8α+ DC. Although plasmacytoid DC did not produce IL-10 upon stimulation, addition of this cytokine exogenously suppressed their production of IL-12, TNF, and IFN-α, showing trans but not autocrine regulation of these cytokines by IL-10 in plasmacytoid DC.

Microglia‐specific expression of microsomal prostaglandin E₂ synthase‐1 contributes to lipopolysaccharide‐induced prostaglandin E₂ production

Microsomal prostaglandin E 2 synthase (mPGES)‐1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E 2 (PGE 2 ) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES‐1 is poorly understood. In this study, we demonstrated the expression of mPGES‐1 in the brain parenchyma in a lipopolysaccharide (LPS)‐induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES‐1 in activated microglia. In neuron‐glial mixed cultures, mPGES‐1 was co‐induced with cyclooxygenase‐2 (COX‐2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia‐enriched cultures, the induction of mPGES‐1, the activity of PGES and the production of PGE 2 were preceded by the induction of mPGES‐1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES‐1 and production of PGE 2 were also either attenuated or absent in microglia treated with mPGES‐1 antisense oligonucleotide or microglia from mPGES‐1 knockout (KO) mice, respectively, suggesting the necessity of mPGES‐1 for microglial PGE 2 production. These results suggest that the activation of microglia contributes to PGE 2 production through the concerted de novo synthesis of mPGES‐1 and COX‐2 at sites of inflammation of the brain parenchyma.

Cutting Edge: Roles of Caspase-8 and Caspase-10 in Innate Immune Responses to Double-Stranded RNA

Upon viral infection, host cells trigger antiviral immune responses by inducing type I IFN and inflammatory cytokines. dsRNA generated during viral replication is recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, which interact with an adaptor, IFN-beta promoter stimulator-1, to activate the transcription factors NF-kappaB and IFN regulatory factor 3. In this article we demonstrate that caspase-8 and caspase-10 are involved in these pathways. Both caspases were cleaved during dsRNA stimulation, and overexpression of a cleaved form of these caspases activated NF-kappaB. Knockdown of caspase-10 or caspase-8 in a human cell line resulted in the reduction of inflammatory cytokine production. Cells derived from caspase-8-deficient mice also showed reduced expression of inflammatory cytokines as well as NF-kappaB activation. Furthermore, the Fas-associated death domain protein interacted with these two caspases and IFN-beta promoter stimulator 1. These results indicate that caspase-8 and caspase-10 are essential components that mediate NF-kappaB-dependent inflammatory responses in antiviral signaling.

Highly pathogenic avian influenza A H5N1 and pandemic H1N1 virus infections have different phenotypes in Toll-like receptor 3 knockout mice

Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3(-/-) mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3(-/-) mice. In contrast, MyD88(-/-) mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.

IL-18 and IL-18 Receptor Knockout Mice

No abstract is provided for this article.

Lipopolysaccharide from<i>Coxiella burnetii</i>Is Involved in Bacterial Phagocytosis, Filamentous Actin Reorganization, and Inflammatory Responses through Toll-Like Receptor 4

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4−/− mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4−/− macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4−/− macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4−/− mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4−/− mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-γ and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.

Regnase-1 controls colon epithelial regeneration via regulation of mTOR and purine metabolism

Significance Idiopathic inflammatory bowel disease (IBD) is associated with the gut microbiota and immune system of the host; however, the precise pathogenesis of IBD is poorly defined. We show that specific deletion of the endoribonuclease Regnase-1 in intestinal epithelial cells relieves the symptoms of experimental colitis during acute inflammation. Regnase-1 deficiency potentiates mTOR signaling and purine metabolism in the colon epithelium. These data provide insight into the role of epithelial Regnase-1 in IBD.

Adiponectin Enhances Insulin Sensitivity by Increasing Hepatic IRS-2 Expression via a Macrophage-Derived IL-6-Dependent Pathway

Insulin resistance is often associated with impeded insulin signaling due either to decreased concentrations or functional modifications of crucial signaling molecules including insulin receptor substrates (IRS) in the liver. Many actions of adiponectin, a well-recognized antidiabetic adipokine, are currently attributed to the activation of two critical molecules downstream of AdipoR1 and R2: AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα). However, the direct effects of adiponectin on insulin signaling molecules remain poorly understood. We show here that adiponectin upregulates IRS-2 through activation of signal transducer and activator of transcription-3 (STAT3). Surprisingly, this activation is associated with IL-6 production from macrophages induced by adiponectin through NFκB activation independent of its authentic receptors, AdipoR1 and AdipoR2. These data have unraveled an insulin-sensitizing action initiated by adiponectin leading to upregulation of hepatic IRS-2 via an IL-6 dependent pathway through a still unidentified adiponectin receptor.