Publications

TLR9 signaling in microglia ensures homeostatic neurogenesis in the adult hippocampus

Reciprocal regulation of STING and TCR signaling by mTORC1 for T-cell activation and function

Stimulator of interferon genes (STING) plays a key role in detecting cytosolic DNA and induces type I interferon (IFN-I) responses for host defense against pathogens. Although T cells highly express STING, its physiological role remains unknown. Here, we show that costimulation of T cells with the STING ligand cGAMP and TCR leads to IFN-I production and strongly inhibits T-cell growth. TCR-mediated mTORC1 activation and sustained activation of IRF3 are required for cGAMP-induced IFN-I production, and the mTORC1 activity is partially counteracted by cGAMP, thereby blocking proliferation. This mTORC1 inhibition in response to costimulation depends on IRF3 and IRF7. Effector T cells produce much higher IFN-I levels than innate cells in response to cGAMP. Finally, we demonstrated that STING stimulation in T cells is effective in inducing antitumor responses in vivo. Our studies demonstrate that the outputs of STING and TCR signaling pathways are mutually regulated through mTORC1 to modulate T-cell functions.

Cutting Edge: Toll-Like Receptor 4 (TLR4)-Deficient Mice Are Hyporesponsive to Lipopolysaccharide: Evidence for TLR4 as the <i>Lps</i> Gene Product

The human homologue of Drosophila Toll (hToll), also called Toll-like receptor 4 (TLR4), is a recently cloned receptor of the IL-1/Toll receptor family. Interestingly, the TLR4 gene has been localized to the same region to which the Lps locus (endotoxin unresponsive gene locus) is mapped. To examine the role of TLR4 in LPS responsiveness, we have generated mice lacking TLR4. Macrophages and B cells from TLR4-deficient mice did not respond to LPS. All these manifestations were quite similar to those of LPS-hyporesponsive C3H/HeJ mice. Furthermore, C3H/HeJ mice have, in the cytoplasmic portion of TLR4, a single point mutation of the amino acid that is highly conserved among the IL-1/Toll receptor family. Overexpression of wild-type TLR4 but not the mutant TLR4 from C3H/HeJ mice activated NF-κB. Taken together, the present study demonstrates that TLR4 is the gene product that regulates LPS response.

Epistatic Interaction Between TLR3 And Ptger3 Genes In Experimental Allergic Conjunctivitis

No abstract is provided for this article.

Retraction: C-Jun mediates hepatitis C virus hepatocarcinogenesis through signal transducer and activator of transcription 3 and nitric oxide-dependent impairment of oxidative DNA repair

Epistatic Interaction Between TLR3 And Ptger3 Genes In Experimental Allergic Conjunctivitis

No abstract is provided for this article.

Augmented TLR9-induced Btk activation in PIR-B–deficient B-1 cells provokes excessive autoantibody production and autoimmunity

Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate immune system. However, excess stimulation of TLR pathways is supposed to be connected with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses TLR9 signaling via Bruton's tyrosine kinase (Btk) dephosphorylation, which leads to attenuated activation of nuclear factor κB p65RelA but not p38 or Erk, and blocks the production of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb−/−) mice was further augmented in combination with the Faslpr mutation, which might be linked to the development of autoimmune glomerulonephritis. These results show the critical link between TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing pathogenic natural autoantibody production.

An epigenetic switch induced by Shh signalling regulates gene activation during development and medulloblastoma growth

No abstract is provided for this article.

IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions

Atopic dermatitis (AD) is a pruritic inflammatory skin disease. Because IL-18 directly stimulates T cells and mast cells to release AD-associated molecules, Th2 cytokines, and histamine, we investigated the capacity of IL-18 to induce AD-like inflammatory skin disease by analyzing KIL-18Tg and KCASP1Tg, which skin-specifically overexpress IL-18 and caspase-1, respectively. They spontaneously developed relapsing dermatitis with mastocytosis and Th2 cytokine accumulation accompanied by systemic elevation of IgE and histamine. Stat6-deficient KCASP1Tg displayed undetectable levels of IgE but manifested the same degree of cutaneous changes, whereas IL-18-deficient KCASP1Tg evaded the dermatitis, suggesting that IL-18 causes the skin changes in the absence of IgE/stat6. KIL-18Tg and IL-1-deficient KCASP1Tg took longer to display the lesion than KCASP1Tg. Thus, AD-like inflammation is initiated by overrelease of IL-18 and accelerated by IL-1. Our present study might provide insight into understanding the pathogenesis of and establishing therapeutics for chronic inflammatory skin diseases including AD.

Interferon-γ production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-γ plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-γ-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-γ gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-γ was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-γ production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, γδT and dendritic cells. Neutralization of IFN-γ or TNF-α by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-α mAb partially attenuated IFN-γ synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-γ, which was secreted from helper T cells.

Faculty Opinions recommendation of TRIM5 is an innate immune sensor for the retrovirus capsid lattice.

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice. PMID: 21512573 Funding information This work was supported by: NIAID NIH HHS, United States Grant ID: R01AI59159 NIAID NIH HHS, United States Grant ID: R01 AI059159 NIAID NIH HHS, United States Grant ID: R21AI087467 NIAID NIH HHS, United States Grant ID: R01 AI059159-06 NIAID NIH HHS, United States Grant ID: R21 AI087467

Role of Lipoteichoic Acid in the Phagocyte Response to Group B<i>Streptococcus</i>

Group B Streptococcus (GBS) cell walls potently activate phagocytes by a largely TLR2-independent mechanism. In contrast, the cell wall component lipoteichoic acid (LTA) from diverse Gram-positive bacterial species has been shown to engage TLR2. In this study we examined the role of LTA from GBS in phagocyte activation and the requirements for TLR-LTA interaction. Using cells from knockout mice and genetic complementation in epithelial cells we found that highly pure LTA from both GBS and Staphylococcus aureus interact with TLR2 and TLR6, but not TLR1, in contrast to previous reports. Furthermore, NF-κB activation by LTA required the integrity of two putative PI3K binding domains within TLR2 and was inhibited by wortmannin, indicating an essential role for PI3K in cellular activation by LTA. However, LTA from GBS proved to be a relatively weak stimulus of phagocytes containing ∼20% of the activity observed with LTA from Staphylococcus aureus. Structural analysis by nuclear magnetic resonance spectrometry revealed important differences between LTA from GBS and S. aureus, specifically differences in glycosyl linkage, in the glycolipid anchor and a lack of N-acetylglucosamine substituents of the glycerophosphate backbone. Furthermore, GBS expressing LTA devoid of d-alanine residues, that are essential within immune activation by LTA, exhibited similar inflammatory potency as GBS with alanylated LTA. In conclusion, LTA from GBS is a TLR2/TLR6 ligand that might contribute to secreted GBS activity, but does not contribute significantly to GBS cell wall mediated macrophage activation.

Autophagy in infection, inflammation and immunity

No abstract is provided for this article.

h Interacts with the lmmunoglobulin V-J Recombination Sequences

of Microbiology and Immunology University of California Berkeley, California 94720 Summary DNA-nuclear protein interactions were studied with synthetic recombination signal sequences (RSSs) for immunoglobulin V-J joining. With a gel retardation as- say, a DNA-binding protein that specifically interacts with RSSs was detected in nuclear extracts from a pre- B cell line, 3889. This protein was found in all the recombination-competent pre-B cell lines tested in this study, but not in myeloma, mature T cell, mono- cyte, or fibroblast cell lines. DNA footprint analysis with dimethyl sulfate demonstrated that the 7-mer re- gion of the RSS was strongly protected when com- plexed with the binding protein. Furthermore, a single base substitution in the Fmer region totally abolished the binding. The molecular mechanism of V-J joining is discussed in the context of the RSS-binding protein. Introduction Antigen receptor genes are generated by site-specific DNA recombination both in T cells and 6 cells during the differentiation of lymphocytes (reviewed by Kronenberg et al., 1986; Tonegawa, 1983). On the germ-line chromo- some, variable region genes for the antigen receptor are split into variable (V), diversity (D), and joining (J) seg- ments. These gene segments are brought together by a site-specific DNA recombination process known as V-(D)- J joining. This DNA rearrangement plays a key role not only in the activation of the antigen receptor genes, but also in the diversification of the gene sequences. To study the molecular mechanism of V-(D)-J joining, three different approaches have been taken. One ap- proach is the structural study of the recombination region by DNA cloning and sequencing. From these analyses it has been revealed that both immunoglobulin (lg) and T cell receptor (TCR) genes contain two highly conserved sequences adjacent to the recombination sites; they are a palindromic 7-mer, CACTGTG, and a T-rich 9-mer, GGTTTTTGT, separated by a spacer of constant length (Sakano et al., 1979, 1980, 1981; Max et al., 1979; Early et al., 1980; Davis, 1985). The second approach is to in- troduce artificial recombination substrates with appropri- ate selection markers into recombination-competent pre-B cells (Lewis et al., 1984, 1985; Blackwell and Alt, 1984; Yancopoulos et al., 1986; Hesse et al., 1987). Alt and Balti- more and their colleagues have demonstrated that V-(D)-J joining can take place on exogenous lg or TCR genes in- troduced into the host chromosomal DNA with a retroviral vector or by DNA transfection. The third approach is to characterize the enzymatic machinery responsible for V-(D)-J joining. We assume that at least three activities are involved in the recombinase complex for V-(D)-J joining (Hope et al., 1986): a DNA-binding activity, an endonu- cleolytic activity, and a ligase activity. The DNA-binding activity would recognize the recombination sites and bring the two substrates together into the proper orientation; the endonucleolytic activity would cleave the germ-line seg- ments at the recombination sites; and finally, the ligase activity would join the two cleaved DNA substrates cova- lently to complete the joining reaction. Endonucleases presumably involved in the DNA-cutting activity of recombinase have been previously described (Desiderio and Baltimore, 1984; Kataoka et al., Hope 1986). However, little is known about other activities, and as yet the identification of a whole recombinase com- plex has not been reported. In the present study we have attempted to identify a DNA-binding protein(s) that interacts specifically with recombination signal sequences (RSSs). As a source of the protein, we have used nuclear extracts from an immature pre-B cell line, 3889, kindly provided by Dr. F. W. Alt. In this cell line lg gene rearrangement actively takes place during in vitro culture (Alt et al., 1981; Black- well and Alt, 1984). We synthesized DNA substrates con- taining the Fmer and 9-mer separated by either a 12 bp or a 23 bp spacer. Using the synthetic recombination sub- strates, we first verified that two sets of RSSs are sufficient to cause the V-J-type joining provided the 12/23 bp spacer rule (Sakano et al., 1980; Early et al., 1980) is satisfied. We then searched for a specific DNA-binding protein(s) in the pre-B cell nuclear extracts by using the synthesized RSS probes. With a gel retardation assay (Singh et al., 1986; Schneider et al., 1986), we have identified a nuclear pro- tein that can specifically interact, particularly in the Fmer region, with the RSS probe containing the 12 bp spacer. In this report we characterize this RSS-binding activity, which may be one of the DNA-binding components as- sociated with V-(D)-J joining. Results Synthetic Substrates Containing the Fmer and 9-mer Can Cause V-J-Type Recombination Two double-stranded oligonucleotides containing the Fmer and 9-mer were chemically synthesized (Figure 1A). One contained a 12 bp spacer separating the recombina- tion signals (12 bp-RSS), and the other contained a 23 bp spacer (23 bp-RSS). To study whether these two RSSs are sufficient substrates to mediate V-J-type recombina- tion, we constructed a plasmid, pKOdel-1. It contains a ne- omycin resistance gene (near) (Beck et al., 1982) flanked by two sets of RSSs, and a thymidine kinase gene (tk) (McKnight, 1980) further downstream (Figure 1B). Expres- sion of these genes is mediated by a promotor complex consisting of the SV40 and t/r gene promoters (McKnight, 1980; Southern and Berg, 1982).

Intestinal Lin-c-Kit+NKp46-CD4- population highly produces IL-22 upon IL-1β stimulation (P3260)

Small intestinal innate lymphoid cells (ILCs) regulate intestinal epithelial cell homeostasis and prevent pathogenic bacterial infections by producing IL-22. NKp46+ ILC (ILC22) and CD4+ ILC (CD4+ LTi) are known as main sources for IL-22 in the small intestine. We found that Lin-c-Kit+NKp46-CD4- (CD4- LTi), ILC22, and CD4+ LTi cells comprised 0.30 ± 0.03%, 1.10 ± 0.12%, and 0.07 ± 0.01%, respectively, of the total lymphocytes in the small intestinal lamina propria of adult mice (BALB/c). We next compared the induction of IL-17F and IL-22 among CD4- LTi, ILC22, and CD4+ LTi cells under stimulation with IL-1β or IL-23 or IL-1β and IL-23 stimulation, by real-time PCR, Bio-Plex analysis, and intracellular staining. We found here that CD4- LTi cells that expressed RORγt and IL-7Rα strongly expressed the transcripts for Il17f and Il22, and produced the proteins for IL-17F and IL-22 after stimulation with IL-1β and IL-23 stimulation compared with ILC22 and CD4+ LTi cells. However, none of CD4- LTi, ILC22, and CD4+ LTi cells produced IL-17F and IL-22 after stimulation with Pam3CSK4 (TLR2 ligand), Poly(I:C) (TLR3 ligand), or LPS (TLR4 ligand) for 24 hrs. Taken together, these findings indicate that the Lin-c-Kit+NKp46-CD4- population may control intestinal homeostasis and provide intestinal protection by producing high levels of IL-17F and IL-22 under the stimulation with IL-1β and IL-23.

Correction for Hiramatsu et al., “The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model”

Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis

Background & Aims : We tested whether the attenuation of experimental colitis by live probiotic bacteria is due to their immunostimulatory DNA, whether toll-like receptor (TLR) signaling is required, and whether nonviable probiotics are effective. Methods : Methylated and unmethylated genomic DNA isolated from probiotics (VSL-3), DNAse-treated probiotics and Escherichia coli (DH5α) genomic DNA were administered intragastricly (i.g.) or subcutaneously (sc) to mice prior to the induction of colitis. Viable or γ-irradiated probiotics were administered i.g. to wild-type mice and mice deficient in different TLR or in the adaptor protein MyD88, 10 days prior to administration of dextran sodium sulfate (DSS) to their drinking water and for 7 days thereafter. Results : ntragastric and sc administration of probiotic and E. coli DNA ameliorated the severity of DSS-induced colitis, whereas methylated probiotic DNA, calf thymus DNA, and DNase-treated probiotics had no effect. The colitis severity was attenuated to the same extent by i.g. delivery of nonviable γ-irradiated or viable probiotics. Mice deficient in MyD88 did not respond to γ-irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of γ-irradiated probiotics, whereas, in TLR9-deficient mice, γ-irradiated probiotics had no effect. Conclusions : The protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon. TLR9 signaling is essential in mediating the anti-inflammatory effect of probiotics, and live microorganisms are not required to attenuate experimental colitis because nonviable probiotics are equally effective.

IL-6 and IL-10 Induction from Dendritic Cells in Response to <i>Mycobacterium tuberculosis</i> Is Predominantly Dependent on TLR2-Mediated Recognition

The initial TLR-mediated interaction between Mycobacterium tuberculosis and dendritic cells is critical, since the cytokine production that ensues can greatly influence the class of adaptive immunity that is generated to the pathogen. In this study, we therefore determined the dependency on TLR2 and TLR4 for M. tuberculosis-induced cytokine production by murine dendritic cells. A key new finding of this study is that production of IL-6 and IL-10 from dendritic cells in response to M. tuberculosis is principally dependent on TLR2. The study also indicates that M. tuberculosis can induce IL-12 production in the absence of either TLR2 or TLR4, suggesting redundancy or possibly involvement of other receptors in IL-12 production. In addition, the data also reveal that lack of TLR2 or TLR4 does not impact on dendritic cell maturation or on their ability to influence the polarity of differentiating naive T cells. Collectively, data presented here provide a mechanistic insight for the contribution of TLR2 and TLR4 to tuberculosis disease progression and offer strategies for regulating IL-6 and IL-10 production in dendritic cell-based vaccine strategies.