Publications

Faculty Opinions recommendation of IFIT1 is an antiviral protein that recognizes 5'-triphosphate RNA.

Targeted Disruption of the IL‐6 Related Genes: gp130 and NF‐IL‐6

No abstract is provided for this article.

Targeted lipidomics reveals mPGES-1-PGE2 as a therapeutic target for multiple sclerosis

The arachidonic acid (AA) cascade produces eicosanoids, such as prostaglandins (PGs), that regulate physiological and pathological functions. Although various nonsteroidal anti-inflammatory drugs have been developed, blocking upstream components (cyclooxygenase-1 and -2) of the AA cascade leads to severe side effects, including gastrointestinal ulcers and cardiovascular events, respectively, due to the complexity of the AA cascade. Here, using an AA cascade-targeted lipidomics approach, we report that microsomal PGE synthase 1 (mPGES-1) plays a key role in experimental autoimmune encephalomyelitis (EAE). Eicosanoids (mainly PGD(2)) are produced constitutively in the spinal cord of naive mice. However, in EAE lesions, the PGE(2) pathway is favored and the PGD(2), PGI(2), and 5-lipoxygenase pathways are attenuated. Furthermore, mPGES-1(-/-) mice showed less severe symptoms of EAE and lower production of IL-17 and IFN-gamma than mPGES-1(+/+) mice. Expression of PGE(2) receptors (EP1, EP2, and EP4) was elevated in EAE lesions and correlated with clinical symptoms. Immunohistochemistry on central nervous systems of EAE mice and multiple sclerosis (MS) patients revealed overt expression of mPGES-1 protein in microglia/macrophages. Thus, the mPGES-1-PGE(2)-EPs axis of the AA cascade may exacerbate EAE pathology. Our findings have important implications for the design of therapies for MS.

The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction

TRIF-related adaptor molecule (TRAM) is the fourth Toll/IL-1 resistance domain-containing adaptor to be described that participates in Toll-like receptor (TLR) signaling. TRAM functions exclusively in the TLR4 pathway. Here we show by confocal microscopy that TRAM is localized in the plasma membrane and the Golgi apparatus, where it colocalizes with TLR4. Membrane localization of TRAM is the result of myristoylation because mutation of a predicted myristoylation site in TRAM (TRAM-G2A) brought about dissociation of TRAM from the membrane and its relocation to the cytosol. Further, TRAM, but not TRAM-G2A, was radiolabeled with [ 3 H]myristate in vivo . Unlike wild-type TRAM, overexpression of TRAM-G2A failed to elicit either IFN regulatory factor 3 or NF-κB signaling. Moreover, TRAM-G2A was unable to reconstitute LPS responses in bone marrow-derived macrophages from TRAM-deficient mice. These observations provide clear evidence that the myristoylation of TRAM targets it to the plasma membrane, where it is essential for LPS responses through the TLR4 signal transduction pathway, and suggest a hitherto unappreciated manner in which LPS responses can be regulated.

Protein kinase C zeta plays an essential role for Mycobacterium tuberculosis-induced extracellular signal-regulated kinase 1/2 activation in monocytes/macrophages via Toll-like receptor 2

This study characterized the upstream signalling molecules involved in extracellular signal-regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)-α expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF-α expression in human primary monocytes and monocyte-derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB-induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) ζ decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF-α induction caused by MTBRv or MTBRa. Toll-like receptor (TLR) 2 was found to play a major role in MTB-induced TNF-α expression and PKCζ phosphorylation. Co-immunoprecipitation experiments showed that PKCζ interacts physically with TLR2 after MTB stimulation. Moreover, PKCζ phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCζ interacts with TLR2 to play an essential role in MTB-induced ERK 1/2 activation and subsequent TNF-α expression in monocytes/macrophages.

CXCL10-CXCR3 Enhances the Development of Neutrophil-mediated Fulminant Lung Injury of Viral and Nonviral Origin

Patients who developed acute respiratory distress syndrome (ARDS) after infection with severe respiratory viruses (e.g., severe acute respiratory syndrome-coronavirus, H5N1 avian influenza virus), exhibited unusually high levels of CXCL10, which belongs to the non-ELR (glutamic-leucine-arginine) CXC chemokine superfamily. CXCL10 may not be a bystander to the severe virus infection but may directly contribute to the pathogenesis of neutrophil-mediated, excessive pulmonary inflammation.We investigated the contribution of CXCL10 and its receptor CXCR3 axis to the pathogenesis of ARDS with nonviral and viral origins.We induced nonviral ARDS by acid aspiration and viral ARDS by intratracheal influenza virus infection in wild-type mice and mice deficient in CXCL10, CXCR3, IFNAR1 (IFN-α/β receptor 1), or TIR domain-containing adaptor inducing IFN-β (TRIF).We found that the mice lacking CXCL10 or CXCR3 demonstrated improved severity and survival of nonviral and viral ARDS, whereas mice that lack IFNAR1 did not control the severity of ARDS in vivo. The increased levels of CXCL10 in lungs with ARDS originate to a large extent from infiltrated pulmonary neutrophils, which express a unique CXCR3 receptor via TRIF. CXCL10-CXCR3 acts in an autocrine fashion on the oxidative burst and chemotaxis in the inflamed neutrophils, leading to fulminant pulmonary inflammation.CXCL10-CXCR3 signaling appears to be a critical factor for the exacerbation of the pathology of ARDS. Thus, the CXCL10-CXCR3 axis could represent a prime therapeutic target in the treatment of the acute phase of ARDS of nonviral and viral origins.

Monocytic Cell Activation by Nonendotoxic Glycoprotein from<i>Prevotella intermedia</i>ATCC 25611 Is Mediated by Toll-Like Receptor 2

Lipopolysaccharide (LPS) preparations from gram-negative black-pigmented bacteria such as Porphyromonas gingivalis and Prevotella intermedia activate cells from non-LPS-responsive C3H/HeJ mice, but it is still unclear whether this activity is due to the unique structure of LPS or to a minor component(s) responsible for the activity in the preparation. A nonendotoxic glycoprotein with bioactivity against cells from C3H/HeJ mice was purified from a hot phenol-water extract of P. intermedia ATCC 25611 and designated Prevotella glycoprotein (PGP). Treatment of human monocytic THP-1 cells with 22-oxyacalcitriol (OCT) induced maturation and marked expression of CD14 on the cells, but the cells constitutively expressed Toll-like receptor 2 (TLR2) and TLR4 on the cells irrespective of the treatment. PGP induced a high level of interleukin-8 production at doses of 100 ng/ml and higher in OCT-treated THP-1 cells compared with Salmonella LPS, and the production was significantly inhibited by anti-CD14 and anti-TLR2 but not anti-TLR4 antibodies. Consistent with this, TLR2-deficient murine macrophages did not respond to PGP. It was also shown that PGP activity on the THP-1 cells was LPS-binding protein dependent and was inhibited by a synthetic lipid A precursor IV A . These results indicate that PGP activates monocytic cells in a CD14- and TLR2-dependent manner.

Contribution of IL-33–activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice

When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33 −/− mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2 −/− mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33–stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.

Requirement for DNA CpG Content in TLR9-Dependent Dendritic Cell Activation Induced by DNA-Containing Immune Complexes

Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.

Toll-like receptor ligands synergize through distinct dendritic cell pathways to induce T cell responses: Implications for vaccines

Toll-like receptors (TLRs) may need to cooperate with each other to be effective in detecting imminent infection and trigger immune responses. Understanding is still limited about the intracellular mechanism of this cooperation. We found that when certain TLRs are involved, dendritic cells (DCs) establish unidirectional intracellular cross-talk, in which the MyD88-independent TRIF-dependent pathway amplifies the MyD88-dependent DC function through a JNK-dependent mechanism. The amplified MyD88-dependent DC function determines the induction of the T cell response to a given vaccine in vivo. Therefore, our study revealed an underlying TLR mechanism governing the functional, nonrandom interplay among TLRs for recognition of combinatorial ligands that may be dangerous to the host, providing important guidance for design of novel synergistic molecular vaccine adjuvants.

CD44 Participates in IP-10 Induction in Cells in Which Hepatitis C Virus RNA Is Replicating, through an Interaction with Toll-Like Receptor 2 and Hyaluronan

The mechanisms of induction of liver injury during chronic infection with hepatitis C virus (HCV) are not well understood. Gamma interferon (IFN-γ)-inducible protein 10 (IP-10), a member of the CXC chemokine family, is expressed in the liver of chronic hepatitis C (CHC) patients and selectively recruits activated T cells to the sites of inflammation. Recently, it was shown that a low plasma concentration of IP-10 in CHC patients was closely associated with the outcome of antiviral therapy. In this study, we examined the role of the Toll-like receptor (TLR) pathway on IP-10 production in cells replicating HCV. Among the CXC chemokines, the expression of IP-10 was specifically increased in cells replicating HCV upon stimulation with conventional TLR2 ligands. The enhancement of IP-10 production upon stimulation with TLR2 ligands in cells replicating HCV induced CD44 expression. CD44 is a broadly distributed type I transmembrane glycoprotein and a receptor for the glycosaminoglycan hyaluronan (HA). In CHC patients, the expression of HA in serum has been shown to increase in accord with the progression of liver fibrosis, and HA also works as a ligand for TLR2. In the present study, IP-10 production upon HA stimulation was dependent on the expression of TLR2 and CD44, and a direct association between TLR2 and CD44 was observed. These results suggest that endogenous expression of HA in hepatocytes in CHC patients participates in IP-10 production through an engagement of TLR2 and CD44.

Deletion of the kinase domain from death-associated protein kinase attenuates p53 expression in chronic obstructive uropathy

Death-associated protein kinase (DAPK) is a Ca(2+)/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We previously showed that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) caused by a unilateral ureteral ligation. Here, we used DAPK-mutant mice, generated by the deletion of 74 amino acids from the catalytic kinase domain, to investigate the role of the DAPK kinase domain in regulating the p53 level following COU. The p53 expression levels in obstructed kidneys of wild-type and mutant mice were determined during the course of COU. Western blot analysis revealed that the p53 protein levels were significantly increased at 5 days after a ureteral ligation. This increase in the p53 level was significantly attenuated in mutant kidneys compared to wild-type kidneys. The obstructed kidneys of DAPK-mutant mice showed a significantly lower number of p53-expressing renal tubule cells than wild-type mice. These results are consistent with the hypothesis that DAPK stabilizes p53 protein in response to apoptosis-inducing stimuli. Thus, the present results suggest that the DAPK kinase domain is crucial for stabilizing p53 protein in renal tubular cell apoptosis in a mouse model of COU.

Neutrophils Modulate Osteoblastic Niche for Hematopoietic Progenitors Via Prostaglandin E2 Production Triggered By Sympathetic Nervous System

The mobilization of hematopoietic stem/progenitor cells (HSC/HPCs) from the bone marrow (BM) to peripheral blood by granulocyte colony-stimulating factor (G-CSF) is an essential method in clinic. We have shown that the suppression of osteoblastic niche by β-adrenergic signal is critical for this phenomenon (Cell 2006; Cell Stem Cell 2013). Because G-CSF administration causes fever and back pain and these symptoms are ameliorated by non-steroidal anti-inflammatory drugs, we investigated the role of prostaglandin E2 (PGE2) in the BM microenvironment during G-CSF treatment. First, HPC (CFU-C) mobilization by G-CSF (125μg/kg/dose, every 12 hours, 8 divided doses) was significantly augmented in microsomal PGE syntase-1-deficient (mPGES-1-/-) mice (42% increase, n=16, p<0.05), and strongly inhibited by exogenous administration of PGE2 (6mg/kg/day for 2 weeks) to wild-type (WT) mice (52%, n=8-9 p<0.05). These data suggest that G-CSF induces mPGES-1-mediated PGE2 production, which suppresses the HPC mobilization. In the early phase (at 4 doses) of G-CSF administration, mPGES-1 mRNA in BM cells was upregulated (34% increase, n=5, p<0.05). Furthermore, WT mice reconstituted with mPGES-1-/- BM showed higher HPC mobilization than control mice reconstituted with mPGES-1+/+ BM (2.2-fold increase, n=4, p<0.05), which indicated that blood cells might be responsible for additional PGE2 synthesis. To address this, we examined the PGE2 production by ELISA from various lineage cell lines, such as neutrophil precursor 32D, macrophage RAW264.7, B cell Ba/F3, and T cell EL4. No increase was observed by G-CSF in any cell lines; whereas, isoproterenol induced PGE2 production significantly only in 32D culture supernatant (2.4-fold increase compared to vehicle treatment, n=4, p<0.05) accompanied with drastic increase of mPGES-1 mRNA in the cells and norepinephrine showed a similar effect. Primary neutrophils sorted from the BM also demonstrated prompt PGE2 production by isoproterenol (3-fold increase compared to vehicle treatment, n=4, p<0.05) but not by G-CSF. These data suggest that G-CSF-triggered high sympathetic tone stimulates the BM neutrophils to lead PGE2 production. We next assessed the exact roles of PGE2 in HPC mobilization. The inhibitory effect of PGE2 on HPC mobilization was completely abrogated in PGE2 receptor EP4-deficient (EP4-/-) mice, and the chimeric model generated by the reciprocal BM transplantation revealed that it was EP4 in microenvironment, but not in hematopoietic cells, that was critical for this effect. Since PGE2 did not change the CXCL12 behavior, we speculated that PGE2 increased another anchor in the niche, osteopontin (OPN). Immunofluorescence staining demonstrated upregulation of OPN by PGE2 and/or G-CSF in the endosteum, which was abolished in EP4-/- mice. Indeed, the inhibitory effect of PGE2 on HPC mobilization was canceled partially in OPN-/- mice and almost completely in anti-OPN antibody-treated WT mice. PGE2 also inhibited AMD3100-induced HPC mobilization, and this effect was canceled by anti-OPN antibody, which confirmed that PGE2-mediated niche modulation was independent of CXCL12 axis. To assess the induction of OPN by PGE2 directly, we fractionated non-hematopoietic (CD45-CD31-Ter119-) cells isolated from adult femur into three populations, i.e. Sca-1+ALCAM- immature mesenchymal cells, Sca-1-ALCAM- preosteoblasts that favorably support HPCs, and Sca-1-ALCAM+ mature osteoblasts that are most potent to maintain quiescent HSCs in vitro. PGE2 upregulated the OPN protein 2-fold in Sca-1+ALCAM- immature mesenchymal cells in cultures and more dramatically (6-fold) in Sca-1-ALCAM- preosteoblasts as assessed by flow cytometry. In sharp contrast, no OPN induction was observed in Sca-1-ALCAM+ mature osteoblasts. PGE2 failed to induce OPN in all three fractions from EP4-/- mice. In contrast to HPCs, the mobilization of long-term (6 months) repopulating HSCs was not altered in mPGES-1-/- and in PGE2-treated WT mice. This was consistent with the OPN induction profile in fractionated osteoblasts. These results suggest that PGE2 selectively regulates the osteoblastic niche for hematopoietic progenitors, but not for stem cells, by the induction of OPN via EP4 receptor. Collectively, we propose the inter-communication between the mature hematopoietic cells and the niche for their immature progenitors governed by the sympathetic nervous system. Disclosures No relevant conflicts of interest to declare.

Faculty Opinions recommendation of Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants.

Aluminium adjuvants, typically referred to as 'alum', are the most commonly used adjuvants in human and animal vaccines worldwide, yet the mechanism underlying the stimulation of the immune system by alum remains unknown. Toll-like receptors are critical in sensing infections and are therefore common targets of various adjuvants used in immunological studies. Although alum is known to induce the production of proinflammatory cytokines in vitro, it has been repeatedly demonstrated that alum does not require intact Toll-like receptor signalling to activate the immune system. Here we show that aluminium adjuvants activate an intracellular innate immune response system called the Nalp3 (also known as cryopyrin, CIAS1 or NLRP3) inflammasome. Production of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 by macrophages in response to alum in vitro required intact inflammasome signalling. Furthermore, in vivo, mice deficient in Nalp3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) or caspase-1 failed to mount a significant antibody response to an antigen administered with aluminium adjuvants, whereas the response to complete Freund's adjuvant remained intact. We identify the Nalp3 inflammasome as a crucial element in the adjuvant effect of aluminium adjuvants; in addition, we show that the innate inflammasome pathway can direct a humoral adaptive immune response. This is likely to affect how we design effective, but safe, adjuvants in the future. PMID: 18496530 Funding information This work was supported by: NHLBI NIH HHS, United States Grant ID: T32 HL007974 NIAMS NIH HHS, United States Grant ID: T32 AR007016 NIAID NIH HHS, United States Grant ID: K08 AI065517 Howard Hughes Medical Institute, United States

A Novel Toll-Like Receptor that Recognizes Bacterial DNA

In mammalian immune system, activation of innate immunity is crucial for protecting the host from invading pathogens. The innate immune system specifically recognizes structural patterns of microbial components, which are conserved among a wide variety of microorganisms. These structural patterns are termed pathogen associated molecular patters (PAMPs), and include lipopolysaccharide (LPS), peptidoglycan (PGN), bacterial lipoproteins (BLP), mannans, and bacterial DNA (1). All of these are essential for the microbe’s survival and are not expressed on the host. The host immune system has developed the receptors that can specifically recognize PAMPs, so-called pattern recognition receptors (PRRs). As one of PRRs, Toll-like receptor (TLR) family has been well studied (2,3). TLR family has been phylogenetically conserved from insects to mammals and characterized as a type I transmembrane protein with leucine-rich repeats in the extracellular domain and a cytoplasmic Toll/interleukin (IL)-1 receptor homology (TIR) domain. So far, ten members of human TLR are identified (2). Biological functions of some TLR family members are revealed; TLR4 is responsible for the recognition of LPS and TLR2 is involved in that of PGN and BLP (3). The activation of TLRs induces recruitment of the adaptor protein MyD88 and sequential activation of signaling molecules such as IRAK (IL-1 receptor associated kinase) and TRAF6 (TNF receptor associated factor).

Faculty Opinions recommendation of AhR sensing of bacterial pigments regulates antibacterial defence.

Impaired autophagy in macrophages promotes inflammatory eye disease

Autophagy is critical for maintaining cellular homeostasis. Organs such as the eye and brain are immunologically privileged. Here, we demonstrate that autophagy is essential for maintaining ocular immune privilege. Deletion of multiple autophagy genes in macrophages leads to an inflammation-mediated eye disease called uveitis that can cause blindness. Loss of autophagy activates inflammasome-mediated IL1B secretion that increases disease severity. Inhibition of caspase activity by gene deletion or pharmacological means completely reverses the disease phenotype. Of interest, experimental uveitis was also increased in a model of Crohn disease, a systemic autoimmune disease in which patients often develop uveitis, offering a potential mechanistic link between macrophage autophagy and systemic disease. These findings directly implicate the homeostatic process of autophagy in blinding eye disease and identify novel pathways for therapeutic intervention in uveitis.

Activation of Toll-Like Receptor 2 in Acne Triggers Inflammatory Cytokine Responses

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-kappa B activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.