Publications

As initially demonstrated with murine cytomegalovirus (MCMV), plasmacytoid dendritic cells (pDCs) are the major source of IFN-α/β in response to a variety of viruses in vivo. However, contradictory results have been obtained pertaining to the mechanisms promoting IFN-α/β production by pDCs in response to MCMV. In this study we show that TLR7 and TLR9 exert redundant functions for IFN-α/β, IL-12p40, and TNF-α production by pDCs in vivo during MCMV infection. In contrast, we confirm that systemic production of IL-12p70 strictly depends on TLR9. The combined loss of TLR7 and TLR9 recapitulates critical features of the phenotype of MyD88-deficient mice, including a dramatic decrease in systemic IFN-α/β levels, an increase in viral load, and increased susceptibility to MCMV-induced mortality. This is the first demonstration of the implication of TLR7 in the recognition of a DNA virus.

No abstract is provided for this article.

Glomerular disease can be triggered or exacerbated by microbes that activate the immune system by Toll-like receptor (TLR) ligation. TLR9 activation promotes host defenses through the enhancement of innate and adaptive immune responses that facilitate the recruitment of leukocytes to areas of inflammation. We defined the role of TLR9 in experimental crescentic glomerulonephritis. Wild-type mice administered a TLR9 ligand and sheep anti-mouse glomerular basement membrane antibody developed histological injury with impaired renal function, which was attenuated in TLR9 knockout mice. Consistent with enhanced renal injury, wild-type mice exhibited enhanced T helper 1 and T helper 17 cellular immune responses. Kidney mRNA expression of inflammatory cytokines and chemokines as well as leukocyte recruitment were increased in wild-type mice. The use of bone marrow chimeric mice demonstrated that while both bone marrow and tissue cell TLR9 are required for maximal injury, bone marrow TLR9 is more important. Administration of a TLR9 inhibitor before sheep anti-mouse glomerular basement membrane globulin in wild-type mice attenuated cellular nephritogenic immunity that resulted in decreased renal injury. Administration of the inhibitor 7 days after disease initiation decreased glomerular leukocyte recruitment as well as renal injury. These results define the role of TLR9 in experimental crescentic glomerulonephritis and identify therapeutic potential for TLR9 inhibitors in attenuating renal injury, decreasing cellular nephritogenic immunity early in disease, and decreasing kidney effector responses later.

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.

Abstract Natural interferon-producing cells (IPCs) specialize in the production of high levels of type 1 interferons (IFNs) in response to encapsulated DNA and RNA viruses. Here we demonstrate that the secretion of type 1 IFN in response to herpes simplex virus type 1 (HSV-1) in vitro is mediated by the toll-like receptor 9 (TLR9)/MyD88 pathway. Moreover, IPCs produce interleukin-12 (IL-12) in response to HSV-1 in vitro, which is also dependent on TLR9/ MyD88 signaling. Remarkably, though TLR9/MyD88-deficiency abrogates IPC responses to HSV-1 in vitro, mice lacking either MyD88 or TLR9 are capable of controlling HSV-1 replication in vivo after local infection, demonstrating that TLR9- and MyD88-independent pathways in cells other than IPCs can effectively compensate for defective IPC responses to HSV-1.

The cytosolic RNA helicases melanoma differentiation-associated gene 5 and retinoic acid-inducible gene-I and their adaptor IFN-β promoter stimulator (IPS-1) have been implicated in the recognition of viral RNA and the production of type I IFN. Complementing the endosomal TLR, melanoma differentiation-associated gene 5, and retinoic acid-inducible gene-I provides alternative mechanisms for viral detection in cells with reduced phagocytosis or autophagy. The infection route of respiratory syncytial virus (RSV)-via fusion of virus particles with the cell membrane-points to IPS-1 signaling as the pathway of choice for downstream antiviral responses. In the current study, viral clearance and inflammation resolution were indeed strongly affected by the absence of an initial IPS-1-mediated IFN-β response. Despite the blunted inflammatory response in IPS-1-deficient alveolar epithelial cells, pulmonary macrophages, and CD11b(+) dendritic cells (DC), the lungs of RSV-infected IPS-1-knockout mice showed augmented recruitment of inflammatory neutrophils, monocytes, and DC. Interestingly, pulmonary CD103(+) DC could functionally compensate for IPS-1 deficiency with the upregulation of certain inflammatory cytokines and chemokines, possibly via TLR3 and TLR7 signaling. The increased inflammation and reduced viral clearance in IPS-1-knockout mice was accompanied by increased T cell activation and IFN-γ production. Experiments with bone marrow chimeras indicated that RSV-induced lung pathology was most severe when IPS-1 expression was lacking in both immune and nonimmune cell populations. Similarly, viral clearance was rescued upon restored IPS-1 signaling in either the nonimmune or the immune compartment. These data support a nonredundant function for IPS-1 in controlling RSV-induced inflammation and viral replication.

The detection of high titers of antibodies against small nuclear ribonucleoproteins (snRNP) is a diagnostic finding in patients in whom systemic lupus erythematosus (SLE) is suspected. Endogenous RNA molecules within snRNP trigger Toll-like receptor 7 (TLR-7) activation in B cells and dendritic cells, leading to anti-snRNP antibody production, which is associated with the development of immune complex nephritis in SLE. The purpose of this study was to investigate the role of TLR-7 in anti-snRNP antibody production and renal disease in SLE induced by an exogenous factor in the absence of genetic predisposition, using the pristane-induced murine lupus model.Serum autoantibodies, IgG isotypes, and cytokine levels in pristane-treated wild-type and TLR-7-deficient mice were analyzed by enzyme-linked immunosorbent assay. Histopathologic changes in mouse kidneys were determined by light immunofluorescence microscopy. Cell subsets in splenocytes and peritoneal lavage cells from the mice were examined by flow cytometry.We found that anti-snRNP antibody production induced by pristane treatment was entirely dependent on the expression of TLR-7, whereas anti-double-stranded DNA antibody production was not affected by a lack of TLR-7. Impaired anti-snRNP antibody production in TLR-7-deficient mice was paralleled by lower levels of glomerular IgG and complement deposits, as well as less severe glomerulonephritis.TLR-7 is specifically required for the production of RNA-reactive autoantibodies and the development of glomerulonephritis in pristane-induced murine lupus, a model of environmentally triggered SLE in the absence of genetic susceptibility to autoimmunity. Specific interference with TLR-7 activation by endogenous TLR-7 ligands may therefore be a promising novel strategy for the treatment of SLE.

Using interleukin (IL)-18 deficient (IL-18−/−) mice, we examined the role of IL-18 in the host resistance and Th1 response against infection with Cryptococcus neoformans. Fungal clearance in the lung was reduced in IL-18−/− mice, although there was no significant change in the level of dissemination to the brain. The DTH response, as determined by footpad swelling, was also diminished in IL-18−/− mice compared to control wild-type (WT) mice. The levels of IL-12 and interferon (IFN)-γ in the sera were significantly lower in IL-18−/− mice than in WT mice. Spleen cells from infected WT mice produced a high level of IFN-γ upon stimulation with the microbe, while only a low level of IFN-γ production was detected in spleen cells from infected IL-18−/− mice. Administration of IL-18 almost completely restored the reduced response in IL-18−/− mice, while IL-12 showed a marginal effect. These results demonstrated the important role of IL-18 in the resistance and Th1 response of mice to C. neoformans by potentiating the production of IFN-γ.

Established widely metastatic tumor was cured in a transplanted mouse B cell lymphoma model, by the combination of chemotherapy plus intratumoral injection of oligodeoxynucleotides containing unmethylated C-G motifs (CpG). This therapeutic effect required that the CpG be injected directly into the tumor and was dependent on CD8 T cells. Although the efficacy of CpG oligodeoxynucleotides has been thought to depend on the expression of TLR9, we unexpectedly found that tumor rejection did not require host expression of TLR9. By using a TLR9-deficient tumor and a TLR9KO host, we demonstrate that TLR9 expression either by the host or the tumor is required. These results indicate that activation of Ag presentation by cells within the tumor via TLR9 stimulation can be an effective form of immunotherapy. This study forms the basis of an ongoing clinical trial in patients with lymphoma.

Adenovirus vector (Adv) vaccination at a systemic site, such as intramuscular (i.m.) immunization, can induce antigen-specific CD8+ T cell responses in both systemic and mucosal compartments. It remains unclear, however, how antigen-specific CD8+ T cell response is induced in the mucosa. In this study, we found that type-I IFN signaling is required for the induction of mRNA expression of retinal dehydrogenase in the draining lymph nodes following the i.m. Adv vaccination. We show that type-I IFN signaling is required for the induction of antigen-specific CD8+ T cell response in the gut-mucosal compartment following the i.m. Adv vaccination.

Borrelia burgdorferi stimulates a strong inflammatory response during infection of a mammalian host. To understand the mechanisms of immune regulation employed by the host to control this inflammatory response, we focused our studies on adrenomedullin, a peptide produced in response to bacterial stimuli that exhibits antimicrobial activity and regulates inflammatory responses by modulating the expression of inflammatory cytokines. Specifically, we investigated the effect of B. burgdorferi on the expression of adrenomedullin as well as the ability of adrenomedullin to dampen host inflammatory responses to the spirochete. The concentration of adrenomedullin in the synovial fluid of untreated Lyme arthritis patients was elevated compared with that in control osteoarthritis patient samples. In addition, coculture with B. burgdorferi significantly increased the expression of adrenomedullin in RAW264.7 macrophages through MyD88-, phosphatidylinositol 3-kinase (PI3-K)-, and p38-dependent signaling cascades. Furthermore, the addition of exogenous adrenomedullin to B. burgdorferi-stimulated RAW264.7 macrophages resulted in a significant decrease in the induction of proinflammatory cytokines. Taken together, these results suggest that B. burgdorferi increases the production of adrenomedullin, which in turn negatively regulates the B. burgdorferi-stimulated inflammatory response.

Lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α requires the recruitment of two pairs of adaptors to the Toll-like receptor 4 cytoplasmic domain. The contribution of one pair – Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) – to TNF-α expression is not well understood. To clarify this issue, we studied TRAM knockout bone marrow-derived macrophages (BMDM). LPS-stimulated TRAM-deficient BMDM had decreased TNF-α protein expression even at times when TNF-α mRNA levels were normal, suggesting impaired translation. Consistent with this idea, knockdown of TRAM in RAW264.7 macrophages decreased translation of a reporter controlled by the TNF-α 3′ untranslated region, while transfection of TRAM in HEK293T cells increased translation of this reporter. Also consistent with a role for TRAM in TNF-α translation, LPS-induced activation of MK2, a kinase involved in this process, was impaired in TRAM-deficient BMDM. TRIF did not increase translation of the TNF-α 3′ untranslated region reporter when expressed in HEK293T cells. However, BMDM that lacked functional TRIF produced reduced levels of TNF-α protein in response to LPS despite normal amounts of the mRNA. Unlike BMDM, LPS-stimulated TRAM-deficient peritoneal macrophages displayed equivalent reductions in TNF-α protein and mRNA. Our results indicate that TRAM- and TRIF-dependent signals have a previously unappreciated, cell type-specific role in regulating TNF-α translation.

No abstract is provided for this article.

No abstract is provided for this article.

Infection of influenza A virus in mammals induces hyper lung pneumonia, which often causes lethal diseases. FasL is a specific ligand of Fas, which is a type-I transmembrane protein to induce cell death. Previously, it has been reported that the hyper induction of gene expression associated with Fas signal is observed in lethal influenza A virus infection. More importantly, it was also reported that functional mutation of the FasL gene protects the host against influenza A virus infection. These observations suggest that induction of FasL signal is functionally associated with the severity of influenza. However, regulation of the induction of FasL or Fas by influenza A virus infection is still unknown. Here, we demonstrated that FasL is induced after the viral infection, and inhibition of the Fas/FasL signal by treatment with a recombinant decoy receptor for FasL (Fas-Fc) increases the survival rate of mice after lethal infection of influenza A virus as well as functional mutation of the FasL gene in gld/gld mice. In addition, the induction level of FasL gene expression in the lung was correlated with the severity of influenza. We also showed that a variety of types of cells in the lung express FasL after the viral infection. Furthermore, type-I interferon induced by the viral infection was shown to be critical for induction of FasL protein expression in the lung. These findings suggested that expression of FasL protein induced by type-I IFN on the lung cell surface is critical to determine the severity of influenza.