[Show abstract][Hide abstract] ABSTRACT: The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs including 5' -triphosphate RNA and 5' capped 2' -O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2' -O MTase mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5' -triphosphate RNA, but more preferentially associated with 5' capped 2' -O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2' -O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5' structures.
Journal of Virology 07/2013; 87(18). DOI:10.1128/JVI.00883-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.
[Show abstract][Hide abstract] ABSTRACT: Absent in melanoma 2 (AIM2) is a sensor of cytosolic DNA that is responsible for activation of the inflammasome and host immune
responses to DNA viruses and intracellular bacteria. However, the role of AIM2 in host defenses against Mycobacterium tuberculosis is unknown. Here, we show that AIM2-deficient mice were highly susceptible to intratracheal infection with M. tuberculosis and that this was associated with defective IL-1± and IL-18 production together with impaired T
1 responses. Macrophages from AIM2-deficient mice infected with M. tuberculosis showed severely impaired secretion of IL-1± and IL-18 as well as activation of the inflammasome, determined by caspase-1 cleavage. Genomic DNA extracted from M. tuberculosis (Mtb DNA) induced caspase-1 activation and IL-1±/IL-18 secretion in an AIM2-dependent manner. Mtb DNA, which was present in the cytosol, co-localized with AIM2. Taken together,
these findings demonstrate that AIM2 plays an important role in M. tuberculosis infection through the recognition of Mtb DNA.
International Immunology 06/2012; 24(10):637-44. DOI:10.1093/intimm/dxs062 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ROP18 kinase has been identified as a key virulence determinant conferring a high mortality phenotype characteristic of type I Toxoplasma gondii strains. This major effector molecule is secreted by the rhoptries into the host cells during invasion; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, we show that ROP18 targets the host endoplasmic reticulum-bound transcription factor ATF6β. Disruption of the ROP18 gene severely impairs acute toxoplasmosis by the type I RH strain. Because another virulence factor ROP16 kinase modulates immune responses through its N-terminal portion, we focus on the role of the N terminus of ROP18 in the subversion of host cellular functions. The N-terminal extension of ROP18 contributes to ATF6β-dependent pathogenicity by interacting with ATF6β and destabilizing it. The kinase activity of ROP18 is essential for proteasome-dependent degradation of ATF6β and for parasite virulence. Consistent with a key role for ATF6β in resistance against this intracellular pathogen, ATF6β-deficient mice exhibit a high susceptibility to infection by ROP18-deficient parasites. The results reveal that interference with ATF6β-dependent immune responses is a novel pathogenic mechanism induced by ROP18.
Journal of Experimental Medicine 06/2011; 208(7):1533-46. DOI:10.1084/jem.20101660 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin- A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c(+) cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c(+) cells (iDTRDelta mice). We successfully deleted CD11c(+) cells in bone marrow-derived DCs in vitro and splenic CD11c(+) cells in vivo after DT treatment in iDTRDelta mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.
Biochemical and Biophysical Research Communications 06/2010; 397(3). DOI:10.1016/j.bbrc.2010.05.157 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.
Journal of Experimental Medicine 11/2009; 206(12):2747-60. DOI:10.1084/jem.20091703 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88(-/-)Trif(-/-) mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-gamma was normally induced in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca(2+) level. Furthermore, T. cruzi-induced IFN-gamma expression was blocked by inhibition of Ca(2+) signaling. NFATc1, which plays a pivotal role in Ca(2+) signaling in lymphocytes, was activated in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells. T. cruzi-infected Nfatc1(-/-) fetal liver DCs were impaired in IFN-gamma production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells.
The Journal of Immunology 01/2009; 181(12):8521-7. DOI:10.4049/jimmunol.181.12.8521 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secretory leukocyte protease inhibitor (SLPI) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guérin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guérin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.
The Journal of Immunology 04/2008; 180(6):4032-9. DOI:10.4049/jimmunol.180.6.4032 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The
mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is
critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is
mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However,
the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that
a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex
through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like
protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded
phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85
complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results
demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.
Journal of bacteriology 12/2007; 189(22):8241-9. DOI:10.1128/JB.00550-07 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although prostaglandin E2 (PGE2), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES-1) are known to play a role in various inflammatory events, their roles in the pathogenesis of gastro-oesophageal reflux disease are not known.
We examined the dynamics of COX-1, COX-2, mPGES-1, mPGES-2, cytosolic PGES (cPGES), and PGE2 synthetic activity in rat acid reflux oesophagitis and the effects of COX-2 inhibitors on the severity of oesophagitis.
Acid reflux oesophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus. Rats were killed on day 3 (acute phase) or day 21 (chronic phase) after induction of oesophagitis.
Expression of COX-2 and mPGES-1 was markedly increased in oesophagitis while modest changes in COX-1, cPGES, and mPGES-2 expression were observed. COX-2 and mPGES-1 were colocalised in epithelial cells of the basal layer, as well as inflammatory and mesenchymal cells in the lamina propria and submucosa. COX-2 inhibitors significantly reduced the severity of chronic oesophagitis but did not affect acute oesophageal lesions. COX-2 inhibitors significantly inhibited the increase in PGE2 synthesis in oesophageal lesions on both days 3 and 21. Epithelial proliferation was significantly increased in the basal layer on day 21. Inflammatory cells and epithelial cells of the basal layer exhibited reactions for EP4 in oesophagitis.
PGE2 derived from COX-2 and mPGES-1 plays a significant role in the pathogenesis of chronic acid reflux oesophagitis, and possibly in basal hyperplasia and persistent inflammatory cell infiltration.
Gut 05/2006; 55(4):450-6. DOI:10.1136/gut.2005.081943 · 14.66 Impact Factor