Hee Jeong Kong

IFN Regulatory Factor-4 and -8 Govern Dendritic Cell Subset Development and Their Functional Diversity

Dendritic cells (DCs) are bone marrow (BM)-derived APCs central to both innate and adaptive immunity. DCs are a heterogeneous cell population composed of multiple subsets with diverse functions. The mechanism governing the generation of multiple DC subsets is, however, poorly understood. In this study we investigated the roles of closely related transcription factors, IFN regulatory factor (IRF)-4 and IRF-8, in DC development by analyzing IRF-4−/−, IRF-8−/−, and IRF-4−/−IRF-8−/− (double-knockout) mice. We found that IRF-4 is required for the generation of CD4+ DCs, whereas IRF-8 is, as reported previously, essential for CD8α+ DCs. Both IRFs support the development of CD4−CD8α− DCs. IRF-8 and, to a lesser degree, IRF-4 contribute to plasmacytoid DC (PDC) development. Thus, the two IRFs together regulate the development of all conventional DCs as well as PDCs. Consistent with these findings, IRF-4, but not IRF-8, was expressed in CD4+ DCs, whereas only IRF-8 was expressed in CD8α+ DCs. CD4−CD8α− DCs and PDCs expressed both IRFs. We also demonstrate in vitro that GM-CSF-mediated DC differentiation depends on IRF-4, whereas Fms-like tyrosine kinase 3 ligand-mediated differentiation depends mainly on IRF-8. Gene transfer experiments with double-knockout BM cells showed that both IRFs have an overlapping activity and stimulate a common process of DC development. Nonetheless, each IRF also possesses a distinct activity to stimulate subset-specific gene expression, leading to the generation of functionally divergent DCs. Together, IRF-4 and IRF-8 serve as a backbone of the molecular program regulating DC subset development and their functional diversity.

Cutting Edge: Autoantigen Ro52 Is an Interferon Inducible E3 Ligase That Ubiquitinates IRF-8 and Enhances Cytokine Expression in Macrophages

IFN regulatory factor (IRF)-8 is a transcription factor important for the development and function of macrophages. It plays a critical role in the induction of cytokine genes, including IL-12p40. Immunopurification and mass spectrometry analysis found that IRF-8 interacted with Ro52 in murine macrophages upon IFN-γ and TLR stimulation. Ro52 is an IFN-inducible protein of the tripartite motif (TRIM) family and is an autoantigen present in patients with Sjögren’s syndrome and systemic lupus erythematosus. Ro52 has a RING motif and is capable of ubiquitinating itself. We show that IRF-8 is ubiquitinated by Ro52 both in vivo and in vitro. Ectopic expression of Ro52 enhanced IL-12p40 expression in IFN-γ/TLR-stimulated macrophages in an IRF-8-dependent manner. Together, Ro52 is an E3 ligase for IRF-8 that acts in a non-degradation pathway of ubiquitination, and contributes to the elicitation of innate immunity in macrophages.

Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

Toll-Like Receptor 9 Signaling Activates NF-κB through IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein in Dendritic Cells

Unmethylated CpG DNA binds to the Toll-like receptor 9 (TLR9) and activates NF-κB to induce cytokine genes in dendritic cells (DCs). IFN regulatory factor (IRF)-8/IFN consensus sequence binding protein is a transcription factor important for development and activation of DCs. We found that DCs from IRF-8−/− mice were unresponsive to CpG and failed to induce TNF-α and IL-6, targets of NF-κB. Revealing a signaling defect selective for CpG, these cytokines were robustly induced in IRF-8−/− DCs in response to LPS that signals through TLR4. IRF-8−/− DCs expressed TLR9, adaptor myeloid differentiation factor 88, and other signaling molecules, but CpG failed to activate NF-κB in −/− cells. This was due to the selective inability of −/− DCs to activate I-κB kinase αβ, the kinases required for NF-κB in response to CpG. IRF-8 reintroduction fully restored CpG activation of NF-κB and cytokine induction in −/− DCs. Together, TLR signals that activate NF-κB are diverse among different TLRs, and TLR9 signaling uniquely depends on IRF-8 in DCs.

IRF-8/Interferon (IFN) Consensus Sequence-binding Protein Is Involved in Toll-like Receptor (TLR) Signaling and Contributes to the Cross-talk between TLR and IFN-γ Signaling Pathways

Toll-like receptor (TLR) and interferon-γ (IFN-γ) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-γ and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-α (TNF-α). Comparable synergism was observed between IFN-γ and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-γ enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-κB activation. Interestingly, we found that IRF-8–/– macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-γ plus LPS. Retroviral transduction of IRF-8 into IRF-8–/– macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-γ and TLR signal pathways.

Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus

The beta-defensin-like gene and its cloned isoforms (fBDI-1 to -5) were identified in an expressed sequence tag (EST) library from the early developmental stages of the olive flounder, Paralichthys olivaceus. The fBDI cDNA clones show identical amino acid sequences in 24 residues of the signal peptide and 38 residues of the mature peptide; however, the propiece region varies in sequence and length, from 5 to 15 amino acid residues. The predicted molecular weight of the mature peptide is 3.83 kDa, and its predicted isoelectric point is 4.1, showing anionic properties. The genomic organisation of the isoforms was analysed using bacterial artificial chromosome (BAC) DNA containing the fBDI gene. Southern blotting and sequence analyses of fBDI BAC DNA confirmed that the fBDI isoforms cluster at the same locus and exhibit the conserved gene organisation reported for other fish defensin genes. The fBDI mRNA was expressed constitutively in early developmental stages after hatching, and pathogen challenge induced fBDI expression in the head kidney of juvenile fish. We also produced a recombinant fBDI peptide (smfBD) using the expression plasmid pET32 and examined its bioactivity toward Escherichia coli.

Roseovarius halocynthiae sp. nov., isolated from the sea squirt Halocynthia roretzi

A Gram-negative, non-motile, rod-shaped bacterial strain, designated MA1-6T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea, and was characterized to determine its taxonomic position. Strain MA1-6T grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MA1-6T fell within the clade comprising Ruegeria species and exhibited 95.3-96.5 % similarity to the type strains of recognized Ruegeria species. Strain MA1-6T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid, which is consistent with data for Ruegeria species. The major polar lipids detected in strain MA1-6T and Ruegeria atlantica KCTC 12424T were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA1-6T was 58.6 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain MA1-6T can be distinguished from recognized Ruegeria species. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain MA1-6T is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria halocynthiae sp. nov. is proposed; the type strain is MA1-6T (=KCTC 23463T=CCUG 60744T).

Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis

Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923 bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.

Characterization of Paralichthys olivaceus peroxisome proliferator-activated receptor-α gene as a master regulator of flounder lipid metabolism

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles in lipid and energy homeostasis. Paralichthys olivaceus PPARα (PoPPARα) cDNA was isolated by initial reverse transcription-polymerase chain reaction (RT-PCR) using conserved region among fish species and rapid amplification of cDNA ends (RACE). The full-length of PoPPARα cDNA is 2040-bp long encoding a polypeptide with 505 amino acids and containing a DNA binding domain (C4-type zinc finger) and a ligand-binding domain. PoPPARα was detected from 1 day post-hatch and was highly expressed in the stomach, liver, and intestine of continuously fed flounder, approximately 16 cm in size. PoPPARα mRNA expression was down-regulated in the kidney, stomach, and liver of the 4.5-month-old flounder after a 30 day food-deprivation period. PoPPARα activates the PPAR response element (PPRE)-driven reporter, and treatment with Wy14643, a PPARα agonist, augmented PoPPARα-stimulated peroxisome proliferator response element activity in HINAE and HepG2 cells. PoPPARα activated the expression of fatty acid β-oxidation related genes such as carnitine palmitoyltransferase 1A, medium chain acyl-CoA dehydrogenase, and acyl-CoA oxidase 1 and inhibited the expression of sterol regulatory element binding protein and fatty acid synthase by competitively inhibiting LXR/RXR heterodimer formation. These results suggest that PoPPARα plays an important role in lipid metabolism of olive flounder and that it is functionally and evolutionarily conserved in olive flounder and mammals.