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Jdp2 is an AP-1 family transcription factor that regulates the epigenetic status of histones. Previous in vitro studies revealed that Jdp2 is involved in osteoclastogenesis. However, the roles of Jdp2 in vivo and its pleiotropic functions are largely unknown. Here we generated Jdp2 –/– mice and discovered its crucial roles not only in bone metabolism but also in differentiation of neutrophils. Jdp2 –/– mice exhibited osteopetrosis resulting from impaired osteoclastogenesis. Jdp2 –/– neutrophils were morphologically normal but had impaired surface expression of Ly6G, bactericidal function, and apoptosis. We also found that ATF3 was an inhibitor of neutrophil differentiation and that Jdp2 directly suppresses its expression via inhibition of histone acetylation. Strikingly, Jdp2 –/– mice were highly susceptible to Staphylococcus aureus and Candida albicans infection. Thus, Jdp2 plays pivotal roles in in vivo bone homeostasis and host defense by regulating osteoclast and neutrophil differentiation.

Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to the lysine 63-linked polyubiquitin chain through TAK1-binding protein 2 (TAB2) and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and messenger RNA (mRNA) levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in the TLR signaling pathway.

TGFβ1-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of stress and proinflammatory signals. TAK1 can be activated by inflammation-mediating cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1b (IL-1β), as well as by T- and B- cell receptors (TCR/BCR), and Toll-like receptors (TLRs) signals. Activated TAK1 induces the nuclear localization of NF-kB and the activation of JNK/AP1 by stimulating IKKβ and MKK3/MKK6 phosphorylation respectively. TAK1 has been found to play an important role in inflammation, immunity, T- and B-cell activation, and epithelial cell survival. The TAK1−/ − phenotype is lethal in mice at the early embryonic stage. We found higher levels of TAK1 expression and activity in hematopoietic stem cells and progenitors (HSC/Ps), and reduced expression and activity in differentiated mature hematopoietic cells. To study the role of TAK1 in bone marrow hematopoiesis, we generated inducible-TAK1 knockout mice by crossing TAK1loxp mice with Mx1Cre mice, the latter being an interferon-inducible Cre mouse line. After injection of polyI:C to induce the knockout, we found that all the TAK1 knockout mice died within 8 to 10 days after the first polyI:C injection, showing severe hematopoietic and other defects; heterozygotes were phenotypically comparable to wild-type control animals. The TAK1 deletion in these mice resulted in ablation of bone marrow hematopoiesis due to the loss of C-Kit+ HSC/Ps. Annexin-V staining showed a 3-fold increase in apoptosis in the C-Kit+ HSC/Ps from TAK1 mutant mice compared to those from littermate control mice. Almost all of the mutant animals showed intestinal bleeding as well as other hemorrhaging due to the significant reductions in platelet counts. In reciprocal bone marrow transplantation experiments, we found that the TAK1-mutant bone marrow microenvironment was able to support the growth and function of wild-type HSC/Ps, while HSC/Ps from TAK1−/ − mice failed to grow within the wild-type bone marrow microenvironment. These observations suggest that the bone marrow ablation phenotype which develops in TAK1-mutant mice is the result of intrinsic defects in HSC/P's. We propose that TAK1-mutant HSC/Ps might mediate a survival signal for HSC/Ps stimulated by hematopoietic growth factors and cytokines, such as stem cell factor (SCF). The details of possible mechanisms by which this phenomenon might occur is currently under active investigation by our group.

Nonselective inhibition of PG synthesis augments inflammation in mouse models of airway disease, but the roles of individual PGs are not completely clarified. To investigate the role of PGE(2) in a mouse model of airway inflammation induced by a natural allergen, we used mice lacking the critical terminal synthetic enzyme, microsomal PGE(2) synthase (mPGES)-1. Mice lacking mPGES-1 (ptges(-/-) mice) and wild-type C57BL/6 controls were challenged intranasally with low doses of an extract derived from the house dust mite Dermatophagoides farinae (Der f). The levels of PGE(2) in the bronchoalveolar lavage fluids of Der f-treated ptges(-/-) mice were approximately 80% lower than the levels in wild-type controls. Der f-induced bronchovascular eosinophilia was modestly enhanced in the ptges(-/-) mice. Both Der f-treated strains showed similar increases in serum IgE and IgG1, as well as comparable levels of Th1, Th2, and Th17 cytokine production by Der f-stimulated spleen cells. These findings indicated that mPGES-1-derived PGE(2) was not required for allergen sensitization or development of effector T cell responses. Unexpectedly, the numbers of vascular smooth muscle cells and the thickness of intrapulmonary vessels were both markedly increased in the Der f-treated ptges(-/-) mice. These vascular changes were suppressed by the administration of the stable PGE(2) analog 16, 16-dimethyl PGE(2), or of selective agonists of the E-prostanoid (EP) 1, EP2, and EP3 receptors, respectively, for PGE(2). Thus, mPGES-1 and its product, PGE(2), protect the pulmonary vasculature from remodeling during allergen-induced pulmonary inflammation, and these effects may be mediated by more than one EP receptor.

The differentiation of bone-resorbing osteoclasts is induced by RANKL signaling, and leads to the activation of NF-κB via TRAF6 activation. TRAF family member-associated NF-κB activator (TANK) acts as a negative regulator of Toll-like receptors (TLRs) and B-cell receptor (BCR) signaling by inhibiting TRAF6 activation. <i>Tank</i>^−/− mice spontaneously develop autoimmune glomerular nephritis in an IL-6-dependent manner. Despite its importance in the TCRs and BCR-activated TRAF6 inhibition, the involvement of TANK in RANKL signaling is poorly understood. Here, we report that TANK is a negative regulator of osteoclast differentiation. The expression levels of TANK mRNA and protein were up-regulated during RANKL-induced osteoclastogenesis, and overexpression of TANK <i>in vitro</i> led to a decrease in osteoclast formation. The <i>in vitro</i> osteoclastogenesis of <i>Tank</i>^−/− cells was significantly increased, accompanied by increased ubiquitination of TRAF6 and enhanced canonical NF-κB activation in response to RANKL stimulation. <i>Tank</i>^−/− mice showed severe trabecular bone loss, but increased cortical bone mineral density, because of enhanced bone erosion and formation. TANK mRNA expression was induced during osteoblast differentiation and <i>Tank</i>^−/− osteoblasts exhibited enhaced NF-κB activation, IL-11 expression, and bone nodule formation than wild-type control cells. Finally, wild-type mice transplanted with bone marrow cells from <i>Tank</i>^−/− mice showed trabecular bone loss analogous to that in <i>Tank</i>^−/− mice. These findings demonstrate that TANK is critical for osteoclastogenesis by regulating NF-κB, and is also important for proper bone remodeling.

The E2 ubiquitin-conjugating enzyme UBC13 plays pivotal roles in diverse biological processes. Recent studies have elucidated that UBC13, in concert with the E3 ubiquitin ligase RNF8, propagates the DNA damage signal via a ubiquitylation-dependent signaling pathway. However, mechanistically how UBC13 mediates its role in promoting checkpoint protein assembly and its genetic requirement for E2 variants remain elusive. Here we provide evidence to support the idea that the E3 ubiquitin ligase complex RNF8-UBC13 functions independently of E2 variants and is sufficient in facilitating ubiquitin conjugations and accumulation of DNA damage mediator 53BP1 at DNA breaks. The RNF8 RING domain serves as the molecular platform to anchor UBC13 at the damaged chromatin, where localized ubiquitylation events allow sustained accumulation of checkpoint proteins. Intriguingly, we found that only a group of RING domains derived from E3 ubiquitin ligases, which have been shown to interact with UBC13, enabled UBC13-mediated FK2 and 53BP1 focus formation at DNA breaks. We propose that the RNF8 RING domain selects and loads a subset of UBC13 molecules, distinct from those that exist as heterodimers, onto sites of double-strand breaks, which facilitates the amplification of DNA damage signals.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Mucosal immunity is poorly activated after systemic immunization with protein Ags. Nevertheless, induction of mucosal immunity in such a manner would be an attractive and simple way to overcome the intrinsic difficulties in delivering Ag to such sites. Flagellin from Salmonella enterica serovar Typhimurium (FliC) can impact markedly on host immunity, in part via its recognition by TLR5. In this study, we show that systemic immunization with soluble FliC (sFliC) drives distinct immune responses concurrently in the mesenteric lymph nodes (MLN) and the spleen after i.p. and s.c. immunization. In the MLN, but not the spleen, sFliC drives a TLR5-dependent recruitment of CD103(+) dendritic cells (DCs), which correlates with a diminution in CD103(+) DC numbers in the lamina propria. In the MLN, CD103(+) DCs carry Ag and are the major primers of endogenous and transgenic T cell priming. A key consequence of these interactions with CD103(+) DCs in the MLN is an increase in local regulatory T cell differentiation. In parallel, systemic sFliC immunization results in a pronounced switching of FliC-specific B cells to IgA in the MLN but not elsewhere. Loss of TLR5 has more impact on MLN than splenic Ab responses, reflected in an ablation of IgA, but not IgG, serum Ab titers. Therefore, systemic sFliC immunization targets CD103(+) DCs and drives distinct mucosal T and B cell responses. This offers a potential "Trojan horse" approach to modulate mucosal immunity by systemically immunizing with sFliC.

A rearranged human γ 1 immunoglobulin gene was introduced into fertilized mouse eggs. The phage Ch4A-VCE-γ 1 was constructed by ligating an Eco RI and Bgl II fragment of pBR322-CESSV(CE-l) containing the VDJ region with an EcoRI and BamHI fragment of Ch4A-HJgγ 1 -10 containing the y constant region. About 200 copies of Ch4A-VCE-γ 1 genes were introduced into fertilized mouse eggs. Of 489 eggs injected with these genes, 319 survived and were transferred to oviducts of foster mothers. Thirtyeight mice were born and were screened for the presence of human γ 1 immunoglobulin genes by Southern blot hybridization. Five of these 38 mice had integrated human y immunoglobulin genes. None of the humanγ 1 copies in each mouse had undergone deletions or rearrangements as judged by the Southern blotting patterns for several restriction enzymes. Human γ 1 gene was present in several different tissues. All the mice tested so far transmit the human γ 1 gene to a fraction of their offspring in an autosomal dominant manner. Spleen cells from transgenic mice were analyzed for immunoglobulin production by reverse plaque assay or immunofluorescence staining of cytoplasmic immunoglobulin, but synthesis and secretion of human γ 1 chains could not be detected. No human γ 1 immunoglobulin mRNA was detected in the liver and spleen of a transgenic mouse. The presence of the human γ 1 inimunoglobulin gene appeared to have no effect on the expression of endogenous mouse immunoglobulin genes.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls expressions of several genes involved in cell survival, proliferation and differentiation, and tissue inflammation. However, the significance of pancreatic STAT3 in acute pancreatitis remains unclear. We generated conditional STAT3 knockout (stat3 Δ/Δ ) mice by crossing stat3 flox/flox mice with Pdx1-promoter Cre transgenic mice. Caerulein administration activated pancreatic STAT3 and induced acute pancreatitis as early as 3 hours in wild-type mice, and full recovery from the induced pancreatic injury was observed within 7 days. The levels of serum amylase and lipase and histologic scores of pancreatic necrosis and inflammatory cell infiltration were significantly higher at 3 hours in stat3 Δ/Δ mice than in stat3 flox/flox mice. Pancreatic recovery after pancreatitis was significantly delayed in stat3 Δ/Δ mice compared with stat3 flox/flox mice. Although stat3 flox/flox mice had marked production in the pancreas of pancreatitis-associated protein 1 (PAP1), a serum acute phase protein, this induction was completely abrogated in stat3 Δ/Δ mice. Enforced production of PAP1 by a hydrodynamic procedure in the liver significantly suppressed pancreatic necrosis and inflammation and also promoted pancreatic regeneration and recovery in stat3 Δ/Δ mice to levels similar to those observed in stat3 flox/flox mice. In conclusion, pancreatic STAT3 is indispensable for PAP1 production, and this STAT3/PAP1 pathway plays a protective role in caerulein-induced pancreatitis.

Summary Because MyD88 transduces a core set of Toll-like receptor (TLR)-induced signals, microbial-induced host responses can be divided broadly into the MyD88-dependent and MyD88-independent pathways. A specific pathogen induces a distinct pattern of host response dependent upon the signalling pathways employed. Recently, we demonstrated that a MyD88-dependent pathway is essential for the development of early (4–8 h) host response to Pseudomonas aeruginosa lung infection. Here, we show that the development of a delayed (24–48 h) host response to P. aeruginosa is independent of MyD88. Using MyD88-deficient mice, the production of macrophage inflammatory protein 2, tumour necrosis factor and interleukin 1α in the airway was observed following P. aeruginosa lung infection for 24 or 48 h. Moreover, the MyD88-deficient mice recruited sufficient neutrophils in the lung and cleared the bacteria efficiently from the lung after 48 h. Thus, the full development of host responses to P. aeruginosa lung infection involves, in a sequential, stepwise fashion, a MyD88-dependent early response and a MyD88-independent delayed mechanism.

The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIM S , a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.

The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-α and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-κB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner.